The Roles of Interleukin-27 in Tumor Immunity

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University

Zhenzhen Liu

Program: Ohio State Biochemistry Program (OSBP)

* * * * *

The Ohio State University

2012

Dissertation Committee:

Professor Xue-Feng Bai, Advisor

Professor Lai-Chu Wu, Co-advisor

Professor Pravin Kaumaya

Professor Qianben Wang

Abstract

Interleukin-27 (IL-27) is a member of the IL-12 family of cytokines. IL-27 is a heterodimer consisting of an IL-12 p40-related protein subunit, EBV-induced gene 3

(EBI3) and a p35-related subunit, p28. IL-27 is mainly produced by activated antigen presenting cells. It functions through engaging IL-27 receptor, which is expressed on a variety of immune cell types, including CD4+ and CD8+ T cells. Overexpression of IL-27 by tumor cells exerts potent anti-tumor activity through diverse mechanisms, in which

CD8+ T cells were considered to be the main effector cells. However, the exact mechanisms by which IL-27 enhances anti-tumor CD8+ T cell response and leads to tumor rejection remain unclear.

The impacts of IL-27 on the differentiation and activation of CD8+ T cells were studied by stimulating naïve tumor antigen-specific CD8+ T cells (P1CTL) with cognate P1A peptide in the presence and absence of IL-27. First, T cell proliferation and apoptosis were examined by thymidine incorporation and Annexin V/7-AAD staining, respectively.

Second, expressions of activation and differentiation markers of T cells were analyzed by

Real time PCR and Western Blotting. Third, cytokine production was evaluated by

ELISA and flow cytometry. To investigate the in vivo roles of IL-27 in tumor immunity, mouse models involving tumor cells (J558 plasmacytoma and B16 melanoma) expressing

IL-27 and IL-27-deficient (EBI3-/-) mice were used. To delineate the mechanisms by

ii which IL-27 enhances antitumor CTL responses, tumor antigen specific CD8+ T cells were adoptively transferred into various genetically engineered mice with established tumors, and their in vivo proliferation and apoptosis were studied by CSFE staining and flow cytometry analysis. To examine the role of IL-27 in response and function of T regulatory cells, Anti-CD25 antibody was used to deplete Treg cells; IL-27 deficient or

WT CD4+CD25+ Treg cells were adoptively transferred together with effector T cells into tumor-bearing mice; tumor establishment and metastases were evaluated by monitoring s.c. tumor growth and weighing lungs, respectively.

Overall, we have uncovered four novel findings that can explain why IL-27 boosts antitumor CD8+ T cell responses:

1) IL-27 enhances the survival of activated tumor antigen specific CD8+ T cells both

in vitro and in vivo.

2) IL-27 induces a unique memory precursor cell (MPC) phenotype in activated

tumor antigen specific CD8+ T cells, which is characterized by up-regulation of

SOCS3, Bcl-6, Sca-1 and IL-10.

3) IL-27 robustly induces IL-10 production by tumor antigen specific CD8+ T cells,

which contributes to IL-27-mediated tumor rejection in vivo.

4) IL-27 inhibits the expansion and immunesuppressive ability of CD4+FoxP3+ T

regulatory cells, resulting in more potent anti-tumor CTL responses.

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Our findings suggest that:

1) IL-27 has the potential to be used as an adjuvant to boost the efficacy of antitumor

vaccines;

2) IL-27 can be used to culture tumor antigen-specific CTLs for adoptive transfer

therapy of cancer patients.

Dedication

To my Parents, Lierong Chen and Xiancheng Liu, who gave me life, taught me to be independent and grateful, and support me to pursuit my dreams.

To my Advisor, Dr. Xue-Feng Bai, who gave me the chance to continue my Ph.D. study in the U.S., brought me into the exciting world of tumor immunology, and taught me how to employ the power of immunology to do cancer research. His unconditional support, enthusiasm, and encouragement have been instrumental to the success of this project. His style of decision-making, problem-solving and taking responsibility for his students will also serve as my role model for my future career.

