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T cell-dependent antiviral mechanisms in the pathogenesis of mouse hepatitis virus in the central nervous system
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T cell-dependent antiviral mechanisms in the pathogenesis of mouse hepatitis virus in the central nervous system
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T CELL-DEPENDENT ANTIVIRAL MECHANISMS IN THE PATHOGENESIS OF MOUSE HEPATITUS VIRUS IN THE CENTRAL NERVOUS SYSTEM by Beatriz Parra A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Microbiology) May 2001 Copyright 2001 Beatriz Parra R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UMI Number: 3 0 2 7 7 6 3 UMI UMI Microform 3027763 Copyright 2001 by Beii & Howell Information and Learning Company. Ali rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 90007 This dissertation, written by under the direction of h.£T....... Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of re quirements for the degree of & £ f \ T R l Z P A R R A DOCTOR OF PHILOSOPHY Dean of Graduate Studies Date DISSERTATION COMMIT' Chairperson R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Beatriz Parra Stephen A. Stohlman ABSTRACT Antiviral Mechanisms in the Pathogenesis of JHMV: Cell type specific effectors in viral clearance within the central nervous system (CNS) Replication of the neurotropic JHM strain of mouse hepatitis virus (JHMV) in the CNS is controlled by virus specific CD8+ T cells. CD8+ T cell antiviral effector mechanisms during acute disease were examined to provide insight into the mechanisms responsible for viral persistence within the CNS. CD8+ T cells lyse virus infected cells using the perforin-mediated pathway of cellular cytolysis. Cytolysis can also be mediated via the Fas dependent pathway. In addition, CD8+ T cells secrete cytokines, i.e., interferon gamma (IFN-y), with anti-viral activity. Perforin mediated cytotoxicity contributes to viral clearance from astrocytes and microglia. However, clearance from oligodendroglia in the absence of perforin-mediated cytolysis suggested the participation of a perforin independent mechanism(s). Data in this thesis demonstrate that Fas-mediated cytotoxicity, by either CD8+ or CD4+ T cells, is not essential for clearance of infectious JHMV from the CNS. CD8+ T cell- mediated control of JHMV replication during acute CNS infection is exerted by a combination of IFN-y and perforin-mediated cytolysis in a cell type 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. dependent manner. The relative contributions of IFN-y and perforin-dependent CD8+ T cell function to viral clearance were examined in both gene deficient mice and immunoincompetent SCUD mice following adoptive transfer of virus- specific CD8+ T cells derived from mice in which the BFN-y gene had been disrupted by homologous recombination. Elimination of IFN-y secretion by either vims specific CD8+ or CD4+ T cells abrogated the ability to reduce vims replication in both SCID mice and mice in which both the IFN-y and perforin genes had been disrupted. In both types of recipients histological analysis showed that CD8+ T cells lacking IFN-y, but retaining perforin mediated cytolysis, reduced vims from most major histocompatibility class I expressing cells within the CNS (microglia and astrocytes) but to a significantly reduced extent from oligodendrocytes. Thus, both CD8+ T cell-perforin mediated cytolysis and T cell derived IFN-y play complementary roles in controlling the JHMV infection within the CNS in cell type specific manner. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Dedication To my family, especially to my husband Adolfo and my son Alejandro. Their love, encouragement and support helped me complete the program. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Acknowledgements I wish to thank the people and institutions who encouraged and supported me during the PhD. First and foremost I would like to thank my dissertation director, Dr. Stephen A. Stohlman for his patience, teaching and continuing guidance throughout this graduate career. I would also like to thank my dissertation committee and other members of the Microbiology and Neurology Department, Dr. Stanley Tahara, Dr. Cornelia C. Bergmann, Dr. David Hinton and Dr. Stohlman's laboratory staff for their teaching, support and friendship over the years. I also would like to thank Mrs. Sonia Quiroz Garcia for helping me with the manuscript. Finally, I would like to thank the Microbiology Department at the Universidad del Valle Cali-Colombia and to Colciencias-Colombia for supporting me to complete these advanced studies. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Table of Contents Chapter Page I. Introduction 1 II. Contributions of Fas-Fas Ligand and Interactions to 36 the Pathogenesis of Mouse Hepatitis Virus in the Central Nervous System. III. Kinetics of Cytokine mRNA Expression in the Central 54 Nervous System Following Lethal and Nonlethal Coronavirus-Induced Acute Encephalomyelitis. IV. Gamma Interferon is Required for Viral Clearance 93 from Central Nervous System Oligodendroglia. V. Contributions of CD8+ T Cell-Derived IFN-y to Acute 133 JHMV Infection of the CNS. VI. Contributions of CD4+ derived IFN- y to Viral 185 Clearance and Pathogenesis. VI. Conclusions. 215 VII. Bibliography. 225 iv R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. List of Tables Table Page Chapter V 1. DNA primers sequences used to screen IFNy'7 ’/?'7 " mice. 140 2. Summary of clinical disease in IFNy7'/?'7 'm ice and wt 150 mice. 3. Summary of survival in IFNyv7P_ /' mice and wt mice. 150 4. Summary of T cell infiltration in the CNS of JHMV- 153 infected IFNy7'/?'7 " mice and wt mice. 5. Summary of the histopathological features in the CNS 158 of double deficient IFNy'/7 F /~ mice and control WT mice at various days post infection. 6. Summary of T cell infiltration in the CNS of CD8+ - 160 reconstituted IFNy^TP7' micea (H2d ). Chapter VI 1. Morbidity of T cell adoptive transfer SCID mice. 196 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. List of Figures Figure Page Chapter II 1. Comparison of the morbidity, mortality and kinetic of 43 JHMV replication in the brain of B6.MRL-Faslp r (lpr) and C57BL/6 (wt) mice. 2. CNS inflammation and apoptosis in Fas-deficient 46 B6.MRL Fas (lpr). 3. JHMV replication in the CNS of chimeric mice. 49 Chapter III 1. Comparison of virus replication, accumulative mortality 67 and anti-viral antibody synthesis during a sublethal (2.2v-l) or lethal (JHMV) infection of the CNS. 2. Histologic sections of brains at day 7 post infection with 69 JHMV or 2.2vl. 3. Kinetics of IFN-y (A), iNOS (B) and TNF(C)-a mRNA 71 accumulation in the CNS of mice during a sublethal (2.2v-l) or lethal (JHMV) encephalomyelitis. 4. Kinetic of IL-12 (A), ILl-p (B), IL-loc (C) and IL-6 (D) 74 mRNA expression in the brain at various times after i.e. infection of C57BL/6 mice with JHMV or the neutralization resistant 2.2V-1 variant. 5. Kinetic of accumulation of IL-10 (A) and IL-4 (B) 75 mRNA in the CNS of C57BL/6 mice with a lethal or sublethal encephalomyelitis after infection with JHMV or 2.2v-l variant respectively. vi R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter IV 1. Morbidity (Panel A), mortality (Panel B), and viral replication (Panel C) in IFN-y'7 " and control wt mice following infection with the 2.2vl variant of JHMV. 2. Cytotoxic activity in JHMV-infected IFN-y'7 ' and control wt mice. 3. Specific cytokine secretion from CLN cells and cytokine mRNA expression in brains of 2.2v-l infected IFN-y'7 ' and wt mice. 4. Kinetics of JHMV specific Ab in infected IFN-y'7 ' and wt mice. 5. Encephalitis and JHMV Ag in the brains of control wt mice (A) and IFN-y'7 ' (B) at 7 days p.i. 6. JHMV Ag in the spinal cords of IFN-y'7 ' and control wt mice at 21 days p.i. 7. Viral Ag positive cells in the brain of IFN-y'7 ' mice at 21 days p.i. Chapter V . 1. Strategy to generate double IFN-y- and perforin- deficient mice. 2. Uncontrolled CNS viral replication in the absence of both IFN-y and perforin-dependent cytotoxicity. 3. Increased CD8+ T cells numbers in the CNS of double deficient IFNy'/P'7 ' after 14 days p.i. with JHMV. 4. Specific proliferative T cell response in the periphery of JHMV-infected IFN-y '7 '/P'7 ' mice. 108 111 114 115 117 118 120 139 151 154 156 vii R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 5. CD8+ T cell-derived IFN-y contributes to viral 159 clearance in the CNS. 6. Vims specific CD8+ T cells reduce CNS JHMV 162 replication via IFN-y. 7. Viral antigen in oligodendroglia is reduced via CD8+ 164 T cell-derived IFN-y. 8. Virus-specific CD8+ T cells from IFN-y"7 ' donors are 167 recruited into the CNS of SCID mice. 9. MHC class I expression on microglia during JHMV 170 infection is upregulated independently of CD8+ T cell-derived IFN-y. 10. Demyelination in CD8+ T cell reconstituted SCID 171 mice following infection with JHMV. Chapter VI 1. Characterization of T cell donor cell populations. 192 2. CD4+ T cells infiltrate the brain parenchyma of 197 SCID mice. 3. CD4 T cells induce demyelination during JHMV 198 infection. 4A. Vims specific CD4+ T cells reduce viral replication 201 in CNS via IFN-y dependent and IFN-y independent mechanisms. 4B. IFN-y mRNA levels in the CNS of T cell-adoptive 201 transfer JHMV- infected SCID mice. viii R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter I. Introduction Inflammation within the central nervous system (CNS) plays an important role in a variety of CNS disorders, including viral infections (93,119) and immune-mediated disease (31,33). The initiation and the outcome of inflammation in the CNS is the result of a balance between immune privilege and the immune response (67). The local barrier and immunosuppressive environment constitutes immune privilege within the CNS. The presence of a tight blood-brain barrier (BBB) plays an essential role in restricting the entry of molecules and cells into the CNS (3,20,111). The absence of draining lymph nodes prevents direct communication between the CNS and the immune system. The brain resident cells constitutively express low levels of major histocompatibility molecules (MHC) and adhesion molecules, preventing recognition by T cells (70). However, it is also clear that the CNS is not refractory to T cell mediated immune responses. T cells can enter the brain parenchyma in the absence of obvious pathologies (132) and leukocyte migration is dramatically upregulated during CNS inflammatory reactions (20,33). However, acute immune responses within the CNS differ significantly from acute inflammatory responses in the periphery. Leukocyte recmitment into the CNS is delayed, occurring within days instead of hours, as observed at the peripheral sites (7,33). T cells accumulate initially in the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. perivascular regions, ventricles, and in the subarachnoid space and at late stage of disease T cells accumulate in significant numbers at the parenchymal sites of pathology (33,94,118). Pathological conditions, which may be chronic or fatal, can result from the interaction between viruses and the CNS environment. Examples of these viral-induced diseases include, subacute sclerosing panencephalitis (SSPE), progressive multifocal leukoencephalophathy (PML), tropical spastic paraparesis (TSP) and human immunodeficiency virus (HIV) encephalitis (10,82,114,127). Pathogenesis of these human diseases is associated with persistent viral infections in differentiated cells within the CNS. Furthermore, immune-mediated diseases (72) are also thought to be triggered by persistent viruses. The detailed mechanisms by which viruses maintain long-term persistent CNS infections are unknown. Viral persistence may result from the immune restrictive nature of the nervous system to avoid sustaining damage. Brain cells also encode molecules that attenuate viral transcription (35), thus protecting or limiting antiviral responses allowing virus to persist. Animal models are indispensable to understand the events by which viruses and the anti-viral immune responses cause CNS disease. The immune mechanisms of viral clearance, viral persistence and viral-induced CNS demyelination have been analyzed in several murine models (72,99). The R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. accumulated evidence suggests that, for demyelination to result, a sequence of events must occur. First, the immune response must be sufficient to clear virus and prevent fatal encephalitis (93,96,105,106). Second, the virus is not cleared completely, and persists in the white matter (16,47,57). Finally the immune system contributes to the process of myelin damage (44,77). After a virus has entered the CNS there are different possible outcomes, depending on the balance between the virus and the host immune system (93). Fatal disease may result from rapid viral spreading of virulent strains of viruses in the absence of immune responses [influenza, lymphocytic choriomeningitis virus (LCMV); (102)]. However, fulminant disease also occurs in the presence of inflammatory responses that either clear viral infection [neurotropic strain of mouse hepatitis virus (MHV), JHMV; (56,64,106)] or are ineffective in viral clearance [adenovirus;( 13 6)]; though these responses also lead to extensive CNS tissue damage. By contrast, infection with attenuated viral strains induces acute encephalomyelitis that is resolved by an effective immune response (26,34,38). Various immune effector mechanisms appear to make contributions to the resolution of these CNS viral infections. CD8+ T cells are key effectors of virus clearance in the CNS and they operate primarily via perforin-mediated cytolysis (16,64,101) and via cytokine secretion (25,39,53). Moreover, CNS viral clearance also requires the participation of additional R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. effectors mechanisms mediated by either CD4+ T cells and B cells including cytokines and antibodies (25,34). CD4+ T cells protect and reduce virus loads from the CNS via IFN-y (24,25). CD4+ T cells also provide help to the humoral response critical to the resolution of other virus infections (34). Furthermore, a CD4+ T cell response seems to be essential for maintaining CD8+ T cell antiviral effector function within the CNS (104). Regardless of the predominant immune effector mechanism utilized to resolve acute infection, many viral infections within the CNS become persistent following acute viral clearance. For example, persistent viruses primarily infecting neurons are eliminated either by CD8+ T cell perforin-dependent cytolysis [Theiler’s murine encephalomyelitis virus (TMEV);(88)], CD4+ T cell-derived IFN-y [measles virus;(25)] or humoral responses [alphavirus;(34)]. Nevertheless, these viruses persist, either at the same site of acute infection (alphavirus, measles), or spread to and persist in a different cell type (TMEV). Limited immune responses within the CNS may predispose the virus to persistence, which then results in immunopathology. Although the mechanism(s) of viral persistence remain unclear, sterilizing immune responses during the acute infection appear to be critical in preventing viral persistence. Therefore, defining the relative strength and contribution of distinct immune mechanisms involved in acute viral clearance is one key to R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. clarifying the events by which viruses and the anti-viral immune responses cause CNS disease. CD8+ T cells are critical effectors for the control of many acute viral CNS infections (16,93,96,105,134). Nevertheless, the contribution of distinct mechanisms involved in viral clearance and CNS pathogenesis are less clearly defined compared to viral infections in the peripheral organs. Effector CD8+ T cells are capable of lysing virus infected cells via the perforin/granzyme pathway or the CD95/CD95 Ligand (Fas-FasL) pathway (49,50,51). Both pathways stimulate the caspase cascade in the target cells, leading to apoptosis (95). Studies in perforin- and Fas-deficient mice suggest that only a minor proportion of the cytolytic activity of CD8+ T cells is mediated by the Fas- FasL pathway, whereas the major cytolytic mechanism is perforin-dependent cytotoxicity (50,51,90,100). Upon recognition of infected targets, CD8+ T cells also secrete a variety of antiviral cytokines, such as interferon gamma (IFN-y) and tumor necrosis factor-a (TNF-a) (74,81,89,91) and chemokines that can directly inhibit virus replication or drive the recruitment of additional inflammatory cells (14). Although, TNF-a is a protective cytokine against some viral infections (81,89), IFN-y is believed to be the first line of host defense in the control of most viral infections. The antiviral effects of IFN-y include both direct and indirect activities. It induces proteins and enzymes that R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. directly inhibit viral replication by blocking the accumulation of viral mRNA (46). Indirect effects of IFN-y include increased expression of MHC class I and II molecules on Ag- presenting cells (APC) (22,141), and regulation of Ag processing by augmenting proteolysis in the proteosomal compartment (5,140). Both the cytolytic effector function as well as the expression of cytokines by CD8+ T cells is regulated through T cell receptor (TCR) -dependent signals (95,97). It has been suggested that the predominant effector mechanism utilized in CD8-mediated anti-viral protective responses correlates with the ability of the virus to induce cellular in vitro cytopathogenicity (49,50). For example, immunity to noncytolytic virus is mediated by perforin-dependent cytotoxicity (49). By contrast, noncytolytic effector mechanisms (cytokines, antibody) mediate protection from cytopathic viruses (VV, VSV, Semliki forest virus) (50). Nevertheless, recent studies suggest that cytokines limit infections by both lytic and non lytic viruses including VV, Semliki forest virus, Theiler’s virus (TMEV), influenza virus, herpes simplex virus, measles virus, and cytomegalovirus (CMV) (11,23,25,36,117). Viral clearance without cytolysis may reflect a key strategy to eradicate pathogens without destroying the infected cell. The role of perforin-dependent cytolysis in viral clearance from the CNS is less clear than its role in clearance or control of infections in the periphery. For example, perforin-deficient mice infected with lymphocytic R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. chroriomeningitis virus (LCMV) are protected from otherwise acute severe CNS disease; however, the mice succumb to peripheral infection (50,122). These studies suggest that perforin is involved in both viral-induced acute CNS disease and protection from lethal infection in the periphery. By contrast, perforin deficient mice succumb to acute infection with Theiler’s murine encephalomyelitis virus (TMEV). Death is associated with high CNS viral replication (88), suggesting a crucial role of perforin-dependent cytolysis in CNS viral clearance. Clearance of the neurotropic mouse hepatitis virus (MHV) strain JHM (JHMV) from the CNS during the acute phase induced by either lethal or subacute infections is at least partially dependent on CD8+ T cell perforin-dependent cytolysis (64). CNS viral loads were elevated and clearance of JHMV from CNS was delayed in perforin-deficient mice. However, JHMV-induced fatal encephalitis was delayed in the absence of perforin (64), suggesting that perforin-mediated cytotoxicity contributes to clinical disease. These data indicated that CD8+ T cell perforin-dependent cytolysis is beneficial in mediating viral clearance, but may in the process induce pathology within the CNS. CD8+ T cells are also thought to exert antiviral action by focusing IFN- y locally at the site of infection (55,91). Initial evidence for the antiviral role of IFN-y in the CNS came from studies of LCMV and VV, two infections R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. primarily controlled by CD8+ T cells. Adoptive transfer of CD8+ T cells from immune donors to mice persistently infected with LCMV eliminated virus from the CNS in the absence of any tissue damage or apparent infiltration, suggesting the participation of soluble factors secreted by CD8+ T cells (80). Moreover, LCMV-immune splenocytes from IFN-y deficient mice failed to clear persistent infections of wild type mice (115). Similarly, nude mice cleared infection with a recombinant neurotropic strain of VV expressing IFN- y, but die from infection with the control virus (91). Neutralization of IFN-y abrogated the antiviral action of T cells against vaccinia and vesicular stomatitis virus (VSV) in the CNS but not in peripheral tissues (55,91). IFN-y is also critical to control neuronal infection with measles virus (25), and TMEV (85,87); however, there is no definitive proof that CD8+ T cells were the direct source of IFN-y. These data are consistent with the idea that CD8+ T cells may predominantly use nonlytic effector mechanisms to clear viral infections from tissues where extensive damage may be detrimental to the host such as brain and liver. In agreement with this concept, hepatitis B virus (HBV) infection is cleared from hepatocytes of transgenic mice by virus- specific CD8+ T cell clones in an IFN-y- and TNF-a dependent fashion (36,37). Similarly, persistent liver infection by LCMV appears to be eliminated by IFN-y secreted by virus-specific T cells (115). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. To control viral infections in the CNS, CD4+ T cells also produce a spectrum of cytokines and chemokines involved directly in viral clearance and also in cell recruitment and activation (25,124). CD4+ T cells also provide help to Ab production which appears to successfully resolve alphaviruses- induced acute encephalomyelitis (34) and terminate CNS infection with a neurotropic strain of influenza virus (17). CD4+ T cells modulate CD8+ T cell responses (121), including the priming of CD8+ T cells during viral infections in peripheral tissues (45). However, induction of virus-specific CD8+ T cells can also be T-helper-independent (13). This is in agreement with the CD4+ T cell-independent priming of CD8+ T cells in response to several viral infections of the CNS (TMEV, JHMV, Measles). CD4+ T cells are also essential for sustaining CD8+ T cell cytotoxic function and the control of chronic virus infection in peripheral organs (2,52). Conclusive evidence that CD4+ T cell-mediated help is critical to CD8+ T cell anti-viral function in the CNS is lacking. Consistent with the possible role of CD4+ T cells in the* maintenance of CD8+ T cell effector function in CNS, depletion of CD4+ T cells abrogated the protective role of virus-specific CD8+ T cells to the infection with JHMV (104). CD4+ T cells can also exert anti-viral cytotoxic functions and their major lytic mechanism is mediated by the Fas-FasL system (48,100). Despite the evidence that Fas-FasL interactions contribute to the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. immune-mediated pathology of CNS diseases (18,92), information on the role of this lytic pathway in viral clearance from the CNS is lacking. MHV, a member of the coronaviruses, produces enteric, hepatic, respiratory, and CNS infections. Some strains of MHV are only hepatotropic (41), some are primarily neurotropic (86), and others are both hepatotropic and neurotropic (61,120). The ability of the JHM strain to cause both acute and chronic CNS disease makes it a valuable model to dissect the interactions between the virus and the host’s immune response in the CNS environment. Some features of the chronic CNS infection by JHMV resemble characteristics of the immune-mediated demyelinating disease multiple sclerosis (MS). Therefore, this model is also helpful in elucidating induction mechanisms of both encephalomyelitis and demyelination. The pathogenesis of JHMV has been extensively reviewed (44,56,58,108). In the mouse model, the outcome of the infection depends on virus strain, genetic susceptibility and route of infection (15,56). Infection of susceptible (C57BL/6 or BALB/c) but not resistant (SJL/J) mice with wild type (wt) JHMV results in acute fatal encephalomyelitis with extensive neuronal involvement (15,56,106). By contrast, infections with neuroattenuated variants induce minimum mortality, but increased demyelination (21,26,29,38,86,107,125). During acute infection these attenuated variants preferentially infect glial cells, including R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. oligodendrocytes, microglia and astrocytes, but rarely neurons (44,56). Virus is cleared from the CNS approximately after two weeks after infection; however, viral RNA and occasionally Ag may persist for long periods of time (1,39). Viral persistence is associated with ongoing chronic demyelination (29,38). The immune response to JHMV infection of the CNS involves a number of potential effector mechanisms including cellular and humoral responses. These include CD8+ , and CD4+ T cells (9,105,134), and both neutralizing and nonneutralizing virus-specific antibodies (12,28,62). Whereas T cell responses appear to control acute viral replication (9,134), antibody responses appear to inhibit reactivation of persisting virus during the chronic stages of disease (63). Depletion of either subset of T cells in infected mice during acute infection abrogated virus clearance, emphasizing the role of both CD8+ and CD4+ T cells in protective responses to JHMV in the CNS (113,134). However, a crucial direct role in clearing JHMV from the CNS has been attributed primarily to CD8+ T cells (105,139). By contrast, CD4+ T cells induced during JHMV infection seem to be essential for maintaining continued CD8+ T cell effector capacity and survival (104,113). The crucial role of CD8+ T cells in clearing JHMV from the CNS is based on several observations: 1) Phenotypic analysis of infiltrating cells during JHMV infection showed virus- 11 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. specific CD8+ T cell accumulation within the CNS coincident with viral clearance (9); 2) Depletion of CD8+ T cells with monoclonal antibodies abrogates CNS virus clearance (113); 3) Adoptive transfer of virus-specific CD8+ T cells protect mice from acute infection (105,139). Transferred cells inhibit viral replication in all CNS cell types, although to a lesser extent in oligodendrocytes (105). The role(s) of the individual CD8+ T cell effector mechanisms utilized during resolution of acute JHMV infection is not clear. Virus-specific CD8+ T cells infiltrating the brain parenchyma during the time of maximum viral clearance exhibit perforin-dependent cytotoxicity and produce IFN-y in response to virus-specific CD8+ T cell epitope (9). Moreover, clearance of JHMV from the CNS during the acute phase of either a lethal or subacute infection is at least partially dependent on perforin- dependent cytolysis (64). CNS viral loads were elevated and clearance of JHMV from the CNS was delayed, especially from the MHC-class I expressing microglia and astrocytes following infection of perforin-deficient mice (64). However, eventual control of the infection in these mice also uncovered the potential participation of additional effector mechanisms in viral clearance. JHMV infection also induces vigorous CD4+ T cell responses in the CNS (133,134,68), which provide protection from acute disease (113,134). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The inability of a CD4+ T cell-depleted host to control viral replication suggested that CD4+ T cells are crucial anti-viral effectors during JHMV infections (134,104). However, the precise contribution of this T cell subset in viral clearance is not clear. Adoptive transfer experiments demonstrated the importance of CD4+ T cells in clearance of JHM virus from the mouse CNS. One proposed mechanism was that CD4+ T cells provide help to CD8+ T cell effectors, which clear virus through lysis of infected cells (113). Consistent with this idea, memory CD8+ T cells exhibit decreased survival within the brain parenchyma in a CD4+ T cell depleted host, suggesting CD4+ T cells indirectly contribute to viral clearance by maintaining CD8+ T cell effector function (104). Alternatively, CD4+ T cells may secrete cytokines which act directly on infected cells to mediate clearance. In rats, CD4+ T cells reduced viral load in the absence of CD8+ T cells (54), suggesting direct elimination of virus. In mice, adoptive transfer of CD4+ T cell clones protected from lethal disease via reducing virus replication (139). However, CD4+ T cell clones can also prevent fatal encephalitis without significantly decreasing viral load (110). CD4+ T cells could potentially lyse infected targets via Fas-FasL dependent interactions. However, there is no evidence of direct CD4+ T cell cytotoxic function in the CNS and the role of Fas-mediated cytotoxicity in JHMV pathogenesis has not been investigated. 13 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The notion that T cells may contribute directly to protection via soluble factors such as cytokines is suggested by adoptive transfer experiments into immunocompetent mice (54,105,125). Virus-specific CD8+ T cells eliminated infectious virus from all CNS cell types including the CTL- resistant oligodendroglia (105), suggesting CD8+ T cells may be operating to clear virus from low MHC class I expressing cells through a nonlytic mechanism. Consistent with this idea, virus was completely eliminated from oligodendrocytes in the CNS of perforin-deficient mice (64). Immune competent mice infected with a lethal or wt strain JHMV succumb very early to the infection even though viral titers in the brain have declined to undetectable levels (134,64). These data suggest that the inflammatory response that clears virus may be also implicated in the fatal disease outcome. Therefore, inflammatory cytokines may be involved in both viral clearance and the pathological process. However, the potential role of soluble factors such as cytokines and chemokines as potential mediators of viral clearance or contributors to demyelination has not been carefully examined. Cytokine mRNA expression level were examined in the CNS of mice infected with a strain of JHMV with selective tropism for neurons (83). Infection induced expression of various cytokines during the period of maximal clearance. Particularly relevant was the expression of the inflammatory cytokines IFN-y R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. and TNF-a. However, neutralization of TNF-a did not affect viral clearance or diminish JHMV-induced demyelinating disease (109). By contrast, recombinant IFN-y protected from JHMV-induced liver disease and its neutralization enhanced disease severity (98). When administrated directly into the CNS via a defective-interfering (DI) vector, IFN-y reduced MHV replication and induced a mild disease (142). Additionally, viral clearance of the neuronotropic JHMV strain is delayed in the absence of IFN-y (59). IFN- y is secreted by both CD4+ and CD8+ T cells in response to MHV infection (9,139) and both cell types are crucial effectors of viral clearance. These data support the idea that IFN-y plays a major role in JHMV pathogenesis and prompted the study of the contribution of this cytokine to viral clearance and its role in chronic demyelination. Demyelination is a hallmark of JHMV acute and persistent CNS infection. The immune response associated with viral clearance and protection during the acute phase is thought to be involved in the chronic demyelinating process (43,44,58,123). Several approaches suggest the contribution of T cells in demyelinating JHMV-induced disease. MHV infection of irradiated mice results in uncontrolled vims replication without apparent, or only slight, demyelination (26,43,123). Adoptive transfer of naive splenocytes into SCID mice (43) or transfer of immune anti-MHV splenocytes to irradiated mice R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. restored demyelination (27). The role of T-cell subsets in the demyelinating process is more controversial. Depletion of CD4+ T cells reduces the severity and onset of demyelination (60), supporting a key role of CD4+ T cells in the initiation of demyelination and the contribution of additional mechanism. CD8+ T cells in the CNS parenchyma are found in the parenchyma and in their absence demyelination is diminished but not completely abrogated (32,43). It is unknown whether CD4+ or CD8+ T cells directly mediate demyelination, or provide help to other cells such as macrophages/microglia which may be directly involved in the myelin destruction (137). Demyelination in perforin- deficient mice confirmed that perforin-mediated cytotoxicity is not required for T cell-induced demyelination (64). The contribution of the Fas-FasL cytolytic pathway has not been investigated. Alternatively, damage to oligodendrocytes may result from soluble mediators such as cytokines or chemokines produced by infiltrating T cells or activated macrophages/microglia (129). Chemokines accelerate the inflammatory and demyelinating process in JHMV-induced disease (60). Inflammatory cytokines are also induced during CNS acute MHV infection (83), and staining for tumor necrosis factor - a (TNF-a), IL- 1(3, IL-6 and nitric oxide (NO) is found in demyelinating lesions in mice chronically infected with MHV (112). However, neutralization of TNF-a has no role in modifying the pathogenesis of JHMV-induced demyelination (109). