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Suppressor cell therapy: targeting T regulatory cells in cancer

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Suppressor cell therapy: targeting T regulatory cells in cancer

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Suppressor Cell Therapy: Targeting T Regulatory Cells In Cancer

Saman Shanaya Karimi

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR & EXPERIMENTAL PATHOLOGY)

AUGUST 2013

Committee Members:
Dr. Alan Epstein
Dr. Florence Hofman
Dr. Louis Dubeau

Copyright 2013 Saman Shanaya Karimi
i
Table of Contents

List of Figures--------------------------------------------------ii
List of Tables---------------------------------------------------ii
Chapter 1: Introduction---------------------------------------1-4
Chapter 2: Mouse Immunotherapy--------------------------5-15
Chapter 3: Treg ex vivo drug studies------------------------16-26
Chapter 4: Future Directions---------------------------------27-28
References------------------------------------------------------30-32
Acknowledgements: ------------------------------------------33

ii
List of Figures

Figure 1.1 - Role of Tregs in tumor progression ---------------------------------------------------------- 3
Figure 2.1 - Variable tumor growth rates in vivo -------------------------------------------------------- 9
Figure 2.2 - Response to immunotherapy in six murine tumor models with variable immunogenicity -11
Figure 2.3 - Incidence of autoimmunity with immunotherapy in six tumor models-----------------------12
Figure 2.4 - Co-culture ratio of T regulatory cells with naïve syngeneic splenocytes------------------- 13
Figure 3.1 - Tregs and an overview of immunosuppression-----------------------------------------------17
Figure 3.2 - 4T1 tumor mass formation in BALB/c mice-------------------------------------------------- 18
Figure 3.3 - Assessment of the reversal of suppression via flow cytometry-------------------------------21
Figure 3.4 - Proliferation of T cells in response to CD3/CD28 stimulation beads-----------------------23
Figure 3.5 - IFN- γ sandwich ELISA------------------------------------------------------------------------24

List of Tables

Table 3.1 – List of compounds used in murine ex vivo suppression assays------------------------------- 19

1

Chapter 1.
Introduction

Tumor growth and survival is mediated by many different mechanisms, however recruitment of
tumor-specific inflammatory cells for the purpose of inducing immune tolerance and immune
suppression has recently become a survival mechanism of great interest. Tumors and cancer cells
recruit regulatory T cells (Tregs), myeloid derived suppressor cells, and tumor associated
macrophages to aid in bypassing systemic inflammation and clearance. Tregs, in particular, are
important for mediating tumor immune escape and for promoting angiogenesis necessary for
tumor growth and survival. Tregs have been shown to induce immune tolerance and regulate
immune response in response to cancer cells by suppression of dendritic cells (DC), secretion of
immunosuppressive small molecules, disrupting normal metabolism, and initiating cell lysis. Of
these mechanisms of action, the secretion of immunosuppressive molecules and suppression of
T-cell function have been shown to be among the most important in mediating immune tolerance
(Walsh et al., 2004).

Regulatory T cells have been shown to exist in two different forms, each with a distinct origin.
Natural Tregs (nTregs) are regulatory T cells that are derived from the Thymus, and have been
shown to be maintained by TGF-β. Unlike nTregs, induced Tregs (iTregs) are regulatory T cells
that arise during the transformation of naïve CD4 cells into Tregs. Both nTregs and iTregs are
known to express FoxP3, a transcription factor critical for functional Treg lineage (Curotto de
Lafaille and Lafaille, 2009). The tumor microenvironment promotes renewal and regeneration of
regulatory T cells by mediating expansion of nTregs, as well as generation of new iTregs from
naïve CD4 T cells (Colombo et al., 2007).
2

Tregs release inflammatory cytokines that perform distinct functions; in response to activation
and stimulation by immune-evading cancer cells, they secrete cytokines IL-10, IL-35, and TGF-
β, which aid in the inhibition of effector T cells as well as their inactivation (Collison et al.,
2009). Of these cytokines, IL-10 and TGF-β have been shown to be most critical in suppressing
immune-mediated anti-tumor activity. These cytokines suppress this activity by inhibiting
immune cell proliferation, release of inflammatory cytokines IFN-γ and TNF-α, and directly
lysing effector T cells necessary for cancer cell targeting, lysis, and clearance. Tregs accomplish
effector T cell lysis by activating galectin-1, granzyme B, and the TRAIL pathway to induce
apoptosis of effector cells (Croci et al., 2007). In addition to mediating cytolysis of effector T
cells, regulatory T cells have been shown to express CD39 and CD73, thereby allowing the
catalysis of ATP and generation of adenosine, and allowing adenosine-dependent effector T cell
suppression (Deaglio et al., 2007). Tregs have also been shown to mediate transfer of cAMP
through membrane gap junctions, thereby inhibiting effector cell function (Ring et al., 2010).
Tregs have been shown to secrete VEGF and induce angiogenesis and immunosuppression
(Faciabenne et al., 2011).

In addition to inhibiting effector T cells, Tregs also serve to interact with and inactivate dendritic
cells. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) of Tregs undergoes cell-cell interaction with
CD80/86 on dendritic cells, facilitating the release of indoleamine 2,3-dioxygenase (IDO) from
dendritic cells. In doing so, IDO catabolizes tryptophan, resulting in the suppression of effector T
cell function (Munn et al., 2011) by inhibiting the genetics of theta chain required for the
construction of the T cell receptor. A summary of mechanisms by which Tregs mediate tumor
progression and promote immune tolerance and suppression can be found in Figure 1.1.
3
Since Tregs have been shown to play an important role in immune tolerance and immune
suppression, the development of reagents aimed at inhibiting the immunosuppressive potential of
Tregs have become an area of great pharmacological interest. A number of chemical agents have
already been shown to function in this capacity, such as anti-mitotic including mitroxantrone,
gemcitabine, fludarabine, and cyclophosphamide (Zitvogel et al., 2008). Of these,
cyclophosphamide has been shown to suppress tumor growth by depleting CD4
+
CD25
+
Tregs by
alkylating DNA and facilitating Treg apoptosis.