To my Boyfriend, Andreas Schick, who has been my rock, who has kept me going and always been positive and encouraging through the rough patches and the good ones as well.

Acknowledgments

It is with sincerest gratitude that I would like to acknowledge the following people who have been instrumental in helping me to achieve my goal of Ph.D.

 Dr. Jin-Qing Liu, for her assistance with experiments and sharing her experience.  Dr. Lai-Chu Wu, a special thank to you. For your support, your encouragement, and for truly being there for me.  Dr. Fatemeh Talebian, my lab partner, for her friendship, lively discussions, and assistance with experiments and training. It was a much better journey since we were able to share it.  Dr. Pravin Kaumaya, for everything you have done for me. Thank you for believing in me when I was going through that tough time.  Dr. Qianben Wang, for all your support and understanding, for the knowledge and encouragement for making a better future for myself. I am deeply grateful and appreciative

for making me open my mind to my future capacity.  Dr. Shulin Li, for his generousness providing me with Ad-IL-27, for his guidance on my Ph.D. study, and for his unhesitant sharing life experience with me.  Dr. Kevin Foy and Megan Miller, for their helps and kindness that walked me through the darkness. I will always remember the time we were together in Dr. Kaumaya’s lab.  Dr. Zhong Chen and Dr. Wen Yi, for their academic comments and suggestions on my Ph.D. research, for I might not have gone the distance without your guys support.  Dr. Ying An and Dr. Li Ma, for their support and friendship that carried me through some tough times. It would have been much more difficult without them.  I reserve my deepest appreciation and gratitude to my family, friends and loved ones who gave their unlimited stores of love and support. Thank them for reminding me “I Can Do It”.  Last but certainly not least, I thank my mother and father, Lierong and Xiancheng, for everything they have given me, all they have done for me, and all they still do. Thank them for all the meaning they have infused into my life and all that they keep on giving.

VITA

Born: May 14th, 1983 Shiyan, Hubei Province, China

2001-2005: China Agriculture University, Beijing, China

College of Animal Science and Technology

Bachelor’s of Science

Major: Animal Science and Technology

2005-2007: China Agriculture University, Beijing, China

Department of Animal Genetics and Reproduction

Master’s of Science

Major: Animal Genetics

2007-2010: The Ohio State University, Columbus, Ohio USA

Ohio State Biochemistry Program

Master’s of Science

Major: Biochemistry

2007-2012: The Ohio State University, Columbus, Ohio USA

Department of Pathology

Doctor of Philosophy

Fields of Study: Cancer Immunology, Molecular and Cellular biology

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RESEARCH EXPERIENCE Research Lab Work Experience The Ohio State University, Department of Pathology/OBGYN Columbus, OH Graduate Research Associate in Cancer Immunology 2008 – Present Project 1: The roles of Interleukin-27 in tumor immunity  Generated IL-27-overexpression tumor cell lines  Conducted a variety of immuno-based and biochemical in vitro assays to test the effects of IL- 27 in the differentiation and function of tumor specific CD8+ cells  Performed adoptive transfer of tumor specific CD8+ T cells to tumor bearing mice  Analyzed the IL-27-induced immune responses to established local plasmacytoma (J558) and metastatic melanoma (B16) on various transgenic mouse models.  Investigated the mechanisms by which IL-27 enhances antitumor CTL responses and leads to tumor regression Project 2: The interaction of CD200-CD200R in tumor immunity  Examined CD200/CD200R expression on myeloid cells and T cells under various conditions  Studied the impact of tumor expression of CD200 on tumor formation and metastasis, using the CD200-positive tumor cells, various transgenic mouse models, in vivo cell depletion, and adoptive cell therapy  Discovered that high CD200 expression on human cancer cells may lead to long survival time through analyzing microarray data Project 3: Th17 and Treg responses in EBI3-deficient mice Characterized the dynamic changes of CD4+ T cell subsets in EBI3-/- mice with EAE disease  Conducted in vitro and in vivo T cell proliferation assay to evaluate the suppressive effect of EBI3-/- Treg Project 4: Combination treatment with HER-2 and VEGF peptide mimics induces potent anti-tumor and antiangiogenesis responses  Designed and synthesized novel peptide mimics: HER2 and VEGF  Evaluated the efficacy of the two peptide mimics as a combination treatment for breast cancer both in vitro and in vivo.