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The role of IFN-y in JHMV-induced demyelination is not known. Previous results demonstrating that IFN-y is important for protection from MHV- induced liver disease (98) point toward the idea that this cytokine may play a protective, rather than a destructive, role in JHMV-induced CNS pathogenesis. Infection with an attenuated strain of JHMV argues against the idea that T cell responses are sufficient to viral-induced pathology (68). Few data exist in support of alternative hypotheses to explain JHMV-induced demyelination. Macrophage infiltration in JHMV -induced lesions suggests a direct role of these cells in the effector phase of demyelination (57,103). However, unlike other models of demyelination, depletion of blood-borne macrophages also does not prevent MHV-induced demyelination (138). Viral RNA quasispecies (1) and CTL escape mutants (84) persist in oligodendrocytes during chronic infection and this type of infection may directly alter cellular functions or enhance viral replication. However, high viral replication in the absence of demyelination in immunodeficient mice (43,123) argues against a direct viral injury theory. Overall, the data suggest that although either CD4+ or CD8+ T cells are necessary for demyelination to proceed, T cells alone are not sufficient for JHMV immune-mediated demyelination. Pathogenesis of JHMV chronic disease is a complex and multifactorial process. Immune responses during the acute phase are sufficient to clear CNS 17 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. virus and prevent fatal encephalitis, but insufficient to provide sterility thus allowing virus to persist in the white matter. As discussed above, immunity to JHMV involves multiple effector mechanisms. Although T cells appear to be the effectors in controlling viral replication during the acute disease, the relative contribution of the different T cell mechanisms to the protective response has not been delineated. This thesis examines the contribution of lytic and nonlytic mechanism to the pathogenesis of acute JHMV-induced disease with emphasis on the role of IFN-y in resistance to the infection. In Chapter II, the contributions of the lytic Fas-FasL pathway to viral clearance and immunopathology are discussed. An analysis of the temporal induction of cytokines during lethal and non-lethal disease was performed (Chapter III) previous to the appreciation of the function of IFN-y in JHMV-pathogenesis. The contribution of IFN-y to JHMV pathogenesis was analyzed in Chapter IV using genetically deficient mice. In general, the exact analysis of immune effector mechanisms in genetically defficient hosts is complicated by the fact that the same mechanism is employed by multiple cell types in response to infection. For example, in addition to CD8+ T cells, CD4+ T cells as well as NK cells produce IFN-y. Some MHC class II-restricted CD4+ T cells and NK are also capable of perforin-and/or Fas-FasL-dependent cytolysis. Similarly, perforin and IFN-y effector mechanisms both have pleiotropic effects on the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. immune system. In addition to their anti-viral activity they also play regulatory roles in T cell homeostasis (perforin) and enhancement of antigen presentation (IFN-y). 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T- cell-mediated clearance of mouse hepatitis virus strain JHM from the central nervous system. J. Virol. 63:3051-3056. 114. Tardieu, M. 1999. HIV-1-related central nervous system diseases. Cur. Opin. Neurol. 12:377-381. 115. Tishon, A., H. Lewicki, G. Rail, H.M. von, and M.B. Oldstone. 1995. An essential role for type 1 interferon-gamma in terminating persistent viral infection. Virology 212:244-250. 116. Topham, D.J., R.A. Tripp, and P.C. Doherty. 1997. CD8+ T cells clear influenza virus by perforin or Fas-dependent processes. J. Immunol. 159:5197-5200. 117. Topham, D.J., R.A. Tripp , Sarawar,S.R., Sangster, M.Y. and Doherty, P.C. 1996. Immune CD4+ T cells promote the clearance of influenza virus from major histocompatibility complex class IF7 ' respiratory epithelium. J. Virol. 70:1288-1291. 118. Tran, E.H., K. Hoekstra, N. van Rooijen, C.D. Jijkstra, and T. Owens. 1998. Immune invasion of the central nervous system parenchyma and experimental allergic encephalomyelitis, but not leukocyte extravasation from blood, are prevented in macrophage- depleted mice. J. Immuol. 161:3767-3775. 119. Tyler, K.L., and F. Gonzalez-Scarano. 1997. Viral diseases of the central nervous system. In Viral Pathogenesis. Neal Nathanson, et al., editors. Lippincott-Raven Publishers, Philadelphia. 837-853. 32 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 120. Virelizier, J.L., A.D. Dayan and A.C. Allison. 1975. Neuropathological effects of persistent infection of mice by mouse hepatitis vims. Imm. 12:1127-1140. 121. Wakeham, C.J., R.M. Paige, and R.M. Zinkernagel, et al. 1991. Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353:180- 184. 122. Walsh, C.M., M. Matloubian, C.C. Liu, R. Ueda, C.G. Kurahara, J.L. Christensen, M.T. Huang, J.D. Young, R. Hmed, and W.R. Clark. 1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA 91:10854-10858. 123. Wang, F., S.A. Stohlman, and J. Fleming. 1990. Demyelination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated. J. Neuroimmunol. 30:31-41. 124. Ward, S.G., K. Bacon, and J. Westwick. 1998. Chemokines and T lymphocytes: more than an attraction. Immunity 9:1. 125. Wege, H., A. Schliephake, H. Korner, E. Flory, and H. Wege. 1993. Coronavirus induced encephalomyelitis: an immunodominant CD4(+)- T cell site on the nucleocapsid protein contributes to protection. Adv. Exp. Med. Biol. 342:413-8. 126. Weiner, L. 1973. Pathogenesis of demyelination induced by a mouse hepatitis. Arch. Neurol. 28:298-303. 127. Weiner, L., R. Herndon, O. Narayan, R. Johnson, K. Shah, L. Rubinstein, T. Preziosi, and F. Conley. 1972. Isolation of virus related to SV40 from patients with progressive multifocal leukoencephalopathy. N. Engl. J. 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Characterization of brain-infiltrating mononuclear cells during infection with mouse hepatitis virus strain JHM. J. Neuroimmunol. 32:199-207. 134. Williamson, J.S.P., and S.A. Stohlman. 1990. Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells. J. Virol. 64:4589. 135. Wong, G.H., and D.V. Goeddel. 1986. Tumour necrosis factors alpha and beta inhibit virus replication and synergize with interferons. Nature 323:819-822. 136. Wood, M.J., H.M. Charlton, K.J. Wood, K. Kajiwara, and A.P. Byrnes. 1996. Immune responses to adenovirus vectors in the nervous system. Trends Neurosci. 19:497-501. 137. Wu, G.F., and S. Perlman. 1999. Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus. J. Virol. 73:8771. 138. Xue, S.,N. Sun, N. van Rooijen, and S Perlman. 1999. Depletion of bood-bome macrophages does not reduce demyelination in mice infected with a neurotropic coronavirus. J. Virol. 73:6327-6334. 34 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 139. Yamaguchi, K., N. Goto, S. Kyuwa, M. Hayami, and Y. Toyoda. 1991. Protection of mice from a lethal coronavirus infection in the central nervous system by adoptive transfer of virus-specific T cell clones. J Neuroimmunol. 32:1-9. 140. Yang, Y., J.B. Waters, K. Fruh, and P.A. Peterson. 1992. Proteasomes are regulated by interferon y: implications for antigen processing. Proc. Natl. Acad. Sci USA 89:4928. 141. Young, H.A., and K.J. Hardy. 1995. Role of interferon-y in immune cell regulation. J. Leukocyte Biol. 58:373. 142. Zhang, X., D.R. Hinton, D.J. Cua, S.A. Stohlman, and M.M.C. Lai. 1997. Expression of interferon-y by a coronavirus defective-interfering RNA vector and its effect on viral replication, spread, and pathogenicity. Virology 233:327-338. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter II. Contributions of Fas-Fas Ligand Interactions to the Pathogenesis of Mouse Hepatitis Virus in the Central Nervous System Summary Fas-FasL interactions, resulting in apoptosis in nervous tissue can have both an antiviral and pathogenic role in JHMV-induced CNS disease. Fas- deficient (Ipr) mice controlled JHMV infection comparable to wt mice. This result suggested that, in contrast to delayed kinetics of viral clearance in perforin-deficient mice, Fas-dependent cytotoxicity does not contribute to JHMV clearance from the CNS. Furthermore, the lack of Fas-dependent cytotoxicity did not prevent JHMV-induced demyelination or apoptosis of infiltrating mononuclear cells, suggesting that Fas-mediated cytotoxicity is not essential for either demyelination or down-regulation of inflammation during JHMV-induced CNS disease. However, the simultaneous absence of both Fas- and perforin-mediated cytolysis resulted in an uncontrolled infection, suggesting redundancy of cytolytic pathways to control virus replication. Experiments using short-term chimeric mice show that in the absence of perforin-dependent cytolysis, virus is cleared only in the presence of Fas- mediated cytotoxicity. These data suggest Fas-mediated cytotoxicity may contribute to control viral replication under conditions where perforin- mediated cytotoxicity is diminished for example, at late stages of the acute disease, after the majority of infectious virus is cleared. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Introduction CD8+ T cells are crucial for viral clearance during acute CNS infection induced by JHMV (20). In general, CD8+ T cells exert antiviral effector functions vza two basic mechanisms: a nonlytic pathway involving cytokines (2,18) and two mechanisms based on cytotoxicity (10,12,13). Major histocompatibility complex (MHC) class I dependent lysis of infected cells via release of perforin containing granules is the main CD8+ T cell cytotoxic pathway (10,11). However, cell-cell interactions between Fas, expressed on infected cells, and Fas ligand (FasL), expressed on activated T cells, can also lead to cytolysis (17). JHMV clearance from the CNS of IFN-y- (Chapter IV) or perforin-deficient mice (13) demonstrated that both lytic and nonlytic mechanisms contribute to controlling viral replication in a cell type dependent manner. Failure of IFN-y-deficient mice to clear virus from oligodendrocytes suggested a critical role of this cytokine in clearance from this cell type (16). On the other hand, analysis of perforin-deficient mice demonstrated that this cytolytic mechanism is important in controlling replication in microglia and astrocytes (13). Although delayed, infectious virus is completely eliminated from the CNS of perforin-deficient mice. This suggests that perforin mediated cytolysis is at least a partially dispensable mechanism for JHMV clearance (13). A reduction in the majority of infectious virus in the absence of IFN-y R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. and delayed clearance in the absence of perforin confirmed a partial but not exclusive contribution of perforin-dependent cytotoxicity and suggested an additional antiviral effector mechanism(s). Fas-FasL interactions regulate a major pathway of apoptosis, which may play an important role in both mediating anti-viral effects and in the pathogenesis of autoimmune CNS diseases. Although Fas is not normally expressed on CNS cells, its expression is upregulated during inflammation (5- 7,19). Expression of Fas receptor on MHC class I negative cells in the CNS, particularly oligodendrocytes could potentially target this cell type for the antiviral cytolytic effector function of FasL positive CD8+ T cells. To investigate functional Fas-FasL interactions contributing to the pathogenesis of JHMV infection in the CNS, viral replication and immunopathology was analyzed in Fas-deficient mice in the presence or absence of perforin-dependent cytotoxicity. 38 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. M aterial and Methods Mice and virus infection B6.MRL-Faslp r (lpr) mice (Jackson Laboratories, Bar Harbor MA) and congenic wt C57BL/6J mice (Jackson Laboratories) were infected at 6-7 wk of age by intracerebral (i.e.) inoculation with 200 plaque forming units (PFU) of the neutralizing monoclonal antibody (mAb) derived antigenic JHMV variant, designated 2.2v-l (8,9,14,16). Viral replication and clinical disease were compared in both groups for 21 days post infection (pi.). Severity of clinical disease was scored as previously described (8): 0, healthy; 1, ruffle fur and hunched backs; 2, slow mobility and inability to return to upright position when turn in its back; 3, paralysis and wasting; 4, moribud and death. Radiation chimeric mice were prepared as described by Tophatn et al. (21). Briefly, lpr and wt mice were lethally irradiated (95OR) and immediately • 7 reconstituted by intravenous (i.v.) injection with 7x10 naive splenocytes from either perforin deficient (P_ / ") (Jackson Laboratories) or syngeneic wt mice (P+ /+ ). Mice were maintained under steril conditions until the end of the experiment. Four days after reconstitution, mice were infected i.e. with 200 PFU of JHMV variant 2.2v-l in 32 pi of cold modified Dulbeccos’s (DPBS), (pH 7,4). The kinetics of viral replication were compared in the four chimeric groups following 13 days post infection (p.i). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Virus titer determination Virus replication in the CNS was determined by plaque assay on DBT cells (14). Mice were sacrificed by CO2 asphyxiation and brains removed. One half of the brain was homogenized in 4.0 ml of (DPBS), using Ten Broeck tissue homogenizers. After centrifugation at 200 X g for 7 min at 4°C, supernatants were collected and assayed for virus titers or stored at -70°C. Samples were serially diluted (ten fold) in minimum essential medium (MEM) containing 10% tryptose phosphate broth (TPB). DBT cells were grown at 90% confluence in 100 mm plates. Plates were washed once with MEM containing 10% TPB and diluted virus samples added (250 pl/plate). After virus adsorption for 90 min at room temperature, plates were overlaid with 10 ml of MEM supplemented with 10% TPB, 1% penicillin-streptomycin (Gemini Bio-Products, Calabasas, CA) and 0.6% agarose. Virus plaques were counted after 48 h of incubation at 37° C. Data are the average of triplicate determinations for groups of at least three samples assayed individually. Virus titer is expressed as the log 10 PFU per gm of brain tissue. Histology For histopathological analysis, mice were sacrificed at different time points postinfection by CO2 asphyxiation. Brain and spinal cords were removed and embedded in paraffin or prepared for frozen sections as described 40 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. (14,16). Processing and staining of the samples were done by Wen Qian. For paraffin processing, samples were fixed for 3 h in Clark’s solution (75% ethanol and 25% glacial acetic acid) prior to embedding. Paraffin embedded sections were stained with either hematoxilin and eosin or luxol fast blue (20). JHMV antigen (Ag) was examined by immunoperoxidase staining (Vectastain-AB C kit; Vector Laboratories, Burlingame, CA) using anti-JHMV mAb J.3.3, specific for the nucleocapside protein (9). Primary antibody (Ab) was detected with horse anti-mouse mAb (Vector laboratories) and AEC or DAB as the chromogen substrates (Vector laboratories). To examine CD8+ and CD4+ T cell infiltration, immunoperoxidase staining of acetone fixed frozen sections was performed as described previously (16), using anti-CD4 (L3T4, PharMingen, San Diego, CA) and anti-CD8 (Ly-2, PharMingen) monoclonal antibodies (mAbs). Primary antibodies were detected with biotinylated rabbit-anti-rat antibody (Ab) and Vectastain ABC kit (Vector Laboratories). Apoptotic cells were detected by the TUNEL assay using the Oncor ApopTag kit (Gaithersburg, MD) according to the manufacturer instructions. 41 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Results and Discussion Lpr and wt mice infected with JHMV exhibited signs of acute encephalitis and paralysis followed by slowly progressive, but almost complete recovery at day 21 p.i. Lpr mice exhibited neither differences in disease severity nor mortality following infection compared to wt mice (Figure 1A, IB). Analysis of the kinetics of JHMV clearance from the CNS showed high level of virus replication 3 days p.i. in both lpr and wt mice. Viral titers subsequently declined in both groups until day 12 p.i. (Fig. 1C), after which infectious virus could no longer be recovered. Therefore, similar kinetics of virus replication and clearance were found in lpr mice compared to wt mice (Fig 1C). In contrast to the absence of perforin-dependent cytolysis (14), these data suggest that Fas/FasL interactions do not contribute significantly to JHMV clearance when perforin or IFN-y mediated pathways are functional. However, it is possible that Fas-mediated cytolysis may counteract the loss of perforin- mediated cytolytic activity by infiltrating virus-specific CD8+ T cells at later stages of infection (4). To determine whether perforin-mediated cytolysis or IFN-y secretion compensated for the absence of Fas-FasL interactions, the magnitude and effector functions of virus-specific CD8+ T cells were compared in the groups R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A. If) P 8 ‘c b lpr wt 4 3 2 1 0 0 3 5 7 9 12 14 16 18 21 45 Days post infection B. 100 - 13. 80 - £ 60 - c n 15 40 - * 20 - 0 - O'—— ® # • -O' ' O' -O • 0 0 3 5 7 10 12 16 21 45 Days post infection lpr wt C. I c CD “ S E O ) D LL 0l 5-1 lpr F T " ~ r 4 - ' 3 - 2 - T r 1 - — -f-'— — - f -1---------r 0 3 5 7 10 12 16 21 Days post infection Figure 1. Comparison of the Morbidity, mortality and kinetic of JHMV replication in the brain of B6.MRL-Faslpr (lpr) and C57BL/6 (wt) mice. Mice were infected with 200 pfu of JHMV. Mice were score for clinical disease (1A) and mortality (IB) as followed: 0,healthy; 1, hunch back and ruffled fur; 3,paralysis and wasting; 4,moribund and death. Data shown are the average clinical scores of at least four mice per time point. Virus titers were determined by plaque assay (i.e.). Each time point represents the mean titer for groups of at least three mice and standard deviations are expressed as error bars. 43 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. of infected mice (experiments were performed by Dr. Mark Lin and Dr. Cornelia Bergmann). Cytotoxic T-lymphocyte (CTL) activity in splenocytes was measured by a 5 1 Cr release assay after 6 days of in vitro stimulation (3,14). Specific IFN-y secreting CD8+ T cells were compared in CNS mononuclear cell infiltrates, cervical lymph node and splenic cells by ELISPOT assay (4). However, no differences were found in either the frequency of IFN-y secreting virus-specific CD8+ T cells or in the CTL activity specific for the S510 epitope in lpr mice compared with wt mice. Finally, virus-specific CD8+ T cells within the brain mononuclear cell infiltrates were quantitated by FACS analysis using a tetrameric MHC-Db-510 peptide complex as previously described (5). A similar proportion of virus-specific CD8+ T cells infiltrated the CNS of both Fas-deficient and wt infected mice. Fas-FasL interactions play two potential roles in the pathogenesis of CNS diseases. First, activation induced T cell apoptosis is mediated via Fas (1,5) suggesting a role in regulating mononuclear cell infiltration during the resolution of acute infection. Second, Fas-FasL interactions are associated with autoimmune CNS demyelination (6,7,22), possibly via oligodendrocyte apoptosis (7). To determine whether Fas mediated cytotoxicity contributes to JHMV induced inflammation, mononuclear cell infiltrates, demyelination and the frequency of apoptotic cells were compared in infected lpr mice and wt R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mice. Analysis of brain and spinal cord revealed no differences in the extent or distribution of inflammatory cells at any time point examined. Mononuclear cell infiltrates were prominent in the brain at 7 days p.i. (Fig 2 A , B) and in the brain and spinal cords at days 12, 14, and 16 p.i. No perivascular mononuclear cell infiltrates remained at day 21 p.i. Although diffuse foci of mononuclear cells were still present in the parenchyma at 21 days p.i, there was no difference between the two groups (Fig. 2C , D). Although numerous apoptotic infiltrating mononuclear cells were present in the CNS of Fas deficient and wt mice at days 12 and 16 p.i no differences in the amount or distribution of apoptotic cells were observed. The number of apoptotic cells declined with time and even at day 21 p.i. no differences were found (Fig. 2E , F). Furthermore, no differences were found in either the distribution or frequency of CD4+ or CD8+ T cells comparing wt and Fas-deficient mice by immunohistochemistry (data not shown), consistent with the analysis of both total and Ag specific CD8+ T cells derived from the CNS mononuclear cell population. These data suggest that down regulation of immunopathology during acute JHMV infection of the CNS is not primarily exerted through Fas-mediated apoptosis. Extensive but equal demyelination was also present in both groups at days 14, 16 and 21 p.i. (data not shown), suggesting that Fas-mediated cytolysis does not contribute to JHMV induced demyelination. Consistent with 45 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 2. CNS inflammation and apoptosis in Fas-deficient B6.MRL-Fas (lpr) mice. Lpr (B,D,F) and C57BL/6J (A,C,E) mice were infected with 200 pfu of JHMV and sacrificed at days 7,12 and 21 p.i. CNS tissues were fixed in 75% ethanol and 25% glacial acetic acid and embedded in paraffin. At day 7, prominent perivascular inflammatory infiltrates (arrows) were found in the brain tissues of both wt (A) and lpr (B) mice. At day 21 p.i., only scattered foci of inflammatory cells (arrows) were found in brain tissues of wt (C) and lpr mice (D); perivascular infiltrates were not longer present. Similar numbers of apoptotic cells (arrows), shown here in the spinal cord at day 21 p.i., were found in the brains and spinal cords of both wt (E) and lpr (F) mice. Hematoxylin and eosin stain (A-D), TUNEL stain (E,F). Bar =200 pm. 46 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. equivalent demyelination, the identical cellular localization of viral Ags in both groups suggested that, in contrast to mice deficient in either IFN-y (16) or perforin (14), Fas-mediated cytolysis does not regulate JHMV replication in a cell type specific manner. The indistinguishable response of Fas-deficient and wt mice to JHMV infection support the concept that the Fas-FasL cytolytic mechanism does not contribute to the resolution of virus induced inflammation. To examine the role of Fas-dependent cytolysis in the absence of perforin, JHMV replication was analyzed in chimeric mice either deficient in Fas, perforin or both and compared to control chimeric mice. All mice developed clinical disease (encephalitis and limb paralysis) following JHMV infection. However, in contrast to wt mice, wt control chimeric mice exhibit higher mortality and did not recover from disease. Experiments were terminated at day 13 p.i. when the mortality in the control chimeric wt group (Fas+/+/P+ /+ ) approached 80%. Viral titers in reconstituted wt mice (Fas+ /+ /P+ /+ ) were higher at 6 days p.i. (Fig. 3A) compared to untreated wt mice (Fig. 1), demonstrating that replication is enhanced at early time points in the irradiated-reconstituted wt recipients compared to untreated wt mice. Virus was eliminated from the CNS of 75% of the control chimeric wt mice by day 11 p.i. and from 80% of these mice by day 13 p.i., the last time point examined. These data are consistent with R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the kinetics of viral clearance in untreated wt mice (Fig. 1). By contrast, the majority of chimeric mice with either only the Fas (Fas+ /+ /P'/') or only the perforin (Fas‘/7P+ /+ ) pathway intact still harbored significant viral titers at day 11 p.i. (Fig. 3). The disparity between delayed viral clearance in Fas " /7 P+ /+ mice compared to Lpr mice may be attributed to higher virus load at day 5 p.i. in all chimeric mice. However, virus was essentially eliminated from the CNS of both the Fas+ /+ /P'/' or Fas'/7P+ /+ chimeric groups by day 13 p.i., indicating delayed viral clearance compared to the wt chimeric group. These data suggest that neither perforin- nor Fas-dependent cytolysis alone are absolute requirements for clearance of JHMV from CNS, similar to the Fas-dependent clearance of Influenza virus (21). In contrast to mice deficient in a single pathway, chimeric mice lacking both pathways were unable to control infectious virus (Fig. 3D). These results suggest that in the absence of one cytolytic pathway, the remaining lytic mechanism is sufficient to control JHMV replication in CNS. Histological analysis of infected chimeric mice showed maximal mononuclear cell infiltration at day 9 p.i. with a slight decrease by day 13 in all groups despite the absence of either cytolytic mechanism. Similar demyelination was present in all four chimeric groups (data not shown). 4 8 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. OS o .E " ! 5 & » » X I 4- O E O) 3 7 6 ) 5 4 3 2 1 ) 0 A . Fas+/+/P+/+ t j iemyelinationlvas present in i t r ~ 7 6 i 5 3 2 1 i 0 B Fas_ /7P+/^ 1 • • nr chimeric glbups ^data not r • shown). 7 6 5 JP 4 3 2 1 0 13 6 9 11 13 9 11 6 Days post infection Figure 3. JHMV replication in the CNS of chimeric mice. Viral titers in brains of reconstituted wt mice (Fas+ /+ /P+ /+ ) (Panel A), Fas-deficient mice (Fas'/7P+ /+ ) (Panel B), Fas-competent mice (Fas+ /+ /P'/‘) (Panel C), and Fas- and perforin double-deficient mice (Fas'7 "/?'7 ") (Panel D) following infection with 200 pfu of JHMV. Viral titers below the detection level (<26 pfu/gm brain) are expressed as 0 values. 49 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Therefore, the similar extent of both inflammation and demyelination in chimeric mice revealed no correlation between the absence of Fas-dependent cytolysis and the accumulation of inflammatory cells or decreased CNS demyelination. These data are consistent with the pathogenesis of JHMV in lpr mice (Fig. 2) and suggest Fas-dependent cytotoxicity is not required for either inhibiting the extent of mononuclear cell infiltration or demyelination during JHMV-induced acute encephalomyelitis. Moreover, normal viral clearance in lpr mice (Fig. 1) and eventual viral clearance in chimeric Fas'7'/P+ /+ mice, but not in chimeric Fas"7 '/?’7 ' mice (Fig. 3), confirmed that in the presence of an intact perforin pathway, Fas-dependent cytolysis is redundant. However, the absence of perform clearly revealed a functional antiviral role of Fas-dependent cytotoxicity in the CNS. The data suggest that Fas-FasL interactions are an alternative cytotoxic pathway that may predominantly be operative in the absence of perforin-dependent cytolysis, which is down regulated during the clearance of infectious virus from the CNS (4). 50 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. References 1. Alderson, M.R., T.W. Tough,T. D. Smith, S. Braddy, B. Falk, K.A. Scbooley, E.G. Goodwin, C.A. Smith, F. Ramsdell and D.H. Lynch. 1995. Fas ligand mediates activation-induced cell death in human T lymphocytes. J. Exp. Med. 181:71-7. 2. Bergmann, C.C., J.D. Altman, D. Hinton, and S.A. Stohlman. 1999. Inverted Tmmunodominance and Impaired Cytolytic Function of CD8 + T Cells During Viral Persistence in the Central Nervous System. J. Immunol. 163:3379-3387. 3. Bergmann, C.C., Q. Yao, M. Lin, and S.A. Stohlman. 1996. The JHM strain of mouse hepatitis virus induces a spike protein-specific Db- restricted cytotoxic T cell response. J. Gen. Virol. 77:315-25. 4. Biron, C.A. 1994. Cytokines in the generation of immune responses to, and resolution of virus infection. Curr. Opin. Immunol. 6:530. 5. Bonetti, B., J. Pohl, Y.-L Gao and C.S. Raine. 1997. Cell death during autoimmune demyelination: effector but not target cell are eliminated by apoptosis. J. Immunol. 159:5733-5741. 6. Dowling P., G. Shang, S. Raval, J. Menonna, S. Cook, and W. Husar. 1996. Involvement of the CD95 (APO-l/Fas) Receptor/Ligand System in Multiple Sclerosis Brain. J. Exp. Med. 184:1513-1518. 7. Fleming, J.O., M.D. Trousdale, F.A. el-Zaatari, S.A. Stohlman, and L.P. Weiner. 1986. Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies. J. Virol. 58:869-875. 8 . Fleming, J.O., S.A. Stohlman, R.C. Harmon, M.M.C. Lai, J.A. Frelinger, and L.P. Weiner. 1983 Antigenic relationship of murine coronavirus: analysis using monoclonal antibodies to JHM (MHV-4) virus. Virol. 131:296-307. 9. Henkart, P.A. 1994. Lymphocyte-mediated cytotoxicity: two pathways and multiple effector molecules. Immunity 1:343-346. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 10. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K.J. Olsen, E.R. Podack, R.M. Zinkernagel, and H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31-7. 11. Kagi, D., F Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata. efcal. 1994. Fas and perforin pathways as major mechanisms of T-cell-mediated cytotoxicity. Science 265:528-530. 12. Kagi, D., P. Seiler, P. Pavlovic, B. Ledermann, K. Burki, R. M. Zinkernagel, and H. Hengatner. 1995. The roles of perforin and fas- dependent cytotoxicity in protection against cytopathic and non cytophatic viruses. Eur. J. Immunol. 25: 3256-3262. 13. Lin, M.T., S.A. Stohlman, and D.R. Hinton. 1997. Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis. J. Virol. 71:383. 14. Lohman, B.L., E.S. Razvi and R.M. Welsh. 1996. T-lymphocyte downregulation after acute viral infection is not dependent on CD95(Fas) receptor-ligand interactions. J. Virol. 70:8199-8203. 15. Parra, B., D.R. Hinton, N.W. Marten, C.C. Bergmann, M.T. Lin, C.S. Yang, and S.A. Stohlman. 1999. IFN-y is required for viral clearance from central nervous system oligodendroglia. J. Immunol. 162:1641-1647. 16. Rouvier, R., M.-F. Luciani, and P. Golstein. 1993. Fas involvement in Ca2+-independent T-cell mediated cytotoxicity. J. Exp. Med. 177: 195-200. 17. Ruby, J. and I. Ramshaw. 1991. The antiviral activity of immune CD8 + T cells is dependent on interferon-y. Lymphokine Cytokine Res. 10:353. 18. Sabelko, K.A., K.A. Kelly, M.H. Nahm, A.H. Cross, and J.H. Russell. 1997. Fas and Fas ligand enhance the pathogenesis of experimental allergic encephalomyelitis, but are not essential for immune privilege in the central nervous system. J. Immunol. 159:3096- 3099. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 19. Topham. D.J., R.A. Tripp, and P.C. Doherty. 1997. CD8 + T cells Clear Influenza Virus by Perforin or Fas-Dependent Processes. J. Immunol. 159:5197-5200. 20. Troutt, C.S. Raine, and J.P. Antel. 1996. Multiple Sclerosis: Fas signaling in oligodendrocyte cell death. J. Exp. Med. 184:2361-2370. 21. Waldner H., R.A. Sobel, E. Howard and V.K. Kuchroo. 1997. Fas- and FasL-Deficient Mice are Resistant to Induction of Autoimmune Encephalomyelitis. J. Immunol. 159:3100-3103. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter III. Kinetics of Cytokine ; mRNA Expression In The Central Nervous System Following Lethal And Nonlethal Coronavirus-Induced Acute Encephalomyelitis Summary The potential role(s) of cytokines in the reduction of infectious virus and persistent viral infection in the CNS was examined by determining the kinetics of cytokine mRNA expression following infection with JHMV. Mice were infected with an antibody-escape variant, which produces a nonlethal encephalomyelitis and compared to a clonal virus population which produces a fulminant fatal encephalomyelitis. Infection with both viruses induced the accumulation of mRNAs associated with Thl and Th2-type cytokines including IFN-y, IL-4 and IL-10. Peak mRNA accumulations were coincident with the clearance of virus and there were no obvious differences between lethally and nonlethally infected mice. TNF-a mRNA was induced more rapidly in lethally infected mice compared to mice undergoing a nonfatal encephalomyelitis. Rapid transient increases in the mRNAs encoding IL-12, type 2 nitric oxide synthase (iNOS), IL -la, IL-ip and IL- 6 occurred following infection. Nonlethal infections were associated with increased IL-12, IL-lp and earlier expression of IL-6 , while lethal infections were associated with increased iNOS and IL -la mRNA. These data suggest a rapid but differential response within the CNS cells to infection by different JHMV variants. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. However, neither the accumulation nor kinetics of induction provide evidence to distinguish lethal infections from nonlethal infections leading to a persistent infection. Accumulation of both Thl and Th2 cytokines in the CNS of JHMV infected mice is consistent with the participation of both cytokines and cell immune effectors during resolution of acute viral-induced encephalomyelitis. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Introduction The goal of the immune response during viral infection is to limit replication via induction of both nonspecific and specific antiviral effectors. Acute viral infections of the CNS result in vigorous, but in some instances limited, host immune responses (38). In contrast to responses in the periphery, where limiting virus replication can generally be carried out with minimal regard to tissue damage, within the CNS checks and balances minimize inflammatory-mediated damage while limiting viral-induced cytopathology. Although a wide range of immune effectors are often induced, predominant anti-viral mechanisms appear related to the pathogenesis strategy of the individual agent. For example, infection of mice with LCMV induces a predominant CD8 + CTL response (25). By contrast, resolution of measles virus-encephalitis in mice is mediated by CD4+ T cells and correlates with the local production of IFN-y (9). Finally, resolution of Sindbis virus-induced encephalitis is related to induction of neutralizing Ab and a pattern of Th2 cytokines within the CNS (50). Variations between viral infections resulting in CNS inflammation prompted an examination of the temporal induction of CNS cytokines during fatal and nonfatal CNS infections by variants of JHMV. In immunocompromised hosts JHMV replicates unchecked in the CNS R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. demonstrating the importance of immune effectors in limiting GNS virus replication (21,16,24). Effector mechanisms implicated in protection and clearance of JHMV from the CNS include cell-mediated immunity and both neutralizing and non-neutralizing antibodies. JHMV provides an interesting paradigm of acute viral encephalitis not only because of its associated demyelination (22,49) but also because some immune effector mechanisms prevent death via directly reducing CNS virus replication while other immune effectors prevent death without significantly altering virus replication (21,15,24). A common theme appears to be prevention of neuronal infection by reducing viral load or preventing neuronal infection, most likely via cytokines. The exact mechanisms of immune mediated protection and clearance of JHMV from the CNS are not yet clear. The antiviral effects of CD8 + T cells appear to be due to direct lysis of infected cells; however, CD8 + and CD4+ T cells may also exert antiviral activity indirectly via cytokine secretion (3). Neither infected neurons nor oligodendrocytes appear susceptible to MHC class I -mediated cytolysis in vivo, consistent with the inability of JHMV- specific CTL to clear virus from infected oligodendroglia (44). Furthermore, clearance of JHMV from the CNS is inhibited, but not abolished, in mice genetically deficient in perforin-mediated cytolysis (26). These data suggest R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the possibility that cytokines contribute to either clearance or protection from JHMV infection. During JHMV infection of the CNS there is an abrupt increase in mRNA encoding IL -la, IL-1(3, IL-6 , TNF-a and IFN-y, at the time of maximal decrease in virus replication and mononuclear cell infiltration (34). No IFN-y mRNA was detected in immunodeficient mice, suggesting this cytokine may be important during viral clearance (34). Consistent with this concept, mice treated with anti-IFN-y are more susceptible to JHMV, while administration of IFN-y provides protection (40). IL-6 , TNF-a and iNOS have also been detected in the CNS during acute JHMV infection (45,43,23) while IL-1(5, IL-6 , TNF-a and iNOS were detected in the CNS of chronically infected mice (45). The complex interactions of multiple immune effector mechanisms during JHMV infection may reflect both the relative immune privilege of the CNS (38) as well as the specific tropism of the virus for CNS cell types. Neurotropic MHV isolates differ in tropism and include viruses with predominant tropisms for astrocytes, microglia and oligodendroglia as well as neurons (11,35,21,34,43). The balance between limiting viral replication and preserving CNS function occasionally results in incomplete viral clearance and a persistent CNS infection which may or may not involve the continued presence of infectious virus (21,16). Persistence of infectious virus correlates with the presence of CTL escape variants (36). To understand R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the complex interrelationships between encephalitis, protection and viral clearance leading to a persistent infection of the CNS, the expression of pro- and anti-inflammatory cytokine mRNAs in the CNS of mice undergoing either lethal or sublethal JHMV infection were compared. The two JHMV chosen for study infect either primarily microglia and astrocytes, less frequently oligodendroglia and neurons (DM) or primarily oligodendroglia, much less frequently microglia and astrocytes and rarely neurons (2.2v-l). These viruses contrast to the predominantly neuronotropic OBLV-60 variant previously examined (34,23). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Material and Methods Mice and Viruses C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) at 6 week and maintained in the University of Southern California vivarium. To produce a lethal infection, mice were infected i.e. with 100 pfu of the plaque purified DM isolate of JHMV (41) in a volume of 32 pi. This virus has the plaque size and pathogenesis similar to the parental suckling mouse brain pool of JHMV originally described by Weiner (49) and produces a lethal encephalomyelitis with minimal demyelination apparent at the time of death. To produce a sublethal infection, mice were infected with 25 pfu of the 2.2v-l mAb-derived neutralization resistant variant of JHMV (11). This variant replicates predominantly in oligodendroglia producing a flaccid paralysis. Although viral Ag is cleared from survivors by 30 days post infection (p.i.), viral RNA persists for at least 12 months (1). Groups of at least 3 mice were sacrificed at various times p.i. Immunosuppression was induced by lethal irradiation (850R) 24 hr prior to infection. Sham infected mice were injected i.e. with 32 pi of sterile endotoxin free DPBS. Virus titration Virus titers were determined by plaque assay using monolayers of DBT cells as previously described (41; Chapter II). One half of the brain was R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. homogenized using Tenbrock tissue homogenizers in 2.0 ml of PBS, pH 7.4. The remaining half was taken for histopathology or RNA extraction (see below). Following centrifugation at 1,500 g for 7 min at 4° supernatants were assayed immediately, or frozen at -70°. Data presented are the average titer o f groups of three or more mice. Antibody titration JHMV specific IgM, IgGl and IgG2a antibodies (Ab) were quantitated by ELISA as previously described (26). Briefly, serum-free JHMV (DM n > strain) supernatant containing approximately 10 pfu/ml was diluted 1:50 with DPBS to coat ELISA plates (Immulon II, Dynatech Laboratories, Inc., Chantilly, VA) by overnight incubation at 4°C. Plates were blocked with 10% FCS in DPBS, pH 7,2 for lb. Serially diluted serum samples (10% FCS in PBS, pH 7,2) were added and incubated overnight at 4°C. Mouse antibodies were detected with rabbit anti-mouse IgM, IgGl or IgG2a biotinylated antibodies (Cappel, Costa Mesa, CA). Bound biotinylated secondary antibodies were detected with avidin-peroxidase used at 1 : 1 0 0 0 dilution in 10% FCS in DPBS (Sigma Chemical Co.) for 1 h at RT. A solution of o- phenylenediamine (OPD; Sigma Chemical Co) diluted in 0.1 M citrate buffer pH 5.0 as a chromogen substrate was used as per manufacturer instructions. Samples were read on a automated ELISA reader (Dynatech, Minireader II) at 6 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 495 nm. Antibodies titers were expressed as the highest dilution with O.D. values three times above background level. Neutralizing antibodies were tested in serum by plaque reduction neutralization assay as previously described (26). Briefly, sera were heat inactivated at 56°C for 30 min and serially diluted (five-fold dilutions) with serum free MEM. Diluted samples were mixed with an equal volume of DM strain of JHMV containing approximately 200 PFU per each 200pl. After lh at 37°C, the residual nonneutralized virus was quantitated by TCID50 on DBT cells, using Falcon 96 well tissue culture microplates (Becton Dickinson Co, Franklin Lakes, NJ). Briefly neutralization titer was calculated as the reciprocal of the Ab dilution reducing the number of plaques by 50%. Histology Histopathologic analysis was performed as previously described (43; Chapter I). Briefly, tissues were fixed for 3 hr in Clark's solution (75% ethanol, 25% glacial acidic acid) and embedded in paraffin. Sections were stained with hematoxylin and eosin or luxol fast blue. Staining was performed by Wen Qian. Distribution of JHMV Ag was examined by immunoperoxidase staining using the anti-JHMV mAh J.3.3 specific for the viral nucleocapsid protein (10). Analysis of the samples was done in collaboration with Dr. David Hinton. 62 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Cytokine mRNA expression Brains were processed individually to prevent contamination. RNA was isolated from half brains by homogenization at room temperature in guanidinium isothiocyanate using Tenbrock tissue homogenizers as previously described (7). Samples were sheared by repeated passage through at 21 g needle prior to centrifugation through 5.4 M cesium chloride (lOOmM EDTA; pH 7,0) at 100,000 x g for 18 hr to isolate RNA. The cDNA were prepared using avian myeloblastosis reverse transcriptase (Promega, Madison, WI) and oligo dT primers (Promega) for 60 min at 42°C. Expression of cytokine mRNA was determined by semi-quantitative PCR analysis, as previously described (7,8). Briefly, PCR was performed using AmpliTaq DNA polymerase (Perkin Elmer, Branchburg) and specific cytokine primers for IFN-y, IL -la, IL-ip, IL- 4, IL-6 , IL-10, TNF-a (31,8) and IL-12p40. The sequences of the IL-12p40 oligonucleotides primers and probe used are as follows: 5’ primer, GAC CCT GCC CAT TGA ACT GGC, 3’ primer, CAA CGT TGC ATC CTA GGA TCG, oligoprobe, TGT CTG CGT GCA AGC TCA GGA. Amplification was carried out using 35 cycles of one denaturation step at 94° (45s), primer annealing at 59° (45s), extension step at 12° (2.5 min) followed by a final extension step for 7 min. For IL-4 and iNOS a nested PCR was performed by using internal primers in a second round of PCR (25 cycles) under the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. conditions described above. The oligonucleotide primers used in the second PCR for IL-4 were the corresponding sequences for the 5’-primer and the probe described by Cua et al., (8 ). The nucleotide sequence for the IL-4 oligonucleotide probe was TTG AAG GAG GTC AGA GGA GAA GGGA (39)7). The 5’ and 3’ outer primer sequences for iNOS were GCC TTC CGC AGC TGG GCT GT and ATG TGG TAG CCA CAT CCC GAG CC respectively (28). Internal 5’ and 3’ iNOS primers were AGC TAC TGG GTC AAA GAC AAG AGG CT and the 3’ outer primer respectively. The oligonucleotide probe consisted of the sequence CTC CCT TCC GAA GTT TCT GGC AGC A. For quantification, PCR products were two fold diluted in denaturing solution (0.4 N NaOH, 25 mMEDTA) to a volume of 100 pi. Diluted samples were then neutralized with equal volume of Tris HCI (1.0 M; pH 8.0) and transferred to 0.45pm Nytran membranes (Schleicher , Schuell, Keene, NH) using a Minifold I dot blot apparatus (Schleicher , Schuell). Membranes were hybridized (60°; overnight) with 32P-ATP labeled internal oligonucleotide probes. Membranes were washed (three times; 2x SSC, 0.1% SDS; room temperature), exposed to Storage Phosphor Screens (Molecular Dynamics, Sunnyvale, CA) and scanned using a phosphorimaging scanner (Molecular Dynamics). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Radioactive signals of cytokine cDNA were quantified and normalized to the house-keeping enzyme hypoxanthine phosphoriboxyltransferase (HPRT) values to adjust for differences in cDNA as previously described (7,8). The sample with the highest specific activity was designated the 1 0 0 % maximal response and values for the remainder were derived as percentage of the highest value. Data shown are mean values for 3-4 mice at each time point ± 1 standard deviation. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Results Acute and Subacute JHMV-Induced Encephalitis Fatal encephalomyelitis induced by JHMV is associated with minimal demyelination (21,16). This contrasts with infection by 2.2-vl which produces an acute nonfatal encephalomyelitis with extensive demyelination (11,48). Although both viruses replicated rapidly to high titer in the CNS (Fig. 1A), JHMV-infected mice succumbed within 8 days while 2.2-vl-infected mice underwent a subacute disease with little or no mortality (Fig IB). Peak 2.2v-l replication was at day 3 while the peak of JHMV replication was delayed until day 5. 2.2v-l clearance began at day 5 p.i. and by day 7 virus was nearly undetectable. By contrast, titers in JHMV infected mice initially decreased at day 7 p.i. and detectable virus was still present in the CNS of moribound mice at day 8 p.i. (Fig. 1 A). During lethal JHMV infection, virus replication within the CNS is not reduced as rapidly as in mice which survive infection (Fig. 1 A) consistent with the notion that rapid clearance correlates positively with protection. Consistent with these findings, immunohistologic examination of the brains of JHMV-infected mice at day 7 showed abundant viral Ag in regions of encephalitis while only focal residual viral Ag was found in 2.2vl- infected animals (Fig. 2). Encephalitis was prominent in mice infected with 6 6 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. either 2.2vl or JHMV and no differences in the amount or distribution of mononuclear cell infiltrates were found at day 7 (Fig. 2). 2.2V-1 JHMV 5 100 <V-*e 2.2v-1 JHMV 1 3 5 7 9 1 1 Days post infection 1 3 5 7 9 11 13 Days post infection - ® - lgG1 — © — lgG2a 4 3 2 1 0 5 10 15 20 25 30 Days post infection Figure 1. Comparison of virus replication, accumulative mortality and anti-viral Ab synthesis during a sublethal (2.2v-l) or lethal (JHMV) infection of the CNS. Panel A. Kinetics of virus clearance from the CNS. Panel B. Cumulative mortality. Panel C. Kinetics of JHMV-specific serum IgGl and IgG2a in 2.2v-l-infected mice. Each point represents data for 3 or more mice per group. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. No serum neutralizing antibodies were detected in either group by day 9 post infection, even though the virus titer in the CNS had declined over 3 logio (26; data not shown). In contrast to neutralizing antibodies, IgM was first detected at day 5 post 2.2v-l infection (data not shown) and both IgGl and IgG2a were detected as early as 7 day p.i. (Fig. 1C). The IgGl and IgG2a response suggest the absence of a shift toward either a Thl or Th2 type response reported to be involved in the response to Sindbis virus-induced encephalitis (50). Promflammatory Cytokines The mRNA encoding IFN-y increased in both groups of mice through day 5 post infection, consistent with the rapid accumulation of both NK and T cells in the CNS of infected mice (52,53) (Fig. 3A). No IFN-y mRNA was detected in either sham infected mice or in infected immunodeficient mice. During the lethal JHMV infection IFN-y mRNA did not increase between day 5 and day 7. However, in mice undergoing a sublethal infection the level of IFN-y mRNA continued to increase to day 7 and remained elevated suggesting the possibility that IFN-y is important following infection with a JHMV variant tropic for oligodendroglia. Even though IFN-y mRNA increased during the early phase of infection, a sharp transient increase in iNOS mRNA was detected at day 5 p.i. in mice with a lethal encephalomyelitis (Fig. 3B). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 2. Histologic sections of brains at day 7 post infection with JHMV or 2.2vl. One half of the brain was fixed in Clark’s fixative, embedded in paraffin and stained either with hematoxylin and eosin (A,B) or by immunohistochemical methods for viral Ag using mAh J3.3 (C,D). Encephalitis was widespread after infection in either JHMV (A) or 2.2vl (B); no differences in amount or distribution of inflammation were found. Glial cells and neurons positive for viral Ag were present throughout the areas of encephalitis at day 7 after JHMV infection (C) after 2.2v-l infection only very rare degenerating cells were positive for viral Ag (D). Magnification x 80 (insert x 320). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 70 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. £ —o— JHMV Sham —■> -IR-JHMV - * - â– IR-2.2V-1 iNOS TNF-a c u.y — o ' ( / ) < g 0.6 - i— CL 5 S 0 .3 - a ) > nr. . 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15 Days post infection Days post infection Days post infection Figure 3. Kinetics of IFN-y (A), iNOS (B) and TNF(C)-a mRNA accumulation in the CNS of mice during a sublethal (2.2v-l) or lethal (JHMV) encephalomyelitis. RNA was extracted from the brains of groups of C57BL/6 mice infected with JHMV or the neutralization resistant 2.2v-l variant at various times p.i. The levels of cytokine mRNA determined by semi-quantitative dot blot, normalized to the level of HPRT mRNA and expressed as a relative amount value obtained for comparison. Sham infected, irradiated JHMV-infected (IR-JHMV) and irradiated 2.2v-l-infected (IR-2.2v- 1) mice represented as a single point. Each point is the mean value for at least 3 mice per group. Only a slight increase in iNOS mRNA was detected in mice undergoing subacute encephalomyelitis. Interestingly, infection of immunodeficient mice with JHMV induced the accumulation of iNOS mRNA to approximately 50% the level found in infected immunocompetent mice, suggesting a direct response to viral infection. The increase in iNOS and TNF-a mRNA in immunodeficient mice, which showed no evidence of IFN-y mRNA, suggests TNF-a may also contribute to iNOS mRNA induction (5,13). Consistent with R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. this notion, TNF-a mRNA was first detected at day 3 in mice undergoing a lethal infection and at day 5 in mice sublethally infected (Fig. 3C). Similar to the kinetics of IFN-y, TNF-a mRNA increased until death of lethally infected mice. In mice undergoing a sublethal infection, TNF-a mRNA declined following the peak of virus replication and approached baseline levels by 14 day p.i. Similar to both TNF-a and iNOS, IL-12 is secreted from macrophages during the induction of cell mediated immunity and protects from a number of viral infections via a IFN-y-dependent mechanism (33,32). No IL- 12 mRNA was found following sham infection; however, IL-12 mRNA increased rapidly and peaked at 3 days following both infections (Fig. 4A). Increased IL-12 mRNA also occurred in immunodeficient mice at 3 days p.i., suggesting a direct response to infection which may be related to the recently described IFN-y-independent induction of IL-12 (14). IL-12 mRNA levels decreased after day 3 and nearly approached base line levels found in uninfected mice by 14 day p.i. The IL-ip mRNA level found at day 1 p.i. declined by 3 day p.i. consistent with induction of an early transient increase in IL-lp mRNA in sham infected mice (Fig. 4B). IL-lp mRNA peaked at day 5 following sublethal infection and subsequently declined as virus was cleared from the CNS. Following a lethal infection, the quantity of IL-ip mRNA increased R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. from day 3 p.i. until death. IL -la mRNA peaked at day 3 p.i. in the mice undergoing a subacute infection (Fig. 4C) and then declined but never returned to baseline. In lethally infected mice the peak of IL -la mRNA was delayed (day 5 p.i.) and then declined as the animals succumbed to infection (Fig. 4C). IL- 6 mRNA peaked at day 5 post infection in lethally infected mice and declined by day 7 as virus was cleared from the CNS (Fig. 4D). In contrast to the lethal infection, the levels of IL- 6 mRNA increased rapidly and peaked at day 3 p.i. following subacute infection. The level then declined rapidly by day 5 and had reached baseline levels by day 9 p.i. No IL- 6 mRNA was detected in sham infected mice, suggesting a rapid response to virus infection. Very low levels of IL- 6 mRNA were detected in immunodeficient. mice infected with either virus. Th2-Related Cytokines. IgGl and IgG2a virus-specific antibodies were detected in survivors of JHMV infection; however, there appeared to be little relationship between induction of antibody and control of JHMV infection within the CNS. Induction of both isotypes suggest that Thl and Th2 cytokines are induced by JHMV infection. The kinetics of IL-10 mRNA induction was of interest due to the association of IL-10 with reduced Thl activity in vitro and with remission during experimental allergic encephalomyelitis (19). IL-10 mRNA was first R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. on E c o w C /5 0 1_ Q. X 0 0 > jo 0 on B 1.2 IL-12 0.9 0.6 0.3 0.0 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15 Days post infection Days post infection IL-1a > 0.0 JHMV 2.2v-1 Sham IR-JHMV IR-2.2V-1 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15 Days post infection Days post infection Figure 4. Kinetic of IL-12 (A), IL l-p (B), IL -la (C) and IL-6 (D) mRNA expression in the brain at various times after i.e. infection of C57BL/6 mice with JHMV or the neutralization resistant 2.2V-1 variant. Each point is the mean value for at least three mice. 74 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. detected at day 3 p.i. in lethally infected mice, but not until day 5 post infection in the CNS of the mice undergoing a subacute encephalitis (Fig. 5). However, at the time most lethally infected mice were about to succumb to infection (day 7), there was no difference in the peak levels of IL-10 mRNA between the two groups. The kinetics of IL-10 mRNA accumulation differed between the groups; IL-10 mRNA accumulation in mice undergoing a sublethal infection was slower and remained at peak levels until day 9 p.i., prior to declining to near basal levels by day 14. No IL10 mRNA was detected in the CNS of sham infected or infected immunodeficient mice. < z B c c E o IL-4 c n ( / ) a > a. x 0) > 0.0 1 3 5 7 9 11 13 15 - e — 2.2v-1 IL' 10 JHMV 1 3 5 7 9 11 13 15 Days post infection Days post infection Figure 5. Kinetic of accumulation of IL-10 (A) and IL-4 (B) mRNA in the CNS of C57BL/6 mice with a lethal or sublethal encephalomyelitis after infection with JHMV or 2.2 v-1 variant respectively. Each point is the mean value for at least three mice. No IL-4 mRNA was detected following a single amplification during lethal or sublethal JHMV infections. However, after a second amplification, R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. low abundant mRNAs were detected (Fig. 5 A). No IL-4 mRNA was detected in either sham infected or infected immunosuppressed mice following two amplifications (data not shown). The kinetics of IL-4 mRNA expression following acute and subacute infections showed that the levels increased in parallel through day 7 p. i. (Fig. 5 A). In 2.2v-l-infected mice, the level of IL-4 mRNA continued to increase until day 9 p.i. and then declined slightly by day 14. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Discussion JHMV produces an acute CNS infection associated with several immune effector mechanisms including both CD4+ and CD8+ T cells (21,16). Kinetic analysis of cellular CNS infiltrations during JHMV infection of mice shows that NK cells accumulate prior to CD8+ T cells which in turn precede accumulation of CD4+ T cells and macrophages (52,53). There is no direct evidence for a role of NK cells in suppressing JHMV replication (15); however, CD8+ CTL appear to be critical immune effectors (51,44). Recent analysis of JHMV pathogenesis in mice deficient in perforin suggests that in addition to cytolytic effectors other immune components also contribute to sterilizing immunity (26). Similarly, the adoptive transfer of virus-specific CD4+ T cells to JHMV infected mice demonstrates that some clones protect via reducing viral replication (55), while others protect without reducing virus replication (42), suggesting that cytokines may play an important role in providing sterile immunity. In general the kinetics of cytokine mRNA expression correlated with the temporal presence of CNS infiltrating mononuclear cells. Many cytokine transcripts, with the exceptions of IL-12, IL -la, IL-6 were maximally expressed by 7 day p.i., near the peak inflammatory cell infiltration and during the elimination of virus from the CNS (52,53). Previous data using the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. OBLV-60 JHMV variant which has a selective tropism for neurons suggested a correlation between IFN-y induction, T cell accumulation and reduction of virus replication (34). The semi-quantitative kinetic analysis of IFN-y mRNA in the CNS of mice undergoing both lethal and sublethal JHMV infections supports the positive correlation between IFN-y and viral clearance. However, the OBLV-60 JHMV variant is cleared from the CNS of IFN-y deficient mice (23), consistent with IFN-y exhibiting poor in vitro anti-JHMV activity (57) and inability of rIFN-y to inhibit CNS virus replication (40). These data contrast with other viral-induced encephalopathies in which IFN-y plays a significant role (20,9), including some (56) but not all (50) neuronotropic viruses. The kinetics of IFN-y mRNA induction suggests that it may play a more prominent role in the pathogenesis of JHMV variants with predominant tropisms for microglia, astrocytes (JHMV) or oligodendroglia (2.2v-l). The isotype diversity of the anti-JHMV antibody response, suggests that both Thl and Th2 subsets of CD4+ T cells are activated during infection. IL-4 mRNA accumulation in the CNS corresponds to infiltration of Th2 cells (7) and kinetic analysis suggests that T cells expressing Th2 cytokine profiles are recruited into the CNS with nearly equal kinetics in both lethally and sublethally infected mice (Fig. 5 A). IL-4 increases the severity of encephalitis (17), and could potentially play a role in JHMV persistence via inhibition of with perm ission of the copyright owner. Further reproduction prohibited without perm ission. viral clearance (29). In support of the recruitment of Th2 cells, IL-10 mRNA also increased with kinetics similar to those of IFN-y and IL-4. Whether this difference in detection of Th2 cytokines is due to differences in mouse strains or the selective tropism of the virus is not known. It is interesting that although IL-10 is secreted by activated microglia in vitro (27), no IL-10 mRNA was detected in sham infected or immunodeficient mice. This contrasts with other cytokine mRNA detected in either sham infected or virus infected immunodeficient hosts (see below). TNF-a mRNA is induced following JHMV infection (34,43,45) and TNF-a is present during both the acute and persistent JHMV infections. TNF- a mRNA is not translated in JHMV-infected cells (43) although it may be secreted by adjacent but not infected cells. In addition, inhibition of TNF-a, which prevents experimental autoimmune encephalitis (37), has no effect on either JHMV-induced encephalitis or demyelination (43). As anticipated, based on the relative tropism of the two viruses analyzed, TNF-a mRNA accumulated initially in the CNS of mice infected with JHMV. However, by day 5 p.i. there was little difference in the levels of TNF-a mRNA in the two groups. Finally, the level of TNF-a mRNA decreased with increasing time following sub acute infection, consistent with the resolution of encephalitis. It is interesting that the CNS of mice with active macrophage-mediated R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. demyelination (day 14 p.i.) showed little evidence of TNF-a mRNA, consistent with the inability of anti-TNF-a to prevent JHMV-mediated demyelination (43). A surprising number of mRNAs peaked relatively early following JHMV infection. The mRNAs encoding iNOS, IL-12, IL -la, IL-lp, and IL-6 peaked either prior to, or coincident with initiation of viral clearance. In most cases (except iNOS mRNA) the levels were either higher, or increased more rapidly, in the mice undergoing subacute infections. Accumulation of iNOS mRNA was first detected in mice undergoing a lethal infection coincident with the initial detection of IFN-y mRNA. However, the mRNA levels declined as virus replication declined, suggesting a direct effect of virus on iNOS induction. In contrast to lethal infections, iNOS mRNA lagged detection of IFN-y in mice undergoing subacute infections and increased to less than 50% the level detected in mice undergoing a lethal infection. Similar to the recent data demonstrating low levels of iNOS in the CNS of both nude mice and mice deficient in IFN-y (23), iNOS mRNA in immunodeficient mice was approximately 50% the levels detected in the CNS of intact mice at day 3 p.i. Although JHMV is susceptible to inhibition by iNOS in vitro, iNOS is not associated with in vivo protection (23). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. IL-12, predominantly produced by cells of the myelomonocytic lineage, is associated with the induction of Thl CD4+ T cells (4). IL-12 mRNA peaked early (day 3) in mice undergoing both lethal and sublethal JHMV infections. However, no significant differences were found comparing mRNA levels in immunodeficient mice to intact mice. This may suggest that JHMV infection induces transcription of IL-12 mRNA in CNS cells. In addition, 2.2v-l infects predominantly, but not exclusively oligodendroglia while JHMV infects predominantly microglia and astrocytes. The relatively higher level of IL-12 mRNA in 2.2v-l infected mice suggests the possibility that oligodendroglia transcribe IL-12 mRNA in response to JHMV infection, similar to the induction of IL-12 mRNA following measles virus infection of oligodendroglia (54). During both the lethal and sublethal infections the IL -la mRNA peaks appear to coincide with replication and not clearance, suggesting that infection induces a rapid induction of IL -la mRNA. These data contrast to the association of IL -la mRNA and the clearance of the OBLV-60 variant of JHMV (34), suggesting an additional difference in cytokine responses depending on the tropism of the virus analyzed. IL-ip mRNA, previously detected in the CNS of JHMV infected mice (34), increased directly after infection at day 1 p.i. However, the level was approximately the same as the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. level detected in sham infected mice suggesting it was induced by trauma. In all mice the levels subsequently dropped by day 3 p.i. The levels of IL-lp mRNA peaked at day 5 following 2.2v-l infection and at day 7 following JHMV infection suggesting IL-lp mRNA was also induced by infection. Analysis of the levels in immunodeficient mice were consistent with the notion that infection, and not immune infiltrates, contributed the majority of the IL-ip mRNA levels. IL-6, another pleiotropic cytokine with numerous effects on immune responses (47), was also detected early following both lethal and sublethal infections. By contrast, IL-6 mRNA was also only detected at 6 day p.i. with the neuronotropic OBLV-60 JHMV variant (34). Kinetic analysis shows that the levels of IL-6 mRNA peaked at day 3 post 2.2v-l infection and at day 5 post JHMV infection. Interestingly, analysis of the mRNA levels in the immunodeficient mice showed virtually no induction of IL-6 mRNA, suggesting that in contrast to IL -la and IL-lp an intact immune response was required for IL-6 mRNA induction. Rapid induction of IL-6 mRNA following JHMV infection is consistent with other models of viral-induced encephalitis in which it also precedes IFN-y (30). Although both IL-6 and IL-10 are cofactors for CTL induction (6,46), kinetic analysis is consistent with the notion that IL-6, and not IL-10, may be involved in the induction or R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. recruitment of JHMV-specific CTL. JHMV infection induces IL-6 secretion from both brain endothelial cells and astrocytes following in vitro infection with JHMV (18), consistent with data showing that it is produced by resident CNS cells following infection with lymphocytic choriomeningitis virus (12). It is interesting that IL-6 mRNA peaks first in mice infected with the 2.2 v-1 variant compared to JHMV which infects a significantly larger number of astrocytes. The rapid induction of IL-6 and IL-ip following infection with 2.2v-l is consistent with the induction of these mRNA in oligodendroglia infected by measles virus in vitro (54). These data demonstrate that lethal and sublethal infections of the CNS induce mRNAs associated with both Thl and Th2 cytokines. Predominant infections of microglia and astrocyes by JHMV and of oligodendroglia by the neutralization escape variant 2.2v-l results in the accumulation of mRNA encoding IFN-y, IL-4 and IL-10. This is the first demonstration of the induction of IL-4 and IL-10 mRNA following JHMV infection. IL-10 is an immunosuppressive cytokine suggested to play a role in the clinical and histological remission phase of experimental autoimmune encephalitis (19,7). Although IL-10 is increased following both the lethal and sublethal infections, the elevation of IL-10 mRNA at day 7 and day 9 during sublethal infection suggests it may play a positive role in reducing the extent of CNS R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. inflammation thereby inadvertantly contributing to persistent infection. Some aspects of our data, i.e., the rapid induction of IL-12 mRNA in mice infected with 2.2v-l, suggest that infection of specific cell types may influence the induction of cytokine mRNA (54). This supports the notion that the cytokine mRNA patterns more closely reflect diversity of the immune response to an individual agent, although differential secretion of cytokines following infection of unique CNS cell types cannot be ruled out (2,45). While the kinetics of IFN-y, IL-4 and IL-10 showed little difference between the groups undergoing lethal or sublethal infections, mRNAs encoding IL-6 and IL-1(3 either appeared more rapidly (IL-6) or accumulated to higher levels (IL-ip) following infection with 2.2v-l virus. By contrast the induction of iNOS and IL-a mRNAs were increased in mice undergoing a lethal infection. These data suggest that an early induction of IL-6, and possibly IL-1 (3 , are associated with sublethal infection or the different tropisms exhibited by these two JHMV variants. However, during both infections the mRNA levels decreased as virus was cleared. Similarly, there appears to be an inverse correlation between a rapid induction of iNOS mRNA and sublethal disease, consistent with the recent demonstration that although iNOS is protective in vitro, inhibition of iNOS activity in vivo appears to have no effect on JHMV pathogenesis (23). Taken together, kinetic analyses of the induction of cytokine mRNA during the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. lethal and sublethal JHMV infections are consistent with the accumulation of both Thl and Th2 associated cytokines and support the interaction of multiple cellular and soluble effector mechanisms whose balance may be critical in providing protection and sterilizing immunity. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. References 1. Adami, J. Pooley, J. Glomb, E. Stecker, F. Fazal, J.O. Fleming and S.C. Baker. 1995. Evolution of mouse hepatitis virus (MHV) during chronic infection: quasispecies nature of the persisting MHV RNA. Virology 209:337-346. 2. Benveniste, E.N. 1992. Inflammatory cytokines within the central nervous system: sources, function and mechanism of action. Am. J. Physiol. Cell Physiology 263:32, C l-16. 3. Biron C.A. 1994. Cytokines in the generation of immune responses to, and resolution of virus infection. Cur. Opin. Immunol. 6:530-538. 4. Brunda, M.J. 1994. Interleukin-12. J. Leukoc. Biol. 55:280-288. 5. Colasanti, M., T. Persichini, T. Di-Pucchio, F. Gremo, and G.M. Lauro. 1995. Human ramified microglial cells produce nitric oxide upon Escherichia coli lipopolysaccharide and tumor necrosis factor alpha stimulation. J. Immunol. 200:144-146. 6. Chen, W-F, and A. Zlotnik. 1991. IL-10: A novel cytotoxic T cell differentiation factor. J. Immunol. 147:528-534. 1. Cna, D.J., D.R. Hinton and S.A. Stohlman. 