Figure 1.1. Role of Tregs in tumor progression. Tumors recruit regulatory T cells by means of hypoxia-induced
CCL28, DC, and tumor-derived CCL22 secretion. Tregs are regenerated in the tumor microenvironment either by
stimulation of existing Tregs by TGF-β or conversion of naïve T cell precursors by IL-10. Tregs utilize cytokine
secretion, cytolysis, and metabolic disruption to suppress CD4
+
and CD8
+
T cells in order to induce immune
4
suppression and tolerance and promote tumor growth. They also stimulate angiogenesis by inhibiting angiostatic
cytokines IFN-γ and CXCL-10 and upregulation of VEGF. Adapted from Facciabene et al., 2012.

Other than anti-mitotic, other immunotherapeutic agents, such as antibodies specifically targeting
and depleting CD25
+
, have also been successful in enhancing tumor suppression rather than
immune suppression. However, therapeutic approaches relying on CD25
+
depletion have been
met with limitations, since depletion of CD25
+
promotes naïve T cell transformation to Tregs to
compensate for reduced Treg expansion and the depletion of CD25
+
CD8
+
effector cells.

Although Tregs have been shown to play an important role in immune suppression, recent
findings suggest that Tregs may play a dual role in carcinogenesis. While Tregs induce immune
tolerance in most cancers, they may also play a favorable role in other cancers, suppressing
carcinogenesis-promoting bacterial inflammation and resulting in better prognosis in some
cancers, including colorectal cancer (Whiteside, 2012). These findings suggest that our
understanding of regulatory T cells and their role in tumor propagation is still in its infancy, and
that extensive study is still necessary to fully understand its role in cancer pathology.

5

Chapter 2.
Mouse Immunotherapy

INTRODUCTION

It has been well established that cancer cells induce immune suppression and immune tolerance
by the recruitment of regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), and
tumor associated macrophages (TAMs), and that most tumors employ this combination of
suppressor mechanisms in order to prevent being identified as a foreign entity by the immune
system (Marigo et al., 2008). Some cancers have been shown to be highly immunogenic,
exhibiting surface tumor antigens and high HLA expression. To counter this, tumors mount a
more potent immunosuppressive response, resulting in more significant recruitment of Tregs,
MDSCs, and TAMs in order to avoid being targeted and to bypass the immune system.
As shown by Sadun et al., the immune signature of the tumor, as well as the mechanism of
immunosuppression being employed, can be detected at the time of initial biopsy (Sadun et al.,
2007). The importance of immune tolerance and suppression as a mechanism of tumor growth
and survival can not be overstated. As such, one of the most important goals of immunotherapy
has become the reversal of cancer cell immune tolerance and immune suppression, as well as
subsequent immunostimulation without the initiation of autoimmune responses.

Immunotherapy targeting immune tolerance has been shown to be dependent on different factors,
such as age or the type of tumor being treated. Hurez et al. demonstrated that in aged B16 tumor
bearing mice, Treg depletion combined with anti-Gr-1 antibody was necessary to achieve the
same immunotherapeutic effect as the depletion of Treg alone in young B16 mice (Hurez et al.,
2012). Begley et al. demonstrated that Bcl-2 inhibitor ABT-737 was successful in sensitizing
6
cancer cells to antigen specific immunotherapy in CT26 colon carcinoma, but that the
immunotherapy treatment was unsuccessful in B16 melanoma cancer cells (Begley et al., 2009).
Similarly, cetuximab, an anti-EGFR monoclonal antibody used as a immunotherapeutic agent,
has been shown to be successful in the treatment of head and neck squamous cell carcinoma
(Mehra et al., 2008), but completely ineffective in advanced pancreatic cancer (Philip et al.,
2010). Therefore, it is imperative to develop immunotherapeutic agents and regimens that take
into consideration factors such as age and tumor identity in order to achieve favorable responses.

MATERIALS AND METHODS

Cell lines and animals
Mouse tumor cell lines 4T1, CT26, RENCA, B16, and LLC were obtained from ATCC and
maintained in 10% RPMI complete media (RPMI-1640 with 10% FCS, 2mM L-Glutamine, 100
U/ml Penicillin, and 100ug/ml Streptomycin) in a humidified 5% C0
2
at 37
0
C incubator. These
tumor cell lines were injected subcutaneously at a concentration of 2x10
6
cells/200 µl inoculum
into 4-6 week old female C57BL/6 or BALB/c mice (Harlan, Livermore, CA), with control mice
receiving PBS injection instead; resultant tumor masses were measured every 2-3 days with
calipers. Spontaneous and transplantable gliomas were grown in FVBN and C57BL/6 mice as
described elsewhere (Davie et al., 2007). All animals were handled in accordance with USC or
University of Minnesota Institutional Animal Care and Use Committee guidelines.

Suppression assays
Upon growth of tumor mass to 1cm in diameter (tumor volume 300-600 mm
3
), spleens and
TDLN were harvested from tumor-bearing mice. Mouse CD4
+
CD25
+
regulatory T cell isolation
kit (Miltenyi Biotec) was used to isolate Treg population, respectively, from the harvested tumor
7
mass. CD3/CD28 beads (Invitrogen) were used in co-cultures of suppressor cells and CFSE-
labeled (3µM) fresh mononuclear spleen cells from syngeneic naïve mice at a ratio ranging
between 1:1 and 1:20. Spleen cell proliferation was evaluated after four days using BD LSRII
flow cytometer.

