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Gender dependent immune regulation: effect of regulatory t cells on macrophages and potential mechanism of protection from autoimmune disease
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Gender dependent immune regulation: effect of regulatory t cells on macrophages and potential mechanism of protection from autoimmune disease
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GENDER DEPENDENT IMMUNE REGULATION:
EFFECT OF REGULATORY T CELLS ON MACROPHAGES AND POTENTIAL
MECHANISM OF PROTECTION FROM AUTOIMMUNE DISEASE
by
Stefanie Johanna Kirwin
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
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
December 2006
Copyright 2006 Stefanie Johanna Kirwin
ii
Dedication:
To my parents.
Thank you for giving me the skills and confidence to achieve my dreams.
iii
Acknowledgements:
My sincere thanks to all those who guided me during my years at USC, especially Dr
Stephen Stohlman for being my mentor. Thanks also to the members of the Stohlman and
Bergmann laboratories for their help, especially Ni Feng for her excellent technical
support. Furthermore, I’d like to express my gratitude to the members of my dissertation
committee, Dr Stanley Tahara, Dr Michael Lai, and Dr Allan Epstein.
Last but not least thanks to my family for all your love and support and your unwaivering
believe in me. Without you I could not have done it.
iv
Table of Contents:
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
Abbreviations x
Abstract xiii
Chapter 1:
Introduction
1
Chapter 1 References 20
Chapter 2:
T
reg
Control of Macrophage Activity in Naïve Mice
43
Introduction 44
Materials and Methods 47
Results 53
Discussion 66
Chapter 2 References
71
Chapter 3:
Gender dependent changes in macrophage phenotype before and after
antigen encounter
79
Introduction 79
Materials and Methods 83
Results 84
Discussion 109
Chapter 3 References
114
Chapter 4:
Altered Neuroantigen-Specific Cytokine Secretion In A Th2 Environment
Reduces Experimental Autoimmune Encephalomyelitis
121
Introduction 122
Materials and Methods 125
Results 131
Discussion 147
Chapter 4 References 151
v
Chapter 5:
Discussion
157
Chapter 5 References
169
Bibliography
180
Appendix:
IFN- γ expression from DI-particles in MHV infection of the CNS
207
Introduction 207
Materials and Methods 209
Results 213
Discussion 227
Appendix References 230
vi
List of Tables:
Table 1: Ag-specific cytokine release by cells derived from female and male SJL
mice
133
Table 2: Th2-mediated EAE protection 135
vii
List of Figures:
Figure 1: APC derived from CD4 depleted male donors support Th1 induction
55
Figure 2: CD4 depletion before antigen encounter alters APC phenotype
57
Figure 3: CD25 depletion before immunization results in suppression of Th2
polarization
59
Figure 4: APC derived from CD25 depleted male donors support Th1 induction
61
Figure 5: T
reg
are increased in male compared to female SJL
63
Figure 6: In vitro suppressive function of CD25 cells is gender independent 65
Figure 7: Cell populations isolated from spleen and lymph nodes do not vary in
naïve mice
86
Figure 8: Macrophages, Neutrophils, and T and B cells differ in size and density
87
Figure 9: Male SJL have increased neutrophils in the spleen compared to female
mice
89
Figure 10: F4/80
+
macrophage populations are similar in male and female SJL
91
Figure 11: MHC class II expression does not differ significantly between male and
female SJL mice
92
Figure 12: Plasmacytoid dendritic cells do not show gender dependent change in
phenotype
94
Figure 13: Dendritic cell populations are increased in male compared to female
SJL
96
Figure 14: Gender does not influence the percentage of CD11c
+
CD11b
+
CD8α
-
populations in SJL mice
98
Figure 15: CD11c
+
CD11b
-
CD8α
+
dendritic cells do not exhibit a gender
dependent change in phenotype
99
Figure 16: Macrophage numbers are similar in male and female peritoneum,
whereas neutrophils are increased in males
101
viii
Figure 17: CD8a
+
and CD8α
-
dendritic cells are similar in the peritoneum of male
and female SJL
101
Figure 18: Dendritic cells do not ingest OVA in the peritoneum
103
Figure 19: Injection of OVA i.p. results in varying degrees of uptake by
macrophages in individual mice
105
Figure 20: Uptake of OVA by peritoneal macrophages results in proteolytic
cleavage of the protein
106
Figure 21: MHC class II expression is reduced on male derived macrophages after
OVA uptake
108
Figure 22: Th2 environment prevents demyelination and inflammation
136
Figure 23: Th2 protection decreases CNS pro-inflammatory gene expression
138
Figure 24: Reduced CNS inflammation in Th2 protected mice
141
Figure 25: Decreased MHC class II expression on F4/80+ macrophages and
microglia in Th2 protected mice
142
Figure 26: Th2 and CD4
+
CD25
+
cells within the CNS of protected mice
144
Figure 27: Increased frequency of PLP-specific Th2 secreting cells within the CNS
of Th2 protected mice
146
Figure 28: Transfection of JHM-MHV using DI particles encoding IFN- γ results in
cytokine production in vitro
215
Figure 29: Virus titer increase correlates with loss of IFN- γ secretion
216
Figure 30: Clinical symptoms do not differ significantly between mice infected
with MHV-JHM DI- γ and control virus
218
Figure 31: Virus titers are similar in MHV-JHM DI- γ and control virus infected
mice
219
Figure 32: Infection with MHV-JHM DI- γ does not alter the infiltrating cell
populations
221
Figure 33: IFN- γ inducible genes are not preferentially expressed in the CNS of
MHV-JHM DI- γ infected mice
223
ix
Figure 34: MHV-JHM DI- γ infection does not result in upregulation of MHC class
II on microglia
225
Figure 35: MHV-JHM DI- γ infection does not enhance 2’-5’ OAS expression
compared to MHV-JHM DI-x infected mice
226
x
Abbreviations:
Ig = Immunoglobulin
NK = Natural Killer
IL = Interleukin
i.c. = Intracerebral
MHV = Mouse Hepatitis Virus
CNS = Central Nervous System
DTH = Delayed type hypersensitivity
KLH = Keyhole Limpit Hemocyanin
FGG = Fowl Gamma Globulin
Th = T Helper
CD = Cluster of Differentiation
IFN = Interferon
APC = Antigen Presenting Cell
i.p. = Intraperitoneal
LPS = Bacterial Lipopolysaccharide
MHC = Major Histocompatibility Complex
EAE = Experimental Autoimmune Encephalomyelitis
MS = Multiple Sclerosis
MBP = Myelin Basic Protein
PLP = Proteolipid Protein
xi
DNA = Desoxyribo Nucleic Acid
GA = Glatiramer Acetate
T
reg
= Regulatory T Cell
CTLA = Cytotoxic-T Lymphocyte-Associated-Antigen
mAb = Monoclonal Antibody
GITR = Glucocorticoid-Induced Tumor Necrosis Factor Receptor
TGF = Transforming Growth Factor
NOD = Non-Obese Diabetic
DHT = Dihydrotestosterone
TNF = Tumor Necrosis Factor
TLR = Toll Like Receptor
DPBS = Dulbecco’s Phosphate Buffered Saline
PEC = Peritoneal Exudate Cells
RT = Room Temperature
PCR = Polymerase Chain Reaction
RT = Room Temperature
RNA = Ribo Nucleic Acid
DEPC = Diethyl Pyrocarbonate
dd = Double Distilled
NO = Nitric Oxide
NCI = National Cancer Institute
OVA = Ovalbumin
xii
FCS = Fetal Calf Serum
MFI = Mean Fluorescence Intensity
Ag = Antigen
WM = White Matter
DI = Defective Interfering
PFU = Plaque Forming Units
MOI = Multiples of Infection
TPB = Tryptose Phosphate Broth
NBC = Newborn Calf Serum
iNOS = Inducible Nitric Oxide Synthetase
MCP = Monocyte Chemoattractant Protein
RANTES = Regulated Upon Activation, Normal T Cell Expressed and
Secreted
IP = Gamma Interferon Inducible Protein
MIG = Monokine Induced by Gamma Interferon
2’-5’ OAS = 2’-5’ Oligoadenylate Synthetase
xiii
Abstract:
SJL mice exhibit a unique gender-dependent bias in their immune response.
Males mount an anti-inflammatory Th2 response, whereas females react with an
inflammatory Th1 response, which correlates with susceptibility to experimental
autoimmune encephalomyelitis, a mouse model for multiple sclerosis. Castration as well
as macrophage transfer from females reverses the male phenotype. Utilizing this mouse
strain for the study of gender-dependent mechanisms of immune regulation, the role of
CD25
regulatory T cells was examined. These cells maintain a Th2 environment in naïve
males by regulating macrophage responsiveness. Transfer of macrophages from naïve
CD25
+
-depleted males into untreated males results in a Th1 response after immunization
demonstrating that regulatory T cells directly influence macrophage function. Males have
a two-fold increase in the number of regulatory T cells compared to females, but no
difference in cell surface marker expression or in vitro suppressive action was detected.
The macrophage phenotype in naïve mice showed no difference in number or activation
status comparing males and females. Analysis of cell surface molecules after antigen
uptake uncovered a reduction in MHC class II expression on macrophages in males
compared to females. The lack of antigen uptake by dendritic cells further confirmed the
importance of a macrophage antigen presenting cell.
Male-derived Th2 cells suppress experimental autoimmune encephalomyelitis.
Examination of the T cell response in protected mice showed only moderate reduction in
CNS infiltrating cells and no difference in cell composition. However, MHC class II
xiv
expression on microglia was significantly reduced indicating reduction in inflammatory
cytokines such as IFN- γ and possible action of anti-inflammatory cytokines. Neither
regulatory T cells nor Th2 cells were increased in the CNS of protected mice. However,
selfantigen specific T cells in the CNS of protected mice demonstrated a shift in cytokine
pattern from inflammatory Th1 to anti-inflammatory Th2. This suggests that the
induction of a Th2 response to non-self antigen in the periphery can inhibit CNS
inflammatory disease by altering the cytokine response to the potentially
encephalitogenic selfantigen.
1
Chapter 1
Introduction
SJL mice were initially described as an animal model for Hodgkin’s disease due
to the fact that they develop spontaneous reticulum cell neoplasm
139
. Subsequently, a
number of immunological abnormalities were described in this mouse strain. B cell
dysfunction is evident by depressed immunoglobulin (Ig)E levels
211
as well as the
inability to make antibodies to a known autoantigen
84
. Mice of this strain also have a
reduced number of Natural Killer (NK) cells
24,64
and a defect in NK T cells which lack
the ability to produce Interleukin (IL)-4
221
. Furthermore the T cell repertoire in SJL mice
shows deletion of 50% of V
β
genes compared to BALB/c mice. The V
β
chain is part of
the variable region of the T cell receptor β chain
18,77
and one might expect that this
deletion limits the T cell repertoire and therefore the number of different pathogens the
adaptive immune response can recognize. However, SJL mice are not more susceptible to
infections than other mouse strains. They are highly resistant to a number of microbial
infections such as Listeria monocytogenes
31
and Cryptococcus neoformans
160
, whereas
resistance to parasitic infections varies
121
. Mice of this strain are not susceptible to rabies
virus infection
114
, but resistance to measles virus is age-dependent, as demonstrated by
the fact that mortality in infected neonates is 100%, whereas all mice survive the
infection if inoculated later in life
143
. Age-dependent virus susceptibility is also found
when male SJL mice are inoculated intracerebrally (i.c.) with the mouse hepatitis virus
(MHV) strain JHMV. Male SJL mice cannot control virus in the central nervous system
(CNS) at six weeks of age, but if infected at 12 weeks of age, there is no evidence of viral
2
replication in either brain or spinal cord. Further study of this phenomenon demonstrated
that young male SJL are not only unable to clear JHMV infection in the CNS, but they
are equally unable to mount a delayed type hypersensitivity (DTH) response to a variety
of antigens, including vesicular stomatitis virus, but also non-infectious agents such as
sheep red blood cells and protein antigens such as keyhole limpet hemocyanin (KLH) or
fowl gamma globulin (FGG)
120,182
. This age-dependent defect is not observed in female
SJL mice
41
. Further evidence for a role of hormones in the lack of a T helper (Th)1
response in young males is the fact that castration changes the phenotype of the male
mouse to a DTH responder and results in IL-12 production by macrophages in vitro
41,215
.
DTH is an antigen-specific immune response directed by Th1 type Cluster of
Differentiation (CD)4 T cells
33
. A Th1 response is characterized by inflammatory
cytokines such as Interferon (IFN)-γ and IL-12, whereas a Th2 response involves anti-
inflammatory cytokines such as IL-4 and IL-10
34,135,136
. T cell stimulation by antigen
presenting cells (APC) in the presence of IL-12 leads to IFN-γ production and a Th1
response
115
. On the other hand, when antigenic stimulation of the T cell is accompanied
by exposure to IL-4 possibly from NKT
50,222
or T cells
34,161
, the immune response is
shifted to a Th2 phenotype
147
. Th1 and Th2 responses can also be distinguished by the
subclass of antibody in the serum, namely IgA and IgG2a during a Th1 response and
IgG1 and IgE during Th2
179
. DTH reactions occur after immunization, when the animal is
challenged with the same antigen, usually in the footpad or ear. This induces a T cell
dependent
202
influx of lymphocytes and causes edema and swelling
33
, which can be
measured using a micrometer device.
3
In SJL mice, DTH reactivity and the ability to mount a Th1 response, can be
transferred to young males using cells from spleen or the peritoneum of female mice
41
and older males
181,182
. Depletion of T or B cells or irradiation before transfer does not
affect the cells’ ability to induce a DTH response; however, depleting IA
+
, CD11b
+
or
adherent cells does. This indicates that a macrophage is central in the transfer of DTH
responsiveness
121,182
. Large numbers of macrophages can be harvested from the mouse
peritoneum after intraperitoneal (i.p.) injection of thioglycollate
44,182
. These cells have an
activated phenotype, but remain unable to induce a DTH reaction if harvested from male
SJL
41,182
.
Transferring macrophages from older mice into young male SJL 24h after
immunization does not induce DTH reactivity in young males
182
, but transferring cells
from older mice, which are loaded ex vivo with antigen, does
120
. These data indicate that
the difference between DTH responsiveness and lack thereof lies in the induction phase
of the T cell response, not the effector phase. T cell-independent APC stimulation using
bacterial lipopolysaccharide (LPS) results in similar IL-12 production in cultures of
macrophages from both young and older mice
215
and further studies showed that male-
derived T cells are not anergic or suppressed, but rather actively promote a Th2
response
42
. Culture of macrophages from female mice with T cells from either male or
female SJL results in IL-12 release, whereas culture with male derived APC results in IL-
10 production
215
. IL-10 inhibits mixed lymphocyte reaction of naïve T cells
224
and can
inhibit IFN-γ
62,203
, but not IL-4 production
63
. Phenotypic analysis does not reveal a
4
difference in thioglycollate-elicited macrophages from male or female SJL in any of the
assessed cell surface markers
44
.
These findings suggest that there is an age- and gender-dependent difference in
antigen presentation or co-stimulation which results in a lack of a DTH response in male
SJL mice. This difference is unique to the SJL strain and offers the opportunity to study
induction of pro- or anti-inflammatory immune responses in mice with identical genetic
makeup, eliminating confounding issues, such as differences in major histocompatibility
complex (MHC) molecule expression or other less well defined differences in the
genome that can influence the immune response. It also allows transfer of cells between
male and female mice to study the influence of individual cell types or molecules on the
induction of inflammatory immune responses.
The ability to mount a DTH response in female SJL correlates with susceptibility
to experimental autoimmune encephalomyelitis (EAE)
41,107
a mouse model for the human
demyelinating disease multiple sclerosis (MS)
25
. Though first recognized in the early 19
th
century, there are still more questions then answers about MS. Approximately 2.5 million
people world wide suffer from the disease
35
, which is the most common cause for
neurological deficits in young people. It affects women twice as often as men and the
onset in women is usually during early adulthood
214
. There are multiple factors that play
a role in disease penetrance including genetics, the environment and hormones
20,28,35
. One
reason indicating that hormones may play a role in MS, apart from the female to male
ratio, is that pregnancy, especially the third trimester, has been associated with
protection
1,20,36
. This protection can be attributed to the inhibitory effect of high doses of
5
estrogen during late pregnancy
91
. It is likely that this immune suppression is necessary
during pregnancy to avoid rejection of the fetus
131
. Female MS patients show a strong
skewing towards a Th1 phenotype in their peripheral T cell population, whereas male MS
patients do not
150
.
The hallmark of MS is demyelinating lesions in the CNS and the disease leads to
motor, sensory and autonomic dysfunction
35
. MS is often described as a Th1 disease,
because inflammatory infiltrates in the CNS contain mainly T cells as well as activated
macrophages and microglia
15,214
. Also, IL-12 in the cerebrospinal fluid is significantly
increased in patients with CNS lesions
59
.
EAE is an animal model for the human disease. It can be induced either by active
immunization with mouse spinal cord homogenate
22
or myelin antigens such as myelin
basic protein (MBP)
68
or proteolipid protein (PLP)
200
or by passive transfer of T cells
from mice immunized with one of those antigens
127,151
. Young male SJL are resistant to
EAE induced by immunization
41
and T cells from male mice cannot induce
EAE
17,42,46,183
. Castrated males are susceptible to EAE
16
.
In female SJL, active and passive induction results in a relapsing/remitting
disease
25,68,200
. The first symptoms of EAE develop approximately 5-7 days after T cell
transfer
99
. The notion of the CNS as an immune – privileged site that is completely closed
off to circulating lymphocytes has been challenged and it is now clear that activated T
cells can enter the brain and spinal cord
74,78,130
. However, there is little MHC class II
expression in the uninflamed CNS
21,130
and it is not clear what type of APC the
transferred T cells interact with in the recipient. Activated T cells do not simply migrate
6
to the CNS and recognize antigen on resident APC as transgenic expression of MHC
class II only in the CNS is not enough to develop EAE after transfer of activated T cells.
Rather it has been proposed that T cells recognize their cognate antigen on CD11c
+
APC
in the meninges and blood vessels which initiates inflammation and EAE
73
. Transferring
T cells expressing green fluorescent protein have shown that these cells first migrate to
the lymph nodes before entering the CNS
65
. Interestingly, animals devoid of secondary
lymphoid organs are nonetheless susceptible to passive EAE implying that the migration
to lymph nodes is not a necessary step in disease induction
73
. Transferred T cells can be
traced in the recipient mice using monoclonal antibodies if donor and recipient express
different haplotypes of CD90. SJL express the CD90.2 haplotype, but T cells from
transgenic CD90.1 SJL are not rejected and T cell function is not altered
99,177
.
The discovery that IL-17-producing T cells and not IFN-γ-producing Th1 cells are
responsible for the induction of EAE was made recently
109,123
. This also helps to explain
the observation that mice deficient in IFN-γ are not resistant to EAE and even develop
more severe disease then wild type mice
103
. The increased severity is most likely due to
the lack of the downregulatory effect that IFN-γ has on activated T cells and to the
composition of the cellular infiltrate which contains an unusually high number of
neutrophils
47,213,223
. There is an increase in IFN-γ as well as IL-6, IL-1 and IL-4 mRNA
during acute disease in the CNS and increase in IL-10 during recovery
97
. IL-10-deficient
mice develop more severe EAE then wild type mice
6,23
and show increased disease
incidence and a higher frequency of inflammatory cells
23
. IL-4-deficient mice develop
7
also more severe disease then wild type mice, but not as severe as IL-10 deficient
animals, and incidence is not increased
23,58
.
Studies using recombinant IL-4 and IL-10 have not completely resolved the
respective roles of these cytokines. Administration of IL-4 i.p. has been shown to
suppress disease
155
and daily injections
43,140,165
or intranasal administration
218
of IL-10
result in delayed onset and reduction of severity of the disease, but in this case IL-4 can
abrogate protection
140
and a single injection of IL-10 does not protect
38
. In general,
cytokine treatment has to be administered frequently to have an ameliorating effect on
EAE. Therefore, alternative methods of achieving downregulation of the immune
response have been explored.
IL-10-producing cells generated in vitro with the help of Vitamin D3 and
Dexamethasone suppress EAE if they recognize their antigen in the CNS
13
. Intranasal
administration of encephalitogenic peptide also suppresses disease and this protection is
IL-10-dependent
26
.
Intracranial transfer of fibroblasts modified to secrete IL-10 only protects from
disease if IL-10 is produced long term by retroviral infection and not transiently by a
transgenic adenovirus
38
. Direct injection of IL-10-producing adenovirus also protects
mice from EAE
43
, whereas IL-10 delivered by plasmid desoxyribo nucleic acid (DNA)
i.c. had no effect
39
. Using CNS antigen-specific cells and transfecting them to secrete IL-
10 under the IL-2 promoter results in protection
119
as does IL-10 expressed under the
MHC class II promoter
40
. Also retroviral transduction of encephalitogenic cells with the
IL-4 gene can inhibit EAE, whereas transfection of non-antigen specific cells does not
172
.
8
Taken together, these results show that Th2 cytokines can protect from inflammatory
disease in the CNS, but that the site of administration and timing are essential. Th2 cells
are ideal candidates to deliver anti-inflammatory cytokines since they can migrate to sites
of inflammation and then be activated by endogenous stimuli to regulate the response at
the site of inflammation. However, in vitro generated Th2 cells are not effective at
suppressing disease
98,106
. Immunizing male SJL mice is a way to obtain in vivo generated
antigen-specific Th2 cells that can be transferred to EAE-susceptible mice without being
rejected
208
. During acute EAE, PLP-specific cells are most frequent in the CNS and then
decline rather than switching to a Th2 phenotype
194
. Transferring MBP-specific Th2 cells
from male into female SJL at the same time as MBP specific Th1 cells protects mice
from disease
42
. Th2 cells specific for non-self antigen can also inhibit EAE when
activated in the recipient by immunization
57,183
. This protection is dependent on IL-10,
but not IL-4
183
. Though the results of studies investigating the effect of the two
prototypical Th2 cytokines IL-4 and IL-10 on EAE vary according to the method used, it
is evident that IL-10 is more potent than IL-4 in suppressing EAE and that delivery by in
vivo activated Th2 cells is more effective then other methods. However, it is not clear if
these Th2 cells act locally in the CNS or potentially change the cytokine environment in
the periphery to inhibit the inflammatory potential of encephalitogenic Th1 cells.
In MS patients, corticosteroids are used to inhibit the Th1 response and the most
aggressive forms of MS are now treated with chemotherapy to destroy the
encephalitogenic cells. This treatment, however, is very toxic
35,49
. There is evidence that
several new drugs, rather then simply inducing cell death in lymphocytes, can actually
9
change the phenotype of the response from Th1 to Th2. IFN-β treatment reduces relapse
rates and possibly accumulation of disability
35,37,86,93
and it results in an increase of IL-10
production by T cells and monocytes in blood
112,133
. This coincides with an increase in
serum IL-10
4,166
. Also, a reduction in the frequency of IFN-γ-producing cells and an
increase in IL-5 producers
90
has been demonstrated. Glatiramer acetate (GA) is another
treatment option. This molecule is modeled after an encephalitogenic antigen and was
initially believed to inhibit T cell response by competing for binding to MHC
3
. This is
probably not the only mechanism of disease suppression by this molecule since
cyclophosphamide abrogates the beneficial effect of GA treatment indicating an active
protective mechanism that involves T cells. Also, T cells from GA-treated patients
produce Th2 cytokines in response to GA stimulation
52
. High dose estrogen in vitro has
also been shown to enhance IL-10 secretion of T cells from MS patients
70
and oral
administration of estriol resulted in some improvement in a small clinical trial
176
. There is
some controversy over the best way to assess efficacy of drugs in MS patients due to the
variability in criteria used in different studies
138
. Nevertheless, taken together the studies
in mice and humans indicate that Th2 cells and induction of IL-10 might be protective in
CNS inflammation.