China Agriculture University, Department of Animal Genetics Beijing, China Graduate Research Associate in Animal Genetics and Reproduction 2004 – 2007 Project 1: Molecular Mechanism of Chromium in Promoting Chicken Growth In order to understand the molecular mechanism by which chromium enhances the action of insulin and subsequently promotes chicken growth, I examined carcass traits, insulin and glucose in the serum, and mRNA expression of genes in the insulin signaling pathway, such as insulin, IR, IRS and IGF-1. Project 2: Sex-reversed Chickens Induced by Aromatase Inhibitor The purpose of this study is to understand the mechanism of avian sex determination and differentiation. Female chicken treated with aromatase inhibitor can ejaculate fully fertile spermatozoa. In addition, W sperms are present in sex-reversed chicken and they are able to reproduce healthy baby chickens. Project 3: Analysis of SNP Markers for Blue-shelled Gene in Chicken by PCR-SSCP In order to understand the cause of eggshell pigmentation biosynthesis, a bioinformatics approach was first used to blast the genes for enzymes involved in porphyrin pathway. PCR-SSCP was used to search for candidate SNP markers for blue-shelled gene.

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Research Awards  Junior Investigator Research Award. The TRCCC 15th Annual Meeting: Cancer Vaccine and Immune Regulation, Seven Springs, PA. Feb 22-24, 2012.  Best Oral Presentation Award. 6th Annual Interdisciplinary Graduate Programs Symposium, Columbus, OH. May 23th, 2012

PUBLICATIONS 1. Zhenzhen Liu, Jin-Qing Liu, Fatemeh Talebian, Shulin Li, Xue-Feng Bai. IL-27 enhances survival of tumor antigen specific CD8+ T cells and programs them into IL-10 producing memory precursor cells. (European Journal of Immunology in press)

2. Zhenzhen Liu, Jin-Qing Liu, Zhihui Wang, Lai-Chu Wu, Xue-Feng Bai. IL-27 inhibits melanoma tumor establishment and metastasis through suppressing Foxp3+ CD4+ Treg cell development. (manuscript in preparation)

3. Kevin C. Foy, Zhenzhen Liu, Sharad Rawale, Gary Phillips, Megan Miller, Aravind Menon, Nina D. Osafo, Pravin T.P. Kaumaya. Combination treatment with HER-2 and VEGF peptide mimics induces potent anti-tumor and antiangiogenic responses in vitro and in vivo. J. Biol. Chem. 2011 286: 13626-13637.

4. Jin-Qing Liu, Zhenzhen Liu, Joseph W. Carl Jr., Xuejun Zhang, Fatemeh Talebian, Fu-Dong Shi, Caroline C. Whitacre, Joanne Trovich, Xue-Feng Bai. Enhanced Th17 and Treg responses in EBI3-deficient mice lead to marginally enhanced development of autoimmune encephalomyelitis. J. Immunol. 2012; 188(7):3099-106.

5. Fatemeh Talebian, Jin-Qing Liu, Zhenzhen Liu, Mazin Khattabi, Xue-Feng Bai. Cancer cell expression of CD200 inhibits tumor formation and lung metastasis through inhibition of the myeloid cell functions. PLoS ONE. 2012; 7(2):e31442.

6. Zhihui Wang, Jin-Qing Liu, Zhenzhen Liu, Xue-Feng Bai. IL-35 promotes tumor growth via multiple mechanisms. (Journal of Immunology in revision)

7. R. Zhao, G.Y. Xu, Z.-Z. Liu, J.Y. Li, N. Yang. A Study on Eggshell Pigmentation: Biliverdin in Blue-shelled Chickens. Poultry Science. 2006, 85:546–549.