Immunotherapy studies
BALB/c and C57BL/6 tumor-bearing mice were randomized into groups (n=5) for treatment
when resultant tumor volume ranged from 40 to 80 mm
3
. Tumor-bearing mice received a
50mg/kg intraperitoneal injection of chemotherapy agents 5-fluorouracil (5-FU) (Sigma) and
cyclophosphamide (CTX) (Sigma) suspended in PBS to treat MDSC and Treg, respectively.
Dendritic cells (DC) for vaccines were isolated post-7 day culture of GM-CSF (R &D Systems)
and syngeneic naïve mouse bone marrow, and collected using CD11c
+
magnetic beads (Miltenyi
Biotech). Isolated CD11c
+
cells were co-cultured with irradiated tumor cells in a 2:1 ratio in the
presence of 5µg/mL Hiltonol (Oncovir) and 10ng/mL of GM-CSF & IL-4 (R&D Systems)
overnight. Vaccination was accomplished by injecting 1x10
6
DC, collected from culture flasks
using Detachin (Genlantis, San Diego, CA), and 30µg Hiltonol suspended in 200µl PBS i.p. To
chemoattractant antigen presenting cells into the tumor Lec/chTNT-3 The PB3 fusion protein
was administered at a concentration of 30µg/dose suspended in 100 µl of PBS, and injected i.v.
Mouse tumor volume was measured using calipers every 2-3 days, with mice being sacrificed
upon growth of tumor mass to a diameter exceeding 2cm. Tail vein blood samples were collected
5 days post-immunotherapy regimen treatment of experimental and control mice, and
accumulation of anti-nuclear antibodies (ANAs) was determined by Mouse Anti-Nuclear
Antigens Ig’s ELISA kit (Alpha Diagnostic International, San Antonio, TX) to demonstrate the
presence or absence of autoimmune reactions.
8
Statistical Analyses
ANOVA was used to measure change in immune-related gene expression and TIL counts in the
six different tumor models. Student t tests were used to measure change in immune-related gene
expression and TIL counts between tumor models that were highly and poorly immunogenic, and
these tests were corrected using the Holm-Sidak method. Suppression assays were performed
and the change in mean proliferation of suppressor cells to splenocytes was measured using
ANOVA and Dunnett’s test for pair-wise comparisons. Linear regression analysis was performed
to determine tumor growth rates. ANOVA and pair-wise comparisons were used to determine
percent tumor cell positivity for ki-67 and caspases among tumor models. ANOVA and
Dunnett’s test for pairwise comparisons were performed to determine changes in tumor volume
upon treatment of immunotherapy regimens. Log-rank test was used to evaluate changes in
animal survival upon immunotherapy regiment treatment to tumor models. Chi-square tests were
used to compare ANA positivity between immunotherapy regimen treated and untreated tumor
models. Prism 6 software (Graph Pad Software, Inc) was used to perform all tests, quantify and
graph the data. P < 0.05 was considered statistically significant.

RESULTS

Tumor growth rate inversely related with level of immunogenicity
As demonstrated in Figure 2.1, growth of tumor models CT26, RENCA, 4T1, MAD109, LLC,
and B16 in immune-competent mice in the absence of treatment varied greatly. However, tumor
growth rate was indirectly associated with MHC class I expression & overall immunogenicity of
the tumor model, with CT26, RENCA, and 4T1 demonstrating slowed tumor growth and B16,
LLC, and MAD109 demonstrating enhanced tumor growth. Contributions of proliferation and
cell death towards the observed tumor growth variability were evaluated by staining tumor
9
sections for Ki-67 and caspase, which demonstrate proliferation and apoptosis, respectively
(Figure 2.1). Proliferation rates among the six tumor models did not vary as determined by Ki-67
staining, thereby eliminating the possibility that the unique in vivo tumor growth rates among the
six tumor models is a result of enhanced or faster proliferation of some tumors models over that
of others. However, enhanced apoptosis was observed in those immunogenic tumor models
possessing slower tumor mass growth, as determined by caspase staining. A distinct correlation
between tumor growth and necrosis was also observed, and RENCA tumors were shown to
possess higher necrotic tissue potential than the rapidly growing 4T1 and MAD109 tumor
models. Collectively, these data demonstrate enhanced endogenous cell-mediated targeting and
clearance of tumor cells upon enhanced antigen expression in immune competent hosts for the
more immunogenic tumor models.

Figure 2.1. Variable tumor growth rates in vivo. (A) Tumor volumes where measured in groups of inoculated
immune competent mice to determine growth rates of the six tumor models by linear regression analysis. (B) Ki-67
+

immunohistochemically stained cells were quantified, measuring Ki67 positivity per 100 tumor cells. The mean
positive fraction is shown here, with SEM. There was no distinguishable difference between tumor models.

B
10
Success of two immunotherapy regimens correlated directly with tumor immunogenicity

The tumor-host immune interactions of the six transplantable tumor models discussed earlier
have been shown to be highly variable, thereby suggesting a distinct immunotherapeutic
approach for each tumor model. Treg and MDSCs were present in all tumor models, whereas
mature DC and CTLs were noticeable absent in the tumor microenvironment. Thus, inhibition of
Treg and MDSCs, as well as preservation of mature DC and CTL populations, could serve as an
efficacious anti-tumor immune response. The immunogenicity level of the six tumor models
outlined above may also play a role in regulating tumor immunotherapeutic response to distinct
immunotherapy regimens. These regimens consist of either tumor-targeted chemokine LEC
(CCL16) and low dose chemotherapy (CTX and 5-FU) to suppress Treg and MDSC (Regimen
1), or a DC tumor vaccine with toll-like receptor agonist Poly IC:LC (Regimen 2). Due to the
variable immune profiles observed previously in the six tumor models used, variable tumor
response rates were also anticipated across these tumor models. Figure 2.2 demonstrates that
CT26, RENCA, and 4T1 tumors possess the most significant response to both regimens 1 & 2.