The source of IL-10 is also a topic of investigation. Apart from Th2 cells
42,183
or
macrophages
40
, B cells are also an important source of protective IL-10 in mice
61
.
Recently, intense focus has been concentrated on regulatory T cells (T
reg
) and studies
show that during recovery from EAE, IL-10 in the CNS is produced by T
reg
124
and T
reg
transfer can inhibit disease
100,124,225
. By contrast, depletion leads to increased mortality
10
and earlier disease onset as well as abolition of recovery
225
. T
reg
cells from IL-10-
deficient mice do not suppress EAE
225
and transgenic T cells that are directed against
encephalitogenic T cells can inhibit relapse even after onset of symptoms in an IL-10-
dependent manner
128
.
T
reg
are one of several mechanisms that control autoimmunity by preventing
activation of self-reactive T cells which occur naturally in the periphery
150,157,163
.
However, as opposed to T cell anergy
92
and ignorance
170
, which renders cells
unresponsive to antigenic stimuli, they actively suppress the immune response. T
reg
have
been described in mice
167
and humans
145
and they include inducible as well as natural
T
reg
. Inducible T
reg
are very likely important in downregulating ongoing immune
responses
60,92,124,164
, whereas natural T
reg
are present in the naïve mouse
167
and have the
potential to control autoimmunity. The initial observation that thymectomy in 3 day old
mice leads to oophoritis was interpreted as a direct effect of the thymus on ovaries
146
.
Subsequently it was shown, however, that autoimmunity in thymectomyzed mice can be
prevented by the transfer of T
reg
and these cells are found in the thymus until mice are at
least 7 days old
10
.
Transfer of CD25
+
-depleted CD4 cells from wild type BALB/c mice to athymic
nude mice results in spontaneous autoimmune disease, whereas co-transfer of
CD4
+
CD25
+
T
cells does not, confirming a T
reg
population expressing CD4 and CD25 on
the cell surface
167
. T
reg
are poorly proliferating cells which can inhibit proliferation of
CD4 T cells in an antigen-independent manner in vitro
53,191,196,197
. This suppression is
also independent of MHC haplotype as shown by the observation that T
reg
from one
11
mouse strain can suppress proliferation of CD4 from a different strain
196
. T
reg
from T cell
receptor transgenic mice were used to demonstrate that once T
reg
are activated with their
cognate antigen, they are capable of inhibiting proliferation of CD4 T cells stimulated by
an unrelated antigen
191,197
. Inhibition is abolished when cell-cell contact is interrupted
using a transwell system and also by addition of IL-2 or stimulation with CD28
191,196
. The
manifestation of autoimmunity in T
reg
deficient mice is different depending on the genetic
makeup of particular mouse strains
174,190
.
Expression of CD25 on the cell surface as induced by T cell activation is not
enough to confer suppressive activity to CD4 T cells
188,196
and not all regulatory T cells
express CD25
158
. The discovery of the T
reg
-specific transcription factor FoxP3 has made
it possible to distinguish activated CD25 expressing T cells from T
reg
60,81
, though FoxP3
-
T
reg
have also been described
60,206,210
. FoxP3 deficiency leads to a lethal autoimmune
syndrome in both mice and humans and CD4
+
CD25
-
T cells exhibit T
reg
activity after
transfection with the transcription factor
66
. T
reg
also express several other molecules
which are not unique to this T cell subset, but are associated with suppressor function.
Blockade of Cytotoxic-T Lymphocyte-Associated-Antigen (CTLA)-4 in vivo using
monoclonal antibody (mAb) inhibits T
reg
development
88
as well as function
95
most likely
by interfering with maintenance of suppressive activity rather than induction
54
. On the
other hand, mice deficient in CTLA-4 seem to be able to compensate and have normal
T
reg
function
193
and blocking CTLA-4 in vitro does not affect suppressive activity
196
.
Neutralizing the action of CTLA-4 in EAE exacerbates the disease
95
.
12
CD103 is an integrin molecule which is expressed on a wide variety of cells
9
and
a small subset of T
reg
, which show greater suppressive activity in vitro compared to
CD103
-
T
reg
. Transfer of CD103
+
T
reg
inhibits colitis
111
. It is not clear if CD103
expression on T
reg
is necessary for this suppression. Transfer of CD103
-
T
reg
into immune
deficient mice suppresses colitis caused by CD4 T cells, but T
reg
are ineffective if
recipient APC do not express CD103
9
.
Glucocorticoid-Induced Tumor Necrosis Factor Receptor (GITR) is
predominantly expressed on CD4
+
CD25
+
T
reg
and inhibition of this molecule in vitro
abrogates suppression
175
. Neutralizing GITR during the induction phase of EAE
exacerbates disease
101
.
Transforming growth factor (TGF)-β1 deficient mice develop lymphoproliferative
disease similar to mice deficient in FoxP3
105,118
. T
reg
numbers are normal in the thymus in
these mice
118
and T
reg
from TGF-β receptor-deficient mice can inhibit in vitro
proliferation, but not colitis
56
. A reduction in the T
reg
number in the periphery indicates a
role for TGF-β in the maintenance of the peripheral T
reg
population. T
reg
cells from TGF-
β-deficient mice have decreased suppressive capacity in vitro, which is also true for T
reg
that lack the ability to react to TGF-β due to a dominant negative receptor for the
molecule
118
. Suppression of colitis is abrogated by blocking TGF-β
153
. A different study
found no effect of blocking TGF-β in vitro, but did find that in vivo it abrogates
protection from colitis
141
and T
reg
are unable to suppress colitis caused by TGF-β
Receptor-deficient CD4 T cells
56
. Finally, TGF-β-deficient T
reg
suppress normally in
13
vitro and show no defect in suppressing autoimmune disease of the gut
56,152
. In murine
autoimmune pneumonitis, protection is abrogated by antibody blocking TGF-β
83
.
Other cytokines have also been implicated in T
reg
function. T
reg
stimulation in the
presence of IL-2 results in the production of IL-10
14
and T
reg
cells from mice deficient in
this cytokine cannot suppress autoimmune colitis
11
. Suppression of allergic asthma is also
dependent on IL-10 as demonstrated by the fact that blocking IL-10 activity using
antibody leads to abrogation of suppression, but the source of the cytokine is unclear as
IL-10-deficient T
reg
suppress as well as wild type cells
96
. Protection from autoimmune
pneumonitis is also dependent on IL-10
83
. This is in contrast to in vitro suppression
assays where no role for soluble factors have been demonstrated
191,196
. An effect of IL-4
has not been shown in T
reg
-mediated suppression of CD4 T cell responses
152
. T
reg
numbers vary between mouse strains, for example BALB/c mice develop more cancer
and less autoimmunity then C57BL/6 and this has been shown to correlate with an
increased number of T
reg
in BALB/c mice
32
.
Considering the inhibitory effect of T
reg
in autoimmunity and the observation that
women are more susceptible to disease of that nature, the possibility of a direct or indirect
effect of hormones on T
reg
populations exists. SJL mice are useful in exploring this
facultative difference and its potential effect on inflammatory responses. The central role
that macrophages play in inducing a Th1 response in female or a Th2 response in male
mice is demonstrated by the fact that Th1 responsiveness can be transferred to young
male SJL by adoptive transfer. There are several possible interactions between
macrophage and T
reg
that can affect the outcome of an immune response. Expression of
14
hormone receptors has been demonstrated on T cells and macrophages, albeit at varying
levels
17,131,168,180,207
and with low binding affinity
131
, making either cell type a possible
target for hormonal modulation.
CD4 T cells as well as macrophages express androgen receptors
113
and several
observations argue for an anti-inflammatory role of this hormone. Male SJL are unable to
mount a Th1 response after immunization and this state of unresponsiveness can be
reversed by castration
41,215
. In the non-obese diabetic (NOD) strain of mice, females are
more likely to develop diabetes and islet cell destruction can be prevented by androgen
therapy
67
. Adoptive transfer of dihydrotestosterone (DHT)-treated encephalitogenic T
cells causes a lower incidence of EAE as well as decreased mortality and less severe
clinical symptoms than transfer of untreated cells
17
. Lymphocytes which are unable to
produce or respond to androgen show a higher rate of proliferation in a mixed
lymphocyte reaction than wild type cells
212
. Stimulating T cells from female SJL in the
presence of APC and DHT on the other hand does not inhibit their proliferation, but
results in fewer IFN-γ and more IL-10 producing cells
17
. Cells from DHT-treated female
mice produce significantly more IL-10 and less IL-12 in response to TCR-mediated
stimulation by anti-CD3
113
. Androgens also inhibit the recovery of lymphocyte numbers
after chemotherapy
162
. It is unclear if this is due to an effect on T cells directly or on APC
function. APC from males are more efficient in presenting antigen in mixed lymphocyte
reactions than those from females and castration enhances antigen presentation in male
mice from a variety of mouse strains
212
.
15
Suppression of DTH responses in male SJL mice may be influenced by T
reg
action. Direct action of T
reg
on APC has been observed in humans as well as mice.
Human T
reg
suppress mixed lymphocyte reactions early and potently, indicating that they
act directly on APC, rather than activated T cells
71
. Activated T
reg
can inhibit
upregulation of co-stimulatory molecules on APC
82,134
as well as secretion of IL-12
82,125
,
Tumor Necrosis Factor (TNF)-α
82,189
, and IL-6 via an IL-10-dependent mechanism
82,205
.
T
reg
can inhibit diabetes by suppressing APC maturation
171
as well as repress a Th1
response induced by adoptively transferred, antigen-pulsed APC
148
. In SJL mice
androgen may act on T
reg
, which in turn can affect APC function and inhibit Th1
responses in male mice indirectly.
On the other hand, hormones can potentially act on APC directly. The term APC
encompasses all cells capable of presenting antigen to T cells; however, only professional
APC have the potential to stimulate naïve T cells due to their ability to express co-
stimulatory molecules
129
. B cells, macrophages and dendritic cells are considered
professional APC
199
.
Except in a transgenic mouse system where antigen is targeted directly to B cells
in the spleen
29
, this cell type is unable to prime naïve T cells
104
. Though B cells are able
to activate primed T cells
72
, it is unlikely that they play a role in the differential cytokine
profile of T cells stimulated by APC in previously naïve SJL mice.
Lately the belief that only dendritic cells can prime naïve T cells
178,204
has been
challenged by experiments in which both dendritic cell and macrophage transfer resulted
in T cell priming
154
. This may be explained by more recent findings that demonstrate that
16
in mice dendritic cells, granulocytes and macrophages can arise from the same progenitor
cell
87
. Some monocytes, which are contained in the CD11b
hi
population, also express
CD11c as well as F4/80
69,110,186
and they can be cultured and differentiated into F4/80
+
macrophages or CD11c
+
dendritic cells
69
. A common CD11c
+
precursor can furthermore
differentiate into different dendritic cells subsets that were previously thought to be
derived from separate precursors
126,198,217
. It is becoming evident that dendritic cells and
macrophages more likely represent separate differentiation states rather than cells arising
from different ontogeny.
Macrophages are found in many tissues of the body, such as Kupffer cells in the
liver, alveolar macrophages in the lung, and microglia in the CNS and they can also be
found in the reproductive system, the serosal cavities, the lamina propria of the gut,
interstitium of the heart, pancreas, and kidney
195
. Macrophages can direct the
inflammatory response towards Th1 or Th2 depending on their activation state and the
cytokines they secrete
45
. This in turn is influenced by the local microenvironment
surrounding the cell and the type of stimuli they receive. IFN-γ and TNF-α enhance
macrophage activation, whereas IL-4, IL-10, as well as TGF-β inhibit it
27,116,137,184,185,201
.
However, the effects of cytokines are temporary and sequential treatment with different
cytokines results in a phenotype unlike that of a macrophage exposed to either cytokine
alone
184
. Other stimuli can also alter macrophage activation state. Binding to some Toll
like Receptors (TLR) on macrophages result in IL-12 secretion
2,8
, whereas engagement of
Fc receptors is followed by the production of IL-10
8
. Both IFN-γ and LPS act as stimuli
to upregulate MHC class I on macrophages
102
. Uptake of apoptotic cells by macrophages
17
induces TGF-β production which may help in resolution of an immune response
85
.
Encounter with antigen activates macrophages to induce a Th1 response, but if this
antigen is complexed by antibodies, then it results in IL-4 induction
7
. Interestingly, gene
expression patterns in response to activation differ between mouse strains
32,132,149,185
.
Macrophages not only degrade antigens and produce cytokines locally, but they
can take up antigen in the periphery and then migrate to the lymph nodes where they
come in contact with T cells
19,156
. Which cell type takes up antigen depends on injection
site and type of antigen
12,51,144,156
. Injection of fluorescent microspheres i.p. for example
results in uptake mainly by macrophages, but intradermal injection results in uptake by
dendritic cells
144
.
Dendritic cells, as defined by high class II expression and stellate morphology, are
found in a number of organs, for example the heart, liver, thyroid, pancreas, skin, kidney,
ureter, and bladder, but not the brain
76
. Mouse dendritic cells express CD11c on their cell
surface and can be further classified by a variety of markers
130
. There are three major
dendritic cell subpopulations in the spleen
209
. Plasmacytoid dendritic cells, which are
characterized by expression of CD11c, Gr-1, and B220, but lack of CD11b
142
, produce
IFN-α when stimulated by viral antigen and their frequency varies depending on the
mouse strain
142
. Additionally, there are CD4
-
CD205
+
CD11b
-
CD8a
+
lymphoid and
CD205
-
CD11b
+
CD8a
-
myeloid cells, some of which express CD4
+80
. Pulsing purified
dendritic cells with antigen and transferring them into mice results in the production of
Th1 cytokines in recipients of lymphoid CD8α
+
DC, but Th2 cytokines predominate in
recipients of myeloid CD8α
-
dendritic cells
117
.
18
Dendritic cells are further subdivided into immature and mature cells. Despite
their name, immature dendritic cells have the potential to be important regulators in the
immune response. They can present self-peptides on MHC molecules, though these are
quickly internalized
173,216
and can produce IL-10 which limits their capacity to initiate a
Th1 response. Uptake of necrotic cells stimulates dendritic cell maturation, whereas
apoptotic cells do not
169
. This may be an important mechanism in limiting tissue
destruction during inflammation. Culturing human CD4 T cells with immature dendritic
cells results in IL-10-producing T
reg
, whereas culture with mature dendritic cells induces
an inflammatory T cell phenotype
94
. Inhibition of co-stimulatory molecules results in an
immature dendritic cells which can prevent diabetes when transferred to NOD mice
75
. T
cells activated by mature dendritic cells exhibit more blastogenesis, CD25 expression and
intracellular IL-4 then those activated by immature dendritic cells
187
. Immature dendritic
cells efficiently take up small, but not large antigen
12
and once they mature they
downregulate phagocytic activity
159
. Dendritic cells can be activated by stimuli such as
LPS, TNF-α and IL-1
30
which leads them to upregulate MHC
5,30,192
and co-stimulatory
molecules and produce IL-12
192
. This production is transient and later during activation
IL-10 is the predominant cytokine. This may lead to preferential Th1 induction early after
stimulation, whereas later interactions result in Th2 cells
108
. The location of dendritic
cells can also influence their propensity to induce a Th1 or Th2 response. Dendritic cells
in the Peyer’s Patches have increased MHC class II and decreased co-stimulatory
molecule expression compared to those in the mucosa and are more likely to prime Th2
cells
89
. Further complicating the issue is the effect of cytokines from dendritic cells
19
themselves, or other sources, which can also influence dendritic cell maturation. The
presence of IL-10 during in vivo maturation of dendritic cells for example, results in an
APC that is unable to stimulate T cell responses
55,220
, leading to reduced proliferation of
T cells and inhibition of inflammatory cytokine secretion
48,122
. TGF-β1 can also decrease
dendritic cell maturation
219
and transfer of antigen-pulsed IL-12-deficient dendritic cells
induces IL-4 and little IFN-γ in mice in contrast to transfer of wild type dendritic cells,
which induce little IL-4 and large amounts of IFN-γ
79
.
Both dendritic cells and macrophages are very plastic cell populations that have to
be able to adjust quickly to a changing environment to defend the body against invading
pathogens and at the same time protect it from excessive tissue damage or autoimmunity.
This makes it challenging to study the cell type in vitro as removal from
microenvironment and handling of the cells may be enough to change their phenotype.
This dissertation examines a potential role of gonadal hormones on T
reg
in SJL
mice and explores changes in phenotype and function in male and female mice.
Furthermore, regulation of APC activity by T
reg
in naïve mice is addressed. The
phenotype of male- and female-derived APC populations before and after antigen
encounter is investigated for measurable changes brought by T
reg
or hormones. Finally
male-derived T cells are used to elucidate the mechanism of disease suppression using
Th2 cells to suppress the Th1-mediated autoimmune disease EAE.
20
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43
Chapter 2
T
reg
control macrophage activity in naïve mice
Summary
Young male SJL mice do not mount a Th1 response after immunization with a
wide variety of antigens, whereas female SJL mice do. Transfer of APC from females or
older males can overcome this deficit. Depletion of CD25
+
regulatory T cells in male SJL
mice changes their APC phenotype to one that can induce an inflammatory response as
measured by IL-12 production in vitro and induction of a DTH response after APC
transfer into non-responder mice. Furthermore, immunization of male mice after CD25
depletion results in a shift towards a Th1 response evidenced by increased antigen
specific IFN- γ secretion from T cells as well as decreased IL-10 and IL-4 secretion. This
correlates with a 50% reduction in CD4
+
CD25
+
T cells in naïve female SJL mice
compared to young males, while in vitro T cell suppression and expression of T
reg
associated molecules such as GITR, CTLA-4, or CD103 is similar on T
reg
of male and
female mice, as is expression of the T
reg
-specific transcription factor FoxP3. These data
show that CD4
+
CD25
+
T
reg
are involved in the shift from Th1 to Th2 response in young
male SJL mice via regulation of macrophage activity.
44
Introduction
SJL mice react normally to many viral and bacterial infections
14,33
, despite the
fact that they have a variety of defects in the lymphocyte compartment, such as
deficiencies in CD5
+
B cells, NK cells, certain subsets of T cell receptor genes as well as
depressed serum IgE levels
33,62
. A gender- and age-dependent difference in the immune
responses in SJL mice has been reported. Young male SJL are unable to mount a DTH
response to a wide variety of antigens
34
. This defect is not observed in older male or in
female SJL mice
12,34
. Th1 unresponsiveness in males can be reversed by castration
63
or
by transfer of APC from female mice
12,34,35
. Blocking IL-10 in male mice before and
during T cell activation results in DTH responsiveness
11
and treatment of female mice
with recombinant IL-10 suppresses the DTH response
14
.
Phenotypic analysis implies the importance of an APC with properties of a
macrophage in the SJL model
12,35
. No difference in cell surface marker expression on the
APC has been described between male and female mice. The source of the APC can be
spleen, resident peritoneal cells or thioglycollate elicited peritoneal macrophages. Cell
transfer after immunization is ineffective, indicating a difference during T cell activation
rather then in the effector phase
50
. Also, co-culture experiments with APC and T cells
from male and female SJL mice demonstrate that the difference in immune phenotype is
dependent on the gender of the donor of the APC, not the T cell. In vitro activation of T
cells with APC from female mice results in secretion of IL-12. Culturing T cells with
APC from male mice on the other hand results in secretion of IL-10, but not IL-12. T
cell-independent stimulation of APC with LPS results in secretion of IL-12 by APC from
45
either gender, ruling out the possibility that male derived cells are simply incapable of
producing this cytokine and therefore Th1 deficient
63
.
The inability of male SJL mice to mount a Th1 response is not due to T cell
anergy, but rather due to a preferential induction of a Th2 type response. Th2 responses
do not result in a DTH reaction, but can be measured by determining the cytokine profile
of T cells harvested from immunized mice and cultured with antigen. This shows
preferential secretion of the Th2 cytokines IL-4 and IL-10 in cultures of T cells from
immunized male mice and secretion of the prototypic Th1 cytokine IFN-γ from cells of
their female counterparts
11
. The female phenotype also correlates with heightened
susceptibility to autoimmune disease, specifically EAE
5,12,13,15
.
The induction of Th1 versus Th2 response during infection or immunization is
determined by the cytokine environment during the initial T cell stimulation. In the
absence of IL-4, APC can produce IL-12, which leads to induction of a Th1 response
30
.
When antigenic stimulation of the T cell is accompanied by exposure to IL-4, T cells
acquire a Th2 phenotype
40
. The source of IL-4 is not clear. NKT cells have been
suggested as a source of IL-4
16,65
and depleting this cell type in young male SJL mice
results in the induction of a Th1 response
16
. T cells can also secrete IL-4 and stimulate
the induction of an antigen specific Th2 response
10,45
.
One subset of T cells which is present in naïve mice is the T
reg
subset. This cell
type can control potentially autoreactive T cells in the periphery. Natural T
reg
in naïve
mice express CD25 on the cell surface
46
and were first described as preventing
autoimmune disease of the gut resulting from adoptive transfer of CD4
+
CD25
-
cells from
46
BALB/c into athymic nude mice
2,46
. T
reg
do not proliferate well and can inhibit
proliferation of CD4
+
CD25
-
cells via an antigen-independent mechanism
17,54,57,58
. In vitro
this suppression is cell-cell contact dependent, but cytokine independent
54,57
. More
recently the transcription factor FoxP3 has been found to be expressed in T
reg
. Mice and
humans with a mutation in the Foxp3 gene succumb to a lethal autoimmune syndrome
and transfection of T cells with this molecule confers regulatory activity
19
. Several
surface markers are associated with CD25
+
T
reg
function or phenotype. These include
CTLA-4
18,23,25,39
, CD103
29
, GITR
28,48
and surface bound TGF-β
39
.
There is no evidence for the involvement of soluble factors such as IL-4, IL-10
and TGF-β in in vitro suppression by T
reg
57
. However, Th2 cytokines such as IL-4 and
IL-10 are important in limiting various autoimmune diseases such as EAE
1,6,7,51
. Also,
T
reg
secrete IL-10 in response to stimulation via their TCR in the presence of IL-2
4
and
T
reg
from IL-10 deficient mice are unable to protect mice from colitis
3
. On the other hand,
suppression of asthma is abrogated by anti-IL-10 antibody, but not when transferring IL-
10-deficient T
reg
26
. IL-10 is also involved in protection from autoimmune pneumonitis
22
.
T
reg
-derived IL-4 has not been shown to play a role in protection from autoimmunity,
though IL-4 deficient mice develop more severe disease
43
.