1995. Self-antigen- induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice. J. Immunol. 155: 4052-4059. 8. Cua, D.J., R.L. Coffman, and S.A. Stohlman. 1996. Exposure to T helper 2 cytokines in vivo before encounter with antigen selects for T helper subsets via alterations in antigen-presenting cell function. J. Immunol. 157:2830-2836. 9. Finke, D., U.G. Brinckmann, V. ter Meulen and U.G. Liebert. 1995. Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis. J. Virol. 65:469- 5474. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 10. Fleming, J.O., S.A. Stohlman, R.C. Harmon, M.M.C. Lai, J.A. Frelinger and L.P. Weiner. 1983. Antigenic relationships of murine coronavimses: analysis using monoclonal antibodies to JHM (MHV-4) vims. Virology. 131:296-307. 11. Fleming, J.O., M. Trousdale, F. El-Zaatarim, S.A. Stohlman and L.P. Weiner. 1986. Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies. J. Virol. 58:869-875. 12. Frei, K., U.V. Malipiero, T.P. Leist, R.M. Zinkernagel, M.E. Schwab and A. Fontana. 1989. On the cellular source and function of interleukin 6 produced in the central nervous system in viral disease. Eur. J. Immunol. 19:689-694. 13. Gazzinelli, R.T., I. Etoum, T.A. Wynn and A. Sher. 1993. Acute cerebral toxoplasmosis is induced by in vivo neutralization of TNF-a and correlates with the down-regulated expression of inducible nitric oxide synthase and other markers of macrophage activation. J. Immunol. 151:3672-3681. 14. Heinzel, F.P., R.M. Rerko, F. Ahmed and A.M. Hujer. 1996. IFN-y independent production of IL-12 during murine endotoxemia. J. Immunol. 157:4521-4528. 15. Houtman, J.J., and J.O. Fleming. 1996a. Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM. J. NeuroVirol. 2: 101- 111 . 16. Houtman, J.J., and J.O. Fleming. 1996b. Pathogenesis of mouse hepatitis virus-induced demyelination. J. NeuroVirol. 2:361-376. 17. Ikemoto, K., R.B. Pollard, T. Fukymoto, M. Morimatsu and F. Suzuki. 1995. Small amounts of exogenous IL-4 increase the severity of encephalitis induced in mice by the intranasal infection of herpes simplex vims type 1. J. Immunol. 155:1326-1333. 87 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 18. Joseph, J., J.L. Grim, F.D. Lublin and R.L. Knobler. 1993. Interleukin-6 induction in vitro in mouse brain endothelial cells and astrocytes by exposure to mouse hepatitis virus (MHV-4, JHM). J. Neuroimmunol. 42:47-52. 19. Kennedy, M.K., D.S. Torrance, K.S. Picha, and K.M. Mohler. 1992. Analysis of cytokine mRNA expression in the central nervous system of mice with experimental autoimmune encephalomyelitis reveals that IL-10 mRNA expression correlates with recovery. J. Immunol. 149:2496-2505. 20. Kundig, T.M., Hengartner, H. and Zinkernagel, R.M. 1993. T cell- dependent IFN-y exerts an antiviral effect in the central nervous system but not in peripheral solid organs. J. Immunol. 150:2316-2321. 21. Kyuwa, S. and S.A. Stohlman. 1990. Pathogenesis of a neurotropic murine coronavirus strain JHM in the central nervous system. Seminar Virol. 1:273-280. 22. Lampert, P.W., J.K. Sims, and A.J. Kniazeff. 1973. Mechanisms of demyelination in JHM virus encephalomyelitis. Electron microscopic studies. Acta Neuropathol 24:76-85. 23. Lane, T.E., A.D. Paoletti, and M.J. Buchmeier. 1997. Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus. J. Virol. In press. 24. Lane, T.E. and M.J. Buchmeier. 1997. Murine coronavirus infection: a paradigm for virus-induced demyelinating disease. Trends in Microbiol. 5:9-14. 25. Lehmann, G.E., D. Moskophidis, and J. Lohler. 1988. Recovery from acute virus infection. Role of cytotoxic T lymphocytes in the elimination of lymphocytic choriomeningitis virus from spleens of mice. Ann. N.Y. Acad. Sci. 532:238-256. 26. Lin, M.T., S.A. Stohlman, and D.R. Hinton. 1997. Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis. J. Virol. 71:383-391. 88 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 27. Lodge, P.A., and Sriram, S. 1996. Regulation of microglial activation by TGF-(3, IL-10, and CSF-1. J. Leukocyte Biology 60:502- 508. 28. Lyons, C.R., G.J. Orloff and J.M. Cunningham. 1992. Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. J Biol. Chem. 267:6370-6374. 29. Moran, T.M., H. Isobe, A. Fern an dez-S esma, and J.L. Schulman. 1996. Interleukin-4 causes delayed virus clearance in influenza virus- infected mice. J. Virol. 70:5230-5235. 30. Moskophidis, D., Frei, K., Lohler, J., Fontana, A. and Zinkernagel, R.M. 1991. Production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of interleukin-6 (IL-6) but not IL-4, IL-5, and gamma interferon. J. Virol. 65:1364-1369. 31. Murphy, E., S. Hieny, A. Sher, and A. O’Garra. 1993. Detection of in vivo expression of interleukin-10 using a semi-quantitative polymerase chain reaction method in Schistosoma mansoni infected mice. J. Immunol. Methods 162:211-223. 32. Orange, J.S. and C.A. Biron. 1996. An absolute and restricted requirement for IL-12 in natural killer cell IFN-y production and antiviral defense. J. Immunol. 156:1138-1142. 33. Ozmen, L., M. Aguet, G. Trinchieri, and G. Garotta. 1995. The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon. J. Virol. 69:8147-8150. 34. Pearce, B.D., M.V. Hobbs, T.S. McGraw, and M. Buchmeier. 1994. Cytokine induction during T-cell mediated clearance of mouse hepatitis virus from neurons in vivo. J. Virol. 68:5483-5495. 35. Perlman, S., and D. Reis. 1987. The astrocyte is a target cell in mice persistently infected with mouse hepatitis virus, strain JHM. Microb. Pathog. 3:309-314. 89 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 36. Pewe, L., G.F. Wu, E. M. Barnett, R.F. Castro, and S. Perlman. 1996. Cytotoxic T cell-resistant variants are selected in a virus-induced demyelinating disease. Immunity 5:253-262. 37. Ruddle, N.H., C.M. Bergman, K.M. McGrath, E.G. Lingenheld, M.L. Grunnet, S.J. Padula, and R.B. Clark. 1990. An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis. J. Exp. Med. 172:1193-1200. 38. Sedgwick, J.D., and R. Dorries. 1991. The immune system response to viral infection of the CNS. Neurosciences 393-100. 39. Sideras, P., S. Bergstedt-Lindquist, E. Severinson, Y. Noma, T. Naito, C. Azuma, T. Tanabe, T. Kinashi, F. Matsude, Y. Yaoita, and T. Hon jo. 1987. IgGl induction factor: A single molecular entity with multiple biological functions. Adv. Exp. Med. Biol. 213:227-236. 40. Smith, A.L., S.W. Barthold, M.S. de Souza and K. Bottomly. 1991. The role of gamma interferon in infection of susceptible mice with murine coronavirus, MHV-JHM. Arch. Virol. 121:89-100. 41. Stohlman, S.A., P.R. Braton, J.O. Fleming, L.P. Weiner and M.M.C. Lai. 1982. Murine coronaviruses: isolation and characterization of two plaque morphology variants of the JHM neurotropic strain. J. Gen. Virol. 63:265-275. 42. Stohlman, S.A., G.K. Matsushima, N. Casteel and L.P. Weiner. 1986. In Vivo effects of coronavirus-specific T cell clones: DTH inducer cells prevent a lethal infection but do not inhibit virus replication. J. Immunol. 136:3052-3056. 43. Stohlman, S.A., D.R. Hinton, D. Cua, E. Dimacali, J. Sensintaffar, F.M. Hofman, S.M. Tahara and Q. Yao. 1995a. Tumor necrosis factor expression during mouse hepatitis virus- induced demyelinating encephalomyelitis. J. Virol. 69:5898-5903. 44. Stohlman, S.A., C.C. Bergmann, R.C. van der Veen and D. Hinton. 1995b. Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from the central nervous system. J. Virol. 69, 684-694. 90 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 45. Sun, N., D. Grzybicki, R.F. Castro, S. Murphy and S. Perlman. 1995. Activation of astrocytes in the spinal cord of mice chronically infected with a neurotropic coronavirus. Virology 213:482-493. 46. Takai, Y., G.G. Wong, S.C. Clark, S.J. Burakoff, and S.H. Hermann. 1988. B cell stimulatory factor-2 is involved in the differentiation of cytotoxic T lymphocytes. J. Immunol. 140:508-512. 47. Van Snick, J. 1990. Interleukin-6: an overview. Ann. Rev. Immunol. 8:253-278. 48. Wang, F.I., S.A. Stohlman and J.O. Fleming. 1990. Demyelination induced by murine hepatitis virus, JHM strain (MHV-4) is immunologically mediated. J. Neuroimmunol. 30:31-41. 49. Weiner, L.P. 1973. Pathogenesis of demyelination induced by a mouse hepatitis virus (JHM virus). Acta. Neurol. 28:298-303. 50. Wesselingh, S.L., B. Levine, R.J. Fox, S. Choi, and D.E. Griffin. 1994. Intracerebral cytokine mRNA expression during fatal and nonfatal alphavirus encephalitis suggests a predominant type 2 T cell response. J. Immunol. 152:1289-1297. 51. Williamson, J.S.P., and S.A. Stohlman. 1990. Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells. J. Virol. 64:4589-4592. 52. Williamson, J.S.-P., K. Sykes and S. Stohlman. 1991. Characterization of brain infiltrating mononuclear cells during infection with mouse hepatitis virus strain JHM. J. Neuroimmunol. 32:199-207. 53. Williamson, J.S.P. 1992. Virus-specific T cells in the central nervous system following infection with an avirulent neurotropic mouse hepatitis virus. Regional Immunol. 4:145-152. 54. Yamabe, T., G. Dhir, E.P. Cowan, A.L. Wolf, G.K. Bergey, A. Krumholz, E. Barry, P.M. Hoffmann and S. Dhib-Jalbut. 1994. Cytokine-gene expression in measles-infected adult human glial cells. J. Neuroimmunol. 49:171-179. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 55. Yamaguchi, K., N. Goto, S. Kyuwa, M. Hayami and Y. Toyoda 1991. Protection of mice from a lethal coronavirus infection in the central nervous system adoptive transfer of virus-specific T cell clones. J. Neuroimmunol. 32:1-9. 56. Yu, Z., E. Manickan and B.T. Rouse. 1996. Role of interferon-y in immunity to herpes simplex virus. J. Leukoe. Biol. 60:528-532. 57. Zhang, X.M., D. Hinton, D.J. Cua, S.A. Stohlman and M.M.C. Lai. 1997. Expression of gamma interferon by a coronavirus defective- interfering RNA vector and its effect on viral replication, spread and pathogenesis. Virology in press. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter 1Y. Gamma Interferon is Required for Viral Clearance from Central Nervous System Oligodendroglia Summary Infection of the CNS by JHMV is a rodent model of the human demyelinating disease multiple sclerosis (MS). The inability of effective host immune responses to eliminate virus from the CNS results in a chronic infection associated with ongoing recurrent demyelination. JHMV infects a variety of CNS cell types during the acute phase of infection including ependymal cells, astrocytes, microglia, oligodendroglia and rarely neurons. Replication within the majority of CNS cell types is controlled by perforin- dependent virus-specific CTL. However, inhibition of viral replication in oligodendroglia occurs via a perforin-independent mechanism(s). The potential role for IFN-y as mediator controlling JHMV replication in oligodendroglia was examined in mice deficient in IFN-y secretion (IFN-y'7 ' mice). IFN-y'7 ' mice exhibited increased clinical symptoms and mortality associated with persistent virus demonstrating an inability to control replication. Neither anti-viral Ah nor CTL responses were diminished in the absence of IFN-y, although increased IgGl was detected in IFN-y'7 ' mice. Increased virus Ag in the absence of IFN-y localized almost exclusively to oligodendroglia and was associated with increased CD8+ T cells localized R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. within white matter. These data suggest that while perforin-dependent CTL control vims replication within astrocytes and microglia, which constitute the majority of infected CNS cells, IFN-y is critical for control of viral replication in oligodendroglia. Therefore, different mechanisms are utilized by the host defenses to control vims replication within the CNS, dependent upon the phenotype of the targets of vims replication. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Introduction A major responsibility of innate and adaptive immunity is to limit infections prior to the accumulation of sufficient damage to result in death or permanent sequelae. Most tissues are continuously sampled for the presence of foreign Ag; however, the CNS appears to be at a distinct disadvantage in responding to infection. First, there is no specialized lymphatic drainage, potentially limiting or delaying Ag recognition. Second, the normal CNS is devoid of dendritic cells and must rely upon microglia, endogenous cells of the macrophage lineage, for initial Ag recognition and presentation. Finally, MHC molecules are not normally expressed by neurons or glial cells, although they appear to be rapidly upregulated under the influence of IFN-a/(3 (31). CNS viral infections result in vigorous immune responses; however, these inherent limitations appear to contribute to viral persistence or latency (43). Indeed, infection by a variety of both RNA and DNA viruses ultimately results in persistent CNS infections. Although many viruses predominantly infect single CNS cell types, each of the major cell types can provide a reservoir for persistent or latent viral infection. For example, herpes simplex virus and measles virus infect and subsequently persist in neurons (44,41). Other viruses, such as LCMV and the neurotropic coronavirus strain JHMV initially infect a variety of cell types (40,18,28,26) but subsequently persist in only a 95 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. subset of CNS cells (25,35,50,32). Immunity within the CNS appears to arrive at a balance which eliminates infectious virions and minimizes damage by either allowing or actually facilitating viral persistence. A remaining unanswered question is how immune effector mechanisms influence viral infections of the diverse cell types which comprise the CNS, resulting in persistent infection within specific cell types. Infection of the rodent CNS by JHMV provides a model of acute viral infection which progresses to a chronic infection associated with ongoing CNS demyelination (25,19,27). The immune response contributes to both viral clearance (57,48), but also to viral- induced primary demyelination (19,53). Virus-specific Ab and T cell responses (57,48,7) have been implicated in limiting infectious virus. Although, these immune effector mechanisms suppress virus replication, they are unable to completely eliminate virus resulting in persistent infection (1). JHMV replication in oligodendroglia may contribute to demyelination (26); however, increasing evidence suggests that demyelination is immunopathologically mediated and possibly distinct from the immune mechanisms which control virus replication (19,27,53). Defining the roles of the immune effectors which contribute to elimination of infectious virus is clarifying the strategies of JHMV persistence within the CNS. Neutralizing Ab are generally detected only following JHMV clearance (29,33) and are 96 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. suspected of playing a role in establishing or maintaining chronic infection without directly contributing to viral clearance. By contrast, both CD4+ and CD8+ T cells accumulate within the CNS during acute JHMV infection and are associated with viral clearance (29,55,54). Virus-specific CD4+ T cells may (57) or may not (46) contribute directly to viral clearance, possibly via the secretion of soluble mediators. The CTL response limits virus replication in astrocytes and microglia but only to a lesser extent in oligodendrocytes (49) via a perforin-dependent mechanism (29). Indeed, CTL escape mutants of JHMV are associated with persistent CNS infectious virus (36,37). The CTL- mediated reduction of virus replication in oligodendroglia was suggested to be a consequence of the overall successful CTL-mediated elimination of virus producing cells and not due to direct cytolysis of oligodendroglia (48). IFN-y plays important roles in many anti-viral immune responses and is predominantly secreted by CD8+ T cells (41,58). Therefore, the reduction of JHMV infected oligodendrocytes in the perforin-deficient mice (29) could have occurred via IFN-y secretion. Consistent with this interpretation, inhibition of IFN-y which enhances the severity of viral infections (6,13,2,30,20,17) is not correlated with defects in the generation of other anti viral immune effectors (20,17). The notion that IFN-y plays a critical role in the pathogenesis of JHMV with tropism for microglia, astrocytes and R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. oligodendroglia is suggested by several observations. Analysis of the kinetics of cytokine mRNA accumulation within the GNS of mice infected with JHMV, including infection with a neuronotropic variant of JHMV (OBLV-60), suggested a relationship between IFN-y and inhibition of viral replication (33,34). T cell-dependent IFN-y inhibits vaccinia virus replication in meninges but not peripheral organs (24), suggesting that IFN-y secretion within the CNS may be a critical component in controlling virus replication within specific CNS cell types. However, as shown by the clearance of the OBLV-60 variant of JHMV from the CNS of IFN-y'7 ' mice (28), IFN-y is not critical for virus clearance from neurons. An antiviral role of IFN-y is also supported by the elimination of JHMV from the CNS of perforin-deficient mice (29) and following adoptive transfer of either JHMV-specific CD4+ T or CD8+ T cells (57,48). Virus-specific T cells all reduced virus in infected CNS cells including oligodendrocytes potentially via secretion of IFN-y (57,48). Finally, the expression of the IFN-y receptor (51) but not MHC molecules on oligodendroglia (43) suggest that IFN-y may reduce JHMV replication in this cell type. These observations provided the basis for examining the potential contribution of IFN-y in limiting, JHMV infection of oligodendroglia. Infectious virus was not completely cleared from the CNS of IFN-y'7 ' mice, even though CTL and neutralizing Ab responses were induced. Consistent R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. with a vigorous but ineffective CTL response, CD8+ T cells were selectively recruited to white matter areas of the CNS. Survivors showed persistence of viral Ag in oligodendroglia consistent with CTL-mediated clearance from astrocytes and microglia, but not oligodendrocytes (48). These data suggest that although IFN-y is not required for the inhibition of replication in neurons (28), it either directly or indirectly, controls viral replication within oligodendroglia. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. M aterial and Methods Mice and Virus C57BL/6 mice heterozygous for a non functional IFN-y gene (IFN-y7') were obtained at N9 from Genentech (San Francisco, CA) (37) and maintained by homozygote mating. Syngeneic wt C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Male and female 6-8 wk old IFN-y7' and wt mice were used in all experiments. Mice were infected by i.e. injection with 25 pfu of the 2.2v-l mAb derived neutralization escape variant of JHMV (14) and monitored for 21 days p.i.. This strain produces a subacute encephalomyelitis with primary CNS demyelination and paralysis which progresses to chronic infection (19,1,14). Clinical scores were read daily and graded as previously described (29,33,14). Virus titration Brains were divided into thirds in the sagital plane and processed for virus titration, histopathology and RNA extraction. Viral titers were determined by plaque assay on monolayers of DBT cells as previously described in chapter II and elsewhere (48,29,33). Briefly, tissue was homogenized in 4 ml of Dulbeco’s PBS (pH 7.4), centrifuged (200 x g; 7 min; 4°C) and supernatants assayed immediately or stored at -70°. Average titers of groups of at least three mice per time point are presented. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Isolation of CNS-derived mononuclear ceils Mononuclear cells were isolated from the CNS of mice as described previously (). Briefly, brain and spinal cords were removed and homogenized in RPMI 1640 medium supplemented with 25mM BEEPES (pH7.2) using Ten Broeck tissue homogenizers. Cells were suspended in 30% Percoll (Pharmacia) in calcium and magnesium free PBS (pH 7.2), concentrated onto a 1 ml cushion of 70% Percoll by centrifugation at 800 X g for 20 min at 4°C, and collected from the interface. Flow Cytometry Virus-specific CD8+ T cells in the CNS mononuclear population were identified by staining with mAbs specific for CD8+ (Clone 53-6.7; PharMingen) and with the Db major histocompatibility complex (MHC) class I tetramer associated with viral S protein pS510 peptide (Db-S510), as described previously (3). Virus-specific Ab response JHMV-specific IgGl and IgG2a serum Ab were quantitated by ELISA as described previously (Chapter III; 29,33) and expressed as the log of the highest dilution with O.D. values three times above background level. Rabbit anti-mouse IgGl or IgG2a were used as secondary Ab (Cappel, Costa Mesa, CA). Neutralizing Ab titers were determined by TCID50 plaque reduction assay (Chapter III; 29,33). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Cytokines For analysis of cytokine production, CLN cells from days 3, 5 and 7 days p.i were cultured in 96 well plates (8 x Iff cells/well) in the presence or absence of U. V inactivated JHMV infected DBT cell lysate (1x1 Q 8 PFU prior to U.V inactivation) (used at 1:80 dilution). Supernatants were harvested at 24 h for IL-2 and at 48 h for IL-4, IL-10 and IL-5 secretion. Cytokine concentrations were determined by sandwich ELISA and calculated from standard curves using commercially obtained recombinant cytokines as previously described (10). Specific anti-cytokine monoclonal antibodies for IL-2 (JES6-1A12), IL-4 (BVD4-1D11), IL-10 (JES5-2A5) and IL-5 (TRFK5) obtained from PharMingen were used to coat ELISA plates (Immulon II, Dynatech Laboratories, Inc. Chantilly,VA). Biotinylated anti-IL-2 (JES6- 5H4), anti-IL-4 (BVD6-24G2), anti-IL-10 (SXC-1) and anti-IL-5 (TRFK4) were purchased from PharMingen. Avidin-peroxidase (PharMingen) was used as detection system with OPD as chromogen substrate (Sigma Chemical Co). Detection limits for IL-5, and IL-10 were 75pg/ml and 125pg/ml for IL-4. RNA was isolated from brains by homogenization in guanidium isothiocyanate and centrifugation through cesium chloride as previously described (Chapter III; 34,9). cDNAs were prepared using AMV reverse transcriptase and oligo dT primers (Promega, Madison, WI) at 42°C. Cytokine R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mRNA was determined by semi-quantitative PCR as previously described (Chapter III; 20,21,40) using specific primer pairs for IL-5 (5’ oligoprimer, 5’- GGT TAC AGA CAT GCA CVCA TTG CCA CTA GTT - 3’; 3’ oligoprimer S ’- CTA GTG GGT ATT AAA TTG AAG TTA GAT AGG-3’) (44), IL-4, and IL-10 (33). PCR products were quantitated by hybridization with 3 2 P ATP-labelled internal oligoprobes in a dot blot assay. Membranes were exposed to an imaging screen (Molecular Dynamics) and analyzed using a phosphorimaging scanner (Molecular Dynamics). Cytokine cDNAs were normalized to the housekeeping gene HPRT to adjust for cDNA variations (33,9). Cytolytic activity CTL activity was measured using CLN cells obtained at 6 days p.i. and spleen cells at various days p.i. (7, 9 and 14) in a 5 1 Cr release assay as previously described (48,33,51). Direct ex-vivo CTL activity was measured in the mononuclear cells isolated from CNS at day 14 p.i. CLN and spleen cells were incubated for 7 days in the presence of the 1 pM S510 peptide (30,8). EL-4 (H-2b ) target cells, propagated in Dulbecco’s modified MEM with 10% PCS, were labeled with lOOpci of Na5 1 CrC>4 (New England Nuclear, Boston, MA) for 1 h at 37°C and washed three times. 5 1 Cr-labeled EL-4 cells were coated with 1 pM S510 peptide prior to use as targets (20,41). Effectors were R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. added to the target cells at various E:T ratios and 5 1 Cr release measured after 4 h incubation at 37°C. Data are expressed as specific 5 1 Cr release. Spontaneous release values were <20% of the total release values. Sequence analysis of Spike protein CTL epitope ..... Viruses from brain samples obtained at 21 days p.i. were propagated once on confluent monolayers of DBT cells. When approximately 80% of the cells exhibited cytopathology (24-48 h), cells were lysed by addition of guanidine thyiocyanate solution and RNA isolated by phenol/chloroform extraction. RNA prepared from DBT cells infected with parental 2.2v-l virus and uninfected DBT cells were used as controls for mutations incorporated by taq polymerase and for PCR contamination respectively. RNA (5 jug) was reverse transcribed using AMV reverse transcriptase and random hexanucleotide primers (Promega). A 27nt cDNA encompassing the S protein CTL epitope [viral bases 1528-1554] plus surrounding 500 bases [1420-1890] was amplified for 45 cycles using Amplitaq Gold Polymerase (Perkin- Elmer, Branchburg, NJ) and primers JS 1895: 5’- GCA TGC TAC GTT ATG TCC AGG CTG AGT C and JS 1390: 5’- GAT GTT GCC TAC GCC CAG C -3’). Excess primers were removed using the Magic PCR Prep (Promega) and the cDNA sequenced on an ABI Prism automated sequencing apparatus using JS 1390 as primer. Experiments were done by Dr Norman Marten. 1 0 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. H istopathological analysis Brain and spinal cord tissues were fixed in Clark’s solution for 3 h, and prepared for paraffin sections as previously described (48,29). Sections were stained with hematoxylin and eosin or luxol fast blue for routine examination. To examine viral Ag distribution, sections were incubated with the anti-JHMV mAb J.3.3 specific for the nucleocapsid (N) protein of JHMV (47) and immunoperoxidase-labeled anti-mouse mAh as secondary Ab (Vectastain- ABC kit, Vector Laboratory). Multiple serial-step frozen sections of the brain tissue of 2 mice per group at day 14 p.i. were stained with mAb J.3.3 Ab. Ag positive cells were counted to compare the number of infected cells in both study groups. Similarly, 5 serial-step paraffin sections of the spinal cord of 3 to 4 mice per group at day 21 p.i were stained with luxol fast blue and the number of demyelination plaques counted per unit area. To examine CD4+ and CD8+ cells immunoperoxidase staining was performed in acetone fixed frozen sections as previously described (16). In brief, rat anti-CD4 (L3T4, PharMingen) and rat anti-CD8 (Ly-2, PharMingen) were used as primary Ab. Visualization was achieved using biotinylated rabbit-anti-rat Ab, the Vectastain ABC kit and peroxidase substrate kit with 3- amino 9-ethylcarbizole (AEC) as chromogen (Vector Laboratory). For quantitative comparisons, 5 serial-step frozen sections of individual samples 105 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. from groups of 3 mice at day 7 p.i and 2 mice at day 14 p.i. were stained. All CD8+ T cells were counted. To phenotype viral Ag positive cells, sections were stained using the JHMV specific mAh J.3.3 and Ab specific for astrocytes (Glial Fibrillary Acidic Protein [GFAP]; Dako Corporation, Carpenteria, CA), oligodendroglia [Rip; generously provided by Regeneron Pharmaceuticals, Tarrytown, NY (39)] or microglia (CD lib; Pharmingen). JHMV Ag and GFAP double staining was performed on paraffin sections and visualized with peroxidase VIP® and SG® substrate kits (Vector Laboratory) respectively. Viral Ag in oligodendroglia was detected in frozen sections from paraformaldehyde- perfused tissue and detected using the alkaline phosphatase (AP) Vectastain ABC kit for Rip Ab and the peroxidase Vectastain ABC kit with AEC substrate for detecting J.3.3 mAb. Viral Ag in CD1 lb positive cells was detected in acetone-fixed frozen sections, using AP Vectastain ABC kit for CD1 lb and peroxidase Vectastain ABC kit with AEC substrate for J.3.3 mAb. 106 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Results Pathogenesis of JHMV infection in IFN-y"7 " mice Morbidity and mortality were compared in IFN-y"7 " and wt mice infected with JHMV until 21 days p.i. at which time infected wt mice recovered from acute disease (Fig. 1) (29,33,14). Animals in both groups began to exhibit encephalitis at 7 days p.i., which progressed to a paralytic disease (clinical score = 2.0 ± 0.2 for wt and 2.7 ± 0.2 for IFN-y'7 " mice at 14 days p.i.) (Fig. 1A). Wt mice exhibited almost total clinical recovery by 21 days p.i. (clinical score = 1.2 ± 0.2). By contrast infected IFN-y"7 ' mice rapidly progressed to a paralytic disease by 14 days p.i and exhibited both slower clinical recovery and higher mortality (clinical score = 2.6 ± 0.6 at 21 days p.i.). Only 34% ± 8% of JHMV-infected IFN-y"7 " mice survived infection compared to 89% ± 8% of control mice indicating that IFN-y influences both survival and clinical course of JHMV-induced encephalomyelitis (Fig. IB). Higher viral titers were found in the CNS of infected IFN-y"7 " mice compared to controls at all time points examined (Fig. 1C). In contrast to wt mice, in which infectious virus was no longer detected after day 7 p.i., infectious virus (>10 plaque forming units/gm of brain) persisted in the CNS of all IFN-y"7 " mice until 21 days p.i., the last time point examined (Fig. 1C). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 1. Morbidity (Panel A), mortality (Panel B), and viral replication (Panel C) in IFN-y'7 ' and control wt mice following Infection with the 2.2vl variant of JHMV. Panels A and B represents summary of three experiments (15-20 mice per experiment). Viral titers (Panel C) were determined by plaque assay and each time point represents the mean of at least three mice. Increased infectious virus in the brains of IFN-y’7 ' mice was significant (p< 0.05; Student’s t test at all time points examined. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A. M orbidity -o— W T 4 3 2 1 0 4 6 8 10 12 14 16 18 20 22 Days post infection B. M o rta lity m I F N - y 0 '0 4 6 8 10 12 14 16 18 20 22 Days post infection C. Virus titer o ar o re L- 0 Q *>» O E 2 o D u_ CL 5 4 3 H 2 1 0 \ \ \ \ \ s \ \ \ \ W T E\\l I F N - y 0' \ \ 0 3 5 7 9 14 21 Days post Infection R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. To rule out reversion of the 2.2v-l variant to wt JHMV, virus isolated from the brains of IFN-y7 ' mice at 21 days p.i. was assayed for plaque formation in the presence or absence of mAb J.2.2 (used for selection of JHMV neutralizing escape variant 2.2 v-1) (14). No differences in either plaque numbers or morphology was detected, consistent with the absence of in vivo reversion (data not shown). Immune responses in infected IFN-y7" Mice Infected IFN-y7" mice were analyzed for JHMV-specific CTL activity to determine if the inability to clear infectious virus was due to altered effector function. Virus-specific CD8+ T cells were present in the CNS of infected IFN-y"7 ' mice at a similar frequency as in wt controls (Fig. 2A). This result indicate normal priming and recruitment of specific CD8+ T cells in the absence of IFN-y. Moreover, CNS mononuclear cells from both groups exhibited excellent ex-vivo cytolysis against targets pulsed with peptide comprising the virus-specific CTL epitope (Fig. 2B). CTL activity in CLN cells of JHMV infected IFN-y7" mice was identical to the activity in wt mice during virus clearance from the CNS (Fig. 2B). These data indicate that the absence of IFN-y did not impede the generation of specific CTL or diminished their cytotoxic activity. Furthermore, results also suggest that IFN-y is not required for T cell trafficking to the CNS. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A W T o BPoet 1 4 9 8 0 0 5 .005 IFN-Y B Poet 1498003.003 4 W r o r T ! 1000 g ’T l'f i l 't 'f i Pr r t m i 1000 F L 1 -H eig h t> B C < D to ro d ) 15 si O 10 T — L O 5 - 4 — o n - O' o wr IFN-y ’ —v W T no Ag IFN-y No Ag __y - -y — y — y, 50 25 12 6 Effector/Target ratio t v C O C D _ a > C D O 50 40 30 20 10 " A ~ â–¡ ..... '- i' / . Q - W T I F N - y W T /N o A g I F N - y ’ ’/N o A g — I -----------1 ----------1 -----------1 40 20 10 Effector / target ratio Figure 2. Cytotoxic activity in JHMV-infected IFN-Y’7 ’ and control wt mice. A, CNS mononuclear cells from infected mice at day 14 p.i. were stained for expression of CD8 (FITC-labeled anti-CD8; x-axis) and specific TCR (PE-labeled Db-S510; y-axis). B, Ex-vivo cytotoxicity of S510 CD8+ T cells was assayed in CNS cells at the indicated E:T ratios. C. CLN cells were obtained from infected mice at 6 days p.i. and cultured in vitro for 7 days in the presence of 1 uM S510 peptide. Cytolytic activity of CNS and CLN cells were measured using S510 peptide coated EL-4 target cells. Similar results were obtained using recombinant vaccinia virus S510 infected EL4 and IC-21 target cells. Spleen cells at days 7, 9 and 14 p.i. were also examined and no significant differences in CTL activity were detected in splenocytes from infected IFN-y’7 ’ mice compared to wt mice (data not shown). 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. JHMV isolated during infection of neonatal mice partially protected via maternal Ab, exhibit a high mutation rate within the viral H-2b-restricted CTL epitope allowing viral escape from CTL recognition (36,37). To examine the possibility that CTL escape mutants contributed to the persistence of infectious virus within the CNS of IFN-y'7 ' mice, the S protein mRNA encoding the CTL epitope [RNA bases 1528-1554] as well as 500 bases (1420-1890) surrounding the epitope was analyzed for mutations by Dr. Norman Marten. No dominant base substitutions or deletions within these S protein sequences were detected by sequencing of bulk PCR products for virus pools isolated from the CNS of persistently infected IFN-y7' mice at 21 days p.i. after infectious virus had been completely eliminated from the CNS of wt mice. These data are consistent with a direct anti-viral effect of IFN-y on JHMV replication within the CNS. An alternative explanation for reduced virus clearance in IFN-y7' mice may reside in preferential induction of Th2-type responses. No differences in proliferative responses to either JHMV Ag or the CTL-specific S510 peptide were detected in either splenocytes or CLN cells from infected IFN-y7' mice compared with wt mice at 7 or 14 days p.i. (data not shown). However, CLN isolated from infected IFN-y7' mice at 3 and 5 days p.i. secreted more IL-2 in response to JHMV Ag compared to infected wt controls (Fig. 3). IL-4 secretion was not detected from cells derived from either group (data not R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. shown). IL-5 and IL-10 were secreted at higher levels from CLN derived from infected IFN-y'7 " mice at 7 days p.i. (Fig.3). No differences were detected in the quantity of IL-10 mRNA; however, less IL-5 mRNA was detected in the CNS of infected IFN-y"7 ' mice compared to wt controls (Fig. 3). The diminished expression of IL-5 mRNA and transient expression of IL-4 mRNA at day 7 p.i. (Fig. 3) in IFN-y"7 ' mice suggest the absence of increased trafficking or retention of Th2 cytokine secreting cells within the CNS in the absence of IFN-y. This contrasts with an initial increase in Ag-specific secretion of Th2 type cytokines (IL-5 and IL-10) by peripheral T cells. Consistent with the increased secretion of Th2-type cytokines, a 5-fold increase in JHMV-specific IgGl was detected in the serum of infected IFN-y"7 " mice compared to wt controls, while a 10-fold increase in IgG2a was initially detected in wt mice compared to IFN-y"7 " mice at day 9 p.i. (Fig.4). Although the IFN-y"7 " mice continued to have increased serum JHMV-specific IgGl at 14 days p.i., by 21 days p.i., the levels of both isotypes were equivalent in the IFN-y"'" and wt mice (Fig. 4). As previously reported (29,33), no serum neutralizing Ab was detected prior to day 9 p.i. and no differences were detected in JHMV neutralizing titers at 14 or 21 days p.i. (data not shown) suggesting that secretion of neutralizing Ab was not altered in the absence of IFN-y. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Cytokine sec re tio n -Cytokine mRNA e x p re ssio n A IL-2 1 £ I 5 2 1 0 0 3 5 7 Days post infection B. IL-5 0.3 0.2 0.1 0.0 i r~ 0 3 5 7 Days post infection C. IL-10 I 0.3 0.2 - 0.1 - 0.0 0 3 5 7 Days post infection WT IFN -y/' A IL-4 1.2 - 1.0 - 0.8 - 0.6 H 0.4 0.2 0.0 WT . a T 0 3 5 7 Days post infection C. IL-10 1.2 1.0 H 0.8 0.6 0.4 0.2 0.0 IFHy 0 3 5 7 Days post infection 0 3 5 7 Days post infection Figure 3. Specific cytokine secretion from CLN cells and cytokine mRNA expression in brains of 2.2v-l infected IFN-y'/_ and wt mice. At various time points p.i. CLN cells were cultured in the presence or absence of JHMV Ag and cytokine secretion determined by ELISA assay. Supernatants were tested at 24 h for IL-2 (Panel A) and at 48 h for IL-5 (Panel B) and IL-10 (Panel C). The levels of cytokine mRNA in infected brains were determined by RT-PCR and semi-quantitative dot blot and the data expressed as a relative value obtained for comparison. 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. O ) o c o ts t5 E S C D CD 4 3 2 1 15 22 8 lgG2a/IFN-f lgG1/IFN-y SgG2a/WT lgG1/WT Days post infection Figure 4. Kinetics of JHMV specific Ab in infected IFN-y7' and wt mice. Ab titers were measured in sera by ELISA assay as described in Material and Methods. Histopathology Spinal cords and brains from infected IFN-y7' mice were compared to infected wt mice to determine the influence of the absence of IFN-y on viral Ag distribution and extent of pathological changes. Prominent demyelination was found within white matter tracts of both groups after 21 days p.i. Semi- quantitative analysis showed no significant differences in demyelination plaque numbers between IFN-y7' (2.4 ± 1.4) and wt (3.4 ± 2.1) mice suggesting that IFN-y does not influence development of JHMV-induced demyelination. 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Similarly, no differences in the overall amount of mononuclear inflammation were noted; however, an increase in the amount of CD8+ T cells was detected in the white matter tracts of infected IFN-y7' mice at 7 and 14 days p.i. compared to wt mice. Semi-quantitative estimation of CD8+ T cells infiltrating the brain parenchyma of infected mice showed a 4-fold increase in the number of CD8+ cells in IFN-y7' mice at days 7 and 14 p.i. (200 ± 80 and 750 ± 210) compared to wt mice (30 ± 10 and 110 ± 50). Interestingly, increased CD8+ infiltration in the white matter regions correlated with increased Ag positive cells in IFN-y7' mice suggesting that the lack of IFN-y results in increased viral Ag promoting local infiltration of CD8+ T cells. Differences in the amount and type of Ag positive cells were observed beginning at day 7 p.i (Fig. 5 A, 5B). Decreasing numbers of viral Ag positive cells were observed in wt mice, consistent with the clearance of infectious virus (Fig 1C). By contrast, subsequent to day 7 p.i., significant Ag persisted until day 21 p.i. in IFN-y7' mice (Fig. 6A, 6B). By day 14 p.i. the IFN-y7' mice had a 10-fold increase in numbers of viral Ag positive cells (IFN-y7' mice = 1500 ± 780 vs. wt mice = 75 ± 60) compared to wt mice. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 5. Encephalitis and JHMV Ag in the brains of control wt mice (A) and IFN-y7' (B) at 7 days p.i. Immunoperoxidase stain using mAb J.3.3 with hematoxylin counterstain. Note in wt mice (A) the perivascular cuffs (arrow heads) and viral Ag positive cells (arrows) are localized to discrete regions of the section. In IFN-y7" mice, the white matter tracts show increased viral Ag positive cells (arrows) in the same vicinity as the infiltrating mononuclear cells (arrow heads). Magnification x 110. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 6. JHM V Ag in the spinal cords of IFN-y"7 ' and control wt mice at 21 days p.i. In cords from wt mice (Panel A) only very rare Ag positive cells (arrow) and scattered inflammatory loci (arrow head) are seen. In cords from IFN-y7' mice (Panel B) numerous Ag positive oligodendroglia are seen in the white matter (arrows and insert). Immunoperoxidase stain using mAh J.3.3 with hematoxylin counterstain. Magnification xllO (insert x440). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Consistent with the presence of infectious virus in infected IFN-y'7 ' mice, oligodendroglia, astrocytes, microglia and occasional neurons contained viral Ag; however, double staining for viral Ag in astrocytes, microglia and oligodendrocyte revealed that oligodendroglia accounted for the majority of Ag positive cells within the CNS of IFN-y'7 ' mice at day 21 p.i. (Fig. 7A). These data are consistent with the inability of CTL to mediate clearance from oligodendrocytes (48,29) and demonstrate that IFN-y is indeed an important immune effector in controlling JHMV infection of oligodendroglia. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 7. Viral Ag positive cells in the brain of IFN-y'/_ mice at 21 days p.i. Double staining was performed on brain sections with the JHMV specific mAh J.3.3 and Ab specific for oligodendrocytes [Rip (Panel A), astrocytes [GFAP (Panel B)J or microglia cells [CDlib (Panel C)]. Immunoperoxidase stain using mAh J.3.3 was performed for viral Ag without counterstain. In A (thick arrows), Rip staining (blue) is localized on the cell membrane of numerous cells also showing cytoplasmic positivity for viral Ag (red). Virally infected Rip positive oligodendrocytes shown at high magnification in inset. In B, no GFAP positive astrocytes [(blue), thin arrows] were also positive for viral Ag [(red), arrowhead]. Similarly, in C, no CD1 lb positive microglial cells [(blue), thin arrows] were positive for viral Ag [(red), arrowhead]. Magnification, X400. Inset magnification, X I000. 1 2 0 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Discussion Immune responses to viral infection of the CNS contribute to both virus clearance and demyelination (43,25,19,27,39,5). JHMV infects all major CNS cells types resulting in an acute encephalomyelitis with demyelination (28,26,25,35,50,19,27). During acute infection, the immune mechanisms required for viral inhibition appear distinct from those which contribute to primary demyelination (19,55). It is the inability of the immune response to eliminate virus which results in persistent infections associated with ongoing demyelination. CTL eliminate infectious JHMV from infected microglia and astrocytes via a perforin-dependent cytolytic mechanism (48,29). However, virus is still eliminated from the CNS in the absence of perforin-mediated cytolysis (29), suggesting that other mechanisms play important roles in viral elimination, especially from oligodendroglia which appear at least partially refractory to CTL-mediated clearance (48). IFN-y is produced by both CD4+ and CD8+ T cells in response to viral infections (4,42). However, it is essential for vims elimination during some (5,13,30,22) but not all (28,45) infections. Direct antiviral effects of IFN-y appear to be not only dependent upon the type of infection but also the tissue or cell type infected. For example, IFN-y contributes to viral clearance from the periphery but not CNS (12), or from the CNS but not peripheral organs (24). Although JHMV was partially cleared R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. from the CNS of IFN-y7’ mice, infectious virus persisted. Consistent with the ability of the IFN-y7’ mice to exert partial control of JHMV replication, CTL effectors were not inhibited in the absence of IFN-y. These data support previous observations that other cytokines, including IL-2, EL-4, IL-6 and IL- 10, in addition to IFN-y contribute to CTL induction (42). Thus, it appears that CTL play a role in local viral inhibition even in the absence of IFN-y probably via direct perforin-mediated cytolysis of infected astrocytes and microglia (48,29). Infection of IFN-y7' mice with a JHMV variant whose replication is predominantly restricted to neurons demonstrated that IFN-y plays little or no role in the elimination of JHMV from neurons (28), similar to the inability of CTL to mediate the clearance of LCMV from neurons (30). By contrast, elimination of measles virus from neurons is mediated by a CD4+ T cell population (58), possibly due to the local secretion of IFN-y (12). The mechanism(s) of JHMV clearance from neurons is not clear; however, the majority of JHMV is cleared from the CNS of IFN-y7’ mice, supporting the important role of CTL in regulating virus replication in astrocytes and microglia (47). Vector-mediated expression of IFN-y from CNS cells infected with a different strain of neurotropic coronavirus enhanced recruitment of R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. mononuclear cells and decreased virus replication (49). These data suggested that IFN-y acts at multiple levels by influencing cell recruitment as well as serving as an anti-viral effector. Although the majority of virus was eliminated from the CNS of IFN-y'7 ' mice, infectious virus and increased numbers of Ag positive cells (mainly oligodendrocytes, but also a few glial cells and rare neurons) remained in the CNS of IFN-y7' mice in contrast to the complete elimination of infectious virus and dramatic reduction in the number of Ag positive cells in wt mice. In the absence of IFN-y no differences in total infiltrating mononuclear cells were observed in the brains of either neuronotropic OBLV60 variant (28) or JHMV infected IFN-y7' mice compared to wt controls. These data suggest that IFN-y similar to TNF-a (21) is not an absolute prerequisite for inflammatory cell entry into the CNS or the associated loss of blood brain barrier integrity during acute JHMV induced encephalitis. The absence of IFN-y resulted in increased CD8+ T cell infiltration into white matter regions. Increased CD8+ T cells correlated with increased Ag positive oligodendrocytes, suggesting that CTL accumulate within the CNS even though they appear unable to effectively inhibit virus replication within oligodendroglia. By contrast, no increased CD8+ T cells were observed in IFN-y7' mice infected with a neuronotropic (OBLV60) R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. variant (28), suggesting clearance from neurons via a IFN-y-independent mechanism. No differences in the number of demyelinating plaques was observed in the absence of IFN-y suggesting that JHMV-induced lesion formation requires additional inflammatory mechanisms. These data support the notion that IFN-y contributes to viral clearance but that it does not directly influence macrophage-mediated demyelination. Nevertheless it is possible that the equivalent demyelination in JHMV-infected IFN-y"7 ' mice occurs by an effector mechanism which differs from autoimmune demyelination (23) or alternatively it may be due to direct effects of JHMV replication in oligodendroglia (26) associated with necrotic, rather than apoptotic cell death (29). The immune mediated encephalomyelitis induced by JHMV infection is comprised of NK cells, virus-specific CTL and CD4+ T cells and monocytes. The emerging picture of this complex infection which results in viral persistence and chronic demyelination is that specific immune effector mechanisms contribute to the control of virus replication within specific subsets of the major CNS cells types. Although NK cells are rapidly recruited, they appear to play little or no role in this infectious process (18,56). Similarly, neutralizing Ab is detected only after the majority of virus is cleared (29,33), suggesting that it plays little or no role in limiting virus replication. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. By contrast, the virus-specific CTL response appears to limit acute infection of both astrocytes and microglia (48). JHMV-specific induction of CTL was initially reported to be dependent upon CD4+ T cells (55); however, recent data demonstrate that CTL are induced and traffic normally into the CNS during infection of CD4-depleted hosts (16). The inability to limit virus in the absence of CD4+ T cells is related to a requirement for the maintenance of CTL viability with the CNS. Therefore, CD4+ T cells play a critical role in CTL effector function with the CNS, in addition to their potential direct role in virus clearance (57,59). This report demonstrates that IFN-y contributes to the overall inhibition of virus replication specifically in oligodendroglia. Thus both virus-specific cytolytic activity and IFN-y appear to differentially inhibit JHMV replication within the CNS by exhibiting cell type specific effector function. The inability of CTL to inhibit replication in oligodendroglia may reflect the absence of MHC class I expression, similar to the inability of CTL specific for LCMV to limit virus replication in neurons (32). Expression of the IFN-y receptor on oligodendroglia (51) and the recent suggestion that IFN-y may be critical in limiting measles virus expression in neurons (13) both support a vital role for this cytokine in limiting virus infections within specific CNS cell types. 125 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. References 1. Adami, C., J. Pooley, J. Glomb, E. Stecker, F. Fazal, J.O. Fleming and S,C. Baker. 1995. Evolution of mouse hepatitis virus (MHV) during chronic infection: Quasispecies nature of the persisting MHV RNA. Virology 209:337. 2. Baumgarth, M. and A. Kelso. 1996. In vivo blockade of gamma interferon affects the influenza virus-induced humoral and the local cellular immune response in lung tissue. J. Virol. 70:4411. 3. Bergmann, C.C., J.A, Altman, D. Hinton, and S.A. Stohlman. 1999. Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the Central nervous system. J. Immunol. 163:3379-3387. 4. Bergmann, C.C., Y. Qin, M. Lin, and S.A. Stohlman. 1996. The JHM strain of mouse hepatitis virus induces a spike-protein-specific Db restricted cytotoxic T cell response. J. Gen. Virol 77:315. 5. Biron, C.A. 1994. Cytokines in the generation of immune responses to, and resolution of virus infection. Curr. Opin. Immunol. 6:530. 6. Bouley, D.M., S. Kanangat, W. Wire, and B.T. Rouse. 1995. Characterization of herpes simplex virus type-1 infection and herpetic stromal keratitis development in IFN-y knockout mice. J. Immunol. 155:3964. 7. Buchmeier, M.J., H.A. Lewicki, P.J. Talbot, and R.L. Knobler. 1984. Murine hepatitis virus-4 (Strain JHM)-induced neurologic disease is modulated in vivo by mAb. Virology 132:261. 8 . Campbell, H.D., C.J. Sanderson, Y. Wang, Y. Hort, M.E. Martinson, W.Q. Turcker, A. Stellwagen, M. Strath, and I.G. Young. 1988. Isolation, structure and expression of cDNA and genomic clones for murine eosinophil differentiation factor. Comparison with other eosinophilopoietic lymphokines and identity with interleukine-5. Eur. J. Biochem. 174:345. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 9. Castro, R.F., and S. Perlman. 1995. CD8+ T-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenecity. J Virol. 69:8127. 10. Cua, D.J., D.R.Hinton, and S.A. Stohlman. 1995. Self-Ag-induced Th2 responses in experimental allergic encephalomyelitis (EAE)- resistant mice. J. Immunol. 155:4052. 11. Dalton, D.K., S. Pitts-Meek, S. Keshav, I.S. Figari, A. Bradley, and T.A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon-y genes. Science 259:1739. 12. Finke, D. and U.G. Liebert. 1994. CD4+ T cells are essential in overcoming experimental murine measles encephalitis. Immunology 83:184. 13. Finke, D., U.G. Brinckmann, V. ter Meulen, and U.G. Liebert. 1995. Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis. J. Virol. 69:5469. 14. Fleming, J.O., M. Trousdale, F. El-Zaatarim, S.A. Stohlman, and L.P. Weiner. 1986. Pathogenicity of antigenic variants of murine coronavirus JHM selected with mAh. J. Virol. 58:869. 15. Fleming, J.O., S.A. Stohlman, R.C. Harmon, M.M.C. Lai, J.A. Frelinger and L.P. Weiner. 1983. Antigenic relationship of murine coronaviruses: analysis using monoclonal antibodies to JHM (MHV-4) virus. Virology 131:296. 16. Friedman, B., S. Hockfield, J.A. Black, K.A. Woodruff, and S.G. Waxman. 1989. In situ demonstration of mature oligodendrocyte and their processes: An immunocytochemical study with a new monoclonal antibody, Rip. Glia 2:380-390. 17. Graham, M. B., D. K. Dalton, D. Giltinan, V. L. Braciale, T. A. Stewart, and T. J. Braciale. 1993. Response to influenza infection in mice with a targeted disruption in the interferon-y gene. J. Exp. Med. 178:1725. 127 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 18. Houtman, J.J., and J.O. Fleming. 1996. Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM. J. NeuroVirol. 2:101. 19. Houtman, J.J., and J.O. Fleming. 1996. Pathogenesis of mouse hepatitis virus-induced demyelination. J. NeuroVirol 2:361. 20. Huang, S., W. Hendriks, A. Althage, S. Hemm, H. Bluethmann, R. Kamijo, J. Vilcek, R. Zinkernagel, and M. Aguet. 1993. Immune response in mice that lack the interferon-y receptor. Science 259:1742. 21. Jones, L.S., L.V. Rizzo, R.K. Agarwal, T.K. Tarrant, C.-C. Chan, B. Wiggert, and R.R. Caspi. 1997. IFN-y deficient mice develop experimental autoimmune uveitis in the context of a deviant effector response. J. Immunol. 158:5997. 22. Koszinowski, U.H., M.J. Reddehase and S. Jonjic. 1991. The role of CD4 and CD8 T cells in viral infections. Curr. Opin. Immunol 3:471. 23. Krakowski, M. and T. Owens. 1996. Interferon-y confers resistance to experimental allergic encephalomyelitis. Eur. J immunol 26:1641 24. Kundig, T.M., H. Hengartner, and R.M. Zinkernagel. 1993. T cell- dependent IFN-y exerts an antiviral effect in the central nervous system but not in peripheral solid organs. J. Immunol. 150:2316. 25. Kyuwa, S. and S.A. Stohlman. 1990. Pathogenesis of a neurotropic murine coronavirus strain JHM in the central nervous system. Seminar Virol. 1:273. 26. Lambert, P.W., J.K. Sims, and A.J. Rniazeff. 1973. Mechanism of demyelination in JHM virus encephalomyelitis. Electron Microscopic Studies. Acta Neuropath. (Berl.) 24:76. 27. Lane, T.E. and M.J. Buchmeier. 1997. Murine coronavirus infection: a paradigm for virus-induced demyelinating disease. Trends in Microbiol. 5:9. 128 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 28. Lane, T.E., A.D. Paoletti, and M.J. Buchmeier. 1997. Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus. J Virol. 71:2202. 29. Lin, M.T., S.A. Stohlman, and D.R. Hinton. 1997. Mouse hepatitis virus is cleared fro m the central nervous systems of mice lacking perforin-mediated cytolysis. J. Virol. 71:383. 30. Lucin, P., I. Pavic, B. Polic, S. Jonjic, and U.H. Koszinowski. 1992. Gamma interferon-dependent clearance of cytomegalovirus infection in salivary glands. J. Virol. 66:1977. 31. Njenja M.K., L.R. Pease, P. Wettstein, T. Mak and M. Rodriguez. 1997 . Interferon a/p mediates early-virus induced expression of H2° and H2k in the central nervous system. Lab. Invest. 77:71. 32. Oldstone, M.B.A., P. Blount, P.J. Southern, and P.W. Lampert. 1986. Cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system. Nature 321:239. 33. Parra, B., D.R. Hinton, M.T. Lin, D.J. Cua, and S.A. Stohlman. 1997. Kinetics of cytokine mRNA expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis. Virology 233:001. 34. Pearce, B.D., M.V. Hobbs, T.S. McGraw, and M. Buchmeier. 1994. Cytokine induction during T-cell mediated clearance of mouse hepatitis virus from neurons in vivo. J. Virol. 68:5483. 35. Perlman, S., and D. Reis. 1987. The astrocyte is a target cell in mice persistently infected with mouse hepatitis virus, strain JHM. Microbiol. Pathog. 3:309. 36. Pewe, L., G.F. Wu, E.M. Barnett, R.F. Castro, and S. Perlman. 1996. Cytotoxic T cell-resistant variants are selected in a virus-induced demyelinating disease. Immunity 5:253. 129 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 37. Pewe, L., X . Shurong, and S. Perlm an. 1997. Cytotoxic T-cell- resistant variants arise at early times after infection in C57BL/6 but not in SCID mice infected with a neurotropic coronavirus. J. Virol. 71:7640. 38. Pullen, L.C., S.D. Miller, M.C. Dal Canto, P.H. Van der Meide, and B.S. Kim. 1994. Alteration in the level of interferon-y results in acceleration of Theiler’s virus-induced demyelinating disease. J. Neuroimmunol. 55:143. 39. Rodriguez, M., K. Pavelko, and R.L. Coffman. 1995. Gamma interferon is critical for resistance to Theiler’s virus-induced demyelination. J. Virol. 69:7286. 40. Rodriguez, M., M.J. Buchmeier, M.B.A. Oldstone and P.W. Lampert. 1983. Ultrastructual localization of viral antigens in the CNS of mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Am. J. Pathol. 110:95. 41. Ruby, J., and I. Ramshaw. 1991. The antiviral activity of immune CD8+ T cells is dependent on interferon-y. Lymphokine and Cytokine Research. 10:353. 42. Sarawar, S.R., R.D. Cardin, J.W. Brooks, M. Mehrpooya, A. -M. Hamilton-Easton, X. Y. Mo and P.C. Doherty. 1997 Gamma interferon is not essential for recovery from acute infection with murine Gammaherpesvirus 68. J. Virol. 71:3916. 43. Sedwick, J. D., and R. Dorries. 1991. The immune system response to viral infection of the CNS. The Neurosciences 3:93. 44. Simmons, A. and D.C. Tscharke. 1992. Anti-CD8 impairs clearance of herpes simplex virus from the nervous system: Implications for the fate of virally infected neurons. J. Exp. Med. 175:1337. 45. Smith, A.L., S.W. Barthold, M. S. de Souza, and K. Bottomly. 1991. The role of gamma interferon in infection of susceptible mice with murine coronavirus, MHV-JHM. Arch. Virol. 121:89. 130 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 46. Stohlman, S. A., G. K. Matsushima, N. Casteel, and L. P. Weiner. 1986. In Vivo effects of coronavirus-specific T cell clones: DTH inducer cells prevent a lethal infection but do not inhibit virus replication. J. Immunol. 136:3052. 47. Stohlman, S.A., C.C. Bergmann, M.T. Lin, D. Cua, and D.R. Hinton. 1998. Cytotoxic T lymphocyte activity within the central nervous system requires CD4+ T cells. J. Immunol 160:2986. 48. Stohlman, S.A., C.C. Bergmann, R.C. van der Veen, and D.R. Hinton. 1995. Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from the central nervous system. J. Virol. 69:684. 49. Stohlman, S.A., D.R. Hinton, D. Cua, E. Dimacalli, J. Sensintaffar, F.M. Hofman, S. M. Tahara and Q. Yao. 1995. Tumor necrosis factor expression during mouse hepatitis virus-induced demyelination encephalomyelitis. J. Virol. 69:5898. 50. Sun, N. and S. Perlman. 1995. Spread of a neurotropic coronavirus to spinal cord white mater via neurons and astrocytes. J. Virol. 69:633. 51. Torres, C., I. Aranguez, and N. Rubio. 1995. Expression of interferon-gamma receptors on murine oligodendrocytes and its regulation by cytokines and mitogens. Immunol. 86:250. 52. Van Pottelsberghe, C., K.W. Rammohan, H.F. McFarland and M. Dubois-Dalcq. 1979. Selective neuronal, dendritic and postsynaptic localization of viral antigen in measles-infected mice. Lab. Invest. 40:99. 53. Wang, F. I., S. A. Stohlman, and J. O. Fleming. 1990. Demyelination induced by murine hepatitis virus, JHM strain (MHV-4) is immunologically mediated. J. Neuroimmunol. 30:31. 54. Williamson, J. S. P, K. Sykes and S. A. Stohlman. 1991. Characterization of brain infiltrating mononuclear cells during infection with mouse hepatitis virus strain JHM. J. Neuroimmunol. 32:199. 131 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 55. Williamson, J.S.P., and S.A. Stohlman. 1990. Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells. J. Virol. 64:4589. 56. Xue, S., A. Jaszewski, and S. Perlman. 1995. Identification of a CD4+ T cell Epitope within the M protein of a neurotropic coronavirus. Virology 208:173. 57. Yamaguchi, K., N. Goto, S. Kyuwa, M. Hayami, and Y. Toyoda. 1991. Protection o f mice from a lethal coronavirus infection in the central nervous system adoptive transfer of virus-specific T cell clones. ./. Neuroimmunol. 32:1. 58. Young, H.A., and K.J. Hardy. 1995. Role of interferon-y in immune cell regulation. J. Leukoc. Biol. 58:373. 59. Zhang, X.M., D.R. Hinton, D.J. Cua, S.A. Stohlman, and M.M.C. Lai. 1997. Expression of gamma interferon by a coronavirus defective-interfering RNA vector and its effect on viral replication, spread and pathogenesis. Virology 233:327. 132 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter V. Contributions of CD8+ T cell-derived IFN-y to acute JHMV infection of the CNS. Summary The relative contribution of IFN-y secretion and perforin-mediated cytotoxicity by CD8+ T cells to JHMV clearance was examined by adoptive transfer of virus-specific CD8+ T cells into immunodeficient mice. Virus replication and pathogenesis were examined in both IFN-y/perforin double deficient (IFN-y7'/ P7') and severe combined immunodeficient (SCID) recipient mice following adoptive transfer of virus-specific CD8+ T cells either deficient or competent for IFN-y secretion. Uncontrolled virus replication with high mortality in nonreconstituted IFN-y7'/ P7' and SCID mice confirmed that IFN-y and perforin-dependent cytotoxicity are the major protective antiviral effector mechanisms controlling JHMV replication in the CNS. Furthermore, CD8+ T cells derived from wt mice almost completely eliminated virus from the CNS of both immunodeficient recipients. Although viral Ag was reduced in MHC class I- expressing cells in the absence of CD8+ T cell-derived IFN- y, virus clearance was inhibited. These results demonstrate that IFN-y secreted by CD8+ T cells influences CNS clearance and confirms that CD8+ T cells reduce JHMV replication by both perforin-mediated cytolysis and IFN-y secretion. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Introduction CD8+ T cells are critical in the protection from viral infections. They may act as CTL either via perforin- or Fas- dependent cytotoxicity (14,18,19,20). CD8+ T cells also produce cytokines, such IFN-y, that exert anti-viral action in multiple ways (4,37). Contributions of the individual CD8+ T cell specific effector mechanisms in resistance to viral infection have been study in gene deficient mice. These studies are limited by the fact that many of the effector mechanisms employed by CD8+ T cells are also employed by other immune cell types (4,17,18,23). It has been suggested that the preferential usage of either lytic or non-lytic effector mechanisms correlates with viral cytophatogenicity (18,21). CD8+ T-cell mediated cytolysis may be required for immunity to nonlytic viruses (LCMV) but is not a necessary effector mechanism in resistance to lytic viruses (VV, VSV or Semliki Forest virus) (21). However, infections of perforin-deficient mice with more virulent viruses (35,46,48) suggested that perforin mediated cytolysis is also critical for resistance to some lytic viral infections in which CD8+ T cells are the predominant protective immune effector. Non-lytic mechanisms, especially IFN-y, has been suggested to contribute predominantly to the resolution of viral infection of essential organs such as the liver and CNS (7,12,24). IFN-y 134 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. is also essential for the CD8+ T cell-mediated protection during viral persistence (45). Replication of JHMV in the CNS is controlled by vims-specific~CD8+ T "cells (42). However, persistent infection and subsequent chronic demyelination are nevertheless established. Understanding the role of the different CD8+ T cell antiviral effector mechanisms during the acute phase of disease may help to explain the failure of the immune system to control the establishment of viral persistence. Specific CD8+ T cells generated in response to JHMV infection of the CNS, exhibit excellent cytolytic activity and are also potent producers of IFN-y (3). Peak CD8+ T cell infiltration into the CNS coincides with viral clearance (3,53); furthermore, both Ag-specific and non specific CD8+ T cells persist in the CNS thorough chronic stages of the infection (29). However, CD8+ T cell effector mechanisms controlling JHMV replication appear to be tightly regulated within the CNS. Analysis of infected (H-2 d x b ) FI mice showed that CD8+ T cell mediated cytotoxicity is down- regulated coincident with viral clearance during the acute infection (3). In contrast, virus-specific IFN-y secreting CD8+ T cells (3) and IFN-y mRNA (33) are still detected in the CNS during later stages of acute disease. This regulated balance may reflect an attempt to control the infection while reducing CNS immunopathology. R eproduced with perm ission o f the copyright owner. Further reproduction prohibited without perm ission. Infection of immune deficient mice suggested that JHMV replication in astrocytes and microglia is mainly controlled by perforin-dependent cytolysis (28) whereas replication in oligodendrocytes is controlled by IFN-y (Chapter IV). Therefore, both CD8+ T cell immune effector mechanisms together may be more effective in mediating JHMV clearance than either single mechanism. The relative contributions of IFN-y and perforin-dependent CD8+ T cell function to viral clearance and pathogenesis were examined by the adoptive transfer of virus-specific CD8+ T cells deficient in IFN-y secretion into recipients lacking both IFN-y and perforin-mediated cytotoxicity and into immunodeficient SCID mice. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Material and Methods Mice Wild type BALB/c and C57BL6 and BALB/c SCID mice were obtained from The National Cancer Institute (NCI), Frederick, MD. Homozygous IFN-y 'A BALB/c mice were kindly provided by Robert Coffman (DNAX Reseach Corporation, Palo Alto, CA) and bred locally at USC. IFN-y mice on the C57BL/6 background were maintained by homozygous breeding as described in Chapter IV. Breeding pairs of perforin deficient mice on the C57BL/6 background (P'A ) were obtained from the Jackson Laboratory. Dr. William Clark (University of California, Los Angeles) kindly provided homozygous Y 1 ' mice on the 129 (H-2b) background (49). Mice were seronegative for MHV by ELISA. All mouse strains were maintained in the vivarium facility at USC. Immunodeficient mice were maintained under sterile conditions. Mice deficient in both IFN-y secretion and perforin mediated cytolysis (IFN-y^VP'7 ') were generated on both the C57BL/6 and BALB/c backgrounds by crossing IFNy'7 ’ mice with P'7 ' mice. Since the genes encoding IFN-y and perforin are both located to chromosome 10, Fj mice were backcrossed to IFN- y ~ A mice to facilitate homologous recombination. Mice homozygous for the mutation in IFN-y and heterozygous for the mutation in perforin gene (IFN-y~A R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. /P+ 7 ') were selected by PCR and intercrossed to obtain homozygocity in both mutated genes (IFN-y'7 '/P'7 '). Mice were screened by PCR from tail tissue DNA using specific primers. A schematic representation of the screening procedure is showed in figure 1. Primers for detecting the mutated and wild type perforin and IFN-y genes are described in Table I. Amplification was carried out with approximately lpg of DNA using a single initial denaturation step at 94°C for 4 min and 30 cycles of one denaturation step at 94°C (lmin), primer annealing at 60°C (lmin), extension step at 72°C (2.5 min) followed by a final extension step for 7 min. Heterozygous IFN-y‘/7P+ /' mice on the BALB/c background were obtained by backcrossing the initial Fi [IFNy'7 ' (H- 2d ) x P'7 ' (129, H-2b)] with BALB/c IFN-y7 ' mice. IFN-y"7 7P+ /' mice were subsequently backcrossed for nine generations (N9) with IFN-y'7 ' BALB/c mice. Examination of naive single deficient IFNy'7 ' or P'7 ' mice and naive double deficient mice IFNy'7 ' /P'7 ' mice showed normal CD4+ /CD8+ T cells ratios in the spleen, suggesting the absence of an endogenous defect in the CD8+ T cell population of these mice. 138 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 139 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 1 . Strategy t o generate double deficient IFN-y' TP'' mice Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table 1. DNA primers sequences used to screen IFNy^VP"7 ' mice Mouse Strain Gene Primers Sequences Size (bp) Reference C57BL/6 pa wt 5 C 5-AGG TCA GGC CAG CAT AAG AGT AG-3 519 18 3 D 5-CCA CAC AGC CCC ACT GCG GTT TC-3 KO° 5 C 800 3 Neo727 5-ATC GAC AAG ACC GGC TTC CAT CCG A-3 BALB/6 wt 5 P12 5-TGG CCT AGG GTT CAC ATC CAG-3 500 48 3 P17 5-CGT GAG AGG TCA GCA TCC TTC-3 KO 5 P12 350 3 P26 5-ATA TTG GCT GCA GGG TCG CTC-3 C57BL6 or BALB/c IFN- y wt KO 5 y5 5-AGA AGT AAG TGG AAG GGC CCA GAA G-3 240 3 y3 5-AGG GAA ACT GGG AGA GGA GAA ATA T-3 5 Neo 725 5-TCA GCG CAG GGG CGC CCG GTT CTT T-3 340 3 Neo 727 a Perforin gene b Primers identifying wild type (wt) genomic sequences c Primers identifying the seq. knockout with the Neomycin insert V iru s infection Mice were infected i.e. with the attenuated 2.2v-l variant of JHMV (500 pfu in 32 pi of Dulbecco’s PBS, pH 7.4), which produces an acute encephalomyelitis with progressive chronic demyelination (8). Virus titers and pathogenesis were examined until 14 days p i in mice with the H-2d background and following 29 days in mice with H-2b background. BALB/c wt mice and BALB/c IFN-y _ /' donor mice were immunized i.p. with lxlO6 pfu of the DM variant of JHMV. At 4 weeks post-immunization spleens were removed to purify CD8+ cells. Approximately 1-10% of the CD8+ T cell population in the spleens were virus-specific as determined by flow cytometry using class I tetramer technology (3). CD8+ T cell purification CD8+ T cells were purified by negative selection via magnetic cell sorting (MACS) columns as previously described (41). Spleen cells from immunized IFNy7' or wt mice were first partially depleted of B cells by adsorption on to 150 mm plates coated with goat anti-mouse immunoglobulin (5ml/plate at lOOpl/ml in calcium and magnesium free Dulbecco’s PBS; Cappel Laboratories, Costa Mesa, CA). Anti-mouse Ab-coated plates were washed three times with calcium and magnesium free Dulbecco’s PBS prior to o addition of 4x10 spleen cells in 10 ml of calcium and magnesium free Hank’s R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Balance Salt Solution (HBSS) containing 10% FCS and 25mM HEPES buffer, pH 7.4. After 30 min incubation at room temperature, unattached cells were removed. CD4+ T cells and residual B cells were depleted with anti-CD4 (L3T4; Miltenyi Biotec Inc. Auburn, CA) and anti-CD 19 (Miltenyi Biotec Inc. Auburn,CA) mAbs conjugated to magnetic beads according to the manufacturer instructions. Briefly, cells were resuspended in MACS buffer (2.5 mM EDTA, 0.5% BSA in RPMI) at lx l0 8 cells/ml and mixed with the mAbs conjugated to magnetic beads (5 pi each per lxlO7 cells). After lh incubation at 4°C, cells were washed in 10 mis of MACS buffer, centrifuged (200x g; 4°C; 7 min) and resuspended in 500pl of MACS buffer. Cell suspension were then passed through a steel wool column (Miltenyi Biotec) pre-equilibrated with MACS buffer and attached to a MACS magnet (Miltenyi Biotec). The unattached fraction containing the CD8+ T cell enriched population was eluted by three washes with MACS buffer. All magnetic separation procedures were done using MACS buffer at 4°C. Purity was determined by flow cytometric analysis using the following mAbs antibodies from PharMingen: PE- anti-CD8+ (Clone 53-6.7); FITC-anti-CD4+ (clone GK1.5); FITC-anti-CD19 (clone 1D3). CD8+ T cells were enriched to approximately 70-90%. CD4+ T cells were completely depleted. Contamination with CD19+ B cells ranged from 1 to 10%. 