These tumor models underwent notably reduced tumor growth, improved survival rate, and 5/10
of the CT26 tumor bearing mice were cured when treated with these regimens. MAD109 and
LLC tumor models, on the other hand, exhibited moderate response to immunotherapy regimens
1 & 2. Although these tumor models demonstrated notable reduction in tumor growth, they did
not exhibit any significant improvement in survival rate in response to the immunotherapy
regimens. Of the tumor models, B16 possessed the poorest response to immunotherapy, with

11

Figure 2.2. Response to immunotherapy in six murine tumor models with variable immunogenicity. Tumor
volume and survival of tumor-bearing mice were evaluated in the presence of absence of immunotherapy treatment.
Regimen 1 was comprised of 50mg/kg of cyclophosphamide and 50mg/kg of 5-FU. Regimen 1 was delivered
intraperitoneally (i.p.) once on day 4, and 30ug/dose of LEC/chTNT-3 fusion protein was delivered i.p. on days 8-
13. Regimen was comprised of Regimen 1 supplemented with bone-marrow derived DC vaccine primed with
Hiltonol and irratdiated tumor cells. Regimen 2 was delivered on days 7 and 14. On 17, tumor volume of treatment
groups was evaluated. Statistically significant differences are indicated with an asterix. Mice were sacrificed upon
growth of tumor to a diameter of 2cm, and survival was recorded for all tumor model groups. Survival of CT26,
RENCA, and 4T1 tumor model mice treated with regimens 1 & 2 were significantly improved.

12
slight suppression of tumor growth and no change in survival rate in response to treatment with
regimens 1 & 2. Thus, the immunogenicity of these tumor models determined the response of
these mice tumors to the immunotherapy regimens discussed earlier.

Autoimmune reaction related to immunotherapy regimen, not tumor immunogenicity
Autoimmune reactions were measured by examining serum anti-nuclear antibody (ANA) levels
to determine differences in successful immune responses between tumor bearing mice treated in
the presence or absence of immunotherapy regimens 1 & 2 (Figure 2.3 A). Unlike the success of
the immunotherapy regimens, the positive serum ANA levels were independent of
immunogenicity of the tumor model and were relatively consistent among all tumor models. The
induced autoimmunity was immunotherapy-dependent, and was not affected by tumor model
immunogenicity (Figure 2.3 B). Collectively, these data demonstrate that although either
regimen could reverse tumor immune tolerance, treatment of these immunotherapy regimens was
not sufficient for tumor immune clearance in most tumor models.

Figure 2.3. Incidence of autoimmunity with immunotherapy in six tumor models. Serum anti-nuclear antibodies
(ANA) were measured post immunotherapy Regimen 1 or 2 treatment. ANA served as a measure of reversal of
immune tolerance and the generation of immune effector responses, and (A) increased with the increased strength of
the regimen. (B) Serum ANA levels were not tumor model dependent.

13

Figure 2.4. Co-culture ratio of T-regulatory cells with naïve syngeneic splenocytes. Regulatory T cells from 6
tumor models were co-cultured with naïve syngeneic splenocytes at a ratio of 1:1, 1:2, and 1:5.

Ex vivo Suppression Assay
T regulatory cells were isolated from TDLN and spleen from each of the six tumor models using
magnetic isolation techniques. The resultant Tregs were co-cultured with naïve syngeneic
splenocytes activated by CD3/CD28 stimulating beads, and allowed to proliferate for 4 days. The
samples were analyzed using LSRII and Flowjo proliferation index followed by ANOVA and
Dunnett’s comparison. Significant functional suppression was observed at 1:1, 1:2 and 1:5 ratios
(Figure 2.4).
Spleen CT26 4T1 RENCA MAD109
1.0
1.5
2.0
2.5
Proliferation Index
CD4
+
CD25
+
Treg
Tumor Model Source
of Suppressor Cells
1:2 ratio
1:1 ratio
1:5 ratio
Spleen LLC B16
1.0
1.5
2.0
2.5
Proliferation Index
CD4
+
CD25
+
Treg
Tumor Model Source
of Suppressor Cells
1:2 ratio
1:1 ratio
1:5 ratio
14
DISCUSSION

Immunotherapy has generally proven challenging due to the multifactorial effects that impact
tumor responsiveness to the immunotherapy of choice. Unfortunately, to date no
immunotherapeutic agent can successful treat all tumor types, and as such it is necessary to tailor
immunotherapeutics based on the characteristics of the tumor of interest. For instance, Kyeung et
al demonstrated that Glioblastomas exhibiting enhanced expression of MET, a tyrosine kinase
that stimulates cancer survival, proliferation, and invasion, are resistant to radiation therapy,
whereas Glioblastomas endogenously expressing low MET respond exceedingly well to
radiation therapy (Joo et al., 2012).

Although positive in vivo findings may serve as an indication of the potency and success of novel
immunotherapeutic agents, it is crucial to take into account not only the characteristics of the
tumor, but also the gene expression profile of the tumor model being studied. In a study
conducted by Niers et al., a series of anticoagulants, including low molecular weight heparin,
were shown to be effective inhibiting tumor development in B16 melanoma cells, but ineffective
in K1735 melanoma cells and CT26 colon cancer cells (Niers et al., 2009). Upon further
examination, it was determined that K1735 and CT26 cells lack PAR-1 and CD24, and that B16
melanoma cells exhibit enhanced expression of these antigens, suggesting that the anti-
tumorigenic treatment was successful due to B16’s expression of these immunogenic markers.

The findings shown here demonstrate that of the tumor models examined, those that were more
immunogenic exhibited enhanced responsiveness to immunotherapy regimens outline above.
Furthermore, immunogenicity was found to be the most important factor in predicting tumor
15
response to immunotherapies, including DC vaccines, Treg and MDSC inhibitors, and tumor-
targeted chemo attractants, independent of autoimmunity. The findings proposed here suggest
that immune activation is more difficult to achieve exogenously than reversal of immune
tolerance and immune suppression.