A number of studies have focused on the effect of APC on induction of regulatory
cells
20,24,61
or effect of cytokines on the capacity of the APC to induce T cells
responses
49,55,61,64
. However, these studies do not address what cell type might be
responsible for regulating APC activity before antigen encounter. T
reg
have been shown
to inhibit upregulation of co-stimulatory molecules on APC
21,37
as well as secretion of
47
inflammatory cytokines
21,36,53,59
in an IL-10 dependent manner
59
. APC cultured with
anergic T cells lose the ability to stimulate T cells
47
and T
reg
can inhibit Th1 responses
induced by adoptively transferred, antigen pulsed APC
42
.
The present study shows that T
reg
influence the gender-dependent lack of Th1
responsiveness in SJL mice in vivo and that depletion of CD25
+
cells before antigen
encounter changes the phenotype of the APC from a Th2 to a Th1 inducer.
Methods
Mice
Male SJL mice were obtained from The Jackson Laboratory (Bar Harbor, ME) at 4-5
weeks of age. Female SJL mice were obtained from the National Cancer Institute
(Frederick, MD) at 6 to 7 weeks of age. Animals were housed and maintained in the
University of Southern California vivarium. All procedures were performed in
compliance with the University of Southern California Keck School of Medicine
Institutional Animal Care and Use Committee approved protocols.
DTH response
Mice were immunized i.p. with 100 μg of endotoxin free KLH (Calbiochem, La Jolla,
CA) in 500 μl of Dulbecco’s Phosphate Buffered Saline (DPBS; Irvine Scientific, Santa
Ana, CA). Five days post immunization mice were challenged with 150 μg KLH in 25 μl
DPBS in the left hind footpad and an equal volume of DPBS in the right hind footpad.
48
DTH responses were determined 24 h post challenge by measuring the difference
between the thickness of the KLH and PBS challenged footpads with a Mitutoyo
micrometer (VWR Scientific, Cerritos, CA).
Flow cytometric analysis
Surface molecule staining was carried out as described in Chapter 4 using the following
antibodies: anti-CD4-FITC (mAb RM4-5), anti-CD4-PerCp (mAb L3T4), anti-CD25-
APC (mAb PC61), anti-GITR-FITC (mAb DTA-1), anti-CD25-PE (mAb PC61) and anti-
CD103-FITC (mAb M290) (all BD PharMingen, San Diego, CA). Intracellular staining
was performed for FoxP3 and CTLA-4. Following cell permeabilization in 1 ml Foxp3
Fixation/Permeabilization buffer (eBioscience, San Diego, CA) for 2h at 4°C and
blocking of non-specific antibody binding using 1 μl rat serum and 1 μg anti-
CD16/CD32 for 20 min at 4 °C, intracellular staining was performed using anti-FoxP3-
FITC or anti-FoxP3-PE (mAb FJK-16s; eBioscience), CTLA-4-PE (mAb UC10-4F10-
11; BD PharMingen), ratIgG2a-PE (isotype control; BD PharMingen), and ratIgG2a-
FITC (isotype control; BD PharMingen) for 20 min at 4 °C. Samples were washed in
permeabilization buffer (eBioscience), resuspended in FACS buffer and analyzed on
FACS Calibur (Becton Dickinson, San Jose, CA) using FlowJo (Tree Star, Inc, Ashland,
OR) software.
Adoptive transfers
APC activity was examined using peritoneal exudate cells (PEC) as a source of
49
APC
12,35,50
. PEC were induced by i.p. injection of 2 ml of thioglycollate broth (Difco,
Detroit, MI) and harvested from the peritoneal cavity by lavage with 7-10 ml of Joklik-
modified MEM (Gibco, Grand Island, NY) supplemented with 5 U/ml of heparin (Sigma,
St. Louis, MO) and 25 mM HEPES buffer, pH 7.2 72 h after induction. PEC, containing
< 7% Ly-6G
+
neutrophils and >90% CD11b
+
macrophages, were transferred i.p. at
5 x 10
5
to naïve males on the day of immunization. The effect of CD4
+
or CD25
+
cells on
APC activity was examined with APC derived from mice depleted of either cell
population via i.p. injection of 500 μl DPBS containing 250 μg of anti-CD4 (mAb
GK1.5) or 250 μg of anti-CD25 (mAb 7D4) on days -7, -4, and 0 relative to PEC
induction. Control mice received 500 μl PBS containing 1 mg of anti-β-galactosidase
(mAb GL113). Depletion of CD4
+
or CD25
+
cells was confirmed by flow cytometric
analysis of splenocytes.
Antigen specific cytokine release
Mice were immunized with 100 μg chicken FGG (Sigma) in 500 μl DPBS i.p. and
challenged with 50 μg of FGG in 25 μl PBS in the footpad 5 days later. Antibody was
administered i.p. at 500 μg anti-CD25 (mAb PC61) or anti-b-galactosidase (mAb GL113)
on day -2, 0, and 2 relative to immunization. Draining lymph nodes were removed 24 h
postchallenge and 1 x 10
6
cells were cultured for 72 h in 1 ml RPMI 1640 medium
supplemented with nonessential amino acids, sodium pyruvate, 2-ME (RPMI complete)
containing 10% prescreened FCS and 800 μg FGG. Supernatants were assayed for IFN- γ,
IL-4 and IL-10.
50
Cell purification
Splenic T cells were purified by differential adherence to nylon wool as previously
described
34,50,63
. Purity was assessed by staining with FITC-labeled anti-CD4 (mAb
RM4-5), PE-labeled anti-CD8 (mAb 53-6.7), and CyC-labeled anti-CD19 (mAb 1D3; all
BD PharMingen) and analyzed by flow cytometry. Nylon wool non-adherent fractions
were consistently ≤5% CD19
+
and ≥90% T cells.
In vitro T cell activation
Macrophage-dependent T cell activation was carried out as described
32
. Briefly, nylon
wool purified T cells and APC were cultured at 37 °C in RPMI complete and prescreened
10 % FCS at a concentration of 3 x 10
6
cells/well in 24-well plates at a ratio of 90% T
cells to 10 % APC. Cultures were activated by addition of 10 μg/ml anti-CD3 (mAb 145-
2C11; BD PharMingen). Cytokine secretion was measured in supernatants after an 18-20
h incubation at 37
0
C.
ELISA assay
Cytokine concentrations for IFN-γ, IL-4, and IL-10 were determined as described in
Chapter 4. IL-12 concentration was measured as previously described
11,12,14,63
. Briefly,
Immulon II plates were coated with 2 μg/ml of capture Ab (mAb C15.6) overnight at 4
0
C. Plates were then washed and blocked for 1-2 hours, followed by washing and addition
of samples and standard. Plates were incubated overnight at 4
0
C, washed, and secondary
biotinylated-Ab (mAb C17.8) was added for 1 h at RT. After washing, avidin-peroxidase
51
(Sigma) was added for 30 min at RT. Plates were developed using 2,2’-azino-bis3-
ethylbenzthiazoline-6-sulfonic acid and read at 405 nm using an auto plate reader (Biotek
Instruments, Winooski, VT). Concentrations were determined by constructing a standard
curve with recombinant IL-12. All antibodies and recombinant IL-12 were obtained from
PharMingen. Sensitivity was ~50 pg/ml.
CD4
+
CD25
+
T cell purification
CD4
+
CD25
+
lymph node cells were purified using the EasySep mouse CD4+ T cell
enrichment kit (StemCell technologies, Vancouver, CA). Lymphocytes were incubated
with 50 μl EasySep negative selection CD4
+
T cell enrichment cocktail, 100 μl Biotin
selection cocktail, and 50 μl magnetic nanoparticles per 1 x 10
8
cells in 3 consecutive 15
min incubations at 4 °C. Subsequently, the single cell solution was placed in a magnet for
5 min at room temperature (RT), followed by removal of the non-bound fraction. The
negatively selected CD4 T cells were then stained with anti-CD25-FITC and anti-CD4-Pe
and sorted by flow cytometry into CD4
+
CD25
-
effector cells and CD4
+
CD25
+
T
reg
using
FACS DiVa (Beckton Dickinson, Franklin Lakes, NJ).
T
reg
suppression assay
To assess CD4
+
CD25
+
T cell mediated suppression, 5 x 10
4
irradiated (2500 rad) CD4
depleted lymph node APC were co-cultured with an equal number of CD4
+
CD25
-
effector cells in 200 μl RPMI complete supplemented with 10 % prescreened FCS and 1
μg/ml anti-CD3. CD4
+
CD25
+
T
reg
were added at the indicated concentrations and cells
52
were cultured for 72 h at 37 °C and pulsed with [
3
H]TdR (ICN Radiochemicals, Irvine,
CA) at 1 μCi/well for the last 6 h of culture. Incorporation was measured by liquid
scintillation spectroscopy.
Real-time Polymerase Chain Reaction (PCR)
For RNA extraction CD4
+
CD25
-
and CD4
+
CD25
+
T cells were isolated from naïve mice
using CD4
+
CD25
+
regulatory T cell isolation kit (Miltenyi Biotec, Auburn, CA). RBC
depleted splenocytes (1 x 10
7
) were incubated with 10 μl Biotin antibody cocktail in 40 μl
PBS containing 0.5% BSA and 2mM EDTA (MACS buffer) for 15 min at 4 °C followed
by the addition of 20 μl anti-biotin magnetic beads, 5 μl anti-CD25-PE and 30 μl MACS
buffer. Following 20 min incubation at 4 °C the cell suspension was applied to a
depletion column and unbound CD4
+
cells were collected. CD4 cells were separated into
CD25
+
and CD25
-
fractions using positive selection following incubation with 90 μl
MACS buffer and 5 μl anti-PE magnetic beads at 4 °C for 20 min. Purity was routinely
≥ 95% for CD25
+
and ≥ 93% for CD25
-
CD4 cells. At least 5 x 10
5
cells were
homogenized in 1ml of TRIzol reagent (Invitrogen, Carlsbad, CA) and incubated 5 min at
RT followed by addition of 200 μl chloroform. After 15 min centrifugation at 12 000 x g,
the aqueous phase was harvested and total ribo nucleic acid (RNA) was precipitated
using 500 μl isopropyl alcohol and 5 μg RNase free glycogen (Invitrogen). After
incubation at RT for 10 min and 10 min centrifugation, samples were washed once using
75% ethanol and resuspended in 10 μl diethyl pyrocarbonate (DEPC; Sigma) treated
double distilled (dd)H
2
O. DNA digestion and reverse transcription were performed as
53
described in Chapter 4. FoxP3 mRNA expression, was quantitated using TaqMan Gene
expression assay for FoxP3 (Applied Biosystems, Foster City, CA) and GAPDH
(Applied Biosystems). Real time PCR was performed on the DNA Engine Opticon
system (MJ Research, Waltham, MA). FoxP3 expression levels were normalized to
GAPDH and converted to a linearized value using the following formula: expression unit
=1.8^
(Ct
ubiquitin
-Ct
gene x
)
x 10
5
. Samples were analyzed in duplicate with two or more mice
per group.
Statistical analysis
Statistical significance was determined by two-tailed Student’s t test. A value of P ≤ 0.05
was considered statistically significant.
Results
CD4
+
T cells influence APC activity in vivo
The presence of IL-4 during priming of naïve T cells by APC results in a Th2 type
response in mice
40
. One possible source of this cytokine are NK cells
65
and depletion of
that cell type results in a change in APC phenotype from Th2 to Th1 in male SJL mice
16
.
T cells can also produce IL-4 and to determine if CD4 T cells influence APC phenotype
before antigen encounter, PEC were used as a source of APC and transferred to young
male SJL mice. Male and female donor mice were injected with anti-CD4 mAb (GK1.5),
control antibody (GL113) specific for an irrelevant antigen, or PBS. FACS analysis
54
confirmed that GK1.5 mAb treatment eliminated > 98% of splenic CD4
+
T cells.
Following PEC harvest and transfer, recipient male SJL were immunized and challenged
with antigen 5 days later. DTH response was measured 24 h after challenge. Transfer of
APC from donor males treated with PBS only did not result in a DTH response in
immunized male recipients; however, depleting CD4
+
T cells in donor males changes the
phenotype of the APC to one capable of inducing a DTH response in recipient males.
Treatment with control antibody does not alter the APC phenotype. Furthermore,
depleting CD4
+
T cells in female donors does not result in a change of the ability of APC
to induce a DTH response in recipient males nor does treatment with control antibody
(Fig. 1). These data support a role for CD4
+
T cells in influencing the phenotype of APC
prior to antigen encounter in young male SJL.
55
Figure 1. APC derived from CD4 depleted male donors support Th1 induction.
Donor mice were treated with anti-CD4 (GK1.5), anti-β-gal (GL113) or PBS.
Thioglycollate elicited PEC (5x10
5
) were transferred to recipients on the day of
immunization. Mice were challenged 5 days postimmunization and DTH responses were
measured 24 h later. Representative of 4 experiments. *p ≤ 0.05 compared to male
control. Data kindly provided by Stephen A. Stohlman.
*
*
*
*
0 10 20 30 40
Male Anti-CD4
Male GL113
Female Anti-CD4
Female GL113
Naïve Female -
Naïve Male -
swelling (x 0.01mm)
APC Donor Treatment
*
*
*
*
56
In vivo depletion of CD4 T cells affects APC directly
To confirm that CD4 T cells inhibit the ability of APC to induce a Th1 response
by secreting IL-12, APC from antibody-treated mice were activated in a T cell-dependent
manner. APC activation in the presence of T cells, using either soluble anti-CD3 or
ConA, results in IL-10 secretion by APC derived from male SJL mice. By contrast,
identical conditions result in IL-12 secretion by APC derived from age-matched females.
This effect is dependent on the APC source not that of T cells
63
. Culture of APC from
CD4 T cell-depleted males with T cells results in a significant increase of IL-12
compared to APC from control antibody-treated male mice. IL-12 in the supernatant of
CD4-depleted males reaches a concentration similar to that induced by female derived
APC (Fig. 2). Culture of APC from male donors treated with control antibody results in
little IL-12 production, whereas the level of this cytokine is high in cultures containing
APC from control antibody treated females
63
. These data indicate that CD4 T cells inhibit
induction of a Th1 response in male SJL and that depletion of CD4 T cells changes the
phenotype of the APC stimulating T cells.
57
Figure 2. CD4 depletion before antigen encounter alters APC phenotype. Purified T
cells and PEC (ratio of 9:1) were cultured in 24 well plates at 3 x 10
6
cells/well. Cultures
were activated with 10 μg/ml anti-CD3. Supernatants from cultures containing male T
cells and APC from CD4 depleted or control treated male and female SJL were collected
after 18 h and IL-12 measured by ELISA. Representative of 3 separate experiments. *p ≤
0.01 compared to male control. Data kindly provided by Kenichi C. Dowdell.
APC Treatment
Male GL113
Female GL113
Male anti-CD4
Female anti-CD4
*
*
*
1.0 2.0 3.0 4.0
1
2
3
4
IL-12 (ng/ml)
58
CD4
+
CD25
+
regulatory T cells inhibit Th1 responses in male SJL mice
One subpopulation of CD4
+
T cells capable of suppressing immune responses are
T
reg
2,46
. To determine if Th1 responses in male SJL mice are suppressed by CD4
+
CD25
+
T
reg
, mice were treated with CD25 depleting (PC61) or control antibody (GL113) and
compared to female SJL. This treatment resulted in a more then 90% reduction of CD25
+
cells in naïve mice; however, when measured 5 days after immunization, the depletion
rate was only 50%. Culture of lymph node cells from female mice with their cognate
antigen revealed a predominance of IFN-γ and low levels IL-4 or IL-10 (Fig 3), in
agreement with previous studies which demonstrated the induction of a Th1 type immune
response in female SJL
11
. Cultures derived from control antibody-treated males on the
other hand showed reduced IFN-γ as well as an increase in IL-4 and IL-10 when
compared to levels in female derived cultures. Inhibiting the action of CD25
+
T
reg
cells
during T cell priming using anti-CD25 antibody treatment resulted in a shift towards a
Th1 phenotype in male SJL mice. Evaluation of culture supernatants showed an increase
in IFN-γ levels compared to control treated males as well as a significant decrease in IL-4
and a reduction in IL-10 levels (Fig. 3). These data suggest that CD25
+
cells contribute to
the maintenance of Th2 responsiveness in male SJL mice.
59
Figure 3. CD25 depletion before immunization results in suppression of Th2
polarization. Male SJL mice were treated with anti-CD25 (PC61) or anti-β-gal (GL113)
antibody and immunized with FGG. Mice were challenged 5 days postimmunization and
draining lymph nodes removed 24 h hours postchallenge. 3 x 10
6
lymph node cells were
cultured with 800 μg/ml FGG. Cytokines were measured by ELISA. Results are average
of at least 3 experiments with 2 mice per group. *p ≤ 0.01 compared to male control.
IL-4
*
IFN-γ
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
male female CD25 depleted
male
ng/ml
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
male female CD25 depleted
male
pg/ml
IL-10
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
male female CD25 depleted
male
ng/ml
60
The CD25
+
subset of CD4 T cells controls DTH responsiveness in male SJL mice
To examine if the effect of CD25 depletion is due to a change in APC phenotype,
age matched male and female SJL mice were depleted of CD25
+
cells resulting in
elimination of > 96% of CD25
+
cells in the spleen. APC were harvested from treated
mice and transferred into naïve male SJL, which were then immunized. The DTH
response was measured 24 h after antigen challenge. CD25 depletion in male mice before
APC harvest resulted in the induction of a DTH response in recipient male mice
comparable to that induced following transfer of female derived APC. Recipients of APC
from untreated mice exhibited the same lack of DTH responsiveness as evident in
untreated male mice, indicating that APC harvest and transfer in itself does not change
the APC phenotype. CD25 depletion in female mice does not alter the APC phenotype
(Fig. 4).
These data indicate that CD25
+
cells regulate naïve APC function in vivo before
antigen encounter.
61
Figure 4. APC derived from CD25 depleted male donors support Th1 induction.
Donors were treated with anti-CD25 (7D4), anti-β-gal (GL113) or PBS. APC (5 x 10
5
)
were transferred to recipients on the day of immunization. DTH responses were
measured 6 d later, 24 h after in vivo antigen challenge. Representative of 3 experiments.
Data kindly provided by Stephen A. Stohlman
510 15 20 25 30
*
*
*
*
swelling (x0.01mm)
Recipient APC Donor
Male None
Female None
Male Male
Male Female
Male αCD25 Male
Male αCD25 Female
62
The T
reg
population is increased in male compared to female SJL
The effect of CD25 depletion on APC function in vivo in male SJL and the lack of
an enhanced response in females indicates a gender-dependent difference in T
reg
in SJL
mice. FACS analysis of CD4
+
CD25
+
cells showed that the CD25
+
population in female
SJL constitutes ~ 5% of CD4
+
cells in the spleen (Fig. 5A). This is consistent with a
reduction of about 50% compared to other mouse strains such as C57BL/6
9
. Interestingly,
male SJL mice show a higher frequency of CD25
+
cells in the spleen compared to female
SJL to levels similar to other mouse strains (Fig. 5A). However, the CD25 molecule is
also expressed on activated T cells and the possibility exists that not all CD25
+
cells are
T
reg
. Therefore FoxP3 expression in CD4
+
CD25
+
T cells was assessed. Real Time PCR
did not show a significant difference in Foxp3 mRNA levels in purified CD4
+
CD25
+
T
cells. Foxp3 mRNA expression in the CD4
+
CD25
-
T cell population is negligible and
most likely due to low level contamination with CD25
+
cells (Fig. 5B). Analysis of
FoxP3 protein levels by flow cytometry confirmed the PCR data. Additionally, this
revealed that the majority of CD4
+
CD25
+
T cells in both male and female mice express
FoxP3 (Fig. 5C). Furthermore, no differential expression of the T
reg
associated surface
molecules CTLA-4, GITR, and CD103 was found comparing male and female SJL (Fig
5D). These data suggest that a higher frequency in CD4
+
CD25
+
T
reg
cells in males, rather
then a difference in the expression of FoxP3 or other T
reg
associated proteins, contributes
to suppression of Th1 responses in male SJL mice.
63
Figure 5. T
reg
are increased in male compared to female SJL. Splenocytes were
isolated from naïve mice and analyzed by flow cytometry. A) Cells were gated on CD4
and percentage of CD25
+
was determined. B) Splenocytes were purified and FoxP3
mRNA expression was measured in CD25
-
and CD25
+
fractions of CD4 T cells C)
Percent of FoxP3 positive cells within the CD4
+
CD25
+
population was assessed. D)
Expression of co-stimulatory molecules on CD4
+
CD25
+
FoxP3
+
cells. Data are average of
3 experiments with 2 mice per group. Error bars denote standard deviation. *p < 0.02
compared to male mice.
C D
*
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
male female
% of CD25
+
within CD4 cells
0
50000
100000
150000
200000
250000
CD4 CD25
relative expression
of FoxP3 mRNA
0.0
20.0
40.0
60.0
80.0
100.0
male female
%FoxP3
+
within CD4CD25
0.0
20.0
40.0
60.0
80.0
100.0
CTLA-4 GITR CD103
% of CD25
+
FoxP3
+
male
female
64
In vitro suppressive activity of T
reg
in SJL mice is not influenced by gender
To examine regulatory activity on a per cell basis, suppression of CD4
+
CD25
-
T
cell proliferation by CD4
+
CD25
+
T cells was assayed. No difference in suppressive
activity between male and female derived cells was found at any of the suppressor to
effector ratios (Fig. 6). These data indicate that the in vivo inhibition of Th1 induction by
APC in male SJL is an effect of the number of T
reg
cells present in the naïve mouse,
rather then being due to increased suppressive function on a per cell basis.
65
Figure 6. In vitro suppressive function of CD25 cells is gender independent.
CD4
+
CD25
+
cells from male and female mice were purified and assayed for their ability
to inhibit proliferation of CD4
+
CD25
-
cells. Data shown as percentage of suppression
compared to CD4
+
CD25
-
culture alone. Data are average of 2 experiments with at least 5
mice per group. Error bars denote standard deviation.
0
10
20
30
40
50
60
70
80
90
100
1:1 1:2 1:4 1:8 1:16 1:32
effector to T
reg
ratio
% suppression
male
female
66
Discussion
Numerous observations have been made about the effect of APC on T
cells
20,24,49,55,61,64
, but only a limited number of studies have addressed the possibility that
T cells can affect APC function. These studies have shown that T cells can reduce the
ability of APC to stimulate T cell responses
52,60
, possibly by inducing downregulation of
co-stimulatory molecules
8
.
The unique gender difference in the immune response to protein antigen in SJL
mice enables the study of the effect of T
reg
cells on APC in vivo before antigen encounter.
Transfer of APC from a DTH responder female to a non-responder male, followed by
immunization, results in a DTH response in males
12,34,35
. However, the non-responsive
male phenotype cannot be transferred to female mice via APC
34
. Furthermore, male APC
secrete IL-12 in response to LPS stimulation
63
, an indication that there is no inherent
defect in the APC leading to the inability to initiate the Th1 pathway. Taken together,
these observations indicate that the specific microenvironment in the male mouse
suppresses DTH responsiveness in vivo.