8. Zhao Rui, Liu Zhenzhen, Xu Gui-yun, YANG Ning. Analysis of SNP Markers for Blue-shelled Gene in Chicken by PCR-SSCP. Chinese Journal of Agricultural Biotechnology. 2006, 14(5): 673- 676.

9. J.X. Zheng, Z.Z. Liu, N. Yang. Deficiency of Growth Hormone Receptor Does Not Affect Male Reproduction of Dwarf Chickens. Poultry Science. 2007, 86:112-117.

10. Zheng Jiang-xia, Liu Zhenzhen, Yang Ning. Effects of Growth Hormone Receptor Gene Mutation on the Semen Quality of Sex-linked Dwarf Chicken. Chinese Journal of Animal Science. 2006, 42(23): 1-3.

Table of Contents

Abstract ...... ii Dedication ...... v Acknowledgments...... vi VITA ...... vii List of Tables ...... xiii List of Figures ...... xiv Abbreviations ...... xv Chapter 1 Introduction ...... 1 1.1 Immunotherapy for the treatment of cancer ...... 1 1.1.1 General principles of cancer immunotherapy ...... 1 1.1.2 Cytokine therapy of cancer ...... 2 1.2 Interleukin (IL)-27 ...... 7 1.2.1 The history of IL-27 ...... 7 1.2.2 The structure and expression of IL-27 ...... 8 1.2.3 The structure and expression of IL-27 Receptor ...... 11 1.2.4 IL-27/IL-27R signaling ...... 12 1.3 The roles of IL-27 in immune regulation ...... 13 1.3.1 The controversial roles of IL-27 in Th1 response ...... 13 1.3.2 The inhibitory role of IL-27 in Th2 response...... 16 1.3.3 The inhibitory role of IL-27 in Th17 response ...... 17 1.3.4 The role of IL-27 in the development of T regulatory cells ...... 18 1.3.5 The immunostimulatory role of IL-27 in CD8+ cell response...... 20 1.3.6 The roles of IL-27 on other cells ...... 21 1.4 The antitumor activities of IL-27 ...... 23 1.4.1 Murine colon carcinoma ...... 24 1.4.2 Murine neuroblastoma TBJ ...... 25 1.4.3 Murine melanoma B16F10 ...... 27

1.4.4 Murine head and neck squamous cell carcinoma SCCVII ...... 29 1.4.5 Murine Lewis lung carcinoma LLC-1...... 30 1.4.6 Murine Prostate Tumor ...... 30 1.4.7 Human Multiple Myeloma ...... 31 1.4.8 Pediatric B-acute lymphoblastic leukemia ...... 32 1.4.9 Pediatric acute myeloid leukemia ...... 32 1.4.10 Intracranial Gliomas GL-26 ...... 33 1.5 The biological roles of interleukin-10 in tumor immunity ...... 34 1.5.1 The pro-tumor effects of IL-10 ...... 35 1.5.2 The antitumor effects of IL-10 ...... 37 1.6 Tumor infiltrated lymphocytes (TIL) in the tumor microenvironment ...... 38 1.6.1 The anti-tumor effector T cells in the tumor microenvironment and adoptive cell therapies (ACT) ...... 38 1.6.2 Memory CD8+ T cells in tumor immunology and immunotherapy ...... 40 1.6.3 T regulatory (Treg) cells in the tumor microenvironment ...... 42 1.7 Hypothesis and goals of the study...... 45 Chapter 2 Experimental Materials and Methods ...... 47 2.1 Mice ...... 47 2.2 Real time RT-PCR ...... 48 2.3 Antibodies and flow cytometry ...... 49 2.4 ELISA ...... 50 2.5 Western Blot ...... 50 2.6 Cancer cell lines and tumor establishment in mice ...... 51 2.7 T cell culture ...... 52 2.8 T cell adoptive transfer ...... 52 2.9 In vivo T cell proliferation assay ...... 53 2.10 CD8+ T cell and NK cell depletion ...... 53 2.11 CD25+ Treg cell depletion ...... 53 2.12 Isolation of CD4+CD25+ Treg cells from spleens ...... 54 2.13 Treg-mediated suppression assay...... 54 2.14 Flow cytometry-based cytotoxicity assay (FloKA) ...... 54 2.15 Lentivector immunization and Adenovector treatment ...... 55 2.16 Statistical analysis ...... 56