One of the challenges of studying current immunotherapy approaches in vivo, is the inability to
utilize heterotransplanted human tumor models to examine the efficacy of immunotherapy
regimens. Thus, current methods are limited to syngeneic animal tumor models for in vivo/ex
vivo experiments and in vitro human assays. While the results of murine studies often may not
directly translate to bedside therapies, they offer insights into our understanding of the
mechanisms governing tumor growth and survival. Immunotherapy success has already been
observed in the clinic with the monoclonal antibody, Trastuzumab, against the HER2/neu
receptor. Tumors positive for Her2 overexpression respond to this monoclonal antibody via
receptor blockade. Nonetheless, no two individuals are exactly alike; hence, no two tumors
arising from those individuals would have identical gene expression, survival mechanisms, and
tumor microenvironment. Therefore, tumor immune signature, histopathology, tumor
microenvironment, the individual as well as the etiology of the tumor must be considered when
designing an effective cancer therapy regimen.

16

Chapter 3.
Treg ex vivo drug studies

INTRODCUTION

Tumor growth and propagation has been attributed to many important physiological processes,
such as angiogenesis and epigenetic mechanisms. Immune suppression and tumor immune
escape, however, are among the most highly utilized and regulated mechanisms of tumor growth
and cancer cell proliferation. Myeloid Derived Suppressor Cells (MDSCs), Tumor Associated
Macrophages (TAMs), and T regulatory Cells (Tregs) enhance cell to cell interactions and
cytokine release in order to abrogate cytotoxic T cell activity as well as the activation and
proliferation of CD4+ helper T cells, thereby permitting enhanced immune-mediated tolerance of
tumor growth.

Tregs are immune cells that co-express CD4 and CD25, and are responsible for regulating
autoreactive T-cells in vivo in order to accomplish immune homeostasis. Adaptive Tregs have
been shown to be induced by adenosine, IL-10, as well as indoleamine 2,3-dioxygenase (IDO)
and TGF-b (Figure 3.1 A, Colombo et al., 2007) and are known to constitute between 5 – 10% of
all CD4
+
T cells in humans. Tregs are primarily regenerated by expansion of existing Tregs, but
can be regenerated by the conversion of naïve T cells, a process that becomes the predominant
means of Treg regeneration upon CD25
+
depletion (Figure 3.1 B, Colombo et al., 2007).
Peripheral tumor tolerance is known to be regulated by Tregs via inhibition, identification,
destruction, and removal of neoplastic cells. By identifying potential chemical agents capable of
selectively inhibiting Treg-dependent immune suppression, this study sought to partially reverse
tumor cell immune tolerance.
17

Figure 3.1 Tregs and an overview of immunosuppression. (A) Tumor cells release TGF-β and indoleamine 2,3-
dioxygenase (IDO) to stimulate pre-existing Treg cells and induce the conversion of naïve T cells into Treg cells to
suppress both the adaptive and innate immune response. (B) Tregs are regenerated by expansion of existing Treg
cells or conversion of naïve T cells into Tregs. Upon CD25
+
depletion, conversion of naïve T cells into Tregs
becomes the primary means of Treg regeneration. Adapted from Colombo et al., 2007.

A

B
18

MATERIALS AND METHODS

Cell lines and tissues

Mouse tumor cell line 4T1 was obtained from ATCC and maintained in 10% RPMI complete
media (RPMI-1640 with 10% FCS, 2mM L-Glutamine, 100 U/ml Penicillin, and 100ug/ml
Streptomycin) in a humidified 5% C0
2
at 37
0
C incubator and expanded after a few growth
cycles.

Heterotransplantation in BALB/c mice
Groups of 6-8 week old BALB/c mice received subcutaneous injections of 1x10
6
tumor cells in a
200μl inoculum. Groups of 4T1 tumor bearing BALB/c mice were sacrificed when tumors
reached approximately 1cm (400-600mm
3
), typically 23-28 days post-injection. The spleen and
the TDLN were excised from the sacrificed BALB/c mice.

19
Figure 3.2. 4T1 tumor mass formation in BALB/c mice. 1. 6-8 week old BALB/c female mice were given
subcutaneous injections of 1x 10
6
tumor. The mice were sacrificed when the tumor reached 1cm (400-600mm
3
in
volume). The spleen and the TDLN were excised for the purpose of CD4+CD25+ T regulatory cell isolation.

Regulatory T cell isolation
Tregs were isolated from spleens and TDLN via mouse CD4+CD25+ Regulatory T Cell isolation
kit (Miltenyi Biotec) using MACS columns. The splenocytes from naïve BALB/c were flushed
out of the spleen, washed and CFSE-labeled. T regulatory cells obtained from the 4T1 tumor-
bearing BALB/c mice were then co-cultured with splenocytes isolated from naïve BALB/c mice.

Analysis of surface markers by flow cytometry
The CD4+CD25+ T regulatory cells and the splenocytes obtained from the naïve mice were co-
cultured in a 96 well plate at a 1 Treg: 4 CFSE-labeled Responder cells ratio in the presence of
activating CD3/CD28 beads (Invitrogen) and complete media.
Drug Name Low Dosage High Dosage Function
DTA-1 1 µg/ml 10 µg/ml Agonistic DTA-1 monoclonal antibody
reacts with mouse GITR, Glucocorticoid-
Induced TNFR family gene.
FGK 45.5 1 µg/ml 10 µg/ml Agonistic CD40 antibody
TY25 1 µg/ml 10 µg/ml PD-L1 antibody
OX86 1 µg/ml 10 µg/ml Agonistic OX40 antibody
RA8 1 µg/ml 10 µg/ml Human anti-CD25 antibody
RMPI-14 1 µg/ml 10 µg/ml PD-L1 antibody
LS69 50 ng/ml 500 ng/ml HIF-1alpha inhibitor
Fc-IFN-β 1 µg/ml 10 µg/ml IFN-beta fusion protein
Anti-CTLA 1 µg/ml 10 µg/ml Anti-CTLA monoclonal antibody
PC61 1 µg/ml 10 µg/ml Rat anti-mouse CD25 monoclonal
antibody
BSO 1 µg/ml 10 µg/ml L-Buthionine-sulfoximine (BSO), a
glutathione biosynthesis inhibitor
Fc-muOX40L 1 µg/ml 10 µg/ml Murine OX40L fusion protein
Fc-muCD40 1 µg/ml 10 µg/ml Murine CD40 fusion protein
Fc-muGITR 1 µg/ml 10 µg/ml Murine GITR, Glucocorticoid-Induced
TNFR family gene, fusion protein.