In SJL non-Th1 responsive APC from young male mice can be manipulated in
vivo and then transferred into a naïve male recipients, which are subsequently immunized
and assessed for DTH responsiveness in the footpad, as a measure for Th1 induction by
the APC
14
. Previous data suggested that the lack of a DTH response in male SJL is not
due to T cell anergy, but instead due to a diversion of the immune response from Th1 to
Th2
11
. Initial production of IL-12 by APC in response to pathogens via pattern
recognition receptors is important in inducing a Th1 response
30
, whereas the presence of
67
IL-4 during initial antigen presentation can lead to a Th2 response. However, the source
of IL-4 is unclear, though it has been proposed that both NK cells
16,65
or T cells could be
responsible
10,45
. Depletion of NK cells in male SJL leads to a change in phenotype of the
APC from a Th2 to a Th1 response inducing cell
16
, indicating a role for NK cells in
maintaining a Th2 environment. However, this does not exclude a role for T cells in
suppressing the Th1 response in male SJL. CD4 depletion in male SJL mice prior to APC
transfer into naïve male recipients results in DTH responsiveness. This supports that
CD4
+
T cells act on naïve APC in male SJL to inhibit the initiation of the Th1 pathway
when antigen is encountered. Study of T cell-dependent cytokine release in vitro further
demonstrates that this treatment results in the APC from male mice secreting IL-12 at
levels similar to those from female mice. In this culture system only the APC source
varies demonstrating an effect on the APC rather then other cell types.
An increase in IL-12 mRNA expression by APC can also be seen after anti-IL-10
treatment of male SJL before APC harvest and reciprocally treatment with recombinant
IL-10 leads to a suppression of DTH responsiveness in female mice
14
. IL-10 and TGF-β
downregulate APC function
41,60
and can influence the type of immune response initiated
following antigen stimulation of the APC
60
.
T
reg
function involves IL-10 in vivo and T
reg
are present in naïve mice, making this
CD4 T cell subset an interesting candidate to examine in SJL mice. Treatment of SJL
mice with anti-CD25 antibody results in depletion of more then 90% of CD25
+
cells from
the spleen of naïve mice. It has recently been suggested that this antibody treatment does
not physically deplete, but rather it may stimulate downregulation of CD25 on the cell
68
surface, functionally inactivating the cells in the process and resulting in blockage of
regulatory function
27
. Immunization of male SJL mice results in a T cell response which
displays Th2 phenotype and activates T cells, which upon restimulation induce the
production of IL-4 and IL-10. In female SJL, however, the same protocol results in
production of the Th1 cytokine IFN-γ
11
. CD25 depletion in male SJL skews the cytokine
profile of the T cell response from predominantly IL-4 and IL-10 and little IFN-γ toward
a more Th1 type response as evidenced by an increase in IFN-γ and decrease in IL-4 and
IL-10 production upon antigen stimulation. The observation that the phenotype in males
is not completely reversed to that seen in females may be due to the incomplete depletion
of CD25
+
cells in these experiments.
Anti-CD25 antibody treatment in male SJL results in APC that can initiate a DTH
response when transferred to naïve male mice. The same treatment in female SJL donors
does not change the scope of the DTH response after transfer to male SJL indicating that
there is a differential effect of the innate T
reg
population on APC in male and female mice
and that CD4
+
CD25
+
cells are involved in controlling Th1 responses in vivo. In vitro T
reg
activity is cell-cell contact dependent and not inhibited by antibody to various
cytokines
54,57
. The cytokine-independent action of T
reg
does not hold true in vivo,
however. There is evidence that IL-10 is important, but the source of the cytokine is
unclear
3,26
. Furthermore, TGF-β may be involved in T
reg
maintenance
31
and
function
22,38,44
, though these findings are controversial
43
. IL-10 and TGF-β have both
been shown to modulate APC function
41,60
and secretion of these cytokines by T
reg
offer a
plausible mechanism of action by which these cells may regulate APC activity.
69
The tendency of female SJL to mount a Th1 response as opposed to the Th2
responsiveness of male mice, may be due to a lack of T
reg
cells in female SJL mice or a
difference in suppressive activity. SJL have been described as having an approximately
50% reduction in CD25
+
cells compared to other mouse strains such as C57BL/6
9
.
However, this is true only for female SJL. Evaluation of the frequency of CD4
+
CD25
+
T
cells in the spleen reveals a two-fold increase of CD25
+
cells within the CD4 T cell
population in male mice compared to females. FoxP3 expression within the CD4
+
CD25
+
T cell population is similar in mRNA expression and translated protein. The majority of
CD25
+
cells in both male and female mice also express FoxP3, confirming their T
reg
phenotype
19
. This indicates that there is an overall reduction in CD25
+
FoxP3
+
CD4 T
cells in female SJL, rather then a loss of FoxP3 expression within the CD25
+
population
that could indicate a loss in suppressor function
19
.
Several cell surface markers, which are also expressed to varying degrees on
effector T cells, are involved in T
reg
function. CTLA-4 is important for both T
reg
development and function
18,23,39,56
and blockage of GITR not only blocks in vitro
suppression, but also exacerbates autoimmune disease
28,48
. CD103 is only expressed on a
small percentage of CD4
+
CD25
+
cells, but it has been shown to be a marker for a highly
suppressive T
reg
subset
29
. Expression of GITR, CTLA-4, and CD103 do not differ
between male and female derived CD4
+
CD25
+
cells indicating that the difference in T
reg
function is not due to the expansion of a more suppressive subset of T
reg
in male mice or
due to impaired function as a result of the lack of an effector molecule on T
reg
in female
mice.
70
Though in vitro suppression by T
reg
cells is most likely different from in vivo
mechanisms, inhibition of T cell proliferation has nonetheless been shown to correlate
with in vivo suppressive activity. T
reg
cells from female SJL are equally capable of
suppressing T cell proliferation as cells from male SJL mice.
These data indicate that the CD4
+
CD25
+
T
reg
subset of T cells influences APC
activity before antigen encounter in vivo. It demonstrates that depletion of CD4
+
T cells
changes the phenotype of the APC in male SJL mice, from a Th2
34
to a Th1 responder.
Furthermore these data show that this effect can be attributed to the CD25
+
subset and
that the same treatment has no effect on APC from female SJL mice. This indicates that
T
reg
can actively control APC function and that they inhibit the ability to mount a Th1
response in male, but not female SJL mice. Finally, this suppressive effect seems to be
highly dependent on the frequency of suppressor cells in an animal, rather then
suppressive activity on a per cell basis.
71
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+
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+
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62. Watanabe, N. and Ovary, Z., Suppression of IgE antibody production in SJL mice.
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78
Chapter 3
Gender-dependent changes in macrophage phenotype before and after
antigen encounter
Summary
The inability of young male SJL mice to mount a Th1 response to antigen can be
overcome by transfer of APC from Th1 responder mice such as female SJL. Additionally,
male-derived APC, when stimulated in culture in a T cell dependent manner, secrete IL-
10, whereas the stimulation of female derived APC results in the secretion of IL-12.
Examination of lymphocytes in spleen, lymph nodes, and the peritoneal cavity of naïve
SJL mice shows a two-fold increase in CD11c
+
dendritic cells in the spleen of male mice,
but no difference in F4/80
+
macrophages. Injection of fluorescently labeled antigen i.p.
reveals that most of the injected antigen is taken up by macrophages. Furthermore, there
is an increase in MHC class II expression on peritoneal macrophages of female SJL
compared to male mice after antigen uptake. This may be due to an inflammatory
environment including IFN- γ in the female mouse or the effect of IL-10 in the male.
These data show that there is no detectable difference between Th1 and Th2 inducing
macrophages in the SJL mouse before antigen encounter. However, the Th2 environment
in male mice and its effect on macrophages becomes evident after antigen uptake.
79
Introduction
Professional APC are important regulators of the immune response. These cells
not only express MHC molecules that enable them to present antigen to T cells, but also
have the ability to upregulate co-stimulatory molecules. This group of cells encompasses
B cells as well as macrophages and dendritic cells
55
. The latter two are part of the
mononuclear phagocyte family, defined by their ability to take up a large variety of
antigen by non-specific means as well as by receptor-mediated endocytosis
53
.
Presentation of antigen on the cell surface of APC allows T cells to recognize antigen and
the subsequent interaction between the two cell types directs the immune response to one
of several possible results such as a Th1 or Th2 response, T cell anergy or cell death. The
outcome also depends on several factors such as the cell type presenting the antigen. In
vitro only dendritic cells are effective at stimulating naïve T cells
46,57
, whereas
macrophages and B cells can only induce a response in primed T cells that have already
seen antigen presented by dendritic cells
20,24,26,54
. However, in vivo experiments have
shown that macrophages are also capable of inducing an immune response in naïve
mice
44
.
F4/80 is expressed exclusively on macrophages
22
and dendritic cells are defined
by CD11c in combination with other surface markers
6
. However, both macrophages and
dendritic cells can be further classified depending on their maturation and activation state
as well as their location. Both cell types are sensitive to stimulation with LPS and
upregulate MHC class II as well as other co-stimulatory molecules such as CD80 and
CD86
2,8,25
. Activated macrophages can also upregulate F4/80
46
.
80
Macrophage populations are divided into immature monocytes which express
CD11b, but little F4/80
51
and differentiated tissue macrophages such as Kuppfer cells in
the liver, alveolar macrophages in the lung or microglia in the nervous system
53
.
Different mouse strains have different propensities to react to antigen with a Th1 or Th2
response and it has been demonstrated that macrophages from Th1 responder strains are
more easily activated and stimulate T cells to make IFN-γ, whereas macrophages from
Th2 responder strains are not only less easily activated
42
, but also do not produce the
effector molecule nitric oxide (NO)
36
. However, no phenotypical differences between
macrophages that are Th1 or Th2 inducers have been described.
Dendritic cells have been extensively classified by their cell surface markers.
They can be divided into immature and mature dendritic cells depending on their
upregulation of MHC class II, CD80 and CD86 following stimulation. Immature
dendritic cells are more phagocytic than mature dendritic cells, but express only low
levels of CD80, CD86, and CD40. Antigen presentation by immature dendritic cells
typically results in anergy or IL-10 production by the T cell specific for the antigen.
Dendritic cells that have matured or been activated by inflammatory stimuli migrate to
the lymph nodes
23,59
.
In the mouse, lymphoid dendritic cells have been described. In addition to CD11c
these cells express high levels of CD8α and intermediate levels of CD11b. Myeloid
dendritic cells on the other hand do not express CD8α, but high levels of CD11b
16
.
Adoptive transfer experiments have shown that T cells isolated from mice that received
antigen pulsed CD8α
+
cells show increased proliferation and secrete IFN-γ. If CD8α
-
81
cells are transferred; however, it results in production of IL-10 as well as IL-4 and IL-5
30
.
Plasmacytoid dendritic cells in the mouse have a CD11c
lo
B220
hi
GR-1
lo
phenotype, are
weakly phagocytic and produce little IL-12. However, they produce large amounts of
IFN-α in response to virus infection
3,41
. Other factors influencing APC responses are
cytokine environment and maturation state, as well as hormones. IL-10 can inhibit
dendritic cell activation of T cells
14,19,29,34,63
and TGF-β is known to downregulate
macrophage activity
12,56
.
Multiple approaches have shown that APC activity is influenced by gonadal
hormones. Exposing dendritic cells to estrogen inhibits their ability to induce T cell
proliferation
27,62
. APC from male mice of various mouse strains are less efficient at
stimulating T cells than those from female mice, but castration of males enhances antigen
presentation
60
. Addition of estrogen- to hormone-free medium results in increased
CD11c
+
CD11b
int
dendritic cells in vitro
43
and estrogen treatment of females before
dendritic cell harvest results in APC with improved T cell stimulatory capacity
31
.
However, hormone levels are intricately balanced and interconnected in an organism and
are therefore difficult to study in a physiologically relevant setting.
SJL mice exhibit a unique gender and age specific defect in the APC population.
Young male mice are unable to mount a Th1 response to a variety of antigen. This is
apparent in the lack of a DTH response compared to older males or female mice
32,48
. The
defect can be overcome by the transfer of splenocytes, resident peritoneal cells, or
thioglycollate elicited PEC from older males. Cell transfer is only effective during the
first 24 h of immunization, indicating that hormones play a role during the activation
82
rather then effector phase. Antibody-mediated depletion of CD11b
+
, Mac-3 or MHC class
II
+
cells before transfer results in a population unable to induce a DTH response.
Removing adherent cells also removes the responsive cells, whereas T or B cell depletion
or irradiation did not have an effect
33,48
. This indicates that a macrophage is the cell type
most likely involved in the transfer of DTH responsiveness
47
, though there is no obvious
difference in cell surface markers on macrophages from thioglycollate induced male and
female SJL
10
. Macrophages express hormone receptors and it is therefore possible that
they are directly affected by hormones
28,35
. Alternatively, other hormone sensitive cell
types could assert their effect on the APC before antigen encounter. NK cell depletion
before antigen encounter results in DTH responsiveness of APC from male mice
17
as
does depletion of CD4
+
CD25
+
cells (Chapter 4). Transfer of APC from young males does
not result in suppression of the DTH response in female mice
10,32
. APC from young male
mice are capable of producing IL-12 in vitro in response to LPS
61
and splenocytes from
immunized male and female mice proliferate equally well to antigen
4,11
. Rather then an
immune deficiency, there is a difference in the type of reaction resulting in a Th2
response in male versus Th1 in female mice
61
. This has been observed in vivo
11
as well as
in vitro
4,9,13,61
and is dependent on the APC rather then T cell
61
.
The following data show that macrophage and dendritic cell populations do not
differ in phenotype between naïve male and female SJL mice. Furthermore, antigen
uptake is concentrated in F4/80
+
macrophages, not dendritic cells or F4/80
lo
monocytes.
Finally, upregulation of MHC class II due to macrophage activation after antigen uptake
is suppressed in male SJL mice.
83
Materials and Methods
Mice
Male SJL mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 5 weeks
of age. Female SJL mice were purchased from the National Cancer Institute (NCI,
Frederick, ME) at 6 weeks of age. Mice were housed locally. All experiments were
performed according to the Institutional Animal Care and Use Committee protocol of the
University of Southern California.
Immunization
Mice were injected i.p. with 100 μg FITC- or TexasRed- labeled Ovalbumin (OVA) or
DQ-OVA (Invitrogen, Carlsbad, CA), in 500 μl DPBS (Irvine Scientific, Santa Ana, CA).
Peritoneal cells were collected 20 h post injection.
Cell purification
Spleen and lymph node APC were extracted from tissue using collagenase digestion.
Organs were cut into small pieces using a razor blade and incubated for 30 min at 37°C in
5 ml prewarmed RPMI 1640 and 10% fetal calf serum (FCS) containing 1 mg/ml
collagenase type IV (Roche, Indianapolis, IN) and 0.1 mg/ml DNase 1 (Roche). EDTA
pH 7.2 was added to a final concentration of 0.01 M for an additional 5 min. Any
remaining tissue was dispersed using the frosted end of glass microscope slides.
Following digestion, remaining erythrocyte depleted cells were washed before being
resuspended at 3 x 10
7
cells/ ml in FACS buffer.
84
Resident peritoneal cells were harvested by injection of 7 ml of 5 Units/ml Heparin
(Sigma, St Louis, MO) in Versene. The animal was agitated before cell harvest.
Flow Cytometry
Staining for flow cytometry was performed as described in Chapter 4 using the following
monoclonal antibodies: anti-CD11b-PerCp (mAb M1/70), anti-CD11c-APC (mAb HL3),
anti-CD8α-Pe (mAb 53-6.7), anti-CD19-FITC (mAb 1D3), anti-CD19-Pe (mAb 1D3),
anti-Ly6G/Ly6C-Pe (mAb Gr-1 and RB6-8C5), anti-B220-FITC (mAb RA3-6B2), anti-
IA
P
-FITC (mAb 7-16.17), anti-Ly6G-Pe (mAb 1A8), anti-CD4-FITC (mAb L3T4; all
BD PharMingen, San Diego, CA), and F4/80-APC (mAb CI:A3-1; Serotec, Raleigh,
NC).
Results
Splenic and lymph node cell populations are similar in naïve mice.
Peripheral lymphoid organs such as the spleen and lymph nodes are sites of T cell
priming. APC travel to these organs after antigen encounter and uptake and present
antigen to naïve T cells
5,15
. Evaluation of density and size of cells isolated from naïve
spleen (Fig 7. A) and lymph nodes (Fig. 7 B) by flow cytometry shows that there is little
difference between male and female SJL mice.
Focusing on the live cell population (Fig. 8 A) and using F4/80 expression to
identify macrophages demonstrates that about 10% of splenocytes are of this cell type.
85
Analysis of forward and side scatter reveals a heterogeneous population that consists of a
group of small, very dense cells (Fig. 8 A broken line) and a second group, that contains
cells that vary widely in granularity and size (Fig. 8 A solid line). Ly6G
+
neutrophils are
a small proportion of splenocytes which are also granular and vary in size. Naïve
lymphocytes such as CD4
+
and CD8
+
T cells and CD19
+
B cells make up the majority of
splenocytes and closer examination of these cells confirms that they are almost
exclusively found in the low density portion of the cell analysis plot (Fig. 8B).
86
Figure 7. Cell populations isolated from spleen and lymph nodes do not vary in
naïve mice. Spleen (A) and lymph node (LN) (B) cells mice from male and female SJL
were isolated and analyzed for their size and density by flow cytometry forward (FSC)
and side scatter (SSC) respectively. Cell populations from individual mice did not differ
significantly. Data is representative of three experiments with two mice per group.
SSC
FSC
male
female
Mouse 1 Mouse 2
SSC
FSC
SSC
FSC
male
female
Mouse 1 Mouse 2
SSC
FSC
A
B
87
Figure 8. Macrophages, Neutrophils, and T and B cells differ in size and density.
Splenocytes from male and female SJL were isolated as in figure 1. Live cells were gated
on F4/80
+
macrophages and Ly6G
+
neutrophils (A) as well as CD8
+
T and CD19
+
B cells
(B) followed by forward and side scatter analysis of the individual cell populations.
SSC
FSC
SSC
FSC
13%
B
30%
CD8
CD19
SSC
FSC
SSC
FSC
SSC
FSC
8%
A
1%
F4/80
Ly6G
SSC
FSC
88
Comparison of neutrophil populations in the spleen revealed a two-fold increase in male
compared to female mice (Fig. 9). It is not clear how this difference may contribute to the
gender dependent difference in T cell activation as neutrophils do not express MHC class
II molecules and are therefore incapable of engaging the T cell receptor of CD4 T cells.
No difference can be found in the lymph nodes (Fig. 9), which may partially be due to the
small number of neutrophils in this organ.
89
Figure 9. Male SJL have increased neutrophils in the spleen compared to female
mice. Ly6G
+
percentage of live cells in spleen and lymph nodes were compared in male
and female SJL. Data is representative of three experiments with two mice per group.
F4/80
Ly6G
spleen
LN
male female
F4/80
Ly6G
1.1% 0.6%
0.3%
0.2%
90
Macrophages do not show a gender-dependent difference in phenotype
Th1 responsiveness of female SJL mice can be transferred to male mice by
adoptive transfer of macrophages, which have not encountered antigen or been activated
by other means. Therefore, the frequency of F4/80 expressing cells was analyzed in
lymph nodes and spleens. Both macrophages and monocytes express F4/80 albeit at
varying levels depending on their maturation state
1
. Monocytes express low F4/80 levels
(Fig. 10 broken circle) compared to macrophages, which have upregulated this molecule
(Fig. 10 white circle). There are few F4/80
+
cells in the lymph node of a naïve mouse and
no discernible F4/80
hi
population. Overall, the number of F4/80
+
cells in spleen or lymph
nodes does not differ significantly between male and female mice (Fig. 10). Furthermore,
expression of MHC class II was analyzed to explore the possibility of a difference in
antigen presenting capacity or activation state. This analysis showed similar MHC class
II expression in male and female SJL (Fig. 11). In a single experiment, expression of the
APC activation markers CD54, Mac-3, CD80, CD86, and ICOS L was analyzed and
showed no difference in splenocytes or lymph node cells between male and female SJL.
91
Figure 10. F4/80
+
macrophages are similar in male and female SJL. F4/80
+
macrophage populations within the live cell gate were compared in spleen and lymph
node of male and female SJL mice. Splenic monocytes/macrophages display F4/80
hi
(white circle) and F4/80
lo
(broken circle) expression. Data is representative of three
experiments with two mice per group.
F4/80
Ly6G
spleen
LN
male female
F4/80
Ly6G
5% 4%
1.1% 0.8%
92
Figure 11 MHC class II expression does not differ significantly between male and
female SJL mice. MHC class II expression on macrophages as identified in figure 4 was
compared between male and female SJL mice in spleen and lymph nodes. Data is
representative of three experiments with two mice per group.
spleen
LN
male female
MHC class II
68% 73%
68% 65%
MHC class II
93
Plasmacytoid dendritic cells
Despite the fact that plasmacytoid dendritic cells are well characterized as being
involved in anti-viral responses
3,41
, they are of interest due to their ability to secrete
significant amounts of IFN-α which can be involved in an inflammatory response.
Plasmacytoid dendritic cells are characterized by expression of CD11c, but not CD11b
and co-expression of Gr-1 and B220. Plasmacytoid dendritic cell numbers are similar in
male and female mice in the spleen (Fig. 12 A) as well as the lymph nodes (Fig. 12 B).
94
Figure 12. Plasmacytoid dendritic cells do not show gender dependent change in
phenotype. CD11c
+
dendritic cells within the live cell population were gated on
CD11b
lo
. Subsequently B220
+
Gr-1
+
plasmacytoid dendritic cell populations were
compared in male and female spleen (A) and lymph nodes (B). Data is representative of
three experiments with two mice per group.
GR-1
B220
B
CD11c
SSC
23%
18%
CD11b
male
CD11c
female
GR-1
GR-1
B220
A
CD11c
SSC
10%
9%
CD11b
male
CD11c
female
GR-1
B220
B220
95
CD11c
+
dendritic cells are increased in male compared to female SJL mice
Numerous dendritic cell populations have been described in humans and mice and
it is not entirely clear if these represent different lineages or merely diverse
differentiation or activation stages of the same cell type. Nonetheless, most dendritic cells
described share the expression of CD11c on the cell surface. Comparison of CD11c
+
cell
splenic cell populations revealed an almost 2-fold increase of dendritic cells in male
compared to female SJL. There was also a small, but reproducible increase of CD11c
+
cells in the lymph nodes, though this difference did not reach statistical significance (Fig.
13).
96
Figure 13. CD11c
+
dendritic cell populations are increased in male compared to
female SJL. Analysis of CD11c
+
dendritic cell populations was carried out on the CD19
-
portion of live splenocytes and lymph node cells of male and female SJL. Data is
representative of three experiments with two mice per group.
CD11c
CD11b
spleen
LN
male female
CD11c
CD11b
2.7% 1.6%
0.23% 0.18%
97
CD11c
+
cells can be further subdivided into lymphoid dendritic cells which are
CD8α
+
CD11b
lo
, and myeloid dendritic cells which are CD8α
-
CD11b
hi
. Myeloid dendritic
cells have been implicated in the induction of Th2 responses
30
, but comparison of this
cell type in SJL mice does not uncover a gender dependent difference in population size
in spleen or lymph node (Fig. 14). Transfer of antigen-pulsed CD8α
+
CD11b
lo
lymphoid
dendritic cells results in Th1 type T cells
30
, however, no change in this cell population
was evident in female SJL mice compared to males (Fig. 15). This shows that both
myeloid and lymphoid dendritic cell populations are 2-fold increased in male SJL mice
compared to females and that the increase in CD11c
+
cells is not due to the preferential
expansion of a single dendritic cell subset.