1 4 2 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CSFE labeling To confirm trafficking of CD8+ T cells into the CNS of the adoptive transferred IFN-y recipient mice, purified CD8+ T cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CSFE) [(CFDA SE cell Tracer Kit); Molecular Probes, Eugene,OR] prior to adoptive transfer , according to the manufacturer instructions. Briefly, a lOmM stock solution of CFSE in DMSO was diluted 1:1000 with PBS (pH 7.2) to a final concentration of lOpM. Cell suspensions (2x107 cells/ml of PBS) were mixed with an equal volume of the diluted CSFE, vortexed and incubated for 10 min at room temperature (RT). The reaction was terminated by addition of an equal volume of FCS for 1 min at RT. Labeled cells were then washed twice with 10% FCS in RPMI medium and once with serum free RPMI. Adoptive Transfers Recipient IFN-y'7 '/?'7 ' mice and SCID mice received purified CD8+ T cells from either IFNy'7 ' or wt immunized mice. Mice were reconstituted intravenously with l-2xl07 CD8+ T cells and infected i.e. with JHMV (as described above) approximately 5 hours later. Viral replication and pathogenesis were determined at days 10 and 14 p.i. at the time when the non reconstituted control mice started to succumb. Animals were sacrificed by asphyxiation with C02 and brains and spinal cords removed. One half o f the R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. brain tissue was processed for histology and the other half homogenized in RPMI medium containing 25 mM hepes using glass Ten Broeck tissue homogenizers (as described in Chapter IV). Homogenized samples were centrifuged at 200 x g for 7 min and supernatants collected for virus titer determination. Virus titers were determined in brain homogenates by plaque assay on DBT cells as described in chapter II. Pellets were used for isolating mononuclear inflammatory cells. Characterization of the CNS mononuclear cell population. Mononuclear cell populations were isolated from brains by tissue homogenization and centrifugation on Percoll gradients as previously described (2; Chapter IV). In brief, halves of brains were homogenized in RPMI supplemented with 25 mM of HEPES, pH 7.2 and adjusted to 30% Percoll (Pharmacia). The suspension was underlayed with 1.0 ml of 70% percoll. After centrifugation at 800x g for 20 minutes at 4° C, mononuclear cells were recovered from the 30%/70% interface. CD4+ T cells and CD8+ T cells in the isolated mononuclear cell populations were characterized by flow cytometry using PE labeled anti-CD4 (GK1.5) and FITC labeled anti-CD8 (53- 6.7) mAbs (PharMingen). Virus-specific CD8+ T cells were detected by double staining with FITC anti-CD8 and the PE-labeled Ld major histocompatibility complex (MHC) class I tetramer reagent associated with 1 4 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. pN318-326 peptide (LdN-318). Brain infiltrating cells were also assayed for ex-vivo cytolytic activity using th e 5'Cr release assay (Chapter III, 2). MHC class I expression on microglia was analysed in infected, non reconstituted and CD8+ T cell reconstituted SCID mice. Double staining with PE anti- CD45 (Ly-5) and FITC anti-CD lib (Ml/70) specific mAbs. CD45lo w CD1 lb+ was used to characterize microglia (5). MHC class I Ld expression on the double stained CD45lo w C D llb+ microglial population was determined by the mean fluorescence intensity of staining with a biotinylated anti- H2Ld (H- 2Ld /H2Db) specific mAb using streptavidin-Cy-chrome as the detection reagent. All antibodies and secondary conjugates used for flow cytometric analysis were obtained from PharMingen. Cytotoxic activity assay Ex-vivo cytolytic activity of brain mononuclear infiltrating cells was assayed in a 5 1 Cr release assay as described in Chapter III and elsewhere (2,3). CNS cells from pooled groups of six to seven mice were tested for cytolytic activity against peptide pulsed (pN 31 8- 3 3 5; l.OpM) 5 1 Cr -labeled J77.4 target cells at various E:T ratios. Data are expressed as percent specific release defined as [(experimental release)-(spontaneous release)]/[(total release- spontaneous release)]. Maximun spontaneous release values were < 20% of the total release value. 1 4 5 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. T cell proliferation Infected- IFNy'/7P'/' mice and control wt mice were compared for CD4+ and CD8+ T cell proliferative responses to viral Ags. Cervical lymph node (CLN) and spleen cells were isolated from IFNy7 ' P'7 ‘ and wt control mice at days 7 and 14 p.i. Cells were cultured in 96 well plates (8x105 cells/well; RPMI 1640 medium supplemented with 1% normal mouse) in the presence of various dilutions of a UV- inactivated lysate of JHMV infected DBT cells (approximately 5xl07 -108 pfu prior to UV inactivation) or virus-specific peptides (pN47 [H2d] or p510[H2b]). Cells were pulsed with 1 pCi /well of 3 [H]-thymidine (ICN Radiochemicals, Irvine,CA) after 60 hr incubation at 37°C and harvested after 10 to 15 hr later. Radioactive incorporation was measured by liquid scintillation spectroscopy and data expressed as the mean counts per minute of triplicate wells. Proliferation indices (P.I.) were calculated as the ratio of mean counts and the mean spontaneous proliferation. Histopathological Analysis To determine inflammation and demyelination, brains and spinal cords were removed and fixed with Clark’s solution (75% ethanol and 25% glacial acetic acid) and embedded in paraffin. Sections were stained with either hematoxilin and eosin or luxol fast blue as described (28). All staining procedures were performed by Wen Qian. To examine viral Ag distribution R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. sections were incubated with mAb J3.3 specific for the viral nucleocapsid protein as described in Chapter III. To phenotype virus Ag positive cells, mice were transcardiacally perfused with 4% paraformaldehyde in PBS (pH, 7.4). Brain and spinal cords were removed, postfixed with 4% paraformaldehyde at 4°C for 24 hr, and then equilibrated with 30% sucrose in PBS (pH,7.4) at 4°C by overnight incubation. Frozen sections made from the perfused-glucose equilibrated tissue were double stained using the JHMV specific mAb J3.3 and mAb specific for astrocytes (Glial Fibrilar Acidic Protein [GFAP]; Dako, Carpinteria, CA) or microglia (CD lib; PharMingen) as described in chapter IV. JHMV Ag and GFAP double staining was visualized with peroxidase Vector VIP and Vector SG substrate kits (both from Vector Laboratories) respectively. Viral Ag in CD lib-positive cells was detected using alkaline phosphatase Vectastain ABC kit for J.3.3 mAb and peroxidase Vectastain ABC kit with AEC substrate for CD1 lb mAb. CD8+ and CD4+ T cell infiltration was examined by immunoperoxidase staining of frozen and acetone-fixed sections of non-perfused brain and spinal cord tissues using rat anti-CD8a (Ly-2) and anti-CD4 (L3T4) mAbs from PharMingen as primary Ab. Primary Ab were detected by immunoperoxidase staining with biotinylated rabbit-anti-rat Ab (Vector Laboratory) and Vectastain-ABC kit. AEC was used as chromogen substrate (Vector 147 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Laboratories). Apoptotic cells were determined in acetone-fixed frozen sections by Tunel assay using the Oncor Apop Tag kit (Gaithersburg, MD) with terminal deoxinucleotidyltransferase (TdT), as indicated by the manufacturer. All the histology slides were examined with the collaboration of Dr. David Hinton and Dr Roscoe Atkinson. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Results Infection in the absence of perforin-dependent cytotoxicity and IFN- y secretion. Pathogenesis of JHMV in mice deficient in either perforin-mediated cytolysis or IFN-y secretion suggested that both mechanisms contribute to inhibit virus replication within the CNS. However, these studies also suggested that the anti-viral function of these two effector mechanisms can be partially substituted by each other or by another mechanism(s) (28;Chapter IV). To determine the contribution of both mechanisms to viral clearance and pathogenesis, double-deficient IFN-y'7 ’/?'7 " mice were generated and infected with the 2.2v-l variant of JHMV. Wt mice usually controlled the infection and recovered from clinical disease by day 21 p.i. (Table 2). In contrast, double-deficient mice succumbed to disease after day 14 p.i. (Table 2; Table 3) Although double deficient H-2b mice exhibited prolonged survival, they showed no signs of recovery by day 29 p.i, the last time point examined (Table 2). These results are consistent with the high mortality and morbidity exhibited by IFN-y '7 ' mice following JHMV infection (Chapter IV). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Table 2. Summary of Clinical Disease in IFNy"/'P'/" mice and wt mice. Day 7 Day 14 Day 21 Day 29 WT 1.0±0a 2.6±0.5 2.1+0.4 1.7+0.5 C57BL/6 IFNy^TP'7 ' 1.0±Q 3.0±0.5 3.1+ND 2.7+1.2 WT 1.0+0 1.810.2 NDb ND BALB/c IFNy'A /P‘ A 1.710.2 3.510.4 ND ND Clinical scores were graded as followed: 0, healthy; 1, hunched back and raffled fur; 2, slow mobility and inability to upright; 3, paralysis and wasting;4, moribund and death. Data shown are the average clinical scores of at least 3 mice per time point. Experiment was done once in the BALB/c and twice in the C57BL/6 background. ND = Not determined Table 3. Summary of Survival in IFNy^'/P"7 " mice and wt mice. Day 7 Day 14 Day 21 Day 29 WT c 3 o © 100 88 75 C57BL/6b IFNy/T r/~ 100 88 38 25 WT 100 100 NDd ND BALB/cc IFN yV ' 100 40 ND ND Numbers represent the percentage survival Summary of two independent experiments (12-15 mice /experiment) Summary of one experiment (15 mice total) Not determined 150 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Virus was completely cleared before day 14 p.i. from the CNS of wt mice as opposed to double deficient mice in which virus still remained at high titers in the moribund animals at days 14 (H-2d mice) and 29 p.i. (H-2bmice) (Figure 2). These results confirm that perforin-dependent cytotoxicity and IFN-y are the mune effector mechanisms primarily controlling the replication of JHMV in the CNS. C57BL/6 BALB/c â– IFN ^'P"7 " â–¡ W T 8s 5 4 - c 2 * 5 E O J 3 U , a. 2 - 1 - - 5 7 14 21 29 s c " r a £ i * 5 E a> s 5 4 3 21 1 14 Days post infection Figure 2. Uncontrolled CNS viral replication in the absence of both IFN-y and perforin-dependent cytotoxicity. IFN-y'" /7P'/' mice were generated and JHMV replication examined in the CNS by plaque assay at various time points p.i. Dashed line represents the detection limit of the plaque assay. Virus titers are the mean of at least 3 mice per group. Experiments were performed once in BALB/c (H-2d ) mice. Data for C57BL/6 (H-2b) mice are the composite of two independent experiments. 151 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Enhanced CD8+ T cells numbers in double-deficient IFNy‘/_ /P^'mice To determine the magnitude of T cell responses in the CNS in the absence of the two major anti-JHMV effector mechanisms, T cell infiltration into the CNS was examined by flow cytometry comparing wt and IFNy'/7P'/“ infected mice. Similar numbers of CD4+ T cells infiltrated the brains of both groups. In contrast, higher frequencies of CD8+ T cells infiltrated the brain of double deficient mice compared to the wt controls (Table 4; Figure 3). This finding was consistent in both H-2d and H-2b mice. Although the same proportion of virus-specific CD8+ T cells relative to the total CD8+ T population were present in the CNS of normal and double deficient mice, the absolute number of virus-specific CD8+ T cells was higher in the double deficient mice (Table 4; Figure 3). Consistent with the flow cytometry data, immunohistochemistry staining showed higher numbers of infiltrating CD8+ T cells in the brain parenchyma and spinal cords of the double deficient mice compared to wt mice. These results suggest either increased CD8+ T cell expansion, recruitment or accumulation as a consequence of defective perforin-dependent cytotoxicity and/or the absence of IFNy'/’ secretion (30,48). Tunnel staining revealed no decrease in the number of apoptotic cells in the CNS of double deficient mice. These data are consistent with increased CD8+ T cell expansion and /or recruitment rather than accumulation due to decreased R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. apoptosis. Interestingly, virus-specific CD8+ T cells, usually not detected in the peripheral tissues of wt infected mice (3), were detected in both CLN and spleens of IFNy'7 '/ P‘ A mice (Figure 3), suggesting increased expansion of virus-specific CD8+ T cells in the periphery. Table 4. Summary of T cell infiltration in the CNS of JHMV-Infected IFNy^VP"7 " mice and wt mice. CD4+ Day 7 CD8+ Tetramer+ b CD8+ CD4+ Day 14 CD8+ Tetramer+ CD8 WT 8a 9 35 6 7 46 BALB/c IFNy-/7 p-/- 12 24 54 4 23 56 WT NDC ND ND 24 16 52 C57BL/6 IFNy'7 Y ' ND ND ND 12 51 35 Numbers represent the percentages within the total population Numbers are the percentages of tetramer+ cells within the CD8+ T cell population Not determined R eproduced with perm ission o f the copyright owner. Further reproduction prohibited without perm ission. A . BR A IN CLN SPLEEN B. 00 v H Z B . 3.0 â– f ^ 46) i n k j j l .... FL1 -Height CD8+ 4.0 â– -A 200 400 600 800 1000 FLi-Height :47.0 - v * 1 7- 0 200 400 600 800 1000 FLi-Height . 15.0 200 400 600 800 1000 FL1 -Height 0.1 200 400 600 800 1000 FLi-Height 200 400 600 800 1000 FLi-Height 200 400 600 800 1000 FL1-Height \ T 7 P . q.. j . 3 # 1 4 0 200 400 600 800 1000 FL1 -Height 200 400 600 800 1000 FL1-Height â– â– . 200 400 600 800 1000 FL1 -Height 1.0 • (6) .jjv • 200 400 600 800 1000 FLi-Height 0 200 400 600 800 1000 FLi-Height CD8+ Figure 3. Increased CD8+ T cells numbers in the CNS of double deficient IFNT7 7P'7 ' after 14 days p.i with JHMV. CNS mononuclear cells, CLN and spleen cells from WT, A) or IFN-y'7 ' P’7 ', B) infected-mice were stained for the expression of CD8+ (FITC), CD4+ (PE) and N epitope specific TCR (LdN318). Numbers represent percentages of the total population. Similar results were obtained at day 7 p.i. Experiment was done once. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. The frequency of total CD8+ T cells in peripheral tissues of IFNy'7 '/ P‘A was similar to wt mice (Figure 3). However, consistent with the presence of tetramer positive CD8+ T cells, splenic T cells from IFNy^TF7 ' exhibited better proliferative response to peptides comprising the CD8+ T cell epitopes compared to spleen T cell from wt mice (Figure 4). These results suggest enhanced priming or activation in the periphery. Therefore, enhanced CD8+ T cell numbers in the CNS of IFNy'/7P~A may result from a combination of both increased local recruitment and/or retention and to increased peripheral activation. To examine CD4+ T cell responses in the double deficient mice, specific proliferation to viral Ag was examined in peripheral tissues. CD4+ T cells from spleen and CLN of JHMV-infected double deficient mice proliferate to UV-inactivated JHMV in a similar manner compared to cells derived from wt mice (Figure 4). Interestingly, background proliferation in the absence of Ag is higher in IFNy^VP7" (Figure 4), suggesting either increased activation or the presence of viral Ag in these tissues. Similarly, in one preliminary experiment, IFNy'7 '/?"7 ' mice serum Ab titers to JHMV were similar to those of wt mice as determined by ELISA. These data indicate that the magnitude of anti-JHMV CD4+ T cell responses, as opposed to CD8+ T cell responses, is not modified by the simultaneous absence ofboth IFN-y and perforin. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. W T I N F - f / - / p - / - a. o 10000 (5.1) 8000 - 6000 - (5.1) 4000 - 2000 - o CM O O CD 0 0 CL O Dilution of u.v JHMV lysate pN47 peptide Figure 4. Specific proliferative T cell response in the periphery of JHMV- infected IFN-y'/7P"/' mice. Spleen cells at day 7 p.i. were cultured with either UV inactivated JHMV lysate or the specific N protein CD8+ T cell epitope peptide (pN47) at various concentrations. Proliferation was measured after 60 h. Numbers in parenthesis represent the maximum proliferation index value (P.I). Similar results were obtained at day 14 p.i. IFN-y and perforin-dependent cytotoxicity are not required for JHMV- induced demyelination. Histopathological findings in the CNS of IFNy'/‘/P'/' mice at different time points p.i. are summarized in Table 5. Numbers of virus Ag positive cells, as determined by staining with the specific J3.3 mAb, were consistent R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. with the kinetics of viral replication. Very few virus positive cells remained in the brains of wt mice after 14 days p.i. In contrast, numerous Ag positive cells were still present in the CNS of double deficient mice at the same time points p.i. (Table 5). Inflammation was decreased in CNS tissues of wt mice at days 14 and 21 p.i compared to the acute phase of infection. By contrast, inflammation remained slightly increased in the double deficient mice at the same time points, consistent with higher CD8+ T cell infiltration (Table 5). Demyelination was present at a similar extent in double deficient mice compared with wt mice. Numerous areas of demyelination were observed in either group examined after 14 days p.i. These results confirm the findings in single IFN-y or perforin-deficient mice, suggesting that neither IFN-y nor perforin-dependent cytotoxicity are key mediators for JHMV-induced demyelination. IFN-y Secreted by CD8+ T Cells Contributes to CNS Viral Clearance. To examine the relative contribution of IFNy7' secretion by virus- specific CD8+ T cells to CNS clearance, virus-specific CD8+ T cells derived from IFN-y7' or wt IFN-y+ /+ donors were adoptive transferred into syngeneic IFNy7'/?7' (H-2d ) recipients. Therefore, viral replication and pathogenesis were examined in the sole presence of CD8+ T cell perforin- dependent cytotoxicity. Viral titers were determined in brains ofboth reconstituted R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table 5. Summary of the histopathological features in the CNS of IFNy'^/P"7 ' mice and control wt mice at various days post infection. Inflamm.a Day 7 p.i Demyeln.b Ag° Inflamm. Day 14 p.i. Demyelin Ag Inflamm Day 21 p.i. Demyelin. Ag BALB/c WT 2+ to 3+ 0 2+ to 3+ + to 3+ + to 2+ + NDd ND ND IFNy_ / 7P‘ A 2+ to 3+ 0 to + 2+ 2+ to 3+ ± to 3+ 2+ to 3+ ND ND ND C57BL/6 WT 2+ to 4+ 0 + to 2+ + to 2+ 0 to 2+ 0 to ± + + to 2+ 0 to ± IFNy " /7P‘ /' 3 + to 4+ 0 2+ to 4+ 3+ to 4+ + to 3+ 2+ to 3+ 2+ + to 2+ 2+ to 3+ Formalin fixed section were stained with hematoxilin and eosin. Cells were semi- quantitated as followed: 0, no infiltration; 1+, 10 cells; 2+, 10-50 cells; 3+, 50-250cells; 4+, >250. Luxol fast blue stained sections. Immunoperoxidase staining for the JHMV nucleocapside protein. Viral antigen positive cells were semi-quantitated as followed: 0, no positive;+ <10 cells; +, 10 cells; 2+, 10-50 cells; 3+, 150-250 cells; 4+ >250 cells. At least three mice per time point were examined. Not determined 0 0 groups at days 7 and 14 p.i. Recipients of wt CD8^ T cells completely cleared virus by day 14 p.i. By contrast, infectious virus was still present in the brains of mice reconstituted with IFN-y'7 ' CD8+ T cells (Figure 5). Nevertheless, viral replication was reduced by 1.5 logio compared to unreconstituted mice (Figure 2), consistent with the intact perforin dependent cytolytic activity. O f 5 c re 4 O Q O , (3 2 3 u . 1 I WT CD8+ â–¡ IFN-y-'- CD8+ f t 7 14 Days post infection Figure 5. CD8 T cell-derived IFN-y contributes to viral clearance in the CNS. CD8+ T cells from JHMV immunized IFNy'7 ' or wt mice were isolated by immunomagnetic cell sorting (negative selection). CD8+ T cells were adoptively transferred into IFNy'7 '/?'7 ' mice and recipients infected with 2.2v-l variant of JHMV. Virus replication in brain was determined by plaque assay on DBF cells. Titers in each group represent the mean of at least 4 mice per group. Dashed line represents the limit of the assay. Experiment was done only once. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Transferred CD8+ T cells trafficked into the brain parenchyma as determined by the presence of CSFE- labeled CD8+ T cells by flow cytometric analysis. CD4+ and CD8+ T cells were recruited to the CNS of both groups of recipients. However, increased numbers of CD8+ T cells were detected in the brains of mice adoptively transferred with IFN-y_/~ CD8+ T cells at day 14 p.i. compared with mice reconstituted with IFN-y+ /+ CD8+ T cells (Table 6). Table 6. Summary of T cell infiltration in the CNS of CD8+ -reconstituted IFNy'/7P/' micea (H2d ). Donor T cells Day 7 p.i Day 14 p.i CD4+ CD8+ Tetramer+ b CD8+ CD4+ CD8+ Tetramer+ CD8+ WT CD8+ 9 28 26 23 28 30 IFNy-/- CD8+ 10 26 25 22 42 30 a CNS mononuclear cells were isolated from brain and spinal cords pooled from groups of three mice. Expression of surface markers was determined by flow cytometric analysis as indicated in Material and Methods. Numbers represent the percentages in the total population. b Numbers represent the percentage of tetramer+ cells in the CD8+ T cell population. Experiment was done once. The inability of IFN-y_ /' CD8+ T cells to mediate clearance, despite increased infiltration, suggests perforin-mediated cytotoxicity alone is ineffective to eliminate JHMV from CNS. These data are further consistent 160 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. with the increased CD8+ T cell infiltration in non T cell transferred double deficient mice (Fig. 3). The presence of viral Ag and/or the absence of IFN-y thus appear to increase CD8+ T cell infiltration in double deficient IFN-y'7 ' and P'7 ' mice. These data confirms that indeed CD8+ T cell derived IFN- y contributes to JHMV clearance in the CNS. IFN-y secreted by CD8+ T cells alone inhibit viral replication in CNS. Although adoptive transfer into double deficient recipients suggested an important role of CD8+ T cell derived IFN-y in viral clearance, the interpretation of outcomes in gene deleted mice is complicated by the presence of other infiltrating lymphocytes, in addition to the increased CD8+ T cell response in these mice. Therefore, experiments were conducted by reconstitution of SCID mice to eliminate potential contributions of the endogenous CD4+ T cell and B cell populations. SCID mice reconstituted with virus-specific CD8+ T cells derived from immune wt mice reduced viral replication at 10 and 14 days p.i. as compared with non-reconstituted SCID mice (Fig. 6). By contrast, virus clearance mediated by virus-specific CD8+ T cells derived from immune IFN-y'7 ' donors was less effective than CD8+ T cells derived from wt donors (Fig. 6). These results demonstrate that IFN-y derived from CD8+ T cells alone influences the clearance of JHMV from the CNS. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 6. Virus-specific CD8+ T cells reduce CNS JHMV replication via IFN-y. CD8+ T cells from JHMV-immunized IFNy'7 " or wt mice were isolated by immunomagnetic cells sorting (negative selection). 2xl07 cells were transferred i.v. into SCID mice. Mice were infected i.e. with JHMV and virus replication quantitated by plaque assay on DBT cells. Virus titers are expressed as Logio pfu per gm of tissue. Titers in each group represent the mean of at least 4 mice per group. Dashed line represents the limit of the assay. Figure is the composite of two experiments. These results are consistent with previous data which suggested that IFN-y participated in clearance of JHMV from CNS (Chapter IV) and the transfer experiments into IFN-y7"/?'7 ' mice (Fig. 5). Nevertheless, virus titers were still reduced by CD8+ T cells from IFN-y"7 ' donors compared to unreconstituted SCID mice (Fig. 6). The ability of these CD8+ T cells to R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. partially clear CNS virus is consistent with the expression of perforin mediated cytotoxicity. These data independently confirm that CD8+ T cells reduce JHMV replication from the CNS by both perforin cytolysis as well as IFN-y secretion. Consistent with the presence of infectious virus in the CNS, viral Ag in recipients of IFN-y'7 ' CD8+ T cells was increased compared to the residual viral Ag positive cells present in the CNS of SCID mice receiving IFN-y+ /+ CD8+ T cells at 14 days p.i. Viral Ag was almost totally cleared from microglia and astrocytes in the brains of both groups of SCID mice (Fig. 7). Furthermore, viral Ag was mainly localized to oligodendrocytes within the spinal cords of either reconstituted SCID groups (Fig. 7), as opposed to the multiple infected cell types present in the spinal cord of the unreconstituted SCID mice (Fig. 7). In contrast to reduced viral Ag positive cells present in the CNS of recipients reconstituted with IFN-y-secreting CD8+ T cells, increased numbers of infected oligodendrocytes were present in the CNS of SCID recipients of CD8+ T cell derived from IFN-y7' donors at day 14 p.i. (Fig. 7). These data confirm the notion that CD8+ T cells control JHMV replication in oligodendrocytes via secretion of IFN-y (Chapter IV). 163 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 7. Viral Ag in oligodendroglia is reduced via CD8+ T cell-derived IFN-y. Viral Ag positive cells in brains of wt CD8+ T cell- (A,C), and IFN-y 7' CD8+ T cell- reconstituted SCID mice (B,D) at day 14 p.i. Double staining was performed on brain sections with the JHMV specific mAh J.3.3 (thin arrows) and Ab specific for microglia (CD lib) in A, B panels [(red); thick arrows] or astrocytes (GFAP) in C and D [(blue); thick arrows]. Magnification, X 110. Viral Ag in spinal cords of unreconstituted, (E); wt CD8+ T cell reconstituted, (F) and IFN-y7' CD8 reconstituted,(G) SCID mice at day 14 p.i. Arrowhead points indicate oligodendrocytes. Magnification, X 200. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. , : - i . •<* . ^ « i t * f e • '* •: *■’ â– , f ............................... "V * / - r - . v . . - f . . -.**. h - â– .â– *e * £ "â– , --* » "* 4 ' i *1 " X X : ' X V 'J» : v â– .. \ ,: ' ::;:; g :|T .x . . . ; p t : .. " ' ...*...» ^ . ; : i , . . . , â– }. - l ? ' . A V 9,........... * * »X . . . . . . : f £ _ s ...: A * .. x . . . . . » .^ ...., : ..= i . . 1 ..... ' . . ..........;. * . â– a * * • " v ** J|i as * ' â– â– ' » â– â– ' - â– : â– â– â– * â– â– . * * • r V " * ^ ' ' â– * ; x :â– , * ' â– - • • * â– ** * - ’• * . • ; . . * * X * ‘ V 4 , - . * t * ^ * * # • % ' > * ' 4 ^ / - â– â– --â– : - " g â– * , , ’ â– - : j , * * 5 - " r= - R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Functional virus-specific IFN-y'/'CD8+ T cells are recruited to CNS. To determine if the reduced ability of virus-specific CD8+ T cells from IFN-y"7 ’ donors, compared to wt donors, to clear infectious virus was due to defects in trafficking or lack of perforin-dependent cytolysis, rather than to a direct effect on virus replication, mononuclear cell populations were isolated from infected brains and analyzed for phenotype by flow cytometry and for function by ex vivo cytolysis. This analysis showed that similar proportions of virus-specific CD8+ T cells were recruited to the brains of both groups of reconstituted SCID mice (Fig. 8). These data confirm that virus-specific CD8+ T cells from IFN-y’ 7 ’ donors traffic normally into the infected CNS in the absence of other potential immune effects. Analysis of mice taken at different time points p.i. revealed that IFN-y'7 ’ CD8 + T cells were recruited into the infected CNS as early as 4 days p.i. and accumulated with both the same kinetic and to similar numbers in both groups of reconstituted mice. Consistent with the phenotypic analysis by flow cytometry, staining of CNS tissue showed equivalent numbers of CD8+ T cells within the brain parenchyma and along the white matter tracts in the spinal cords in both groups. Importantly, the CD8+ T cells deficient in IFN-y secretion infiltrating the infected CNS at day 10 p.i. retained ex vivo perforin mediated cytolysis (Fig. 8). R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. WTCD8* IFN-y/ CD8+ Q C r> i £ i " S A 9(47%) 1 04 o _l o U . T 200 400 600 800 1000 FL1 -Height CD8+ 6(38%) n 200 400 600 800 1000 FL1-Height B o V) ( 0 o o q : â– o o C o 0 ) a. to 16 14 12 10 8 6 4 - 2 â– W+£t>8+ . . o IFN-y-/- CD8 O 25:1 12:1 6:1 3:1 E:T Figure 8. Virus -specific CD8+ T cells from IFN-Y _ /"donors are recruited IFN-Y ^ ’donors are recruited into the CNS of SCID mice. CNS infiltrating cells were prepared from infected, adoptively transferred recipients at day 10 p.i. Cells were stained for CD8 and virus-specific TCR (Ld-N318 tetramer) (Panel A). Numbers represent the percentages of the total population. Data is representative from two experiments. (Panel B), the ex-vivo cytolytic activity of CD8+ T cells from the CNS on pN peptide-coated J774.1 targets at the indicated E:T ratios. Cytolysis is shown as the percentage specific lysis of peptide-coated minus untreated J774.1 targets. Nonspecific cytolysis was zero as determined with uncoated targets. Experiment was done once. 167 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. These data are consistent with elimination of virus from most of MHC class I expressing cells, i.e., astrocytes and microglia (Fig. 7). These results support previous data which indicated that JHMV specific CD8+ T cells exert immune effector functions in the CNS through the concerted action of both perforin mediated cytotoxicity and IFN-y secretion (28; Chapter IV). CD8+ T cell derived IFN-y-independent upregulation of MHC class I expression. IFN-y potentially contributes to viral clearance by increasing expression of MHC class I molecules, thereby contributing to more effective CD8+ T cell stimulation (perforin mediated cytolysis of virus infected cells or higher IFN-y). To determine if the absence of IFN-y secretion by the CD8+ T cells influenced their ability to clear JHMV, expression of the MHC class I Ld molecule, the restriction element for the immunodominant N epitope (2), was examined on microglia/macrophages within the infected CNS. Compared to naive mice, MHC class I expression was increased on CD45,0 W C D llb+ microglia isolated from the CNS of non reconstituted SCID mice after the infection (Fig. 9A, 9B), possibly by IFN-y secreted by NK cells. Moreover microglia derived from both CD8+ T cell recipient groups exhibited similar levels of increased MHC class I expression (Fig. 9C, 9D). These data clearly 168 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. indicate that IFN-y from CD8+ T cells is not required to increase MHC class I expression on the macrophage/microglia population during JHMV infection and support a critical direct role of CD8+ T cell derived IFN-y in controlling viral replication in CNS. CD8+ T cells enhance demyeiination CD8+ T cells are required for virus clearance, however their role in demyeiination is controversial. To examine the role of CD8+ T cells in JHMV- induced demyeiination, brains and spinal cords of unreconstituted and SCID mice reconstituted with CD8+ T cells were stained with luxol fast blue and demyeiination scored as described previously (28). SCID mice exhibited slight demyeiination after JHMV infection (Fig. 10A). However, adoptive transfer of virus-specific CD8+ T cells resulted in increased demyeiination (Figure 10B, IOC). In addition, transfer of CD8+ T cells from IFN -y^ donors resulted in the same amount of demyeiination as observed after the transfer of CD8+ T cells from wt donors. These results are in agreement with previous data suggesting that CD8+ T cells are involved in the demyeiination process (6) and IFN-y is not required for JHMV induced demyeiination. However, experiments in IFN-y"/7P'/" mice or P'/_ (28) suggested that perforin-mediated cytolysis is not required either to induce demyeiination, implying the potential participation of additional CD8+ T cell mechanism in this process. R eproduced with perm ission o f the copyright owner. Further reproduction prohibited without perm ission. A. O S S n ov1299O 01 018 O . j C + -5 > in O f Tf X Q J. o < s H i g h ffjjf lo w 1 & “ m i i t ' t n r i rm ff rrii im p PEPMfcht 1000 SSnovI 2990O 1.012 c 3 O V o - 1000 0 B . 