Table 3.1: List of compounds used in murine ex vivo suppression assays. Each drug was added to co-culture-96
20
well plates at high concentration (10 µg/ml) and low concentration (1 µg/ml). *LS69 is an exception due to
cytotoxicity. In addition, the high dose of each compound was tested on the stimulated splenocytes to confirm that
observed effects are independent of T cell activation.
Suppression of proliferation assay
In addition to setting up control wells for this suppression assay, 15 compounds were tested at
1µg-10µg/mL with the exception of LS69, which was tested at 50ng-500ng/mL for being highly
cytotoxic at concentrations higher than the recommended dose. The co-cultures were incubated
in humidified 5% C0
2
at 37
0
C. On day 3, additional 100µL of complete media was added to each
well for survival and nutritional purposes. Spleen cell proliferation (30,000 events) was analyzed
after 4 days on a BD LSRII flow cytometer. The results were analyzed using Flowjo software
followed with ANOVA and Dunnett’s comparison tests.
IFN-γ ELISA
In order to examine the effects of chemical agents on the co-cultures and confirm selectivity to
Tregs, supernatants from each ex vivo suppression assay were frozen 4 days post-proliferation
and stored at -20
o
C. The supernatants were thawed and subject to BD Biosciences Murine IFN-γ
ELISA Kit II in order to measure functionality of cytotoxic effector cells in the co-cultures.

21
RESULTS
Density gradient and MACS columns were used to isolate CD4
+
CD25
+
T regulatory cells from
4T1 tumor-bearing mice spleen and tumor draining lymph nodes. Suppression assays were then
performed using a ratio of 1 Treg : 4 responder cells (isolated from the spleens of naïve BALB/c
mice) and cells were allowed to proliferate over a 4 day period in the presence of CD3/CD28
stimulation beads.

Figure 3.3. Assessment of the reversal of suppression via flow cytometry. (A) Proliferation of stimulated
splenocytes from naïve BALB/c mice in the presence of CD3/CD28 stimulation beads and the absence Tregs. (B)
Proliferation of non-stimulated naïve BALB/c splenocytes. (C) Suppression of proliferation of splenocytes from
naïve BALB/c mice via the CD4+CD25+ Tregs in the presence of CD3/CD28 beads and the absence of any
reagents. (D) CD4+CD25+ T regulatory cells obtained from 4T1 tumor-bearing BALB/c mice alone in complete
media. (E) Partial reversal of suppression induced via CD4+CD25+ Tregs/reduced proliferation of splenocytes in
the presence of Tregs, 1µg of OX86, CD3/CD28 beads. (F) Partial reversal of suppression induced via CD4+CD25+
Tregs/reduced proliferation of splenocytes in the presence of Tregs, 10µg of TY25, CD3/CD28 beads. (G) Partial
A
B
)
)
C
)
)
D
)
)
E
)
)
G
)
)
F
)
)
22
reversal of suppression induced via CD4+CD25+ Tregs/reduced proliferation of splenocytes in the presence of
Tregs, 1µg of RMPI-14, CD3/CD28 beads.

Fourteen reagents were tested at a concentration of 1ug-10ug/mL in order to test for selective
inhibition of Treg-dependent immune suppression, and intracellular CFSE dilution and flow
cytometry were utilized to quantify and examine effector cell proliferation (Figure 3.3). The
results were analyzed using Flowjo proliferation index followed by ANOVA and Dunnett's
comparison test. Of the 14 drugs tested, 3 drugs showed significant partial reversal of
suppression induced by T regulatory cells. OX86 at 1µg/mL, an agonist OX40 (a member of
Tumor Necrosis Factor Receptor family) antibody, 10µg/mL of TY25, a PDL1 antibody,
10µg/mL L-Buthionine-sulfoximine (BSO), a glutathione biosynthesis inhibitor, and 1µg/mL
RMPI-14, a PDL1 antibody demonstrated significant reversal of suppression when compared to
the control co-cultures (Figure 3.4).