98
Figure 14. Gender does not influence the percentage of CD11c
+
CD11b
+
CD8
-
populations in SJL mice. Spleen and lymph node cells were gated on CD19
-
CD11c
hi
as
in figure 7 and CD11b
+
CD8
-
myeloid dendritic cell populations compared in male and
female mice. Data is representative of three experiments with two mice per group.
CD8α
CD11b
spleen
LN
male female
CD8α
CD11b
22% 29%
19% 19%
99
Figure 15. CD11c
+
CD11b
-
CD8
+
DC populations do not exhibit a gender dependent
change in phenotype. CD11b
lo
CD8
+
lymphoid dendritic cells within the CD19
-
CD11c
hi
populations from spleen and lymph node were compared in male and female SJL. Data is
representative of three experiments with two mice per group.
CD8α
CD11b
spleen
LN
male female
CD8α
CD11b
22%
28%
27%
17%
100
Resident peritoneal cells are similar in male and female SJL
Antigen presenting cells home from the site of antigen encounter to the
peripheral lymphoid organs where they can prime naïve T cells. Following i.p. injection,
the first cells encountering antigen are resident peritoneal cells. Analysis of this cell
population confirmed that a large proportion of peritoneal cells in the naïve mouse are
monocytes/macrophages as determined by their CD11b
hi/int
and MHC class II
+
phenotype.
No difference between male and female SJL was observed in size of this population (Fig.
16). As in the spleen, there was also an increase in Ly6G
+
neutrophils in the peritoneum
of male mice compared to females (Fig. 16), although the significance of this finding is
unclear. Analysis of myeloid and lymphoid dendritic cells found no gender dependent
difference in the CD8α
-
CD11b
hi
or CD8α
+
CD11b
lo
subsets (Fig. 17).
101
Figure 16. Macrophage numbers are similar in male and female peritoneum,
whereas neutrophils are increased in males. Resident peritoneal cells from male and
female mice were gated on the live cell population and F4/80 and Ly6G populations
analyzed.
Figure 17. CD8
+
and CD8
-
DC populations are similar in the peritoneum of male
and female SJL. Cells were isolated from naïve male and female SJL and the live cell
population was gated on CD19
-
CD11c
hi
and myeloid and lymphoid cell populations were
compared. Data are representative of three experiments with two mice per group.
male female
CD8
CD11c
52% 55%
2% 2%
Ly6G
male female
MHC
Class II
CD11b
56%
1.2%
0.6%
43%
MHC
Class II
102
Intraperitoneal administration of antigen results in uptake and retention by
peritoneal macrophages, but not dendritic cells
Previous data showed that Th1 responsiveness can be transferred to young male
SJL mice by transferring macrophages
33,48
. No difference between Th1 and Th2 inducing
macrophages is observed in naïve mice. Though there is an increase in CD11c
+
cells in
male SJL compared to female mice, it is only found in peripheral lymphoid organs and
not the peritoneum which is the site of antigen encounter with APC. To investigate if
antigen uptake triggers differential phenotypes in APC of male and female mice
fluorescent OVA was injected i.p. and APC analyzed 20 h postinjection. Antigen injected
i.p. can potentially be taken up by several resident peritoneal cells such as neutrophils,
macrophages and dendritic cells. Analysis of CD11c
+
dendritic cells after injection of
fluorescently labeled OVA shows that very few dendritic cells ingest i.p. administered
antigen (Fig. 18). This argues against a central role of dendritic cells in inducing a Th2
versus a Th1 response in male and female SJL mice, in agreement with the previous data
showing that adoptive transfer of macrophages
33,48
results in a Th1 phenotype.
103
Figure 18. Dendritic cells do not ingest OVA in the peritoneum. Peritoneal cells from
male and female mice 20 h after FITC-OVA injection were gated on live CD19
-
cells and
CD11c
hi
cells that have taken up OVA were compared. Data are representative of 5
experiments with at least 2 mice per group.
FSC
male
female
SSC
FSC
0.4%
1%
SSC
OVA
OVA
CD11c
CD11c
104
Injecting SJL mice i.p. with FITC-labeled OVA shows that antigen is found almost
exclusively in F4/80
hi
expressing cells (Fig. 19). Furthermore, macrophages from both
male and female SJL are capable of digesting protein antigen as demonstrated by the i.p.
administration of DQ-OVA. DQ-OVA is protein labeled with BODIPY dye resulting in
quenching of the fluorescence until hydrolysis to single dye-labeled peptides, for example
during antigen processing in APC. Injection of DQ-OVA and subsequent examination of
F4/80
+
cells demonstrates that both male and female derived macrophages are not only
able to phagocytose antigen, but also have the ability to cleave it proteolytically (Fig. 20),
an important step in antigen processing. Comparison of individual mice showed that
OVA uptake by macrophages is variable between animals, making it difficult to detect
gender-specific differences in uptake or digestion.
105
Figure 19. Injection of OVA i.p. results in varying degrees of uptake by
macrophages in individual mice. Cells were isolated from peritoneum 20 h after
injection of TexasRed-OVA. Live cells were analyzed by F4/80 expression and OVA
uptake. Data are representative of 4 experiments with 2 mice per group.
female
male
OVA
OVA
F4/80
F4/80
106
Figure 20. Uptake of OVA by peritoneal macrophages results in proteolytic cleavage
of the protein. Peritoneal cells were analyzed for F4/80 expression and cleaved DQ-
OVA 20 h after protein injection.
female
male
F4/80
F4/80
DQ-OVA
DQ-OVA
107
To eliminate the variability caused by protein uptake in individual mice, analysis
was concentrated on macrophages positive for fluorescent OVA. To assess the activation
state of these cells after protein uptake, expression of MHC class II was analyzed. All
macrophages express MHC class II after antigen uptake, but there is a reproducible
decrease in the percentage of male derived macrophages expressing high levels of MHC
class II compared to cells from female mice as well as a reduction in fluorescence
intensity (Fig. 21). MHC expression can be inhibited by anti-inflammatory cytokines
such as IL-10
37
and induced by IFN- γ
7,25
indicating an increased inflammatory
environment in female SJL compared to males.
108
Figure 21. MHC class II
+
expression is reduced on male derived macrophages after
OVA uptake. F4/80
+
cells that have taken up TexasRed OVA were gated on as shown in
figure 12 and MHC class II expression between male and female derived cells was
compared by percentage of MHC class II
hi
cells as well as mean fluorescence intensity
(MFI).
male female
MHC
Class II
F4/80
15% 21%
MFI:
320
MFI:
610
109
Discussion
Young male SJL mice do not mount a Th1 response after immunization with
antigen
32,48
. However, the defect can be overcome by transfer of APC from naïve female,
but not young male SJL
33,48
. This observation indicates a gender dependent difference in
the APC before antigen encounter precipitated either by direct action of gonadal
hormones or their influence on other hormone sensitive cells
17 and Chapter 4
.
Though initial studies did not reveal a difference in cell surface marker expression
in the thioglycollate induced macrophages
10
, renewed interest has been focused on the
role of different types of antigen presenting cells in inducing Th1 or Th2 responses,
warranting the reexamination of APC in male and female SJL mice. Transfer of APC
before antigen encounter demonstrates that the different phenotype is due to a difference
in the naïve APC and not due to influences during activation. Therefore it is reasonable to
assume that there is a difference in the phenotype of antigen presenting cell populations
in naïve male and female mice. Both lymph nodes and spleen are sites of antigen
presentation to naïve T cells by APC and are therefore of interest. Forward and side
scatter analysis by flow cytometry illustrates size and granularity of cells respectively.
This shows a difference in cell populations between lymph nodes and spleen, but no
heterogeneity between individual mice or mice of either gender. Closer investigation
reveals two F4/80
+
macrophage populations. One population contains small cells with
high side scatter, indicating very dense cytoplasm, whereas the other population is made
up of less dense cells with higher forward scatter, which identifies them as large cells.
Neutrophils also fall into the less dense, but relatively large side scatter region.
110
Lymphocyte analysis confirms that they are small and not granular in the resting state.
Macrophages can influence induction of Th1 or Th2 responses depending on their
activation state and cytokine production
12
and in SJL mice transfer of this cell type
directs Th1 responsiveness depending on the gender of the donor mouse
33,48
. Furthermore
macrophage activation in mouse strains that preferentially mount a Th1 response differs
from those in mice that mount Th2 responses. C57BL/6 mice exhibit Th1 responses
36
and
their macrophages are more easily activated in vitro then those derived from the BALB/c
mice
42
, which are more likely to react to antigen by mounting a Th2 response
36
. No
surface marker has been described that can be used to distinguish Th1 or Th2 inducing
macrophages. In agreement with this observation there is no difference in F4/80
+
populations between male and female SJL mice in either spleen or lymph nodes.
Furthermore, the activation state of those cells as measured by MHC class II expression
is similar. Macrophages are very sensitive to the action of pro- and anti-inflammatory
cytokines
7,29,38,49,50,56
and the similar expression profiles of F4/80 as well as MHC class II
in naïve SJL indicate that induction of Th2 responses in males versus Th1 responses in
females is not due to a difference in number or activation state of macrophages before
antigen encounter.
In contrast to macrophages, different subtypes of dendritic cells have been
identified that preferentially induce Th1 and Th2 responses and these can be
distinguished by differential expression of cell surface markers. Three major dendritic
cell populations have been defined, including plasmacytoid, lymphoid and myeloid
dendritic cells
3,21,39,41,58
though frequency of these cells vary considerably in different
111
mouse strains
39
. Plasmacytoid dendritic cells express CD11c as well as Gr-1 and B220,
but not CD11b and can produce type I IFN in response to viral stimuli
3,39,41
. Young male
SJL mice succumb to virus infection in the CNS whereas female mice do not
48
raising the
possibility that the defect in mounting a Th1 response is connected to this cell type.
However, data from naïve SJL mice show similar numbers of plasmacytoid dendritic
cells and therefore do not support this hypothesis, further correlating with the observation
of a broader defect in Th1 induction, rather then a virus specific deficit.
All three subtypes of dendritic cells express CD11c on their cell surface. Analysis
of cells expressing this molecule reveals an increase in CD11c
+
cells in the spleen of
male compared to female SJL mice prompting further investigation to determine if this is
due to a relative increase in myeloid or lymphoid dendritic cell subsets. Transfer of
antigen pulsed CD8α
+
CD11b
lo
lymphoid dendritic cells results in induction of Th1
response, whereas transfer of antigen pulsed CD8α
-
CD11b
hi
myeloid cells results in Th2
induction
30
. Although there is an overall increase in CD11c
+
cells in male mice, this is
not due to a predominance of either dendritic cell subset.
Peripheral lymphoid organs are the site of T cell-APC interaction and contain a
relatively large number of APC compared to other organs providing a large number of
cells to study. However, during antigen challenge, local or bone marrow-derived cells
take up antigen and then transports it to the lymph nodes or spleen rather then cells from
the secondary lymphoid organs migrating to the site of inflammation
5,45
. Considering
this, analysis of APC at the site of antigen deposition is critical. DTH responses are
induced in SJL mice by immunizing the mouse i.p.
32
. Therefore resident peritoneal cells
112
in the naïve mouse were analyzed. This also allows study of APC that have not come into
extensive contact with other cells of the immune system as is the case in the peripheral
lymphoid organs. Almost 50% of peritoneal cells are of the monocyte/macrophage
lineage as determined by CD11b
hi/int
and MHC class II
+
expression, but there is no
significant difference between male and female SJL mice. Myeloid and lymphoid
dendritic cell populations also do not show gender dependent changes in phenotype or
size.
Antigen uptake elicits a change in activation state in both macrophage and
dendritic cell APC. Fluorescently labeled protein allows analysis of only those cells that
have taken up antigen. Antigen uptake by phagocytes 20 h postinjection varies
considerably in individual mice possibly representing small variations in injection site
relative to various organs and to APC. This heterogeneity makes it difficult to assess a
gender dependent difference in antigen uptake. However, it does demonstrate that antigen
can be found almost exclusively in F4/80
hi
macrophages and it is also evident that
macrophages from both genders can process antigen to a similar degree. Comparing
MHC class II expression on macrophages that have taken up antigen reveals a small, but
highly reproducible increase of this molecule on the cell surface of macrophages derived
from female SJL mice. MHC molecules on macrophages are induced by inflammatory
stimuli such as IFN-γ
7,25
whereas IL-10 can downregulate their expression
37
. Reduced
expression indicates an anti-inflammatory cytokine environment in male SJL mice
evident after antigen dependent APC activation. These data also confirm that i.p.
injection of antigen results in uptake mostly by macrophages, not dendritic cells
18,40
. It is
113
unlikely that the lack of detectable antigen uptake by CD11c
+
cells is due to degradation
of the antigen as macrophages express a much higher level of lysosomal proteases then
dendritic cells
15,52
and the antigen is present in macrophages. Taken together these data
confirm the importance of macrophages in inducing Th1 or Th2 responses in SJL mice.
Furthermore it shows no difference in phenotype of Th1 or Th2 inducing macrophages
and underscores the difficulty of assessing inflammatory potential of resting or newly
activated APC.
114
Chapter 3 References:
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Perretti, M., Endogenous monocyte chemoattractant protein-1 recruits monocytes
in the zymosan peritonitis model. The Journal of Leukocyte Biology, 1998. 63(1):
p. 108-116.
2. Akira, S., Takeda, K., and Kaisho, T., Toll-like receptors: critical proteins linking
innate and acquired immunity. Nature Immunology, 2001. 2(8): p. 675-680.
3. Asselin-Paturel, C., Brizard, G., Pin, J.-J., Briere, F., and Trinchieri, G., Mouse
strain differences in plasmacytoid dendritic cell frequency and function revealed
by a novel monoclonal antibody. Journal of Immunology, 2003. 171(12): p. 6466-
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4. Bebo, B.F., Jr., Schuster, J.C., Vandenbark, A.A., and Offner, H., Androgens alter
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Journal of Immunology, 1999. 162(1): p. 35-40.
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nodes. Journal of Immunology, 1996. 157(6): p. 2577-2585.
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producing B220
+
CD11c
-
APC in mouse spleen. Journal of Immunology, 2004.
173(4): p. 2362-2372.
7. Cao, H., Wolff, R., Meltzer, M., and Crawford, R., Differential regulation of class
II MHC determinants on macrophages by IFN-γ and IL-4. Journal of
Immunology, 1989. 143(11): p. 3524-3531.
8. Cella, M., Engering, A., Pinet, V., Pieters, J., and Lanzavecchia, A., Inflammatory
stimuli induce accumulation of MHC class II complexes on dendritic cells.
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121
Chapter 4
Altered Neuroantigen-Specific Cytokine Secretion In a Th2
Environment Reduces Experimental Autoimmune Encephalomyelitis
Summary
Activation of Th2 cells suppresses clinical experimental autoimmune encephalitis
(EAE), demyelination and expression of genes associated with Th1 mediated
inflammation. Despite both reduced central nervous system inflammation and IFN-γ
induced MHC class II expression by microglia, the composition of CNS infiltrates in Th2
protected mice were similar to mice with EAE. Analysis of the CNS infiltrating cells by
flow cytometry suggests that protection did not correlate with abrogation of CD4
+
T cell
recruitment, preferential recruitment of donor Th2 cells or an increased frequency of
CD25
+
CD4
+
T cells. By contrast, protection correlated with an increased frequency of
neuroantigen-specific Th2 cells infiltrating the CNS. These data suggest that a peripheral
Th2 cytokine environment influences both potential antigen presenting cells as well as
recruitment and/or retention of neuroAg-specific Th2 CD4
+
T cells.
This chapter is published in the Journal of Neuroimmunology 178 (2006) pp 30-
39 with the following contributing authors: SJ Kirwin, KC Dowdell, C Hindinger, N
Feng, CC Bergmann, DR Hinton, SA Stohlman.
122
Introduction
Experimental autoimmune encephalomyelitis (EAE) is a CD4
+
T cell mediated
autoimmune disease of the central nervous system (CNS) associated with recruitment of
CD4
+
T cells and macrophages, activation of microglia and demyelination
3,7
. Based on
numerous common characteristics, EAE is studied as a model of the human CNS
demyelinating disease, multiple sclerosis (MS). EAE is induced either actively via
immunization with neuroantigen (neuroAg) or passively via the transfer of neuroAg-
specific Th1 cells
7,30,40,54
. Resistance to EAE and recovery from disease is regulated via
increased anti-inflammatory Th2 cytokines within the CNS secreted by Th2 cells, CD4
+
CD25
+
regulatory T cells and by B cells
21,27,34
. Consistent with the role of these cytokines
in recovery from acute EAE and their increased expression within the CNS
3,27
, mice
deficient in Th2 cytokines exhibit increased EAE severity
5,14,20
, suggesting a role for Th2
cytokines in both regulating EAE and as potential therapeutic approaches to MS.
Treatment of MS patients with Interferon (IFN)β-1b suppresses relapse frequency and
severity
26
and is associated with a shift in cytokine profile towards a Th2 type cytokine
response
6,36,43
. A reduction in number and severity of MS relapses following glatiramer
acetate treatment also corresponds with a shift toward anti-inflammatory Th2
responses
17,18,39
. These data suggest that immuno-modulatory approaches, especially
those resulting in a Th2 environment provide potential therapeutic strategies for MS.
In contrast to the association of Th2 cytokines with effective MS therapies,
attempts to ameliorate EAE via either directly activating Th2 cells or increasing the Th2
cytokine environment, have met with disparate outcomes. Th2 cells and Th2 associated
123
cytokines protect from EAE in some models and reduce subsequent relapses
15,41,46,48
.
However, efficacy appears to correlate with both the model, method of Th2 cell
activation and anatomical location of Th2 cytokine secretion. For example, co-transfer of
myelin basic protein (MBP)-specific Th2 cells with encephalitogenic MBP-specific Th1
cells protects from EAE in the absence of additional Ag induced activation
15
. These data
suggest that activation of Th2 cells within the CNS is protective and are consistent with
both the presence of interleukin (IL)-10 secreting B cells and suppressive effects of Th2
cytokine secretion within the CNS. Adjuvant induced activation of Th2 cells
19
and
transfer of non-neuroAg specific Th2 cells, followed by subsequent activation also
suppresses EAE
48
. Peripheral secretion of Th2 cytokines or inhibition of protection via
anti-IL-10 treatment coupled with the absence of mRNA encoding Th2 cytokines in the
CNS suggested suppression was mediated by peripheral events or possibly alterations in
antigen presenting cell (APC) activity. By contrast, Th2 cell lines generated in vitro,
especially those derived by expansion in the presence of Th1 inhibitors or via the addition
of Th2 cytokines, failed to provide protection
1,28,53
, even when the activating Ag was
delivered directly into the CNS
1
. Similar to Th2 cells, disparate results have also been
reported for direct administration of Th2 cytokines or expression mediated via gene
therapy approaches. IL-10 injection both prevents, and fails to prevent, EAE
8,42
. IL-4 was
not only unable to provide protection, but abrogated IL-10 mediated protection
38
.
Injection of an adenovirus vector expressing IL-10 was unable to suppress disease
compared to intracranial transplantation of fibroblasts expressing IL-10 in Biozzi mice
12
.
By contrast, intracranial, but not peripheral, injection of an adenovirus vector expressing
124
IL-10 suppressed both acute and relapse EAE
16
. These data indicate that an increased
understanding of Th2 mediated protection is warranted.
To explore the mechanism(s) of IL-10-dependent Th2 mediated protection in SJL
mice, the characteristics of the adoptively transferred populations, as well as the
inflammatory responses were examined. In contrast to Th1 activation following
immunization of EAE susceptible female SJL mice
2,13,15,33
, immunization of young adult
male SJL mice elicits a predominant Th2 response associated with reduced secretion of
IL-12, correlating with the lack of induction of delayed type hypersensitivity
responsiveness and resistance to active, but not passive EAE
33,47,51
. Consistent with
previous reports demonstrating protection from MBP-induced EAE
15,48
, simultaneous
transfer of proteolipid protein (PLP)-specific encephalitogenic Th1 cells and keyhole
limpet hemocyanin (KLH)-specific Th2 cells suppressed EAE. Although protected mice
exhibited no demyelination, flow cytometric analysis demonstrated only a modest
reduction in CNS inflammatory cell infiltration, with slightly reduced frequencies of
CD4
+
T cells in the CNS of the protected mice. Protection correlated with reduced
mRNA levels of pro-inflammatory cytokines within the CNS; however, neither enhanced
CNS recruitment of CD25
+
CD4
+
T cells nor prominent KLH-specific Th2 donor cell
recruitment into the CNS was detected. Analysis of cytokines secreted by CD4
+
T cells
recruited into the CNS of Th2 protected mice demonstrated a shift from encephalitogenic
PLP-specific cells secreting Th1 cytokines in unprotected mice to a population of PLP-
specific cells with a prominent Th2 secretion pattern. Consistent with the participation of
IL-10 in protection
48
, a Th2 environment within the CNS was supported by reduced
125
major histocompatibility complex (MHC) class II expression on microglia and infiltrating
monocytes. These data show that one mechanism of Th2 cytokine mediated protection is
to facilitate activation of T cells specific for an encephalitogenic Ag whose subsequent
activation suppresses EAE via secretion of Th2 cytokines.
Materials and Methods
Mice
Female SJL mice were purchased from The Jackson Laboratory (Bar Harbor, ME) or the
National Cancer Institute (Frederick, MD) at 6-8 weeks of age. Male SJL were obtained
at 4-5 weeks of age from The Jackson Laboratory. Breeder pairs of SJL Thy1.1 (CD90.1)
mice
29
were kindly provided by Dr William Karpus (Northwestern University, Chicago,
IL) and maintained locally. All procedures were performed in compliance with The
University of Southern California Keck School of Medicine Institutional Animal Care
and Use Committee approved protocols.
Immunizations
Female donors were immunized s.c. with 200 μl containing 1.5 mg/ml PLP
139-151
peptide
(Microchemical Core Facility, Keck School of Medicine, USC, Los Angeles, CA) in
Dulbecco’s phosphate buffered saline emulsified with an equal amount of incomplete
Freund’s adjuvant (Difco, Detroit, MI) supplemented with 4 mg/ml heat inactivated
Mycobacterium tuberculosis H37Ra (Difco) distributed into four sites on the flanks. Male
126
donors were immunized s.c. at 4 sites on the flanks with 200 μl containing 500 μg/ml
KLH (Calbiochem, La Jolla, CA) in alum (Accurate Chemical & Scientific Corp.,
Westbury, NY).