0 0 342 c 3 O o o 1000 FLPMfht SSapr240(B01,014 538 o 1000 F L 3 1 11111111 n T n T7 j 1000 F L 31 H2L (PFI) Figure 9. MHC class I expression on microglia during JHMV infection is upregulated independently of CD8+ T cell-derived IFN-Y. CNS mononuclear cells were isolated from SCID mice and stained for CD45, GDI lb and H2 Ld expression. A) Representative microglia gate (CD45 o w and CDllb ). B) Expression of MHC class I (H2L ) on gated microglia from (a) aive, (b), JHMV infected, and (c) infected IFN-y+ /+ CD8+ - or (d) IFN-y'7 ' CD8+ T cell reconstituted SCID mice. Class I expression was determined as peak mean fluorescence intensity (PFI). Data is representative two independent experiments done at day 10 p.i. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 10. Demyeiination in CD8+ T cell reconstituted SCID mice following infection with JHMV. Spinal cords from infected unreconstituted SCID (A); wt CD8+ T cell-reconstituted (B) and IFN-y'7 ' CD8+ T cell - reconstituted SCID mice at day 10 p.i. Sections were stained with luxol fast blue as indicated in material and methods. Arrow shows demyelinating plaques. Magnification, X200. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Discussion Previous data suggested a predominant role of the anti-viral CD8+ T cell response in viral clearance (28,33,42); however, the effector mechanisms involved remained undefined. Analysis of JHMV infection of the CNS of mice in which either the perforin or IFN-y genes had been disrupted suggested that both these immune effector mechanisms played critical roles in viral clearance (28,33). IFN-y is secreted by multiple cell types including CD8+ T cells, CD4+ T cells and NK cells in response to viral infection (4,23); however, evidence that CD8+ T cells are a source of the IFN-y required for JHMV clearance has not been provided. Therefore, virus-specific CD8+ T cells were adoptively transferred into either IFN-y'/7P'/' mice or SCID mice to measure the relative contribution of CD8+ T cell derived IFN-y secretion to viral clearance and pathogenesis. The results are consistent with previous data suggesting that effective anti-viral function of CD8+ T cells is exerted by a combination of IFN-y and perforin-dependent cytotoxicity. Despite a marked increase in CD8+ T cell infiltration into the CNS, mice lacking both IFN-y and perforin-dependent cytotoxicity were unable to control JHMV replication. No additional mechanisms appear to compensate for the simultaneous absence of these CD8+ T cell effector mechanisms. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. However, complete viral clearance was only observed when double knockout recipients were adoptive transferred with virus-specific CD8+ T cells competent for both IFN-y secretion and perforin-mediated cytolysis. These data confirm the critical role of CD8+ T cells in controlling the acute infection of JHMV in CNS as suggested by previous CD8+ T cell depletion experiments (52) or adoptive transfers into immunocompetent recipients (41,42). In contrast to CD8+ T cells from wt donors, CD8+ T cells lacking IFN-y cleared virus less effectively. Although these data suggested a predominant role of CD8+ T cell-derived IFN-y in viral clearance, the analysis of gene knockout mice is complicated by the presence of CD4+ T cells, intact Ab responses and alter traffic into infected tissues. Results of the CD8+ T cell adoptive transfers into SCID mice provide a direct demonstration that, in addition to perforin- dependent cytotoxicity, IFN-y is a key CD8+ T cell effector mechanism important for the control of the acute viral CNS infection, even in the absence of CD4+ T cells and antibodies. JHMV is a highly oligodendrocyte tropic strain of MHV and CD8+ T cells may have an absent or diminished cytotoxic action on this low MHC class I expressing cell type. However, in wt mice infectious virus is still eliminated from microglia, astrocytes and oligodendrocytes by a vigorous CTL response (42). This observation may reflect an overall rapid decrease in virus, R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. limiting extensive oligodendroglia infection. In this present experiment virus- specific CD8+ T cells competent for IFN-y secretion efficiently reduced virus from oligodendrocytes of infected SCID mice compared to partial elimination in recipients of IFN-y'/' CD8+ T cells. IFN-y has multiple immunomodulatory activities (37) that could account for the inability of JHMV specific IFN-y’7 ’ CD8+ T cells to effectively reduce virus in the CNS. For example, IFN-y can upregulate MHC class I expression and modulate proteasome activity for generation of peptides and enhancement of Ag presentation (1,9,55). IFN-y can also induce the differentiation of precursor CD8+ T cells to CTL. IFN-y is not required for the generation of CTL (11, Chapter IV) or for the homing of effectors cells to the site of viral infections (31). Indeed, CD8+ T cells from INF-y‘/' donors with intact perforin-dependent cytotoxicity trafficked normally into the CNS (Fig. 8) and reduced virus from most of the MHC class I expressing CNS cells (Fig. 7). IFN-y is potentially required to increase expression of MHC class I molecules on the target cells, thereby promoting effective CTL recognition (9,55). However, MHC class I was expressed at similar levels by microglia of SCID mice reconstituted with IFN-y'/’CD8+ T cells compared to the recipients of CD8+ T cells derived from wt donors. The observation that MHC class I expression increased on microglia after JHMV infection of SCID mice, even in R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the absence of T cells, is consistent with the early recruitment of INF-y- secreting NK cells (data not shown; 53) and the IFN type I (a/p) mediated increase in MHC molecule expression following CNS infection by other viruses (32). Although these data are consistent with the concept that virus- specific CD8+ T cells inhibit virus replication in oligodendrocytes via a IFN- y dependent mechanism, CD8+ T cell derived IFN-y may also inhibit spreading of virus from other CNS cell types by limiting the number of susceptible cells. Therefore, the accumulation of virus in oligodendrocytes during infection in which the CD8+ T cells are unable to secrete IFN-y could result from both an overall increase in virus spreading, in addition to the limited action of the rapidly down regulated perforin-mediated cytotoxicity. However, it is equally likely that the inability of CD8+ T cells to lyse JHMV infected oligodendroglia, in addition to the lack of IFN-y mediated direct anti viral activity, results in the infection of other CNS cell types seeded by the uncontrolled infection of oligodendroglia. The basis for the augmented CD8+ T cell infiltration into the CNS of infected double knockout mice is not clear. Perforin-dependent cytolysis has been suggested as a CD8+ T cell regulatory mechanism in activation-induced cell death during allogeneic responses (38,40) and CD8+ T cell homeostasis during viral and bacterial infections (30,49). However, no major differences in R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. the amount of infiltrating cells undergoing apoptosis in the CNS of IFN-y'7 '/?'7 ' mice compared to wt mice were detectable, suggesting that accumulation of CD8+ T cells in the CNS of IFN-y77P7' was not due to decreased cell death. Interestingly, adoptive transfer of wt CD8+ T cells, which cleared virus to undetectable levels, did not result in local increase of CD8+ T cells observed in non-reconstituted or IFN-y7" CD8+ T cell reconstituted double deficient recipients. These data suggest accumulation of CD8+ T cell results from increased expansion or recruitment rather than decreased perforin-mediated apoptosis. High viral titers coincided with increased CD8+ T cell infiltration in infected IFN-y7"?/7" mice and in double negative recipients of INF-y7" CD8+ T cells . By contrast, SCID mice reconstituted with CD8+ T cells exhibited equal CD8 infiltration regardless of the ability of the donor cells to secrete IFN-y and the quantity of viral Ag. The reason for this discrepancy is not clear. One possibility is that activated CD4+ T cells in the CNS of IFN-y7"P/7" mice, but not SCID mice, may release chemokines such as RANTES, MIP-la, MCP-1 and IFN-y-inducible protein-10 (IP-10), which serve to recruit CD8+ T cells (24,51). JHMV infection induces a spectrum of chemokines in the CNS (27), that are potentially involved in CD4+ T cell induced inflammation and pathogenesis (26). However, the role of CD4+ T cell secreted chemokines in regulating CD8+ T cell infiltration in double deficient mice remains undefined. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Primary demyeiination is a prominent characteristic of JHMV pathogenesis (50) and T cells are involved in this pathological process (15,25,54). However, it is unclear whether CD8+ T cells play a direct role in JHMV-induced demyeiination. The absence of demyeiination in infected SCID (16) or irradiated mice (50) suggest the involvement of CD8+ T cells in the demyelinating process. In contrast, prominent demyeiination was present in CD8+ T cell deficient mice infected with JHMV [nude mice; (16)] or MHV- A59 strain [(3-2 microglobulin deficient mice; (10)]. In addition, the induction of a potent local virus-specific CD8+ T cell response during the infection with a non-demyelinating JHMV strain (29) all argue against the idea that CD8+ T cells are prominent mediators of demyeiination. Experiments in this chapter demonstrate that CD8+ T cells contribute directly to JHMV associated demyeiination. The slight demyeiination present in the infected-non reconstituted SCID mice was enhanced after the transfer of virus-specific CD8+ T cells. However, the nature of the tissue damage appears to be independent of either IFN-y or perforin-mediated cytolysis. Demyeiination was also not decreased or prevented by the simultaneous lack of INF-y and perforin-dependent lysis confirming previous results in perforin (28) and IFN-y knockout mice (Chapter IV). Moreover, infection of SCID mice reconstituted with IFN-y"7 ' CD8+ T cells induced demyeiination equal to the adoptive transfer R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. with CD8+ T cells from wt donors. TNF-oc is thought to have an important effector role in mediating tissue damage in experimental alergic encephalomyelitis (EAE) (34,39). However, JHMV-induced demyeiination is independent of TNF-a (44). An alternative potential CD8+ T cell mechanism contributing to demyeiination may be direct apoptosis of oligodendrocytes by Fas-FasL interactions. Neverthless, Fas-deficient mice developed demyeiination during JHMV infection (Chapter II) suggesting that Fas- dependent cytolytic pathway is not required for demyeiination. The possibility exists, however, that in the absence of one effector pathway, the missing function is overtaken by the remaining mechanisms. Although the precise role of these CD8+ T cell immune effector mechanisms is complicated by the dynamics of an ongoing infection, these data support the hypothesis that separate effector mechanisms are functioning at the single cell level within the CNS. Lytic and nonlytic mechanisms may both be operative to clear JHMV infection from CNS. Therefore, perforin- mediated cytolysis appears to be the predominant CD8+ effector mechanism in clearing infectious vims from astrocytes and microglia/macrophages (28). By contrast, IFN-y appears to be the predominant CD8+ T cell immune effector mechanism that limits JHMV replication in oligodendroglia. 178 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. References 1. Beninga, J., K.L. Rock, and A.L. Goldberg. 1998. Interferon-y can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J. Biol. Chem. 273:18734-18742. 2. Bergmann, C., M. McMillan and S. A. Stohlman. 1993. Characterization of the Ld-restricted cytotoxic T-lymphocyte epitope in the mouse hepatitis virus nucleocapsid protein. J. Virol. 67:7041-9. 3. Bergmann, C.C., J.A. Altman, D. Hinton, and S.A. Stohlman. 1999. Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system. J. Immunol. 163:3379-3387. 4. Biron C.A. 1994. Cytokines in the generation of immune responses to, and resolution of virus infection. 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Fleming. 1996a. Dissociation of demyeiination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM. J. NeuroVirol. 2: 101- 111 . 17. Ju, S.-T. 1991. Distinct pathways of CD4 and CD8 cells induce rapid target DNA fragmentation. J. Immunol. 146:812-818. 18. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K.J. Olsen, E.R. Podack, R.M. Zinkernagel, and H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31-7. 19. Kagi, D., B. Ledermann, K. Burki, R.M. Zinkernagel and H. Hengartner. 1995. Lymphocyte-mediated cytotoxicity in vitro and in vivo: mechanisms and significance. Immunol. Rev. 146:95-115. 20. Kagi, D., F Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata. etal. 1994. Fas and perforin pathways as major mechanisms of T-cell-mediated cytotoxicity. Science 265:528-530. 1 8 0 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 21. Kagi, D., P. Seiler, P. Pavlovic, B. Ledermann, K. Burki, R. M. Zinkernagel, and H. Hengatner. 1995. The roles of perforin and fas- dependent cytotoxicity in protection against cytopathic and non cytophatic viruses. Eur. J. Immunol. 25: 3256-3262. 22. Kalams, S.A., and B.D. Walker. 1998. The critical need for CD4 help in maintaining effective cytototoxic T lymphocyte responses. J. Exp. Med. 188:2199-204. 23. Koszinowski, U.H., M.J. Reddehase and S. Jonjic. 1991. The role of CD4 and CD8 T cells in viral infections. Curr. Opin. Immunol 3:471. 24. Kundig, T.M., Hengartner, H. and Zinkernagel, R.M. 1993. T cell- dependent IFN-y exerts an antiviral effect in the central nervous system but not in peripheral solid organs. J. Immunol. 150:2316-2321. 25. Lampert, P.W., J.K. Sims, and A.J. Kniazeff. 1973. Mechanism of demyeiination in JHM virus encephalomyelitis. Acta Neuropathol. 24:76-85. 26. Lane, T.E., M.T. Liu, B.P. Chen, V.C. Asensio, R.M. Samawi, A.D. Paoletti, I.L. Campbell, S.L. Kunkel, H.S. Fox, and M.J. Buchmeier. 2000. A central role for CD4 T cells and RANTES in virus-induced central nervous system inflammation and demyeiination. J. Virol. 74:1415-1424. 27. Lane, T.E., V.C. Asensio, N. Yu, A.D. Paoletti, I.L. Campbell, and M.J. Buchmeier. 1998. Dynamic regulation of alpha- and beta- chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease. J. Immunol. 160:970- 978. 28. Lin, M.T., S.A. Stohlman, and D.R. Hinton. 1997. Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis. J. Virol. 71:383. 29. Marten, N.W., S.A. Stohlman, and C.C. Bergmann. 2000. Role of viral persistence in retaining CD8+ T cells within the central nervous system. J. Virol. 74:000-000. 181 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 30. Matloubian, M., M. Suresh, A. Glass, M.Galvan, K. Chow, J.K. W hitmire, C.M. Walsh, W.R. Clark, and R. Ahmed. 1999. A role for perforin in downregulating T-cell responses during chronic viral infection. J. Virol. 73:2527-36. 31. Nansen, A., J.P. Christensen, C. Ropke, O. Marker, A. Scheynius, and A.R. Thomsen. 1998. Role of interferon-y in the pathogenesis of LCMV-induced meningitis: unimpaired leukocyte recruitment, but deficient macrophage activation in interferon-y knock-out mice. J. Neuroimmunol. 86:202-212. 32. Njenja M.K., L.R. Pease, P. Wettstein, T. Mak and M. Rodriguez. 1997 Interferon a/J3 mediates early-virus induced expression of H2° and H2k in the central nervous system. Lab. Invest. 77:71. 33. Parra, B., D.R. Hinton, M.T. Lin, D.J. Cua, and S.A. Stohlman. 1997. Kinetics of cytokine mRNA expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis. Virology 233:001. 34. Renno, T., M. Krakowski, C. Piccirillo, J.Y. Lin, and T. Owens. 1995. TNF-alpha expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis: regulation by Thl cytokines. J. Immunol. 154:944-953. 35. Rossi, C.P., A. McAllister, M. Tanguy, D. Kagi, and M. Brahic. 1998. Theiler’s virus infection of perforin-deficient mice. J. Virol. 72:4515-4519. 36. Rubio, N., de Felipe C. 1991. Demonstration of the presence of specific interferon-gamma receptor on murine astrocyte cell surface. J. Neuroimmunol. 35:111-117. 37. Ruby, J. and I. Ramshaw. 1991. The antiviral activity of immune CD8+ T cells is dependent on interferon-y. Lymphokine Cytokine Res. 10:353. 1 8 2 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 38. Sad, S., D. Kagi, and T.R. Mosmann. 1996. Perforin and Fas killing by CD8+ T cells limits their cytokine synthesis and proliferation. J. Exp. Med. 184:1543-1547. 39. Selmajj, K., C.S. Raine, and A.H. Cross. 1991. Anti-tumor necrosis factor therapy abrogates autoimmune demyeiination. Ann. Neurol. 30:694-700. 40. Spaner, D., K. Raju, B. Rabinovich, and R.G. Miller. 1999. A role for perforin in activation-induced T cell death in vivo: Increased expansion of allogeneic perforin-deficient T cells in SCID mice. J. Immunol. 162:1192-1199. 41. Stohlman, S., C. Bergmann, M. Lin, D. Cua, and D.R. Hinton. 1998. CTL effectors function within the central nervous system requires CD4+ T cells. J. Immunol. 160:2896-2904. 42. Stohlman, S., C. Bergmann, R. van der Veen, and D.R. Hinton. 1995a. Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from the central nervous system. J. Virol. 69:684-694. 43. Stohlman, S.A., C.C. Bergmann, M.T. Lin, D. Cua, and D.R. Hinton. 1998. Cytotoxic T lymphocyte activity within the central nervous system requires CD4+ T cells. J. Immunol 160:2986. 44. Stohlman, S.A., D.R. Hinton, D. Cua, E. Dimacali, J. Sensintaffar, F.M. Hofrnan, S.M. Tahara and Q. Yao. 1995b. Tumor necrosis factor expression during mouse hepatitis virus- induced demyelinating encephalomyelitis. J. Virol. 69:5898-5903. 45. Tishon, A., H. Lewicki, G. Rail, H.M. von, and M.B. Oldstone. 1995. An essential role for type 1 interferon-gamma in terminating persistent viral infection. Virology 212:244-250. 46. Topham. D.J., R.A. Tripp, and P.C. Doherty. 1997. CD8+ T cells Clear Influenza Virus by Perforin or Fas-Dependent Processes. J Immunol. 159:5197-5200. 183 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 47. Torres, C., I. Aranguez, and N. Rubio. 1995. Expression of interferon-gamma receptors on murine oligodendrocytes and its regulation by cytokines and mitogens. Immunol. 86:250-255. 48. Tran, E.H., E.N. Prince, and T. Owens. 2000. IFN-y shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164:2759-2768. 49. Walsh, C.M., M. Matloubian, C.C. Liu, R. Ueda, C.G. Kurahara, J.L. Christensen, M.T. Huang, J.D. Young, R. Ahmed and W.R. Clark. 1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. U.S.A. 91:10854-8. 50. Wang, F., S.A. Stohlman, and J. Fleming. 1990. Demyeiination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated. J. Neuroimmunol. 30:31-41. 51. Ward, S.G., K. Bacon, and J. Westwick. 1998. Chemokines and T lymphocytes: more than an attraction. Immunity 9:1. 52. Williamson, J.S.P., and S.A. Stohlman. 1990. Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells. J. Virol. 64:4589-4592. 53. Williamson, J.S.-P., K. Sykes and S. Stohlman. 1991. Characterization of brain infiltrating mononuclear cells during infection with mouse hepatitis virus strain JHM. J. Neuroimmunol. 32:199-207. 54. Wu, G.F., and S. Perlman. 1999. Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyeiination following murine infection with a neurotropic coronavirus. J. Virol. 73:8771. 55. Young, H.A., and K.J. Hardy. 1995. Role of interferon-y in immune cell regulation. J. Leukoc. Biol. 58:373-381. 1 8 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. C hapter VI. Contributions of CD4+ derived IFN- y to Viral Clearance and Pathogenesis. Summary The precise role of CD4+ T cells as effectors of-JHMV clearance is poorly understood, CD4+ T cells seem to prolong the survival of CD8+ T cells in the brain parenchyma, indirectly contributing to the resolution of acute viral infection. However, this T cell subset is a potent producer of IFN-y and could potentially limit CNS infection via the direct anti-viral action of IFN-y. Experiments were undertaken to investigate the role of CD4+ T cells, as a primary source of IFN-y, in JHMV clearance from the CNS. Adoptive transfer of CD4+ T cells into SCID mice suggest that virus-specific CD4+ T cells contribute to JHMV clearance via IFN-y. CD4+ T cells deficient in IFN-y secretion were unable to control the CNS infection in SCID mice as opposed to complete viral clearance mediated by CD4+ T cells competent for IFN-y secretion. However, CD4+ T cells also appeared to help CD8+ T cell cytolytic function, in an IFN-y independent fashion. CD8+ T from IFN-y7" donors alone were ineffective at reducing virus replication. However, the simultaneous presence of CD4+ T cells from IFN-y7" donors also defective in viral clearance, resulted in a dramatic decrease in viral titers, suggesting enhancement in the anti-viral function of the CD8+ T cells. CD4+ T cell R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. infiltration markedly influence clinical disease. CD4+T cell -reconstituted mice exhibited earlier clinical signs of encephalitis, paralysis and demyeiination progressing to a lethal disease, in contrast to the protected CD8+ T cell recipient. These data suggest CD4+ T cells both directly via IFN-y7' secretion and indirectly via altering CD8+ T cell function participate in viral clearance. In contrast to the protective role they also induce clinical disease during acute JHMV infection in the CNS. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. In tro d u ctio n Acute JHMV infection of the CNS is accompanied by an extensive infiltration of mononuclear cells. Among the infiltrating cells, virus-specific CD8+ and CD4+ T cells have been shown to participate in both the pathogenesis and the resolution of the acute disease. Virus-specific CD8+ T cell responses has been rigorously examined during the acute (3,57) and persistent phases of the CNS disease caused by JHMV (3,31). Virus-specific CD8+ protect from lethal acute disease and reduce viral replication. However, CD4+ T cell responses to JHMV in the CNS have not been studied as extensively. In other viral models of CNS infections, such as measles virus, TMEV and influenza virus CD4+ T cells are critical to controling viral replication (8,11) and are also a key mediators of the pathological changes associated with disease such as encephalitis and demyeiination (16,37,40). CD4+ T cell anti-viral functions can potentially be exerted in multiple ways. CD4+ T cells inhibit the CNS replication of measles virus, TMEV and herpes viruses via IFN-y and cytokines secretion (12,15,54). CD4+ T cells mediate cytotoxicity of infected targets via Fas-FasL interactions (44). CD4+ T cells also secrete chemokines involved in attraction and extravasation of monocytes/macrophages (29,30,53,) and help B cell Ab production. Finally, CD4+ T cell help is critical to maintain CD8+ T cell CTL responses to viruses (1,26,33,52), essentially during persistent 187 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. infections. CD4+ T cells also help the priming of CD8+ T cells in few viral models (25). Therefore, CD4+ T cells can be involved at all stages of CD8+ T cell differentiation Little is known regarding the CD4+ T cell Ag specificity and the exact role of this T cell type in JHMV clearance and pathogenesis. At least three I-Ab restricted CD4+ epitopes, one within the transmembrane M protein (M l34-137) and two within the S protein have been characterized (20,59,60). More recently one additional I-Ed restricted CD4 epitope within the N nucleocapside protein (residues 266-279) was identified during peripheral infection with JHMV (51). The presence of CD4+ T cells reacting to the M epitope correlate with induction of encephalitis and demyeiination (55,60). The potential role role of CD4+ T cells in reducing infectious virus is less clear. Depletion experiments have shown a key role for CD4+ T cell in viral clearance. Virus-specific CD4+ T cell clones protect from disease with (61) or without reducing viral replication (46). Although, CD4+ T cells are not necessary for the priming and induction of JHMV specific CD8+ responses they are required for the maintenance of an optimal anti-viral CD8+ T cell function within the CNS (45,47). CD8+ T cells undergo increased apoptosis after infection with a lethal strain of JHMV in the absence of CD4+ T cells (45). In addition to their effects on CD8+ T cells, CD4+ T cells can have direct antiviral function. For example, virus-specific 1 8 8 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Class II-restricted CD4+ T cells with cytolytic activity have been detected during the course of acute responses in mice infected with viruses [Sendai, LCMV; (22,36)]. Similarly, CD4+ cytotoxic T cells contribute to viral clearance during the infection of the liver with the A59 hepatotropic strain of MHV (21,55). However, normal viral clearance in Fas-deficient mice and delayed, but total clearance in perforin deficient mice ruled out the possibility that CD4+ T cells exert crucial antiviral function during JHMV infection in the CNS via Fas-FasL interactions or perforin mediated cytolysis. However, adoptive transfer experiments suggested that CD4+ T cells mediate antiviral function via soluble factors (28,61), since the majority of CNS infiltrating CD4+ T cells localize to perivascular areas, but affect viral clearance from cells within the brain parenchyma (DM variant). Previous studies in IFN-y deficient mice suggested a key role of this cytokine in viral clearance. However the relative contribution of CD4+ T cells as potential INF-y secreting cells recruited to the CNS following JHMV infection is unknown. Previous chapters demonstrated that CD8+ T cell derived IFN-y efficiently reduced JHMV replication especially from oligodendrocytes. In contrast, NK-derived IFN-y appears to play no role in CNS viral clearance (Chapter V; 23,43). To examine the contribution of CD4+ T cell derived IFN-y, virus replication and pathogenesis was studied in SCID mice following adoptive transfer of CD4+ T cells. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. M aterial and Methods Mice Male BALB/c mice at 7 to 8 weeks (Jackson Laboratories. Bar Harbor, ME) and age matched IFN-y"7 ' B ALB/c mice (DNAX Research Institute, Palo Alto, CA) were injected i.p. with lxlO6 PFU of the DM strain of JHMV. Three to four weeks later, spleen cells obtained from immunized mice were used to purify CD4+ and CD8+ T cells for adoptive transfer into SCID mice. SCID BALB/c recipient mice were purchased from Jackson Laboratories or NCI and maintained under sterile conditions. T cell purification and adoptive transfer Virus-specific memory CD4+ T cell populations were expanded from the spleen cells of immunized wt or IFN-y'7 " mice in vitro using a UV- n inactivated lysate of JHMV infected DBT cells. Approximately 7x10 cells were cultured with UV-inactivated viral lysate (1:80 dilution) in 40 ml of complete RPMI 1640 medium supplemented with 10%FCS and 5% of a rat ConA stimulated-spleen cells supernatant (RCS) as a source of IL-2 in 75cm falcon flasks for 5 days at 37°C. To enrich for viable cells, 10 ml aliquots were centrifuged at 800 x g onto 1 ml cushions of Lympholyte-M medium (Cedarlane, Ontario, Canada) in 15 ml Falcon conical tubes. B cells were depleted by adsorption to goat anti-mouse immunoglobulin-coated plates as R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. described in chapter V. CD4+ T cell populations were enriched by CD8+ T cell depletion specific anti-CD8 mAh (Ly-2) coupled to magnetic beads (Miltenyi Biotec) and separated by negative selection in MACS columns (Miltenyi Biotec) using the protocol described to purify CD8+ T cells in Chapter V. The resulting population contained 70-80% CD4+ T cells as determined by flow cytometry using FITC-labeled anti-CD4 (L3T4; PharMingen). Preparations were completely depleted of CD8+ T cells as determined by flow cytometry using PE-labeled anti-CD8 (Ly-2; PharMingen), but contained a minimal contamination comprising CD19+ cells (1D3; PharMingen) (Fig. 1) and NK cells as determined with panNK mAh (0X5; PharMingen). Virus-specific CD8+ T cells were enriched from the spleen of immunized donor mice following in vitro expansion. Cells (7xl07 ) were cultured for 5 days with the specific CTL epitope pN47 peptide in 40 ml of complete RPMI 1640 medium supplement with 10% FCS and 5% of RCS. After lympholyte M purification CD8+ T cells were separated by negative selection after immunomagnetic depletion of CD4+ T cells (Chapter V). CD8+ T cells were 80-90% enriched as determined by flow cytometry and exhibited excellent in vitro cytolytic activity to pN47 peptide-coated (lpM ) J774.1 cells (Fig. 1). Cell populations containing either lx l 07 CD4+ T cells alone or a mixture of both CD4+ T cells (lxlO7 ) and CD8+ T cells (lxlO7 ) were adoptively transferred into SCID mice by i.v. injection. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. A 0 0 0 . 5 U *T O WT CD4+ IFN-y ©. /"CD4+ o ' H r £OC: ‘5 jrr: . < M - M M * 7 3 % L L O - iSl B. 200 400 600 800 1000 FLI-Height CD4+ ____ 200 400 600 800 1000 FLt-Beight o o u. o 0 200 400 600 800 1000 FL1-Height CD19+ O o L i. o 1000 c. WT CD8 DCiul0707006.006 A " 75% â– IV* 1*11111 1 ’ ?r IFNV CD8 D C { m 1Q79700S.Q08 o o © 80% o FLI-Height CD4+ FLI-Height - WT CD4* • IFN-y â– /'C D 4 + 1 0 0 - § 80 4 0 - 20 1 o o 05 < e o o o D ilu tio n o f J H M V l y s a t e D. WTCD8 4) a t ro j» 2 IFN-y CD8H 50 - 30 - o I Q . t/i 10 - 20:1 10:1 5:1 2.5:1 EffectorTarget Figure 1. Characterization of T cell donor cell populations. Virus-specific CD4+ and CD8 cells were expanded in vitro from spleen cells of JHMV immunized donor mice. Cells were negatively enriched for CD4 or CD8 T cells by MACS procedure. Staining for specific CD4, CD8 and CD 19 markers in the CD4 enriched population (A) and CD8 and CD4 markers in the CD8 enriched population (C) are shown. Numbers (upper quadrants) in A and C panels represent the proportion of T cells. Remaining B cells in the CD4 T cell enriched population are represented as percentage of CD 19 cells. Specific proliferation of in vitro expanded CD4 T cells before enrichment (B), and specific cytotoxic activity of the CD8 T cell enriched population (D), are shown. Results are representative of two independent experiments 192 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Virus infection SCID mice were infected by i.e. with 500 PFU of the 2.2v-l variant of JHMV approximately 4 hours after T cell transfer. Viral replication and pathogenesis were analyzed after 8 days p.i. Clinical disease was graded as previously described. 0, healthy; 1, ruffle fur and hunch back; 2, slow mobility and inability to upright; 3,paralysis and wasting; 4,moribund and death. Virus titers in the CNS were determined by plaque assay on DBT as described in Chapter II. Ag specific proliferation. Proliferation to UV-inactivated JHMV infected DBT cell lysate was tested in spleen cells from immunized donors after in vitro expansion by 3[H]- thymidine (ICN, Radiochemical) incorporation as described in chapter V (Fig. 1). Briefly, 8xl05 spleen cells/well were cultured in 96 well plates in 200 pi of complete RPMI 1640 medium supplement with 2% mouse serum. U.V. inactivated lysate of JHMV as Ag was added at different concentrations (5 fold dilutions). Cells were pulsed w ith3 [H]-thymidine (1 pCi/well) after 60 hr incubation at 37°C and harvested 10-15 h later. Thymidine incorporation was measured by liquid scintillation spectroscopy and data are expressed as mean count per minute of triplicate wells. 193 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CTL assay Enriched CD8+ T cells were assayed for cytotoxic activity in a 4 hr 5 1 Cr release assay using pN47 peptide-pulsed J774.1 target cells as described in Chapter III. Cytokine mRNA determination RNA was prepared from brain tissue by guanidinium isothiocyanate purification and ultracentrifugation through 5.4 M cesium chloride (CsCl) as described in Chapter II. IFN-y mRNA was analyzed by semi-quantitative R.T- PCR analysis using primers and internal probes as described in Chapter II. Histology Brains and spinal cords from mice sacrificed at 10 days p.i. were collected and fixed in Clark’s solution (75% ethanol and 25% glacial acetic acid) and embedded in paraffin. Sections were stained with hematoxilin and eosin and luxol fast blue to determine inflammation and demyelination. Viral Ag positive cells were stained with the J.3.3 mAh specific for the viral nucleocapside protein (14) by immunoperoxidase staining as described in previous Chapters. CD4+ and CD8+ T cell infiltration into the CNS was determined by staining frozen sections of spinal cord and brains with specific anti-CD4+ (L3T4) or anti-CD8+ (Ly-2) mAbs from PharMingen as described in Chapters IV and V. 1 9 4 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Results Virus-specific CD4+ T cells contribute to encephalitis and demyelination. CD4+ T cells are effectors of delay-type hypersensitivity (DTH) responses and CD4+ T cell infiltration into the CNS has been associated with clinical manifestations of encephalitis. To examine the role of CD4+ T cells in JHMV-induced encephalomyelitis, SCID mice reconstituted with either CD4+, CD8+ or both T cells were monitored for clinical disease for 8 days p.i. SCID recipients of CD4+ T cells showed very early signs of encephalitis (day 4 p.i.) compared to untreated mice or recipients of CD8+ T cells or an equal mixture of CD4+ and CD8+ T cells at day 8 p.i (Table 1). Moreover, the increased morbidity mediated by CD4+ T cells was independent of the ability of this T cell subset to secrete IFN-y. SCID mice reconstituted with CD4+ T cells alone showed no signs of clinical recovery and moribund mice exhibited hind limb paralysis at day 8 p.i. To verify that transferred T cells were recruited into the CNS, frozen sections of brain and spinal cord were stained for the CD4+ and CD8+ T cell markers. CD4+ T cells trafficked to CNS, localized to the perivascular areas and also entered the brain parenchyma (Fig. 2). These data directly demonstrate that CD4+ T cell infiltration enhances clinical disease by an IFN-y-independent process. Similarly, luxol fast staining of CNS tissues of infected recipient mice revealed increased demyelination in recipients of CD4+ R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. T cells IFN-y+ /+ or IFN-y7' or both CD4+ and CD8+ T cells compared to recipients of CD8+ T cells alone or untreated mice (Fig. 3). These results suggest that CD4+ T cells influence clinical disease and are sufficient to induce primary demyelination during JHMV infection of the CNS. In addition, these data confirm previous results from IFN-y deficient mice in chapter IV, suggesting that IFN-y is not required for JHMV-induced demyelination or encephalitis. Table 1. Morbidity of T cell transferred SCID mice. Treatment a Number of Mice Clinical Score ± SD None 9 0.8 ±0.5 wt CD4+ 6 2.9 ± 0.6 IFN-y7' CD4+ 6 3.3 ±0.4 wt CD8+ 7 0.2 ±0.3 IFN-y CD8+ 7 0.6 ± 07 IFN-y7' CD8+ + wt 6 1.3 ±0.5 CD4+ IFN-y7' CD8+ + 6 2.6 ± 1.1 IFN-y7' CD4+ aSCID mice were adoptively transferred with CD4+ T cells alone (lxlO7 cells) or together with lxlO7 IFN-y 7'CD8+ T cells. Clinical scores were obtained at day 8 p.i. and graded as described in Material and Methods. Data is the composite of two independent experiments. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 2. CD4+ T cells Infiltrate the brain parenchyma of SCID mice. Acetone-fixed frozen sections were stained for CD4+ T cells using immunoperoxidase staining (Vectastain ABC kit) and the rat anti- CD4+ mAh (GK1.5) as a primary Ab. Arrow indicate positive cells. Data show CD4+ T cells in the brain of SCID mice adoptive transfer with (A) wt CD4+T cells; (B) IFN-y'7 ' CD4+ T cells or a combination of IFN-y'7 ' CD8+ T cells plus (C) wt CD4+ or (D) IFN-y'7 ' CD4+ T cells. Arrows indicate CD4+ T cells infiltrating the brain parenchyma. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 3. CD4 T cells induce demyelination during JHMV infection. Luxol fast blue stains of spinal cords from non-reconstituted, (A) CD4+ T, (B) or CD8+ T (C) or dually CD4+ and CD8+ (D) reconstituted SCID mice. Arrows show areas of demyelination. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CD4+ T cells alone mediate viral clearance in an IFN-y dependent manner. To correlate virus replication and clinical disease with T cell function, infected SCID recipients were assayed for viral titers in CNS. At 8 days p.i the majority of moribund SCID recipients of CD4+ T cells from wt donors had completely cleared infectious virus (Fig. 4A). In contrast, recipients of CD4+ T cells from IFN-y'7 ' donors had relatively high virus titers, similar to untreated SCID controls. These data suggested that CD4+ T cells alone are efficient at controlling JHMV replication via an IFN-y dependent mechanism. Consistent with the data in Chapter V, SCID recipients of wt CD8+ T cells also showed a marked reduction in viral replication in contrast to recipients of IFNy'7 ' CD8+ T which exhibited only a small reduction in viral titers. Histological evaluation of Ag positive cells revealed few Ag positive cells remained in the brain or spinal cord of mice reconstituted with CD4+ T cells from wt donors. In contrast, numerous multiple cell types expressed viral Ag in the CNS tissues of SCID mice reconstituted with CD4+ T cells from IFNy'7 ' donors (data not shown). To assess the relative levels of IFN-y attributed to CD4+ vs. CD8+ T cells, IFN-y mRNA levels were determined in CNS. Consistent with the presence of CD4+ T cells, mRNA for IFN-y was only detected in the brain of recipients of IFN-y producing T cells. Interestingly, increased levels of IFN-y mRNA were detected in the brains of SCID CD4+ T cell recipients as R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. compared to SCID CD8+ T cell recipients (Fig. 4B). This could contribute to the enhanced viral clearance mediated by wt CD4+ T cells compared to wt CD8+ T cells (Fig.4A). CD4+ T cells contribute to acute viral clearance independently of IFN-y. The experiments described above demonstrate that virus-specific CD4+ T cells alone are sufficient to clear virus from CNS during the acute phase of JHMV infection and that the effector mechanism depends upon IFN-y secretion. JHMV clearance from the CNS is also mediated by virus-specific CD8+ T cells (Fig. 4A; Chapter V). However, effective CD8+ T cell-mediated clearance of a lethal JHMV strain is dependent on CD4+ T cell help (46,48). To examine the contribution of CD4+ T cells to the anti-viral cytolytic function of CD8+ T cells during nonlethal JHMV disease independently of IFN-y, IFNy7' CD4+ T cells were transferred together with an equal number of CD8+ T cells from IFNy'7 ' donors and viral replication examined at day 8 p.i. Compared to recipients of IFN-y'7 ' CD8+ T cells only, virus was reduced by 3 logio in the recipients of both CD4+ plus CD8+ T cells lacking IFNy (Fig. 4B). Furthermore, transfer of IFN-y'7 'CD4+ T cells plus wt CD8+ T cells mediated complete viral clearance, in contrast a mere 2-logio reduction by wt CD8+ T cells alone. These data suggest that CD4+ T cells not only exhibit direct CD8+ T cells within the CNS. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Figure 4A. Virus-specific CD4+ T cells reduce viral replication in CNS via IFN-y dependent and IFN-y independent mechanisms. Virus-specific memory T cells from JHMV immunized donor mice were expanded in vitro and enriched for either CD4+ or CD8+ populations as described in Material and Methods. SCID mice were adoptive transferred with either CD4+ T cells or CD8+ T cells alone or a mixture of equal numbers of CD4+ T cells and CD8+ T. Virus replication in the brains of reconstituted SCID mice was measured following 8 days p.i. by plaque assay. Data are the average of at least three mice per group + standard deviation. Results are representative of two independent experiments. 4B. IFN-y mRNA levels in the CNS of T cell- adoptive transfer JHMV- infected SCID mice. Semiquantitative RT-PCR analysis was performed to measure the expression of IFN-y mRNA in the brain of T cell reconstituted SCID mice following 8 days p.i with the 2.2v-l strain of JHMV. To adjust for differences in the amount of cDNA, IFN-y cDNA levels were normalized to the houskeeping enzyme hypoxanthine phosphorybosyltransferase (HPRT). Data is expressed as relative amount value obtained for comparison. Data for at least three mice per group + standard deviation (SD). UD= undetectable. 201 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. < z a . c 3 o a ; > C D 4 1 UD UD UD UD o - — + W T + + IFN-y"7" - . W T C D 8 .cm -/- IFN-y + + + 202 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Discussion These results suggest that virus-specific CD4+ T cells are direct effectors of virus clearance and also mediators of viral-induced acute CNS disease. Adoptive transfer of JHMV specific CD4+ T cells, regardless of the ability to secrete IFN-y, resulted in severe encephalitis and rapid demyelination within 8 days. These data are reminiscent of previous reports suggesting that CD4+ T cells subset predominantly contribute to viral (37,40) and autoimmune-induced demyelination (34). However, the exact role of CD4+ T cells in JHMV-induced demyelination has been controversial. Demyelination in the absence of CD8+ T cells (19) or in mice protected from lethal disease by CD4+ DTH T cell clones (46). Reduced demyelination in CD4+ T cell depleted mice (29) suggests a role of this T cell subset in the induction and/or augmentation of demyelination. However, the presence of demyelination in CD4+ T cell depleted mice infected with the A59 strain (48) or in MHC class II deficient mice infected with JHMV (23) suggested that CD4+ T cells are not the sole mediator for MHV-induced demyelination. Results presented in this chapter demonstrate that virus-specific CD4+ T cells are indeed sufficient for primary demyelination and stronger inducers of both clinical and pathological disease compared to CD8+ T cells. The mechanisms underlying CD4+ T cell induced encephalomyelitis and demyelination during viral infection are unclear. Previous work suggested a 203 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. key role of CD4+ T cell derived IFN-y in EAE-induced inflammation and demyelination by determining whether macrophages or neutrophils are recruited to CNS (50,62). IFN-y exerted a protective role by restricting neutrophil infiltration and activation via regulation of the chemOkine profile (50). The present results indicate that CD4+ T cell derived IFN-y plays little or no role in the clinical disease outcome during JHMV CNS infection. CD4+ T cells may, however, contribute to JHMV-induced pathology via secretion of chemokines and inflammatory cytokines (29,30). Chemokines, including RANTES, MCP-1, IP 10, are induced during both EAE, MS and viral-induced inflammatory processes, including infection by JHMV. In addition, their expression correlates with CNS infiltration and clinical progression of disease (17,18,24,29). During JHMV infection, CD4+ T cells produce RANTES which accelerates the severity of JHMV-induced demyelination by attracting more T cells and macrophages into the CNS (29). Increased activation of the infiltrating macrophages and/or microglial population could also lead to a more severe or rapid demyelination. The mechanisms by which CD4+ T cells may directly reduce JHMV replication in the CNS are not clear. A direct antiviral role of CD4+ T cells was suggested previously in Lewis rats in which the transfer of JHMV specific CD4+ T cells reduced virus replication in the absence of CD8+ T cells (28). 204 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Transfer of anti-JHMV specific CD4+ T cell clones also protected mice from lethal encephalomyelitis by reducing viral load in the CNS (61). Data in this chapter support an efficient anti-JHMV function of CD4+ T cells in the CNS and suggest that the CD4+ -I cell anti-viral function can be mediated directly through IFN-y and indirectly by an IFN-y-independent effect on CD8+ T cells. CD4+ T cells inhibited JHMV replication in CNS via IFN-y. This data is in agreement with the decreased JHMV clearance after CD4+ T cell depletion (56) or the augmented virus clearance after adoptive transfer of CD4+ T cells into immune competent mice (61). SCID mice reconstituted with CD4+ T cells from IFN-y+ /+ donors cleared virus from most of CNS cell types (data not shown) suggesting that IFN-y has a potential antiviral effect on all CNS cell types. These results are consistent with the presence of IFN-y receptors on all CNS cell types including microglia, astrocytes and oligodendrocytes (38,41,49) and the upregulation of class II molecules during viral or autoimmune-induced CNS inflammation (4,32). Interestingly, CD4+ T cells from wt donors more efficiently reduced virus replication than CD8+ T cells from wt donors at the only time point examined. The reason for this difference is not clear. Both cell types secrete IFN-y in response to JHMV Ags (3). CD8+ T cells appear to clear virus from CNS via both IFN-y and perforin-mediated cytolysis (Chapter V). CD4+ T cells are stronger secretors of IFN-y in response to viral infections, R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. allogeneic or autoimmune inflammation (27). Therefore, higher level of IFN-y secretion could account for a more efficient CD4+ T cell-mediated clearance. Consistent with this idea, mRNA analysis of the CNS showed elevated levels of IFN-y mRNA in recipients of CD4+ T cells compared to CD8+ T cells. Despite the equalivalent numbers of CD4+ or CD8+ T cells transferred into the SCID recipients, the increased IFN-y mRNA levels in recipients of CD4+ T cells may reflect either an earlier or increased CD4+ T cell recruitment rather than higher IFN-y secretion by CD4+ T cells in response to the infection. Measurement of the magnitude of IFN-y response by CD4+ T cells and CD8+ T cells per cell basis requires a more careful analysis and it is complicated by the lack of information in the Ag specificity of CD4+ T cells. If levels of IFN-y secretion are determined by TCR signaling, then irrespective of CD4+ and CD8+ T cell numbers, better class II presentation by infected CNS cell may explain the high levels of IFN-y coming from CD4+ T cells. CD4+ T cells lacking IFN-y appeared to promote a more effective CD8+ T cell function within the CNS. Virus replication was greatly inhibited by virus- specific CD8+ T cells lacking IFN-y secretion, when transferred together with virus-specific CD4+ T cells from IFN-y7' donors suggesting an indirect and IFN-y independent participation of CD4+ T cells to viral clearance within the 2 0 6 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. CNS. CD4 + T cells play an essential role in the long-term maintenance of CD8+ T cell effector function during persistent viral infections including HIV (39) MHV-68 (5) and LCMV (33). CD4+ T cells maintain optimal CD8+ T cell anti-viral effector function in the CNS during acute infection with a lethal strain of JHMV (45), presumably by preventing the exhaustion of CD8+ T cells via secretion of IL-2 or augmenting CTL activity via secretion of other cytokines (45). The data in this chapter suggest that optimal viral clearance of an attenuated variant of JHMV by CD8+ T cell also require CD4+ T cell assisted help. These data also confirm that CD4+ T cells inhibit JHMV replication in CNS via IFN-y. Furthermore, cells contribute to enhance the CD8+ T cell effector function within CNS by an IFN-y independent mechanism. In summary, CD4+ T cells have a dual role in the pathogenesis of JHMV-induced CNS disease. Whereas they protect via reducing virus replication, they also induce clinical disease and demyelination. 207 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. References 1. Ahmed, R., L.D. Butler, and L. Bhatti. 1988. T4+ T helper cell function in vivo: differential requirement for induction of antiviral cytotoxic T cell and antibody responses. J. Virol. 62:2102-2106. 2. Baumgarth, N., and A. Kelso. 1996. In vivo blockade of gamma interferon affects the influenza virus-induced humoral and the focal cellular immune response in lung tissue. J. Virol. 70:4411-4418. 3. Bergmann, C.C., J.A. Altman, D. Hinton, and S.A. Stohlman. 1999. Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system. J. Immunol. 163:3379-3387. 4. Borrow, P., and A.A. Nash. 1992. 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Synchronous synthesis of alpha and beta chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis. Am. J. Pathol. 150:617-630. 209 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 18. Godiska, R., D. Chantry, G. Dietsch, and P. Gray, 1995. Chemokinc expression in murine experimental allergic encephalomyelitis. J. Neuroimmunol. 58:167-176. 19. Gombold, J., R, Sutherland, E. Lavi, Y, Paterson, and S.R. Weiss. 1995. Mouse hepatitis virus A59-induced demyelination can occur in the absence of CD8+ T cells. Microh. Pathogen. 18:211-221. 20. Heemskerk, M.H.M., H.M. Shoemaker, I. DeJong, Y.E.C.J. Schijns, W.J.M. Spaan, and C.J.P. Boog. 1995. Differential activation of mouse hepatitis virus-specific CD4+ cytotoxic T cells is defined by peptide length. Immunol. 85:517-522. 21. Heemskert, M., H. Shoemaker, W. Spaan, and C . Boog. 1995. Predominance of MHC class II-restricted CD4+ cytotoxic T cells against mouse hepatitis virus A59. Immunology 84:521-527. 22. Hou, S., M. Fishman, K . Gopal Murti, and P.C. Doherty. 1993. Divergence between cytotoxic effector functional and tumor necrosis factor alpha production for inflammatory CD4+ T cells from mice with Sendai virus pneumonia. J. Virol. 67:6299-6302. 23. Houtman, J., and J. Fleming. 1996. Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM. J. Neurovirol. 2:101-110. 24. Hvas, J., C. McLean, J. Justesen, G. Kannaourakis, L. Steinman, J.R. Oksenberg, and C.C. Bernard. 1997. Perivascular T cells express the pro-inflammatory chemokine RANTES mRNA in multiple sclerosis lesions. Scand. J. Immunol. 46:195-203. 25. Jenning, S.R., R.H. Bonneau, P.M. Smith, R.M., Wolcort, and R. Chervenak. 1991. CD4-positive T lymphocytes are required for the generation of the primary but not the secondary CD8-positive cytolytic T lymphocyte response to herpes simplex virus in C57BL/mice. Cell. Immunol. 133:234-252. 26. Kalams, S.A., and B.D. Walker. 1998. The critical need for CD4 help in maintaining effective cytototoxic T lymphocyte responses. J. Exp. Med. 188:2199. 210 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 27. Kelso, A. 1990. Frequency analysis of lymphokine-secreting CD4+ and CD8+ T cells activated in a graft-versus-host reaction. J. Immunol. 145:2167-2176. 28. Korner, EL, A. Schliephake, J. W inter, F. Zimprieh, H. Lassmann, J. Sedgwick, S. Siddell, and H. Wege. 1991. Nucleocapsid or spike protein-specific CD4+ T lymphocytes protect against coronavirus- induced encephalomyelitis in the absence o f CD8+ T cells. J. Immunol. 147:2317-2323. 29. Lane, T.E., IV L T . Liu, B.P. Chen, V.C. Asensio, R.M. Samawi, A.D. Paoletti, IX . Campbell, SX. Kunkel, ELS. Fox, and M.J. Buchmeier. 2000. A central role for CD4 T cells and RANTES in virus-induced central nervous system inflammation and demyelination. J. Virol. 74:1415-1424. 30. Lane, T.E., V.C. Asensio, N. Yu, A.D. Paoletti, I.L. Campbell, and M.J. Buchmeier. 1998. Dynamic regulation of alpha- and beta- chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease. J. Immunol. 160:970- 978. 31. Marten, N.W., S.A. Stohlman, and C.C. Bergmann. 2000. Role of viral persistence in retaining CD8+ T cells within the central nervous system. J. Virol. 74:000-000. 32. Massa, P.T., R.Dorries, and V. ter Meulen. 1986. Viral particles induce la antigen expression on astrocytes. Nature 320:543. 33. Matloubian, M., R.J. Concepcion, and R. Ahmed. 1994. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J. Virol. 68:8056-8063. 34. Miller, S.D., and W.J. Karpus. 1994. The imunopathogenesis and regulation of T-cell mediated demyelinating diseases. Immunol. Today 15:356-361. 211 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 35. Mobley, J., G. Evans, M.O. Dailey, and S. Perlman. 1992. Immune response to a murine coronavirus: identification of a homing receptor- negative CD4+ T cell subset that responds to viral glycoproteins. Virology 187:443-452. 36. Muller, D., 8.H. Roller, I.L. Whitton, K.E. LaPan, K.K. Brigman, and J.A. Frelinger. 1992. LGMV-specific, class 1 1 -restricted cytotoxic T cells in (32-microglobulin-deficient mice. Science 255:1576-1578. 37. M urray, P.D., K.D. Pavelko, J. Leibowitz, X. Lin, and M. Rodriguez. 1998. CD4+ and CD8+ T cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis. J. Virol. 72:7320-7329. 38. Otero, G. C., and J.E. Merrill. 1994. Cytokine receptors on glial cells. Glia 11:117-128. 39. Pantaleo, G., and A.S. Fauci. 1996. Immunopathogenesis of HIV infection. Annu. Rev. Microbiol. 50:825-854. 40. Rodriguez, M., W. Lafuse, J. Leibowitz, and C.S. David. 1986. Partial suppression of Theiler’s virus-induced demyelination in vivo by administration of monoclonal antibodies to immune response gene products (la antigens). Neurology 36:964-970. 41. Rubio N, de Felipe C. 1991. Demonstration of the presence of specific interferon gamma receptor on murine astrocytes cell surface. J. Neuroimmunol. 35:111-117. 42. Sedgwick, J.D., and R. Dorries. 1991. The immune system response to viral infection of the CNS. Neurosciences 393:100. 43. Sorenson, O., A. Sarvani, and S. Dales. 1987. In vivo and in vitro models of demyelinating disease. XVII. The infectious process in athymic rats inoculated with JHM virus. Microbial. Path. 2:79. 44. Stalder, T., S. Hahn, and P. Erb. 1994. Fas antigen is the major target molecular for CD4+ T-cell-mediated cytotoxicity. J. Immunol. 152:1127-1136. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 45. Stohlman, S., C. Bergmann, M. Lin, D. Cua, and D.R. Hinton. 1998. CTL effector function within the central nervous system requires CD4+ T cells. J. Immunol. 160:2896-2904. 46. Stohlman, S.A., G.K. Matsushima, N. Casteel, and L.P. Weiner. 1986. In vivo effects of coronavirus-specific T cell clones: DTH inducer cells prevent a lethal infection but do not inhibit virus replication. J. Immunol. 136:3052-3056. 47. Sussman, M., R. Shubin, S. Kyuwa, and S.A. Stohlman. 1989. T- cell-mediated clearance of mouse hepatitis virus strain JHM from the central nervous system. J. Virol. 63:3051-3056. 48. Sutherland, R.M., M.M. Chua, E. Lavi, S.R. Weiss, and Y. Paterson. 1997. CD4+ and CD8+ T cells are not major effectors of mouse hepatitis virus A59-induced demyelinating disease. J. Neurovirol. 3:225-228. 49. Torres, C, I. Aranguez, and N. Rubio. 1995. Expression of interferon-gamma receptors on murine oligodendrocyte and its regulation by cytokines and mitogens. Immunol. 86:250-255. 50. Tran, E.H., E.N. Prince, and T. Owens. 2000. IFN-y shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164:2759-2768. 51. van der Veen, R.C. 1996. Immunogenicity of JHM virus proteins: Characterization of a CD4+ T cell epitope on nucleocapsid protein which induces different T-helper cell subsets. Virology 225:339-346. 52. Wakeham, C.J., R.M. Paige, and R.M. Zinkernagel, et al. 1991. Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353:ISO- 184. 53. Ward, S.G., K. Bacon, and J. Westwick. 1998. Chemokines and T lymphocytes: more than an attraction. Immunity 9:1. 213 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. 54. Welsh, C.J.R., P. Tonks, A.A. Nash, and W.F. Blafcemore. 1987. The effect of L3T4 T cell depletion on the pathogenesis of Theiler’s murine encephalomyelitis virus infection in CBA mice. J. Gen. Virol. 68:1659-1667. 55. Wijburg, O., M. Heemskerk, A. Sanders, C. Boog, and N. Van Rooijen. 1996. Role of virus-specific CD4+ cytotoxic T cells in recovery from mouse hepatitis virus infection. Immunology 87:34-41. 56. Williamson, J.S.P., and S.A. Stohlman. 1990. Effective clearance of mouse hepatitis virus from the central nervous system requires both CD4+ and CD8+ T cells. J. Virol. 64:4589-4592. 57. Williamson, J., K. Sykes, and S.A. Stohlman. 1991. Characterization of brain-infiltrating mononuclear cells during infection with mouse hepatitis virus strain JHM. J. Neuroimmunol. 32:199-207. 58. Wu, G.F., and S. Perlman. 1999. Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus. J. Virol. 73:8 771. 59. Xue, S., A. Jaszewski, and S. Perlman. 1995. Identification of a CD4+ T cell Epitope within the M protein of a neurotropic coronavirus. Virology 208:173. 60. Xue, S., and S. Perlman. 1997. Antigen specificity of CD4 T cell response in the central nervous system of mice infected with mouse hepatitis virus. Virology 238:68. 61. Yamaguchi, K., N. Goto, S. Kyuwa, M. Hayami and Y. Toyoda. 1991. Protection of mice from a lethal coronavirus infection in the central nervous system adoptive transfer of virus-specific T cell clones. J. Neuroimmunol. 32:1-9. 62. Yoshizawa, L, R. Bronson, A. Ben-Nun, J.R. Richert, M.E. Dorf, and S. Abromson-Leeman. 1998. Differential recognition of MBP epitopes in BALB/c mice determines the site of inflammatory disease induction. J. Neuroimmunol. 89:73. 214 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Chapter VII. Conclusions Previous data had shown a crucial contribution of perforin-mediated cytotoxicity to viral clearance; however, they also suggested the participation of additional anti-viral mechanisms (7). Results contained in this thesis demonstrate that the T cell-mediated control of JHMV replication during acute CNS infection is exerted by a combination of IFN-y and perforin-mediated cytolysis. Several observations in this thesis support the validity of this assumption. First, Fas-mediated cytotoxicity, by either CD8+ or CD4+ T cells appears to not contribute to JHMV pathogenesis since mice deficient in this lytic pathway had identical kinetics of viral clearance and disease compared to wt mice (Chapter II). This result is in agreement with the minor anti-viral role previously attributed to Fas-mediated cytolysis (14). However, experiments with chimeric mice uncovered a potential anti-JHMV role of Fas-mediated cytolysis in the absence of the predominant perforin lytic pathway (Chapter II). In this scenario, the Fas- pathway may be redundant during JHMV acute infection, however, it potentially is operative during viral persistance when perforin-mediated cytotoxicity is down regulated (1). Second, multiple cytokine mRNAs, including IFN-y, TNF-a, IL-10, IL-4 with known antiviral or immunomodulatory activities were induced in the CNS by JHMV infection (Chapter III). The peaks of mRNA induction were coincident with both R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. maximum T cell and macrophage infiltration and viral clearance (Chapter III), suggesting T cell-mediated viral clearance may contribute via cytokines. However, by either depletion experiments or infecting mice in which the genes encoding the cytokines had been ablated, it was shown that TNF-a, IL-10, and IL-4 do not contribute to viral clearance or JHMV-induced CNS pathology (8,15), leaving IFN-y as a potential anti-JHMV soluble factor. Third, experiments in gene-deficient mice or T cell transfers into immunodeficient mice demonstrated that whereas IFN-y contributes to the overall inhibition of virus replication in CNS, especially from oligodendroglia, perforin-mediated cytolysis controls virus replication from infected class I-expressing astrocytes and microglia [(Chapters IV, V and VI); (7)]. In contrast to the partially controlled infection in single deficient IFN-y'7 ' (Chapter IV) or P'7 ' mice (7), double deficient mice (IFN-y'7 '/P‘7 ') succumbed to an overwhelming CNS infection (Chapter V). However, when the perforin-mediated cytolytic function was rescued via adoptive transfer of CD8+ T cells, a complete (wt CD8+ T cells) or partial viral clearance (IFN-y'7 ' CD8+ T cells) was reconstituted (Chapter V). Fourth, both CD8+ and CD4+ T cell can serve as sources of IFN-y in the CNS (Chapter V and VI). The elimination of IFN-y from CD8+ or CD4+ T cells abrogated the ability of these adoptively transferred T cell subsets to reduce virus replication in the CNS of SCID mice. Furthermore, CD8+ T cells lacking R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. IFN-y reduced vims from most of class I expressing cells in CNS (microglia and astrocytes) but at lesser extent from oligodendrocytes. Thus, both CD8+ T cell-perforin mediated cytolysis and T cell derived IFN-y appear to play complementary roles in controlling the JHMV infection within the CNS in cell type specific manner. IFN-y is also a cytokine with immunomodulatory functions and either its direct antiviral or immunomodulatory role are critical components in the defense against viral infections (2,5,16). Nevertheless it can be proposed from the results presented in this thesis that the antiviral effects of IFN-y during JHMV infection of the CNS may not rely on its ability to modulate other immune effector mechanisms. The data are more consistent with a direct anti viral role. IFN-y was not required for the induction of virus-specific CD8+ and CD4+ T cell responses (Chapter IV). Moreover, development of CTL effector function was also independent of IFN-y. Alternatively, in vivo CTL function may depend on the IFN-y-induced upregulated expression of MCH class I Ag for efficient target cell lysis (16). However, upregulation of MHC class I expression on the microglial cell population occurs independently of T cell- derived IFN-y (Chapter V), virus replication is reduced in the majority of class I-expressing cells in the absence of IFN-y, and viral clearance is incomplete by perforin-mediated cytotoxicity alone if CD8+ T cells are unable to secrete IFN-y R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. (Chapter IV and V). Therefore, one interpretation of the fact that the effector activity of T cells is abrogated in the absence of IFN-y is that T cell-delivered IFN-y is directly anti-viral, especially within the oligodendrocytes. This idea is in agreement with previous observations in the intracellular inactivation of viruses via cytokines. For example, HBV in transgenic mice is inactivated at the postranscriptional level from hepatocytes via TNF-a and IFN-y (3). VV replication is also inhibited from the CNS of nude mice when IFN-y is expressed locally via a recombinant virus (IFN-y-W ) (11,6). Finally, reduction of MHV replication from CNS via administration of IFN-y in a DI vector of MHV (17), also confirm the potential direct anti-viral role of IFN-y. However, a definitive proof that IFN-y directly inhibit intracellular replication of JHMV in CNS remains to be demonstrated. MHC expression is required for T cell recognition and it is minimal or not seen in oligodendrocytes (9). Since expression of cytokines by T cells requires Ag presentation and signaling from the TCR (12,13), T cells may act by releasing IFN-y in the vicinity of infected oligodendroglia following interactions with infected class I or II expressing cells. IFN-y may reduce viral replication in nearby infected cells as suggested from the case in HBV infection (3). A second possibility is that if cytokine production in vivo is regulated as R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. suggested by in vitro experiments indicating an on/off/on cycling process (12), it may also be possible that low levels of MHC expression in oligodendrocytes are sufficient to trigger IFN-y secretion by virus-specific T cells that have already been in contact with other nearby infected cells. IFN-y may also protect uninfected cells, rendering them less susceptible to the infection by upregulating cellular proteins such as double-stranded RNA activated protein kinase (PKR) or 2’-5 ’-oligoadenylate synthetase (2 '5 OAS) (2,5), which decrease viral transcription and replication. Consistent with this idea, early during acute infection (10 days p.i) recipients of CD8+ T cells lacking IFN-y secretion showed delayed viral reduction from all CNS cell types as compared to higher reduction in viral loads from the same type of cells by CD8+ T cells competent for IFN-y secretion (Chapter VI). These results suggest that IFN-y may act by two non-exclusive mechanisms; 1) control of viral replication in all cell types, thereby reducing spread to oligodendrocytes, 2) direct control of viral replication primarily within oligodendrocytes. However, since infection of oligodendrocytes is not inhibited in the presence of T cells producing IFN- y (Chapter V), despite the eventual clearance of virus, (Chapter IV and V), this would suggest that IFN-y either secreted in the vicinity or directly delivered by T cells may control JHMV replication directly in oligodendroglia. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. T cell adoptive transfer experiments in this thesis confirm that T cells are critical for the protection during JHMV infection of the CNS but are also critical mediators of clinical disease and demyelination. However, as suggested previously (4), the T cell effector mechanisms, which protect mice from infection, may be different from the effector mechanism inducing demyelination. Demyelination was independent of Fas-FasL interactions (Chapter II), IFN-y or perforin-mediated cytotoxicity (Chapters IV, V and VI), implying the participation of an additional mechanism(s). Inflammatory cytokines can cause immunopathology (10). However, TNF-a, which is also induced in CNS following JHMV infection, is not involved in the process (15). Multiple other inflammatory cytokines such IL -la, ILip, IL-6 also increase in response to the infection (Chapter III). However, they are also produced endogenously by CNS brain resident making complicated the study of these cytokines as T-cell effector mechanisms of CNS disease. Furthermore, the cytokine profiles induced by a more virulent, lethal and non-demyelinating variant of JHMV appear to not differ greatly from the profile induced by the 2.2v-l variant which causes extensive demyelination (Chapter III). Alternatively, T cells also may contribute to enhanced demyelination via activation of macrophages/microglia, which are the cells that remove myelin or injured oligodendrocytes. R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. Understanding the dual role of CD4+ T cells in providing protection but at the same time accelerating disease and demyelination is even more complicated. Virus-specific CD4+ T cells eliminate virus from CNS via IFN-y- dependent and independent mechanisms; however, they cause rapid mortality during the infection with anon-lethal variant of JHMV. This detrimental effect of CD4+ T cells appear to be independent of Fas-FasL apoptotic pathway (Chapter II) or perforin-mediated cytotoxicity (7). However, the sole presence of CD4+ T cell in the reconstituted SCID host may circumvent the immune regulatory mechanisms present during a normal infection, changing the course of the normal pathogenesis. Consistent with this idea, the simultaneous presence of CD4+ and CD8+ T cells ameliorated the disease induced by the transfer of CD4+ T cells alone (Chapter VI). These would suggest that the final control of the CD4-induced disease may be tightly regulated by other immune components. The emerging picture of this complex disease that induces both viral persistance and demyelination is that lytic and non-lytic effector mechanisms are functioning within specific subsets of the major CNS cell types. Both CD8+ and CD4+ T cells reduced virus via IFN-y. The finding that cytokines can eliminate JHMV replication without inducing cytolysis of oligodendrocytes and CNS cells may reflect a protective function of the adaptive immune response in 2 2 1 R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission. eradicating virus without destroying the cell, in an effort to protect the host from extensive injury , as has been suggested is the case for viral infections in the liver (3). However, these non-lytic mechanisms could also paradoxically contribute to viral persistence by reducing infectious virus to minimal levels, thereby shielding the persistent infected cells from T cell recognition. 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T cell-dependent antiviral mechanisms in the pathogenesis of mouse hepatitis virus in the central nervous system
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