23

Figure 3.4. Proliferation of T cells in response to CD3/CD28 stimulation beads. 14 compounds at 2 varying
concentrations were tested. The addition of OX86 at 1µg/mL, an agonist OX40 (a member of Tumor Necrosis
Factor Receptor family) antibody, 10µg/mL of TY25, a PDL1 & PDL2 antibody, 10µg/mL L-Buthionine-
sulfoximine (BSO), a glutathione biosynthesis inhibitor, and 1µg/mL RMPI-14, a PDL1 antibody, are shown to
individually cause partial reversal of suppression of responder cell proliferation.
Proliferation of T cells
Splen. +Treg
Stimulated Splen.
Non-stimulated Splen.
High-DTA-1
Low-DTA-1
A-Splen.-DTA-1
High-FGK45.5
Low-FGK45.5
Splen.-FGK45.5
High-TY25
Low-TY25
Splen.-TY25
0
50
100
150
*
*
*
*
*
*
*
*
Proliferation of T cells
Splen. +Treg
Stimulated Splen.
Non-stimulated Splen.
High-PC61
Low-PC61
Splen.PC61
High-BSO
Low-BSO
Splen.-BSO
High-fc-muOX40L
Low-fc-muOX40L
Splen.-fc-muOX40L
0
50
100
150
*
*
* *
*
*
*
* *
Proliferation of T cells
Splen. +Treg
Stimulated Splen.
Non-stimulated Splen.
High-OX86
Low-OX86
Splen.-OX86
High-RA8
Low-RA8
Splen.-RA8
High-RMPI-14
Low-RMPI-14
Splen.-RMPI-14
0
50
100
150
*
*
*
*
*
* *
*
Proliferation of T cells
Splen. +Treg
Stimulated Splen.
Non-stimulated Splen.
High-LS69
Low-LS69
Splen.LS69
High-fc-IFN-beta
Low-fc-IFN-Beta
Splen.fc-IFN-Beta
High-anti-CTLA
Low-anti-CTLA
Splen. anti-CTLA
0
50
100
150
*
*
*
*
*
*
*
Proliferation of T cells
Splen. +Treg
Stimulated Splen.
Non-stimulated Splen.
High-fc-mGITR
Low-fc-mGITR
Splen.fc-mGITR
High-fc-muCD40
Low-fc-muCD40
Splen.fc-muCD40L
0
50
100
150
*
*
*
*
*
*
24

Figure. 3.5: IFN-γ sandwich ELISA. The supernatants from each ex vivo suppression assay were frozen 4 days
post-proliferation and stored at -20
o
C prior to co-culture FACS analysis. The supernatants were thawed and subject
to IFN-γ ELISA. The results were analyzed per kit instructions (BD Biosciences Murine IFN- γ ELISA Kit II).

In order to examine further the effect of these compounds on cytotoxic CD8
+
cells, and to
determine whether the mechanism of action of these chemicals are selective to Tregs, we
performed a sandwich ELISA (BD Biosciences Murine IFN- γ ELISA Kit II) utilizing
supernatants frozen from our suppression assay co-cultures (Figure 3.5). Of the three compounds
that demonstrated partial reversal of suppression of T regulatory cells, TY25 and OX86 showed
significant secretion of IFN-γ. Comparing the results from control samples, there was no
significant changes in proliferation of stimulated splenocytes + TY25 and stimulated splenocytes
+ OX86. These data, as well as the partial reversal of suppression observed in our FACS analysis
of the suppression data suggest that TY25 and OX86 selectively inhibit T regulatory cells.

0.1
1
10
1 10 100 1000
Optical Density (450 nm)
INF-Gamma (pg/ml)
Spl + Treg + No Drugs
Stimulated Splenocytes
Spl + Treg + DTA-1
Spl + Treg + FGK 45.5
Spl + Treg + TY25
Spl + Treg + OX86
Spl + Treg + RA8
Spl + Treg + RMPI-14
Spl + Treg + LS69
Spl + Treg + Fc-IFN-beta
Spl + Treg + Anti-CTLA
Spl + Treg + PC61
Spl + Treg + BSO
Spl + Treg + Fc-muOX40L
Spl + Treg + Fc-muCD40
Spl + Treg + Fc-muGITR
Spl + Treg + Fc-muCD137L
25
DISCUSSION

In order to inhibit effectively immune escape and tolerance by means of immune suppression,
both inhibition of tumor-mediated immune suppression and ectopic enhancement of
physiological immune response would be necessary for effective immunotherapy.
These results demonstrated significant partial reversal of tumor-mediated immune suppression,
using a suppression assay consisting of Tregs to responder T cell ratios of 1:4, with in vivo ratios
of approximately 1:200. Our data collectively suggest the potential therapeutic applications of
several key chemical compounds in reversing Treg suppression as a means of effective
immunotherapy. This is crucial in combating tumor immune escape and tolerance.

Since the data shown here exhibit only partial reversal of immune suppression in response to
OX86, TY25, and RMPI-14, it will be imperative to identify other means of suppressing Treg
function or proliferation, in order to supplement and enhance the reversal of phenotype presented
here. As described by Colombo et al., tumor cells utilize the IDO pathway and TGF-β in order to
stimulate Treg cell expansion and regeneration. . Although TGF-β may serve as a stimulator of
Treg proliferation, its role in many other molecular pathways makes its manipulation
unfavorable in studying reversal of immune tolerance. As such, an IDO inhibitor can be utilized
in addition to treatment of Tregs with OX86, TY25, and RMPI-14 in order to observe whether
reversal of tumor-mediated immune suppression is enhanced when compared to treatment with
OX86, TY25, and RMPI-14 alone. A potent Treg inhibitor can also be linked to siRNA for
inhibition of secondary signal transduction such as a Treg monoclonal antibody linked to FoxP3
targeting siRNA. Selective targeting of Tregs with two simultaneous hits can pose a more
successful approach to reverse their suppressive effect on the immune system.
26
Recent studies have also suggested that while human Mesenchymal Stromal Cells (MSCs)-
generated Tregs and iTregs from ex vivo induction in the presence of ATRA, TGF-beta and IL-2
may share a similar phenotype (CD4+CD25+FoxP3+), their gene expression of immune
regulatory molecules displayed varying levels of FoxP3, CTLA-4, GITR based on the method of
Treg generation (Haddad et al., 2012). More studies are needed to characterize any differences
between Tregs generated beyond what is already known about nTregs vs. iTregs. This is
imperative for studying therapeutic efficacy of inhibitors and deriving conclusions from them for
translational medicine.

27
Chapter 4.
Future Directions

By performing ex vivo suppression assays using a novel panel of chemical agents, several
promising compounds, namely OX86, TY25, and RMPI-14, have been shown to reverse tumor-
mediated immune suppression. Although this ex vivo model provides useful information
regarding regulatory T cell function in response to the above mentioned chemical agents, it does
not take into account physiological interactions that may negate or accentuate the effects of these
drugs, thereby enhancing or minimizing their effectiveness as a potential therapeutic agent.
Therefore, the potential of OX86, TY25, and RMPI-14 to reverse tumor-mediated immune
suppression will be ascertained by treating subcutaneous tumor-implanted 4T1 mice with
systemic dosage of these chemical agents. Resultant tumor size will be measured every other
day, and survival will also be tracked to determine if these compounds possess the ability to
reverse the disease phenotype and immune suppression in vivo.