Adoptive transfer and challenge
Single cell suspensions were prepared from inguinal, axillary, and brachial lymph nodes
10 days after immunization of female donors and 6 days after immunization of male
donors. Cells derived from female donors were cultured at 4 x 10
6
cells/ml with 30 μg/ml
PLP
139-151
. Cells derived from male donors were cultured at 3 x 10
6
cells/ml with 50
μg/ml KLH. Both populations were expanded at 37 °C for 72 h in RPMI 1640
supplemented with 10% prescreened FBS, 2 mM L-Glutamine, nonessential amino acids,
sodium pyruvate, and 5 x 10
-5
M 2-ME. To induce passive EAE female mice received
2 x 10
7
PLP-specific cells i.p. For Th2 protection, female recipients received 2 x 10
7
PLP-specific and 3-4 x 10
7
KLH-specific cells i.p., followed by immunization with a total
volume of 200 μl containing 100 μg endotoxin free KLH in alum at two s.c. sites. As
previously reported
48
, KLH immunization alone did not alter the day of onset or mean
clinical EAE score of PLP-specific T cell recipients (data not shown). IL-10 was
inhibited in the Th2 protected group via a single i.p. injection of 500 μg anti-IL-10
receptor mAb 1B1.3 (kindly provided by Dr. Robert Coffman, DNAX Corp., Palo Alto,
CA) on the day of T cell transfer.
127
ELISA
Supernatants from in vitro activated cells were harvested after 72 h incubation and tested
for cytokine secretion by ELISA. 96-well plates (Dynex Technologies, Chantilly, VI)
were coated for 18 h at 4 °C with 100 μl containing 1.5 μg/ml of anti-IFN- γ (clone: R4-
6A2), anti-IL-4 (clone: BVD4-1D11) or anti-IL-10 (clone: JES5-2A5). Non-specific
binding was blocked with PBS containing 10% FBS. Samples and recombinant cytokines
(BD PharMingen, San Diego, CA) were adsorbed overnight at 4 °C. Color was developed
by sequential addition of 1.5 μg/ml biotinylated detection anti-IFN-γ (clone: XMG1.2), 1
μg/ml anti-IL-4 (clone: BVD6-2462) or 4 μg/ml anti-IL-10 (clone: JES5-16E3; BD
PharMingen) followed by avidin peroxidase and 1.8 mM 2,2’-azinobis[3-
ethylbenzthiazoline-6-sulfonic acid] diammonium salt (ABTS; Roche Diagnostics,
Indianapolis, IN) in 0.1 M anhydrous citric acid buffer, pH 4.35. Color was read at 405
nm using a Bio-Rad Model 680 microplate reader and analyzed using Microplate
Manager 5.2 software (Bio-Rad Laboratories, Hercules, CA).
Clinical Scores
Recipients were monitored daily for EAE clinical signs and graded as follows: 0, no
abnormality; 1, loss of tail tone; 2, paralysis of one hind limb; 3, total paralysis of both
hind limbs; 4, quadriplegia; and 5, moribund or dead.
128
FACS analysis of CNS inflammatory cells
CNS inflammatory cells were obtained from the brains of mice perfused with sterile PBS.
Brains were homogenized in ice-cold Tenbroeck tissue homogenizers and adjusted to
30% Percoll (Pharmacia, Upsalla, Sweden). Following centrifugation at 800 x g for 20
min at 4 °C cells were recovered from the 30%/70% Percoll interface and washed in
RPMI medium prior to further use. Non-specific binding was inhibited by 20 min
incubation at 4 °C with mouse Fc block CD16/CD32 (clone: 2.4G2) and a mixture of
10% normal goat, human, mouse and rat serums. Cells were stained with CD45-APC or
CD45-PerCp (both clone: 30-F11), CD4-FITC (clone: GK1.5), CD90.1/Thy1.1-PerCp
(clone: OX-7), CD25-PE (clone: 3c7), I-A
P
-FITC (clone: 7-16.17), CD8-FITC (clone:
53-6.7), CD19-PerCp (clone: 1D3), Ly6G-PE (clone: 1A8; all BD PharMingen), and
F4/80-PE or F4/80-APC (both clone: CI:A3-1; Serotec, Raleigh, NC). Samples were
analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) using
FlowJo (Tree Star, Inc, Ashland, OR) software.
Cytokine ELISPOT assay
Cultured cells from immunized mice and CNS derived cells were resuspended in RPMI
containing 10% FBS, and added to 96-well plates (Multiscreen Cellulose Ester
Membrane HA plate, Millipore Corp., Bedford, MA) pre-coated for 18 h at 4 °C with
10 μg/ml anti-IFN-γ (clone: R4-6A2) or anti-IL-4 (clone: BVD4-1D11) (BD
PharMingen). Spleen cells (5x10
6
cells/well) from naïve female SJL mice (irradiated at
2500 Rad) and PLP
139-151
peptide at a final concentration of 25 μM were added prior to
129
incubation for 36 h at 37 °C. After washing, plates were incubated with 5 μg/ml
biotinylated anti-IFN-γ (clone: XMG1.2) or anti-IL-4 (clone: BVD6-2462) (BD
PharMingen) for 18 h at 4 °C before adding avidin peroxidase (Sigma, St Louis, MO).
Following addition of 1.9 M 3,3’-Diaminobenzidine color developed for 5-10 min at
room temperature. Plates were washed with water, air dried and spots counted using a
dissecting microscope.
Histological evaluation
For histopathological evaluation spinal cords were fixed with Clark’s solution (75%
ethanol and 25% glacial acetic acid) and embedded in paraffin. Paraffin sections were
stained with luxol fast blue. Demyelination was scored in a blinded fashion on single
longitudinal sections from five separate experiments containing at least 3 mice per group
as previously described
15
. Representative fields were identified based on average scores
of all sections in each experimental group.
Gene expression analysis
Spinal cords were homogenized in 4 M guanidinium isothiocyanate, sheared with a
20 gauge needle and centrifuged overnight at 100,000 g at 4 °C through a 5.4 M CsCl
cushion. RNA was resuspended in H
2
O and 2 μg of RNA was treated for 10 min at
37 °C with 4 units DNAse I (Roche, Indianapolis, IN), in the presence of 12 units
RNAsin (Promega, Madison, WI). Samples were adjusted to a final concentration of
1mM EDTA, heated to 65 °C for 10 min and incubated 4 min at 70 ºC with 0.5 μg
130
oligo(dT)
15
primer (Promega). Reverse transcription was performed using AMV Reverse
Transcriptase (Promega). Semiquantitative real time PCR was performed on the DNA
Engine Opticon system (MJ Research, Waltham, MA) using SYBR green PCR master
mix. Primers for Ubiquitin, TGF-β1 (5’ CCC GAA GCG GAC TAC TAT GC, 3’ CGA
ATG TCT GAC GTA TTG AAG AAC A), TGF-β3 (5’ CAA TTA CTG CTT CCG CAA
CCT, 3’ AGC ACC GTG CTA TGG GTT GT), IL-10 (5’ TTT GAA TTC CCT GGG
TGA GAA, 3’ GCT CCA CTG CCT TGC TCT TAT T), IFN-γ (5’ TGA TGG CCT GAT
TGT CTT TCA A, 3’ GGA TAT CTG GAG GAA CTG GCA A), ICOS (5’ CCT CTG
CGA ACT CAC CAA GAC, 3’ GAT ATA GAC AGA GCA TTG GAT TCT TGA),
ICOS Ligand (ICOS L; 5’ TGC GTG TGG CAG CAA ACT, 3’ CTG GCC CGG GTT
GGA), TNF-α (5’GCC ACC ACG CTC TTC TGT CT, 3’ GGT CTG GGC CAT AGA
ACT GAT G), INOS (5’ CCG ATT TAG AGT CTT GGT GAA AGT G, 3’ TGA CCC
GTG AAG CCA TGA), and CD40 (5’ CCA CTG CAC AGC TCT TGA GAA G, 3’ TCC
ACT GGG CTG AGA ATT CG) were used at 2.5 μM. Expression levels were
normalized to ubiquitin mRNA and converted to a linearized value using the following
formula:
Unit =1.8^(Ct
ubiquitin
-Ct
gene x
) x 10
5
. Samples from individual mice were analyzed in
duplicate with three or more mice per group.
Statistical analysis
Statistical significance was determined by two-tailed Student’s t test. A value of P < 0.05
was considered statistically significant.
131
Results
Th2 Activation of Cells from Male SJL Mice
Adoptive transfer of in vitro activated Th2 cells derived from male SJL donors,
suppresses passive EAE in susceptible female SJL recipients
15,48
. To understand the
mechanism(s) of suppression, T cells specific for a CNS-irrelevant Ag (KLH) were
induced in young adult male SJL mice, activated in vitro and compared to similarly in
vitro activated PLP-specific T cells derived from age matched female SJL donors. PLP-
specific T cells secreted a preponderance of IFN-γ and minimal levels of the Th2
associated cytokines, IL-4 and IL-10 (Table 1). By contrast, KLH-specific T cells
derived from immunized male SJL donors secreted predominantly IL-10 and IL-4,
coupled with reduced IFN-γ secretion (Table 1), consistent with previous results
examining MBP-specific T cells activated in male and female SJL mice
48
. Frequency
analysis of the Ag-specific IFN-γ and IL-4 secreting T cells demonstrated that Ag-
specific Th1 as well as Th2 cytokine secreting cells were present in both populations.
Although ELISpot analysis showed that the majority of PLP-specific T cells secreted
IFN-γ, a small number (~8%) secreted IL-4. By contrast, activated T cells derived from
male SJL donors contained predominantly IL-4 secreting T cells, with a small frequency
(<10%) secreting IFN-γ. These data suggest that in vitro activated T cells from both
female and male derived populations contained T cells secreting pro- and anti-
inflammatory cytokines. However, the dominant phenotypes are consistent with
preferential induction of Th1 cells following immunization of female SJL mice and
132
preferential activation of T cells secreting Th2 cytokines following immunization of
young adult male SJL mice
15
.
133
Table I. Ag-specific cytokine release by cells derived from female and male SJL
mice.
Cytokine
1
Female
2
Male
3
IFN- γ 5865 (±475) 2301 (±469)
IL-10 290 (±35) 5151 (±258)
IL-4 32 (±22) 331 (±257)
1
cytokine release in pg/ml following 72 h incubation determined by ELISA. Results are
average of at least 3 experiments with standard deviation.
2
T cells from female SJL mice immunized with PLP
139-151
.
3
T cells from male SJL mice immunized with KLH.
134
Th2 Activation Reduces CNS inflammation
Adoptive transfer of PLP-specific Th1 cells induces EAE in naive female SJL
recipients (Table II). However, simultaneous transfer of KLH-specific Th2 cells derived
from male SJL mice, followed by immunization with KLH (PKI group), reduced the day
of onset, disease severity, peak clinical disease as well as cumulative disease scores
(Table II). The relative frequencies of Th1 and Th2 cells within the transferred
populations suggests that approximately equal numbers of Ag-specific Th1 and Th2 cells
were transferred into each recipient. Similar to the IL-10 dependent Th2 mediated
protection from MBP-induced EAE
15
, Th2 protection from PLP-induced EAE was also
reversed via IL-10 inhibition (Table II). The majority of the spinal cords derived from the
Th2 protected group exhibited no evidence of either demyelination or inflammatory cells.
However, small foci of demyelination and inflammatory cells were noted in
approximately 30% of spinal cords from the Th2 protected group. Reduced clinical
disease severity correlated with diminished demyelination in Th2 protected recipients
compared to the untreated group (Fig. 22).
135
Table II. Th2-mediated EAE protection.
Number
Tested
Incidence
Mean Day of
Onset
(±std dev)
Cumulative
Score
(±std dev)
Peak
Score
(±std dev)
PLP
1
45 93% 8.7 (±1.5) 8.9 (±5.6) 3.4 (±1.3)
PKI
2
53 45% 10.2 (±1.2)* 1.4 (±2.4)* 1.0 (±1.1)*
PKI +
αIL-10R
3
11 100% 8.3 (±0.9)
+
13.1 (±4.9)
+
4.1 (±0.5)
+
1
Group receiving PLP specific Th1 cells only.
Results are average of 8 experiments with at least 3 mice per group.
2
Mice receiving PLP specific Th1 cells as well as KLH specific Th2 cells and being
immunized with KLH.
Results are average of 8 experiments with at least 3 mice per group.
3
Mouse treatment as in PKI with the addition of anti-IL-10 Receptor blocking antibody
treatment.
Results are average of 3 experiments with at least 3 mice per group.
* denotes statistical significance in respect to PLP group (P ≤ 0.01)
+
denotes statistical significance in respect to PKI group (P ≤ 0.01)
136
Figure 22. Th2 environment prevents demyelination and inflammation. A. Spinal
cords from PLP mice with clinical EAE at the peak of disease (A) compared to PKI mice
protected (B) via Th2 activation. In A, plaque of demyelination and inflammation is
outlined with arrows. In B, a representative section from the Th2 protected group in
which the white matter (WM) appears normal. Paraffin sections stained with luxol fast
blue. Bar = 100 μ.
137
Consistent with protection from clinical disease, significant decreases in the relative
expression of mRNA encoding TGF-β1, ICOS, ICOS-L, TNF-α, and CD40 were
detected in protected mice (Fig. 23). Decreased IFN-γ and iNOS mRNA were also
detected; however, differences did not reach statistical significance. Surprisingly, no
difference in TGF-β3, suggested to correlate with disease protection
31
or IL-10 mRNA,
correlated with remission
27
, were detected comparing the two groups. Although Th2-
mediated protection is prevented via inhibition of IL-10 (Table I)
48
, the absence of IL-10
mRNA induction within the CNS during Th2 mediated protection suggests either an
absence of local IL-10 secretion, or alternatively that IL-10 mRNA levels do not reflect
IL-10 bio-availability
37
. In addition, no evidence for IL-10 secretion by microglia was
found in either group by flow cytometry (data not shown). Therefore, although cytokine
mRNA analysis is consistent with reduced clinical disease in the Th2 protected group, the
patterns of mRNA present do not directly support local Th2 cytokine mediated
protection.
138
Figure 23. Th2 protection decreases CNS pro-inflammatory gene expression. RNA
was extracted at the peak of clinical disease in PLP only recipients (day 12 post transfer)
and a parallel Th2 (PKI) protected group. Expression in relative units normalized to
ubiquitin mRNA. Data are the average of three experiments with at least 3 mice per
group. Error bars denote standard deviations. *P < 0.05 compared to PLP group.
0
500
1000
1500
2000
2500
3000
3500
TGFb1 TGFb3 I L -1 0 I FNg I COS I COS L TNFa iNOS CD40
relative expression
PLP
PKI
β β −γ −α
∗
∗
∗
∗
∗
139
To quantify differences in inflammatory cells associated with Th2 mediated
protection, CNS derived cells were analyzed by flow cytometry at the peak of clinical
disease in the PLP-only recipient group. Reduced clinical disease in the Th2 protected
group was associated with a ~40% reduction in CD45
hi
CNS inflammatory cells (Fig.
24A). In addition to reduced inflammatory cells, the frequency of CD4
+
T cells within the
inflammatory population was also slightly reduced (Fig. 24B). Phenotypic analysis of the
CD4
+
T cell populations demonstrated equivalent percentages expressing CD44 (PLP =
95%+ 6% vs. PKI = 94%+6%); CD62L (PLP = 24%+ 4% vs. PKI = 28%+1%) and ICOS
(PLP = 71%+ 8% vs. PKI = 80%+6%). The frequency of other cell types within the
inflammatory populations, i.e., CD8
+
T cells (PLP = 17%+ 11% vs. PKI = 15%+10%) ,
CD19
+
B cells (PLP = 8%+ 3% vs. PKI = 3%+3%), and Ly6G
+
neutrophils (PLP = 9%+
4% vs. PKI = 11%+8%) were not significantly different comparing the two groups. These
data suggest that protection was not due to specific abrogation of CD4
+
T cell
recruitment. IFN-γ dependent MHC class II expression on CD45
lo
microglia derived from
both groups was compared as a measure of the in situ Th1 response
4
. MHC class II was
expressed by ~85% of microglia derived from recipients of encephalitogenic T cells
(Fig. 24C), consistent with an acute Th1 mediated autoimmune disease. By contrast, only
~33% of microglia derived from the Th2 protected recipients expressed MHC class II
(Fig 24C). Infiltrating CD45
hi
cells also showed an overall reduction in MHC class II
expressing cells (Fig. 24C). The frequency of F4/80
+
macrophages contained within the
CD45
hi
inflammatory cells was also slightly reduced in the protected group compared to
the group with EAE (Fig. 25A). The mean fluorescence index for class II expression was
140
also diminished on macrophages derived from the Th2 protected group (PLP = 552+33
vs. PKI = 450+107). Consistent with the data in Figure 24C, analysis of the microglia
population based on a CD45
lo
F4/80
+
phenotype showed that only 13%+6% of CD45
lo
microglia expressed MHC class II (Fig. 25B) and the mean fluorescence index for class II
expression was also diminished (PLP = 213+128 vs. PKI = 126+69). Although only a
minor decrease in IFN-γ mRNA was detected in the protected mice, diminished class II
expression on both microglia and macrophages is consistent with IL-10 mediated
inhibition of MHC class II expression
37
.
141
Figure 24. Reduced CNS inflammation in Th2 protected mice. CNS inflammation
was examined by flow cytometry at the peak of clinical disease in the EAE (PLP) group
compared to the Th2 protected (PKI) group. The frequency of bone marrow derived
CD45
hi
cells within the CNS mononuclear cell population (A), the frequency of CD4
+
T
cells within the CD45
hi
(B) and the expression of MHC class II on CD45
hi
infiltrating
cells and CD45
lo
microglia (C). Data are representative of 4 experiments with at least
three mice per group.
PLP PKI
A
B
C
42%
25%
19% 14%
CD45
CD45
CD45
SSC
CD4
MHC class II
23 33
6 36
25 13
40 22
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
200
400
600
800 1000
10
1
10
2
10
3
10
4
200 400 600 800 1000
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
142
Figure 25. Decreased MHC class II expression on F4/80
+
macrophages and
microglia in Th2 protected mice. CNS derived cells were gated on CD45
hi
to examine
bone marrow derived cells and CD45
lo
to examine microglia. MHC class II expression
was analyzed on F4/80
+
macrophages within the CD45
hi
population (A) and the CD45
lo
microglia population(B). Data are representative of 2 experiments.
F4/80
MHC class II
A
PLP PKI
19%
B
F4/80
MHC class II
12% 25% 78% 72%
27% 50%
3%
68%
8% 13%
11%
2% 1% 10% 0%
CD45
hi
CD45
low
143
Recruitment of male derived Th2 cells and the frequency of CD25
+
T cells within
CD4
+
populations from the EAE and Th2 protected groups were also compared to
determine if reduced inflammation reflected a selective increase in recruitment of these
populations. To distinguish donor Th2 from donor Th1 cells, Th2 cells derived from SJL
CD90.1 donors were co-transferred with PLP-specific Th1 cells from CD90.2 donors into
CD90.2 recipients. Only ~10% of CD4
+
T cells in the CNS of the Th2 protected
recipients were derived from the CD90.1 donors (Fig 26A). Although these data
demonstrate that activated Th2 cells are recruited into the CNS, the low frequency within
the CD4
+
T cell population suggests that protection was not associated with the
preferential CNS recruitment/retention of donor derived Th2 cells. CD25
+
CD4
+
T cells
secreting IL-10 are associated with EAE remission
34
; however, the frequency of
CD4
+
CD25
+
T cells within the CNS of protected mice was slightly reduced compared to
the frequency detected in the CNS of encephalitogenic mice (Fig. 26B). Consistent with
the inability to detect increased CD4
+
CD25
+
T cells, no increase in FoxP3 mRNA was
detected within the CNS compared to the EAE group (data not shown).
144
Figure 26. Th2 and CD4
+
CD25
+
cells within the CNS of Th2 protected mice. Th2
infiltration into the CNS was examined via co-adoptive transfer of KLH activated T cells
derived from CD90.1 donors and PLP-specific T cells derived from CD90.2 donors into
CD90.2 recipients. Gated on CD4
+
T cells within the CD45
hi
population (A). Frequency
of CD25
+
cells within the CD4
+
T cell population. Gated on CD4
+
T cells within the
CD45
hi
population (B). Data are representative of 3 experiments.
PLP PK
I
A
B
2% 10%
18% 14%
CD90.1
CD4
CD4
CD25
82% 86%
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
10
1
10
2
10
3
10
4
145
Increased CNS PLP-specific Th2 cells
These data suggest reduced clinical disease, myelin loss, partially inhibited
inflammatory cell recruitment and microglia activation, do not correlate with preferential
recruitment of either donor derived Th2 cells or activation/recruitment of CD4
+
CD25
+
T
cells. Previous analysis of Th2 mediated EAE protection demonstrated that the
encephalitogenic neuroAg-specific T cells in the periphery were neither clonally deleted
nor anergic
48
. To examine the possibility of altered cytokine secretion by CNS infiltrating
PLP-specific T cells, the majority of which are derived from female donors (Fig. 26), the
frequencies of PLP-specific IFN-γ and IL-4 producing cells in the CNS were determined
at peak clinical disease in both groups. As expected
49
, a high frequency of PLP-specific,
potentially encephalitogenic, IFN-γ secreting cells were detected within the CNS of mice
with clinical EAE (Fig. 27). However, a considerable proportion (~20%) of CNS-derived
PLP-specific T cells secreted IL-4 (Fig. 27), consistent with minimal expression of IL-4
mRNA in the CNS of mice with acute EAE
3
. Although the total number of CD4
+
T cells
recruited into the CNS of the Th2 protected group was reduced compared to the EAE
group (Fig. 24), the frequency of PLP-specific IFN-γ secreting cells was significantly
reduced, coincident with an increase in the frequency of PLP-specific IL-4 secreting cells
(Fig. 27), constituting ~50% of the PLP-specific T cells. These data suggest that non-
neuroAg-specific Th2 mediated protection is associated with preferential recruitment
and/or expansion of PLP-specific Th2 cells.
146
Figure 27. Increased frequency of PLP-specific Th2 secreting cells within the CNS
of Th2 protected mice. Frequency of PLP-specific Th1 and Th2 cytokine secreting cells
within the CNS derived mononuclear cells isolated from mice with EAE (PLP) and Th2
protected (PKI) groups determined by ELISPOT. Data are represented as number of
cytokine secreting cells per brain and are representative of 4 experiments with at least 3
mice per group (P < 0.01).
500
1000
1500
2000
2500
3000
IFN-γ IL-4
spots per brain
PLP
PKI
*
*
147
Discussion
Induction of an anti-inflammatory Th2 environment, possibly resulting in immune
deviation, is an attractive therapeutic approach to control human autoimmune diseases
believed to be largely dependent upon self-reactive T cells secreting Th1 cytokines. In
addition, current MS therapeutic modalities are associated with an increased Th2
cytokine environment
6,17,18,36,39,43
. Both immunization with an adjuvant designed to
preferentially elicit Th2 cell activation and the transfer of Th2 cells protect against EAE
induced by homologous Ag
15,19,52
. Adoptive transfer of PLP-specific T cells engineered
to secrete IL-10 provides protection
32
, although Th2 cells polarized in vitro are less
efficient
28
. Similar to these observations, both immunization and transfer of Th2 cells
specific for a non-neuroAg also provide protection from heterologous Ag induced EAE;
however, these approaches required concomitant peripheral T cell activation and
protection mediated via the transferred Th2 cells was IL-10 dependent
20,48
. Transgenic
expression of IL-10 by either T cells or APC prevent induction of EAE
14,32
. Together,
these data suggest that in situ activation of Th2 cells, or preferential secretion of IL-10
during T cell activation, provides an environment sufficient to diminish Th1-mediated
CNS autoimmunity. However, it is not clear if IL-10, which has both pro- and anti-
inflammatory properties
23
, only suppresses CNS autoimmune disease when expressed
within the target tissue
16
. Although the mechanism(s) of protection are not clear, the
protection afforded via transfer of neuroAg specific Th2 suggests the possibility that Ag
presentation and T cell activation occur locally within the CNS
22
.