The ex vivo experiments shown previously demonstrate only partial reversal of tumor-mediated
immune suppression. We hypothesize that treatment of tumor-bearing mice with a combination
of the chemical agents identified earlier may serve to reverse more effectively the disease
phenotype and minimize tumor-mediated immune suppression. Different combinations of the
monoclonal antibodies OX86, TY25, and RMPI-14 can be tested and delivered to tumor-bearing
mice, and reversal of immune suppression can be compared for treatment with single agent and
combinations. In doing so, we will be able to identify which combination of these compounds
possess the highest therapeutic potential.

28
Although future experiments will seek to confirm successful reversal of immune suppression by
the chemical agents discussed above, it will be important to ensure that these chemical agents
due so by the inactivation of regulatory T cells, and not by inducing Treg apoptosis. Colombo et
al. have demonstrated that the elimination of CD25
+
Tregs will induce the conversion of naïve T
cells into Tregs in order to replenish this cell type (Colombo et al., 2007). Inducing apoptosis of
Tregs would only serve to use the available naïve T cells necessary to mount and inflammatory
response to generate new Tregs which would result in immune suppression, thereby negating any
positive effect expected from the immunotherapeutic treatment of the above mentioned chemical
agents. As such, it will be critical to confirm that upon treatment with OX86, TY25, and RMPI-
14, Tregs function is altered to allow loss of normal function without destruction or removal of
these cells. Furthermore, it will also be important to test OX86, TY25, and RMPI-14 on
CD4
+
CD25
+
adaptive Treg activity, to ensure that these Tregs are also inactivated or experience
loss of function as a means of ensuring effective reversal of tumor-mediated immune
suppression.

As seen in chapter one, successful immunotherapy treatment is multifactorial and can differ from
tumor model to tumor model. Previous literature demonstrates that tumor response to
immunotherapy regimens can differ widely from tumor to tumor, and that immunotherapy agents
do not necessarily reverse the phenotype among all tumors. The results showed here
demonstrated a distinct correlation between immunogenicity and response to immunotherapy.
Although in vivo treatment of one chemical agent or another may lead to sufficient reversal of
immune suppression to yield a reversal of phenotype, it will be important to confirm which
tumor models are affected by these agents and which ones do not respond to the chemical agents
29
prior to making any generalization about global immunotherapy potential and application as a
treatment regimen for human tumor-mediated immune suppression.

An innovating approach to reversal of tumor-induced immune suppression is the potential for
linking a potent Treg inhibitor with an siRNA that targets a secondary signal such as the
transcription factor FoxP3 or STAT3 pathway. If identified, this combination has the potential to
selectively inhibit tumor associated Tregs at one hit targeting cell surface inhibition as well as
intracellular secondary signal inhibition. There remains a tremendous need for understanding and
discovering an effective approach to cure cancer, and tumor-induced immune suppression as a
potent mechanism for tumor growth and survival cannot be ignored.

30
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33
Acknowledgements
I would like to thank Dr. Epstein for the opportunity to work on this project and his continuous
guidance. I would like to thank my committee members, Dr. Florence Hofman and Dr. Louis
Dubeau for their support. Last but not least, I would like to extend my appreciation to the
members of Epstein Lab in particular, Dr. Melissa Lechner for her excellent teaching skills and
contagious motivation, curiosity and perseverance in the field of medicine and immunotherapy
research.

Abstract (if available)

Abstract Tumor growth and propagation has been attributed to many important physiological processes, such as angiogenesis and epigenetic mechanisms. Immune suppression and tumor immune escape, however, are among the most highly utilized and regulated mechanisms of tumor growth and cancer cell proliferation. Myeloid Derived Suppressor Cells (MDSCs), Tumor Associated Macrophages (TAMs), and T regulatory Cells (Tregs) enhance cell to cell interactions and cytokine release in order to abrogate cytotoxic T cell activity as well as the activation and proliferation of CD4+ helper T cells, thereby permitting enhanced immune-mediated tolerance of tumor growth. ❧ Tregs are immune cells that co-express CD4 and CD25, and are responsible for regulating autoreactive T-cells in vivo in order to accomplish immune homeostasis. Adaptive Tregs have been shown to be induced by adenosine, IL-10, as well as indoleamine 2,3-dioxygenase (IDO) and TGF-b (Figure 3.1 A, Colombo et al., 2007) and are known to constitute between 5 - 10% of all CD4⁺ T cells in humans. Tregs are primarily regenerated by expansion of existing Tregs, but can be regenerated by the conversion of naïve T cells, a process that becomes the predominant means of Treg regeneration upon CD25⁺ depletion (Colombo et al., 2007). Peripheral tumor tolerance is known to be regulated by Tregs via inhibition, identification, destruction, and removal of neoplastic cells. By identifying potential chemical agents capable of selectively inhibiting Treg-dependent immune suppression, this study sought to partially reverse tumor cell immune tolerance.

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Asset Metadata

Creator Karimi, Saman Shanaya (author)

Core Title Suppressor cell therapy: targeting T regulatory cells in cancer

School Keck School of Medicine

Degree Master of Science

Degree Program Experimental and Molecular Pathology

Publication Date 08/06/2013

Defense Date 08/05/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cancer,effector cells,immunotherapy,OAI-PMH Harvest,suppressor cell therapy,Tregs

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Epstein, Alan L. (committee chair), Dubeau, Louis (committee member), Hofman, Florence M. (committee member)

Creator Email sskarimi@usc.edu,ssmnkarimi@gmail.com

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