148
Protection from both clinical EAE and demyelination mediated by adoptive
transfer of non-neuroAg-specific Th2 cells demonstrated both an absolute requirement
for Th2 activation by cognate Ag and dependence upon IL-10
48
. However, whether IL-10
acts via inhibition of TNF-α, implicated in demyelination
45
, or via inhibition of Th1 cell
activation, either in the periphery or within the CNS due to effects on APC activity via
decreased IL-12 secretion and down regulation of MHC class II expression
37
, is unclear.
This report examines potential mechanisms for the ability of Th2 cells to protect from
clinical EAE and inhibit myelin loss. The observation that Th2 protected mice showed
little or no clinical disease or myelin loss suggested that Th2 mediated protection
significantly limited access of self reactive T cells into the CNS
19,48
. In contrast to these
data flow cytometric analysis suggests that protection from clinical disease is associated
with only moderate inhibition of T cell recruitment into the CNS. In addition, with the
exception of the recruitment of a small number of KLH-specific CD4
+
Th2 donor cells,
the composition of the infiltrating cells in both protected and mice with clinical EAE was
similar. Consistent with previous reports
19,48
, reduced clinical disease correlated with
reduced expression of mRNA encoding pro-inflammatory cytokines. Furthermore, MHC
class II expression on both the CNS infiltrating monocytes/macrophages and microglia
was reduced in the protected mice. Although it is not clear if CNS infiltrating monocytes
acquire MHC class II expression before or after entering the CNS, the reduced MHC
class II expression on microglia is consistent with a reduced IFN-γ Th1 cytokine
environment within the CNS of protected mice
4
. Although the possibility that APC which
acquired Ag in the periphery have trafficked into the CNS and provide a potential source
149
of Th2 activation cannot be ruled out, the limited number of donor derived Th2 cells
recovered from the CNS is consistent with the ability of activated T cells to enter the
CNS in the absence of cognate Ag
25
. However, if regulation of Th2 cytokine secretion is
under constraints similar to Th1 cytokine secretion
11
, it is likely that donor derived KLH-
specific Th2 cells are not secreting cytokines within the CNS due to the absence of TcR-
Ag-MHC class II complex interaction. Recent data has suggested that EAE remission is
associated with an increase in CD4
+
CD25
+
T regulatory cells secreting IL-10 within the
CNS
34
. However, no preferential recruitment of either recently activated T cells or T
regulatory cells, both of which express the IL-2Rα chain
44
was noted in the protected
group. Consistent with these flow cytometric data, preliminary analysis suggests the
absence of a preferential increase in FoxP3 mRNA in the spinal cords of the Th2
protected group (data not shown).
Analysis of the frequencies of neuroAg-specific Th1 and Th2 cells suggest that
Th2 mediated protection may be influenced locally within the CNS, with PLP-specific T
cells most likely the source of Th2 cytokines. T cells committed to either the Th1 or Th2
cytokine secretion pattern are fixed
10
, suggesting the minor Th2 population within the
donor cells, or de novo priming of endogenous Th2 cells, is responsible. In vitro analysis
suggested that Th1 cells are inhibited by IL-10 secreted by Th2 cells of the identical Ag
specificity
50
. However, recent in vivo data suggests that T cells committed to either
lineage continue to expand in the presence of the opposite cytokine environment
9
.
Consistent with these data, IL-10 dependent Th2-mediated protection does not result in
deletion or anergy of potentially encephalitogenic neuroAg-specific T cells
48
. These data
150
suggest that the Th2 environment does not prevent expansion of the adoptively
transferred PLP-specific T cells established by activation after recognition of cognate Ag.
Thus, the minor population of PLP-specific Th2 cells within the donor population may
expand preferentially in a Th2 cytokine environment, and provide the derivation of the
increased frequency of Th2 cells within the CNS of protected mice. Although it is not
clear whether autoAg-specific T cells are activated in a peripheral compartment or
directly within the CNS
35
, the relatively early and abundant PLP-specific Th2 cells within
the CNS favors recruitment of activated donor cells rather than priming of endogenous
cells. Whether the Th2 environment influences potential CNS APC directly, alters T cell
trafficking or preferential activation of neuroAg-specific cells in the Th2 favorable
cervical lymph node environment
24
is not clear. It also remains unclear how presentation
of cognate Ag by the limited number of MHC class II
+
macrophages and microglia within
the protected CNS affects CD4
+
T cell effector function. Taken together these data
suggest that a Th2 environment during induction of CNS autoimmunity influences the
overall recruitment of inflammatory cells, inhibits MHC class II expression by both the
recruited and endogenous CNS APC and facilitates recruitment of Th2 cells specific for
neuroAg. This concept is consistent with recent data suggesting that few potentially
encephalitogenic T cells are recruited into the inflamed CNS
49
and that a Th2 cytokine
environment allows preferential recruitment of neuroAg specific Th2 cells.
151
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Chapter 5
Discussion
Despite numerous abnormalities in the lymphocyte compartment
6,10,32,37,43,100,104
,
SJL mice respond normally to many infections
13,59,62,72,79
. An exception to this are young
male SJL mice, which exhibit a unique defect in mounting CD4 T cell mediated Th1
responses to a variety of antigens
61,87
. Instead, young males mount Th2 responses
22,101
.
This establishes a useful system to study the induction of inflammatory versus anti-
inflammatory immune responses in genetically identical mice, as well as allowing for the
study of the influence of hormones on autoimmune disease. It eliminates interfering
factors such as polymorphisms in genes related to the immune response, for example
MHC molecules. It also allows the study of the importance of specific cell types in
directing the immune response by transfer of specific cell populations from a Th1 to a
Th2 responder without tissue rejection
99
.
Adoptive transfer of macrophages from older male
62,86,87
or female mice
21
can
overcome the defect in young male SJL if transfer occurs before or during T cell
activation
87
, pointing to a change in antigen presentation rather then a deficiency in Th1
effector cell population. The regulation is hormone dependent and castration reverses the
Th2 phenotype
21,101
. Initially, these observations indicated that male gonadal hormones
inhibit the development of macrophages capable of inducing an inflammatory Th1
response. However, evaluation of thioglycollate induced macrophages showed no
difference in surface molecules of macrophages from male and female mice
24
and male
derived APC are capable of secreting IL-12 if stimulated with LPS
101
. LPS is a strong
158
activation signal which acts via TLRs rather then by MHC and T cell receptor
interaction
1
. Macrophages in male SJL are capable of mounting an inflammatory
response following stimulation of their TLR, but react in a Th2 type manner when
stimulated by a T cell dependent mechanism via MHC
21,61,87
. This indicates that there is
an active mechanism regulating macrophage responses to T cell mediated stimuli in male
mice. Depletion of NK cells in SJL changes the phenotype of macrophages from young
male mice to Th1 inducers
25
. NK cells have also been shown to be important in
suppressing autoimmune disease, specifically EAE. Their suppressive effect is important
during the induction as well as during the effector phase of EAE
105
. Suppression of
clinical symptoms during the acute disease stage is most likely due to NK mediated
killing of activated T cells
105
. This killing is regulated by a number of activating and
inhibitory receptors on NK cells that regulate their function
35,36,38
. Their suppressive
action during the induction phase on the other hand implies that there is an effect on
antigen presenting cells. However, no convincing mechanism has been described.
Cytokines such as IL-10 can downregulate the ability of macrophages to induce Th1
responses
41,63,68,91,98
, but in contrast to other mouse strains, NK cells from SJL mice do
not spontaneously produce cytokines therefore making it unlikely that this cell type is
solely responsible for the IL-10 dependent
20
suppression of Th1 response in naïve SJL
mice. Interestingly, activated male derived NK cells produce increased IL-10 compared
to female derived cells
25
and there is some evidence of bi-directional signals between NK
cells and APC
33
. In addition to NK cells, T
reg
are also necessary to maintain a Th2
environment in male SJL mice. T
reg
are present in naïve mice
82
and can inhibit T cell
159
proliferation in an antigen independent manner
27,92,94,95
. There is also evidence that T
reg
can act on APC directly and inhibit expression of co-stimulatory molecules
68
as well as
their ability to induce a Th1 response
63,73
. T
reg
are characterized by expression of CD4,
CD25 and FoxP3
31,40,82
. Depletion of T
reg
in male SJL results in a reduction in Th2
cytokines in response to immunization confirming their importance in inhibiting
inflammatory responses. Additionally, depletion of T
reg
in male mice followed by APC
transfer into untreated mice results in a Th1 response. This shows that T
reg
depletion
changes the phenotype of the APC before introduction of foreign antigen and indicates
that T
reg
are involved in maintaining homeostasis in naïve mice by controlling
macrophage phenotype, providing the first direct evidence that T
reg
control APC
responsiveness in vivo. Examination of T
reg
populations in male and female SJL mice
reveals a 50% increase in female derived CD4
+
CD25
+
FoxP3
+
T cells, but no difference in
expression of other T
reg
associated molecules such as CTLA-4
28,45,71,93
, GITR
52,83
, and
CD103
56
or in the ability to suppress T cell proliferation in vitro on a per cell basis.
Though one might expect that such a profound effect on the immune response is due to a
functional difference in T
reg
populations in male and female SJL, rather then simply a
reduction in numbers, it has been shown that differences in the size of T
reg
populations
between different mouse strains correlate with a propensity to mount Th1 or Th2
responses
14
. This observation indicates that T
reg
to APC ratio plays an important role in
regulating APC, a mechanism which is also observed in NK cell interactions with APC
35
.
Both NK cells and T
reg
influence APC to induce Th2 responses. This may be
facilitated by a direct effect of NK cells on T
reg
which in turn might be activated and act
160
on the APC or by the T
reg
acting directly on the NK cell inducing it to secrete IL-10 or
signaling to APC by cell contact or by one cell type binding to the APC and inducing it to
activate the other to in turn signal back to the APC. NK cells express a number of
inhibitory and activating receptors that can react with MHC molecules
35,36,38
. Though NK
receptors have been studied extensively in regard to the ability of NK cells to kill virus
infected or tumor cells
38
, they could also be involved in signaling between NK and target
cells such as APC. If NK cells and T
reg
interact directly with each other, then culturing of
the two cell types should result in either IL-10 production by NK or T
reg
which could be
determined by intracellular cytokine staining using flow cytometry. Alternatively, it
could alter the signaling of NK cell receptors or gene expression in either cell type. Th1
and Th2 associated transcription factors such as T-bet
90
and GATA
106
could be analyzed
or DNA microarray technique could be used to screen a large number of genes in both
cell types. Transwell experiments would further elucidate if this hypothetical interaction
is due to cell-cell contact or soluble factors. Alternatively, since NK cells in SJL mice do
not spontaneously secrete cytokines, but produce increased amounts of IL-10 compared
to females after stimulation
25
, binding of T
reg
to APC may induce a change in the latter
which in turn activates the NK cells by stimulation of its activating or loss of stimulation
of inhibitory receptors to secrete IL-10. This could include IL-10 secretion by NK cells
which can then act on the APC to inhibit its ability to induce inflammatory responses.
Pre-incubation of the APC with T
reg
and subsequent culture of APC and NK cells may
reveal if suppression of Th1 responses necessitates simultaneous binding of T
reg
and NK
cells to the APC or if T
reg
induce changes in the APC which subsequently stimulate NK
161
cells to produce IL-10. The addition of anti-IL-10 antibody during the pre-incubation or
culture phase can reveal during which step the cytokine performs its essential function.
To measure if there is a change in APC phenotype a functional assay such as TCR
mediated APC stimulation with subsequent assessment of IL-12 and IL-10 production
might be used
101
. To address which cell type is the source of IL-10 in vivo, one could
adoptively transfer wild type T
reg
, NK cells, or APC into male SJL mice deficient in IL-
10 and investigate if IL-10 secretion of a single cell type is enough to maintain Th2
responsiveness. T
reg
can also interact with the APC by cell contact via the T cell receptor
and MHC class II probably with the involvement of self-antigen in naïve mice. Both NK
cells and T
reg
can secrete IL-10 when activated
4,25
and it might be an additive effect of
both that maintains the Th2 phenotype of the macrophage. The effect of these accessory
cells may be enhanced by the action of hormones on other components of the immune
response or on the APC directly as macrophages express receptors for androgen
58
, a
hormone which can inhibit inflammation
5,58
.
Previous studies did not detect a difference in cell surface marker expression in
the transferred, thioglycollate elicited macrophage population in SJL
21
. This is in
agreement with findings in other mouse strains that are predominant Th1 or Th2 inducers
and whose macrophages show functional, but not phenotypic differences
66,74
. In contrast
to macrophages, dendritic cells have been classified into different subpopulations
according to expression of cell surface molecules and function
60
. Though previous studies
showed that transfer of macrophages is necessary to change the Th2 phenotype in male
SJL to Th1, there is increasing evidence that both macrophages and dendritic cells can
162
develop from the same precursor
64,96,102
. For these reasons, study of APC in naïve SJL
was undertaken and analysis of different dendritic cell subpopulations included. Though
there is an overall increase in CD11c
+
cells in SJL males, further study shows that this is
not due to a disproportionate increase in of Th2 inducing myeloid dendritic cells
60
in the
male or in Th1 inducing lymphoid dendritic cells
60
in female mice. No difference in
macrophage populations in spleen, lymph nodes, or peritoneum can be found in naïve
mice.
Injection of fluorescent OVA shows that protein is taken up almost exclusively in
macrophages, not dendritic cells. Despite earlier studies which proposed that only
dendritic cells can prime naïve T cells
85,97
it is now clear that macrophages can not only
ingest and degrade protein, which can lead to activation and cytokine release locally, but
are also capable of migrating to the lymph nodes
7,78
and priming naïve lymphocytes
75
.
The ability of macrophages to adapt quickly
55,89
to a changing microenvironment during
infection and ongoing immune responses is an invaluable asset in fighting pathogens
while limiting immune-pathology. However, it also makes it difficult to study this cell
population. The proportion of F4/80
+
cells which have taken up antigen varies widely
between individual mice, possibly due to small variations in injection site location.
Nonetheless, the data shows that macrophages in both male and female SJL mice ingest
antigen and are capable of digesting it. Furthermore, analysis of those macrophages that
have taken up antigen reveals that there is decreased expression of MHC class II on male
derived macrophages. The reduction may be due to the suppressive effect of IL-10 on
expression of this molecule in male mice
69
or due to increased expression in females
163
caused by IFN- γ
12,53
. Alternatively, this may be due to a direct effect of T
reg
on
macrophages either by cell contact or via the action of soluble mediators. Co-culture
experiments in which T
reg
are allowed direct contact with macrophage or are separated in
a transwell system might be used to address this question. One must however keep in
mind the discrepancies between in vivo and in vitro experiments using T
reg
, which act in a
cytokine independent way in suppressing T cell proliferation in vitro
92,94
, but are clearly
dependent on the action of cytokines in vivo
3,4,42,48
. A better understanding of the
mechanism of T
reg
action might be necessary before the answer can be found.
Despite the outward similarity in macrophage phenotype in male and female SJL,
this cell type reacts differently to antigen exposure depending on the gender of the host.
This may be due to an indirect effect of hormones on intracellular signaling pathways or
gene expression. It is difficult to study the effect of hormones in vitro as there are clearly
accessory cells involved in the differential phenotype and also due to the fact that
hormone levels are very tightly controlled and interdependent in an animal. On the other
hand, the speed with which macrophages can change their phenotype in response to
stimuli makes it challenging to study events that take place immediately after antigen
uptake.
Apart from studying differential initiation of Th1 and Th2 responses, the unique
induction of Th2 responses in male SJL mice versus Th1 responses in females can also be
useful in exploring new therapeutic approaches using Th2 cells to treat Th1 mediated
diseases, such as MS. EAE, a mouse model for MS, is characterized by inflammatory
cytokines in the CNS during acute disease
49
. Th2 cytokines are important during recovery
164
and animals deficient in them exhibit more severe disease then wild type animals
9,30
. In
human patients, successful therapies are associated with a shift of a Th1 to Th2 cytokine
profile in peripheral T cells
2,16,17,26,44,46,47,57,67,81
. The success of recombinant cytokine
therapy is dependent on frequent administration as well as precise timing and location of
administration making it a less then ideal treatment method
19,23,70,77,80,103
. In vitro
generated Th2 cells have not proven successful in treating EAE
50,54
. Male SJL mice are
an ideal source of T cells to study Th2 mediated suppression of EAE and transfer of these
cells at the same time as encephalitogenic T cells from female SJL results in disease
suppression
22
. This is true if the Th2 cells are of the same antigen specificity as the
disease causing cells
22
, but also if they recognize a non-self antigen
29,88
. However, in the
latter case they have to be activated by immunization of the recipient with their cognate
antigen
88
. The mechanism of protection is not clear. There are several different
possibilities that may be responsible for disease suppression. In untreated female SJL
transfer of encephalitogenic T cells results in disease about 5-7 days post transfer
51
. There
is little MHC class II expression in the naïve CNS
8,65
and it is therefore unlikely that
these T cells directly enter the CNS and cause inflammation, because CD4 T cell
activation and cytokine release requires interaction with its cognate antigen on MHC
molecules
18
. It has recently been demonstrated that encephalitogenic T cells first
recognize their antigen on CD11c
+
cells in the meninges and blood vessels
34
. This
interaction can initiate inflammation including IFN-γ secretion by the transferred Th1
cells. IFN-γ in turn initiates upregulation of MHC class II on microglia in the CNS
8
starting an inflammatory cascade. CNS infiltration is evident in Th1 recipients and mice
165
exhibit demyelination which results in clinical symptoms such as paralysis. Th2 protected
mice on the other hand show reduced infiltration in the white matter, very little
demyelination by histology and no clinical symptoms. Surprisingly, flow cytometric
analysis only shows about 50% reduction in infiltrating cells. The relatively high number
of infiltrating cells points either to accumulation of cells outside the white matter in the
CNS periphery or to a change in composition or phenotype in the infiltrate. There is no
evidence of a significant increase or decrease in any single cell type, but examination of
local as well as recruited APC shows a decrease in MHC class II expression in protected
mice indicating diminished presence of IFN-γ
8
and possibly an increase of Th2 cytokines
in the CNS
12,69
. More detailed analysis of Th2 cells in the CNS shows that this reduction
in inflammation is not due to accumulation of T
reg
nor due to preferential recruitment of
the transferred male derived Th2 cells. Rather, there is an increase in PLP specific T cells
secreting Th2 cytokine in the CNS concurrent with a decrease in IFN- γ secreting PLP
specific cells. This means that there is a reduction in encephalitogenic T cells compared
to control mice which show symptoms of EAE and CD4 T cells specific for neuroantigen
produce anti-inflammatory cytokines instead. Once murine T cells have differentiated
into Th1 or Th2 cells their cytokine patterns are fixed
15
thus making it unlikely that Th1
differentiated female derived PLP-specific cells secrete Th2 cytokines in the CNS. It is
possible that de novo priming of recipient T cells results in PLP specific Th2 cells.
However, the short time frame of disease induction and suppression even of early clinical
symptoms argues against this scenario. PLP specific Th0 cells contained in the
transferred population can differentiate into Th2 cells in the host. Alternatively, the minor
166
population of cells secreting Th2 cytokines within the transferred PLP specific cells may
be preferentially expanded. This is particularly plausible as only a small percentage of
transferred female derived cells actually secretes Th1 cytokines in culture indicating that
further expansion occurs after T cell transfer in the recipient.
The site of expansion of PLP specific Th2 cells is unclear. Despite the presence of
male derived T cells in the CNS, their role in suppressing disease in situ could not be
confirmed. Their number is relatively small compared to the female derived and
endogenous cells, but more importantly, they probably do not secrete cytokines in the
CNS, because of lack of their cognate antigen, KLH, at this site
18
. This indicates that they
migrate to the brain due to bystander activation
39
, but makes it very unlikely that these
male derived T cells produce significant amounts of cytokines in the CNS
18
. The PLP
specific Th2 population present in the CNS of protected mice most likely arises from the
expansion of a small population of transferred female derived cells due to preferential
expansion of Th2 cells in the CNS of protected mice. The transferred KLH specific Th2
cells produce Th2 cytokines in the lymph nodes in response to KLH being presented by
local APC. It is clear that encephalitogenic Th1 cells first migrate to the peripheral lymph
nodes before moving on to the brain
34
and it is also where the transferred KLH specific
Th2 cells interact with their cognate antigen. Even though there is evidence that PLP can
be expressed in tissues other than the nervous system
11,76,84
there is no indication that
large amounts of this antigen are presented to PLP specific T cells in the lymph node.
Though there is unlikely enough CNS antigen present in the peripheral lymph nodes to
allow significant expansion of PLP specific Th2 cells, it may be enough to allow
167
interaction of some of the cells with APC presenting PLP in a Th2 environment
stemming from the interaction of the male derived KLH specific cells with their cognate
antigen. KLH is transported to the lymph nodes by APC which take it up at the site of
injection and transfer it to the lymph nodes
7
which then allows for activation of the KLH
specific T cells likely resulting in the secretion of large amounts of IL-10. Some female
derived Th2 cells might be activated in this environment before migration to the CNS,
where they expand and inhibit inflammation. A scenario where the PLP specific Th2 cells
are affected by the cytokine environment elsewhere but in the lymph node initially is less
likely, unless cytokine induction by KLH specific Th2 cells is so strong that it affects
cells outside the lymph node in the blood or lymphatics during their migration to the
CNS. However, since inflammatory responses are usually extremely stringently
regulated, it is more likely that there are Th2 inducing APC presenting PLP to T cells in
the lymph nodes and giving the initial signal to allow skewing of the PLP specific
response in the CNS.
The data presented in this dissertation show that T
reg
play an important role in the
regulation of inflammatory reactions in SJL mice by exhibiting an effect on APC before
antigen encounter and that depletion of this cell type results in a shift from Th2 to Th1
responses in young male SJL mice. Furthermore, despite functional differences between
macrophages from naïve male and female SJL there is no evidence of a difference in size
of the populations or cell surface marker expression before antigen encounter in any of
the major APC populations investigated. However, downregulation of MHC class II after
antigen uptake in APC derived from male SJL is indicative of a Th2 environment after
168
APC activation. Finally, co-transfer of encephalitogenic female derived T cells and male
Th2 cells showed that Th2 cells can be a useful therapeutic approach to inflammatory
autoimmune disease. Furthermore, depression of clinical symptoms is due to a Th2
mediated, IL-10 dependent mechanism via the expansion or de novo induction of
neuroantigen specific Th2 cells rather then a complete inhibition of CNS infiltration or
induction of T
reg
.
169
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Appendix:
IFN- γ expression from DI-particles in MHV infection of the CNS
Summary:
IFN- γ plays an important role in some virus infections by controlling viral replication. In
MHV infection of the CNS, IFN- γ is necessary to clear infection of oligodendrocytes, but
it is not clear if it directly inhibits viral replication or acts by other mechanisms such as
recruiting inflammatory cells or stimulating the local production of soluble mediators.
Pseudorecombinant MHV containing defective interfering (DI) particles expressing
IFN- γ was used to determine correlation between in vitro and in vivo expression of
biologically active IFN- γ and to study the effect of IFN- γ expressing DI particles on virus
infection in the CNS. Infection of DBT cells confirms the production of large amounts of
IFN- γ by the pseudorecombinant virus in vitro, though this did not correlate with in vivo
anti-viral activity.
Introduction
The CNS microenvironment protects this vital organ against pathology by
physical barriers such as the skull and the blood brain barrier which impede entry of
foreign material and cells of the immune system into the CNS. Inflammation can occur in
the CNS if the barriers are breached, but even then it is tightly controlled to avoid
permanent damage to an organ with limited capacity to heal itself
12,13,15,22
. IFN-γ is a
208
central molecule in the inflammatory response in many virus infections. In IFN-γ-
deficient mice, the clinical course of herpes virus infection in the eye is similar to wild
type mice, but the virus persists in the absence of IFN- γ
6
. Measles virus clearance from
the brain is impaired in BALB/c mice treated with anti-IFN- γ antibody
10
and the same
treatment results in reduced cellular infiltration in the lung after influenza virus
infection
1
, though IFN- γ knockout mice showed no deficiency in influenza virus
clearance
11
. Studies using cytomegalovirus showed that IFN- γ by itself does not have
anti-viral activity, but it nonetheless acts during the effector phase without being involved
during the induction of the humoral or cellular immune response
19
.
Using i.c. MHV infection as a model for CNS demyelinating disease
9
, the
complicated role of IFN- γ in virus infections has been further explored. Studies confirm
the presence of IFN- γ mRNA in the CNS during MHV-JHM infection correlating with
the accumulation of T and NK cells and the peak of cytokine mRNA at the time of virus
clearance
25
. Furthermore it has been shown that CD8 derived IFN- γ
24
is important for the
control of MHV replication in oligodendroglia, but not neurons
3,26
.
Modified virus particles can be used to further characterize the direct action of
IFN- γ on virus replication and cellular infiltration in the CNS. MHV as well as other
viruses can contain DI particles
7
, which can be manipulated to express foreign genes
17
and can be incorporated into the virion
18
. The recombinant RNA is replicated as well as
transcribed and retained for several replication cycles, but eventually lost
18
. Proteins such
as IFN- γ are expressed from recombinant DI particles early during CNS infection
29
allowing for the study of the effects of IFN- γ before the influx of T cells which are the
209
source of IFN- γ during infection with wild type virus
24
. Initial studies showed that
transfection of MHV containing IFN- γ-encoding DI particles (MHV DI- γ) results in
production of IFN- γ, though the amount varies depending on the strain of MHV used,
with transfected A59 producing larger amounts of IFN- γ and earlier than JHM
29
. This
may be due to the fact that A59 and JHM variants require different signals for replication
of DI particles
20
. The two virus strains also differ in their virulence and JHM has
increased mortality compared to A59
8,23,27,28
. The INF- γ produced by the transfected virus
only has a small inhibitory effect on in vitro replication of the virus, but it may inhibit
spread to neighboring cells. Infection of a small number of C57BL/6 mice with MHV-
A59 DI- γ and control virus (MHV-A59 DI-x) showed a reduction in mortality as well as
viral antigen in the CNS, concurrent with an increase in cellular infiltration in the CNS in
A59 DI- γ infected mice
29
. The data in this appendix shows that DI- γ transfected MHV
particles produce IFN- γ in vitro, but i.c. infection of mice with MHV-JHM DI- γ does not
result in any measurable effect of IFN- γ on viral replication, inflammation, or gene
regulation in BALB/c mice.
Materials and Methods
Mice
BALB/c mice were purchased at 6-7 weeks of age from National Cancer Institute
(Bethesda, MD). BALB/c IFN- γ
0/0
mice were kindly provided by Robert Coffman
(DNAX Research Corporation,
Palo Alto, CA) and bred locally. Mice were housed and
210
maintained in the University of Southern California vivarium. All procedures were
performed in compliance with the University of Southern California Keck School of
Medicine Institutional Animal Care and Use Committee approved protocols.
Recombinant Virus
Mice were infected i.c. with 1000 plaque forming units (PFU) of a pseudorecombinant
variant of the 2.2v-1 strain of JHMV in 30 μl sterile DPBS. DI RNA of mouse hepatitis
virus was used as a vector to express murine IFN-γ as previously described
16
. Briefly, the
murine IFN-γ gene under the control of a coronavirus transcriptional promoter was
inserted into a pBluescript plasmid at the SpeI and EcoRI sites upstream of theT7
promoter of the DI mRNA. After liberalization of the plasmid, the RNA was transcribed
in vitro using T7 RNA polymerase (Promega, Madison, WI). DBT cells, from a mouse
astrocytoma cell line, were grown in 60 mm Petri dishes to approximately 70%
confluence and infected with JHMV 2.2v-1 at a multiplicity of infection (MOI) of 5.
Infected cells were transfected with in vitro transcribed RNA at 1 h post infection using
DMRIE-C lipofection reagent (GIBCO BRL, Gaithersburg, MD) according to
manufacturer’s instructions for transient transfection of adherent cells. The control virus
contained an identical vector containing a 5 base pair insertion after base pair 23 in the
IFN-γ gene disrupting expression and resulting in the lack of functional protein.
211
In vitro passage and ELISA
10
5
DBT cells were seeded overnight in a 24 well plate and infected with 200 μl of
MHV-JHM DI-γ or DI-x for 1 h at 37 °C. Cells were incubated overnight and
supernatants harvested and stored at -70 °C until assayed. The initial infection was
carried out an MOI between 32 and 40. Subsequent passages were carried out at an MOI
of 3 to 6. IFN- γ levels were analyzed by ELISA as described in Chapter 4.
Virus Titers
Virus was measured by plaque assay. DBT cells were grown to confluency on 100 mm
plates in MEM containing 10% Tryptose Phosphate Broth (TPB, Beckton Dickinson,
Franklin Lakes, NJ) supplemented with 7% Newborn Calf Serum (NBC) washed once
with MEM containing 10% TPB and infected with 200 μl supernatant from infected DBT
cells or brain homogenates. Plates were incubated at RT for 1.5 h and rocked every 15
min. 10 ml of 6% SeaKem ME Agarose (BMA, Rockland, ME) in MEM + 10% TPB was
added and plates were incubated 72 h at 37 °C. Plaques were then manually counted.
Clinical Scores
Mice were scored for clinical disease as follows: 0: healthy; 1: hunched back; 2: partial
hind limb paralysis or inability to return to upright position; 3: complete hind limb
paralysis; 4: moribund or dead.
212
Flow cytometry
Flow cytometry was performed as described in Chapter 4 using the following antibodies:
anti-CD45-CyC (mAb 30-F11), anti-Ly6G/Ly6C-FITC (mAb Gr-1), anti-CD4-FITC
(mAb L3T4), anti-CD8-Pe (mAb Ly-2) anti-NK1.1-Pe (mAb PK136) and I-A/I-E – FITC
(mAb 2G9; all BD PharMingen).
Reverse transcription and real time PCR
Reverse transcription and real time PCR were performed as described in Chapter 4 using
primers for Ubiquitin (5’ TGG CTA TTA ATT ATT CGG TCT GCA T, 3’ GCA AGT
GGC TAG AGT GCA GAG TAA), CD4 promoter (5’ TTT GTG GTT CCC AGC GTA
TAA GT, 3’AGC TGG CTT GCC CTT TAA GG), MHC class II (5’ TCA ACA TCA
CAT GGC TCA GAA AT, 3’ AGA CAG CTT GTG GAA GGA ATG G), IFN- γ (5’
TGA TGG CCT GAT TGT CTT TCA A, 3’ GGA TAT CTG GAG GAA CTG GCA A),
Inducible Nictric Oxide Synthetase (iNOS; 5’ CCG ATT TAG AGT CTT GGT GAA
AGT G, 3’ TGA CCC GTG AAG CCA TGA), Monocyte Chemoattractant Protein
(MCP)-1 (5’ GCT GGA GCA TCC ACG TGT T, 3’ ATC TTG CTG GTG AAT GAG
TAG CA), Regulated Upon activation, Normal T Cell Expressed And Secreted
(RANTES; 5’ GCA AGT GCT CCA ATC TTG CA, 3’ CTT CTC TGG GTT GGC ACA
CA) , Gamma Interferon Inducible Protein (IP)-10 (5’ GAC GGT CCG CTG CAA CTG,
3’ GCT TCC CTA TGG CCC TCA TT), Monokine Induced By Gamma Interferon
(MIG; 5’ TGC ACG ATG CTC CTG CA, 3’ AGG TCT TTG AGG GAT TTG TAG
TGG) and 2’-5’ Oligoadenylate Synthetase (2’-5’ OAS; 5’ AAA AAT GTC TGC TTC
213
TTG AAT TCT GA, 3’ TGT GCC TTT GGC AGT GGA T). Samples were tested for
DNA contamination by amplification of CD4 promoter sequence. Expression levels for
all genes were normalized to ubiquitin expression using the following formula: Unit
= 100000 * 8 . 1
int erest geneof ubiquitin−
.
Results
Cells infected with DI- γ transfected MHV-JHM secrete IFN- γ in vitro.
Pseudorecombinant MHV virus can be constructed to secrete foreign proteins
from infected cells
29
. To achieve this, cells are infected with MHV-JHM and transfected
with DI RNA which results in packaging of DI RNA into virions. Infection of the same
cells with wild type and pseudorecombinant virus then leads to expression of genes
encoded within the DI sequence. Transfection of infected DBT cells with DI- γ RNA
leads to expression of the DI gene and therefore to secretion of IFN- γ in vitro (Fig. 28).
DI particles are lost over several infections cycles
18
and the demonstration that IFN- γ
production is reduced six-fold between the first and third passages (Fig. 28), confirms
this observation. The loss of expression also correlates with an increase in virus release
with each consecutive passage
7
(Fig.29). The dip in virus titers after the second passage
in MHV-JHM DI- γ infected cells, but not control virus (MHV-JHM DI-x) infected cells
may be due to the relatively high amount of IFN- γ present in the supernatant as well as
low multiples of infection. The negative effect on viral replication is not visible after the
first passage, since it is the result of an infection at a high MOI, which is less susceptible
214
to the anti-viral effects of IFN- γ
29
. By the third passage the concentration of IFN- γ in the
supernatant was too low to affect viral replication. Transfection of MHV infected DBT
cells with DI particles does not result in reduced virus titers, though subsequent virus
replication is inhibited by the presence of DI- γ or DI-x particles (Fig. 29).
215
Figure 28. Transfection of JHM-MHV using DI particles encoding IFN-γ results in
cytokine production in vitro. The murine IFN- γ gene was inserted into a pBluescript
plasmid upstream of a MHV intergenic sequence under the control of a T7 promoter. In
vitro transcripts were transfected into DBT cells infected with MHV(passage o). DBT
cells were infected for 8 h and supernatants collected (passage 1) and used for infection
of a second set of DBT cells (passage 2). Supernatants from passage 2 where then used to
infect a third set of cells (passage 3). IFN- γ concentrations in the supernatants were
measured by ELISA. Data are representative of two independent experiments.
γ
100
200
300
400
500
600
0 1 2 3
ng/ ml IFN- γ
Passage
216
Figure 29. Virus titer increase correlates with loss of IFN- γ secretion. DBT cells were
infected with MHV DI- γ as described in Figure 1 and the number of infectious virus
particles was determined by plaque assay. Data is representative of two independent
experiments.
5
10
15
20
DI- γ DI-x
pfu/ml x 10
6
passage 0
passage 1
passage 2
passage 3
217
MHV-JHM DI- γ infection does not alter clinical symptoms or virus replication in
vivo compared to control virus infection.
BALB/c mice infected with control MHV-JHM DI-x virus i.c. develop clinical
symptoms with similar kinetics to wild type MHV-JHM infection
21
. Infection with MHV-
JHM DI- γ infection resulted in a moderate reduction in clinical scores but the difference
did not reach statistical significance (Fig. 30). No difference in virus replication in CNS
tissue of MHV-JHM DI- γ compared to control virus infected mice was found(Fig. 31),
indicating the absence of biologically active IFN- γ in vivo.
218
Figure 30. Clinical symptoms do not differ significantly between mice infected with
DI- γ and control virus. BALB/c mice were infected i.c. with 1000 pfu of MHV-JHM
DI- γ or MHV-JHM DI-x control. Mice were examined for clinical symptoms daily. Data
are average of 3 experiments with 3 mice per group.
1
2
3
4
12 3 45 6 7 8
DI- γ
DI-x
days postinfection
clinical score
219
Figure 31. Virus titers are similar in MHV-JHM DI- γ and control virus infected
mice. Mice were infected as described in Figure 3 and CNS virus titers were determined
on day 3, 5, and 7 postinfection. Data are average of 3 experiments with 3 mice per
group.
1
2
3
4
5
3 5 7
days postinfection
Log PFU/g brain
DI- γ
DI-x
220
Composition of inflammatory infiltrates is not altered by infection with MHV-JHM
DI- γ.
Previous results showed that i.c. infection of C57BL/6 mice with MHV-A59 DI- γ
results in an increase in cellular infiltration compared to mice infected with control
virus
29
. However, analysis of inflammatory cells in the CNS of infected BALB/c mice
did not show an effect of MHV-JHM DI- γ infection on either NK cell, neutrophil, or
CD4 or CD8 T cell infiltration compared to control virus infection (Fig. 32).
221
Figure 32. Infection with MHV-HMV DI- γ does not alter the infiltrating cell
populations. CNS mononuclear cells were isolated from mice infected as described in
Figure 3 and NK cell, neutrophil, CD4 and CD8 T cell populations were assessed 3, 5,
and 7 days postinfection. Data are average of 3 experiments with 3 mice per group.
5
10
15
20
25
30
NK cells
Neutrophils
CD4 T cells
CD8 T cells
5
10
15
20
25
30
5
10
15
20
25
30
Day 3
Day 7
Day 5
DI- γ DI-x
% of CNS infiltrating cells
222
Expression of IFN- γ inducible genes in the CNS is similar in mice infected with
IFN- γ pseudorecombinant and control virus.
Given that there was no measurable impact of DI derived IFN- γ on virus titer,
pathogenesis, or cell infiltration, a more sensitive method of detecting the action of this
cytokine was employed. IFN- γ strongly and consistently induces a number of genes
including those for the chemokines IP-10, RANTES, MCP-1
5
, as well as iNOS and MHC
class II
4
. IFN- γ, RANTES, MCP-1, MIG, iNOS, and MHC class II mRNA levels were
compared in the CNS of MHV-JHM DI- γ and control virus infected mice by Real Time
PCR. Analysis shows that expression of these genes increases over time, due to
endogenous IFN- γ expression, however, no difference between the two experimental
groups was detected indicating the absence of an effect of DI derived IFN- γ (Fig. 33).
223
Figure 33. IFN- γ inducible genes are not preferentially expressed in the CNS of
JHMV DI- γ infected mice. Mice were infected as described in Figure 3 and CNS
mRNA was analyzed for expression of several IFN- γ inducible genes.
1
10
100
1000
relative expression
1
10
100
1000
10000
MHC class II
IFNg
iNOS
MCP-1
RANTES
IP-10
MIG
Day 5 Day 2
224
Secretion of biologically active IFN- γ by MHV-JHM DI- γ cannot be detected in vivo.
No evidence of virus derived IFN- γ was found in wild type mice infected with
MHV-JHM DI- γ when analyzing cell infiltration, virus titer, or gene expression.
However, endogenous IFN- γ could mask the effect of virus derived cytokines. Therefore
BALB/c mice deficient in IFN- γ (BALB/c IFN- γ
0/0
) were infected with IFN- γ producing
and control virus. IFN- γ stimulates the upregulation of MHC class II on microglia. Since
MHC class II molecules are not constitutively expressed in the CNS this upregulation is a
marker for biologically active IFN- γ in the brain
2
. Neither microglia from MHV-JHM
DI- γ nor control virus infected mice exhibited expression of MHC class II. These data
indicate a lack of IFN- γ action on these cells, suggesting that there is no biologically
active IFN- γ in either group (Fig. 34). To confirm these results, Real-Time PCR was
employed to detect expression levels of 2’-5’ OAS. Transcription of this molecule, which
activates RNaseL, which in turn degrades ssRNA, is induced by IFN- γ
5
. Though
expression of 2’-5’ OAS is increased compared to the levels in the CNS of uninfected
mice due to expression of other inflammatory cytokines, it does not differ between JHM-
MHV DI- γ and DI-x infected mice (Fig. 35).
225
Figure 34. MHV-JHM DI-γ infection does not result in upregulation of MHC class
II on microglia. BALB/c IFN- γ
0/0
mice were infected with MHV-JHM DI- γ or control
virus and CNS monocular cells were isolated 24 h and 48 h postinfection, stained with
anti-CD45 and anti-MHC class II antibody and examined by flow cytometry. Cells were
gated on CD45
int
microglia and the percent of MHC class II positive cells established.
Data is representative of 2 experiments with 3 mice per group.
CD45
MHC class II
Day 8
CD45
MHC class II
Day 2
DI-x
DI-γ
1% 3%
3% 3%
226
Figure 35. MHV-JHM DI- γ infection does not enhance 2’-5’ OAS expression
compared to MHV-JHM DI-x infected mice. CNS mRNA was isolated from MHV-
JHM DI- γ, DI-x, and uninfected BALB/c IFN- γ
0/0
mice two and three days postinfection
and expression of 2’-5’ OAS was compared. Data are average of 2 experiments with 3
mice per group.
days postinfection
200
400
600
800
1000
1200
1400
1600
1 2
Relative units
DI- γ
DI-x
Uninfected
227
Discussion
The role of IFN- γ during virus infection depends on type of virus and route of
infection
1,6,10,11,19
. MHV infection of the CNS is a model for demyelinating disease
9
. In
this infection CD8 T cells are the major source of IFN- γ
24
and the cytokine is important
for control of MHV infection in some cell types, but not others
3,26
.
Pseudorecombinant MHV can be utilized to explore the role of IFN- γ in virus
infection. Production of IFN- γ by the virus would result in the presence of this cytokine
in the CNS at an earlier time point than during infection with the wild type virus and may
allow the study of its effect on the innate and subsequently adaptive immune response.
Transfection of MHV infected cells results in the production of IFN- γ confirming that the
gene encoded in the DI particles is transcribed and translated in co-infected cells in
vitro
29
. Furthermore, DI particles are retained over several passages, but rapidly decline
in concentration as evidenced by the reduction in IFN- γ production over several passages.
Though loss of DI particles usually correlates with an increased ability of the virus to
replicate
7
, virus titers are actually lower after the first passage in MHV-JHM DI- γ
infected cells. However, this is most likely due to the effect of IFN- γ in the virus
supernatant in concert with low MOI
29
. This decrease after the first passage is not seen in
control virus, further supporting the hypothesis of a direct effect of IFN- γ on virus
replication or spread.
Contrary to results in C57BL/6 mice using a pseudorecombinant A59 strain of
MHV
29
, i.c. infection of mice with MHV-JHM DI- γ does not result in reduced clinical
symptoms compared to mice infected with the control virus. Analysis of virus titers from
228
the CNS of infected mice also does not show a difference in viral replication.
Furthermore, there is no difference in infiltrating inflammatory cells between the CNS of
MHV-JHM DI- γ and control virus infected mice. This could indicate that IFN- γ has no
direct effect on MHV-JHM replication when given early during infection, or alternatively
that the virus fails to produce IFN- γ in vivo. Assessment of expression of IFN- γ itself as
well as IFN- γ inducible genes by Real Time PCR as a more sensitive method for
detecting the action of IFN- γ in the CNS also did not reveal a difference between the
experimental and control group. The absence of an obvious effect may be due to the
action of endogenous cytokines from BALB/c wild type mice. However, infection of
IFN- γ
0/0
mice revealed no expression of MHC class II molecules on microglia during
early infection with MHV-JHM DI- γ and control infection. This indicates a lack of virus
derived biologically active IFN- γ in the CNS. Real Time PCR analysis of the IFN- γ
inducible gene 2’-5’ OAS in IFN- γ
0/0
mice confirmed this result.
These data show that DI particles can be used to engineer pseudorecombinant
MHV secreting non-viral proteins such as IFN- γ in vitro. However, there is no evidence
of virus derived IFN- γ following infection. This contrasts with previously published data
showing decreased virus replication and clinical disease in mice infected with MHV-A59
DI- γ
29
. The basis for this discrepancy is unclear. However, there are several differences
between the virus strains that could contribute to the divergent results. The immune
response to JHM differs to that to A59
8
for example in the type of cytokine production it
stimulates
8
partially because of differences in the spike gene
14
. Also, a 10 x lower MOI
was used to inoculate the mice with MHV-JHM than with MHV-A59. Therefore the
229
likelihood of wild type virus and DI containing viral particles entering the same CNS cell
allowing for production of IFN- γ is reduced. Furthermore, DI particles in A59 require
different replication signals compared to JHM
20
and in vitro IFN- γ production by MHV
DI- γ is higher in pseudorecombinant A59 then JHM
29
. Any of the above mentioned
differences in MHV-JHMV as opposed to the A59 strain may contribute to the lack of
IFN- γ production during CNS infection with MHV-JHM DI- γ.
230
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Abstract (if available)
Abstract
SJL mice exhibit a unique gender-dependent bias in their immune response. Males mount an anti-inflammatory Th2 response, whereas females react with an inflammatory Th1 response, which correlates with susceptibility to experimental autoimmune encephalomyelitis, a mouse model for multiple sclerosis. Castration as well as macrophage transfer from females reverses the male phenotype. Utilizing this mouse strain for the study of gender-dependent mechanisms of immune regulation, the role of CD25 regulatory T cells was examined. These cells maintain a Th2 environment in naïve males by regulating macrophage responsiveness. Transfer of macrophages from naïve CD25+-depleted males into untreated males results in a Th1 response after immunization demonstrating that regulatory T cells directly influence macrophage function. Males have a two-fold increase in the number of regulatory T cells compared to females, but no difference in cell surface marker expression or in vitro suppressive action was detected. The macrophage phenotype in naïve mice showed no difference in number or activation status comparing males and females. Analysis of cell surface molecules after antigen uptake uncovered a reduction in MHC class II expression on macrophages in males compared to females. The lack of antigen uptake by dendritic cells further confirmed the importance of a macrophage antigen presenting cell.
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Kirwin, Stefanie Johanna
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Core Title
Gender dependent immune regulation: effect of regulatory t cells on macrophages and potential mechanism of protection from autoimmune disease
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Molecular Microbiology
Publication Date
11/21/2006
Defense Date
10/19/2006
Publisher
University of Southern California
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Tag
autoimmunity,macrophage,multiple sclerosis,OAI-PMH Harvest,regulatory T cells,Th1/Th2
Language
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Tahara, Stanley M. (
committee chair
), Epstein, Alan L. (
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), Lai, Michael L. (
committee member
), Stohlman, Stephen (
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)
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