Chapter 3 IL-27 enhances tumor antigen specific CD8+ T cell responses and leads to tumor rejection via multiple mechanisms ...... 57 3.1 IL-27 enhances survival of tumor antigen specific CD8+ T cells and programs them into IL-10 producing memory precursor-like effector cells ...... 57 3.1.1 IL-27 enhances survival of tumor antigen specific CD8+ T cells ...... 57 3.1.2 IL-27 stimulates a large amount of IL-10 production by tumor antigen specific CD8+ T cells ...... 60 3.1.3 IL-27-induced CTL IL-10 production is not essential for its pro-survival effect .. 64 3.1.4 IL-27-induced CTL IL-10 production contributes to tumor rejection ...... 66 3.1.5 IL-27/IL-10 axis induces a memory precursor/effector phenotype in tumor antigen specific CD8+ T cells ...... 69 3.1.6 IL-27-induced CTL IL-10 production contributes to T cell memory ...... 72 3.2 IL-27 inhibits tumor growth and metastasis through suppressing the response and function of Treg cells ...... 76 3.2.1 Tumor-derived IL-27 reduces the frequency of tumor infiltrated Treg cells ...... 76 3.2.2 IL-27 deficiency results in more infiltrated Treg cells and faster tumor establishment...... 78 3.2.3 IL-27 suppresses Treg cell response through inhibiting IL-2 production ...... 80 3.2.4 Depletion of Treg cells enhances antitumor immune response and tumor rejection in EBI3-/- mice ...... 82 3.2.5 Enhanced suppressive function of Treg cells in EBI3-/- mice ...... 85 3.3 Using IL-27 is a promising strategy for cancer immunotherapy ...... 88 3.3.1 EBI3-deficiency impairs the efficacy of tumor antigen vaccination...... 88 3.3.2 Intra-tumoral injection of IL-27 producing adenovirus enhances anti-tumor immunity 90 Chapter 4 Discussion ...... 93 4.1 IL-27 enhances survival of tumor specific CD8+ T cells both in vitro and in vivo ...... 95 4.2 IL-27 programs activated CD8+ T cells to become memory precursor-like effector cells 96 4.3 IL-27 induces a large amount of IL-10 production by activated tumor antigen specific CD8+ T cells, which contributes to CTL-mediated tumor rejection ...... 99 4.4 IL-27 suppresses T regulatory cell response in the tumor microenvironment ...... 101 4.5 Concluding remarks and future directions ...... 105 Reference ...... 109

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List of Tables

Table 1 Current cytokine therapies in clinical trials ...... 4 Table 2 List of studies of IL-27 in tumor immunity ...... 34

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List of Figures

Figure 1 The Structures of IL-12 family members and their receptors ...... 10 Figure 2 Progressive T cell differentiation diminishes proliferative and antitumor capacities...... 42 Figure 3 The role of IL-27 in the survival of activated P1CTLs ...... 58 Figure 4 The role of IL-27 in P1CTL IL-10 production in tumor microenvironment ...... 61 Figure 5 The roles of IL-27 in CTL IL-10 production and effector functions ...... 63 Figure 6 The role of IL-27 in the survival of activated IL-10-/- P1CTL cells ...... 65 Figure 7 The role of IL-27-induced CTL IL-10 production in tumor rejection ...... 68 Figure 8 Phenotypes of IL-27 stimulated tumor antigen specific CD8+ T cells ...... 71 Figure 9 The role of IL-27-induced IL-10 production in T cell memory ...... 74 Figure 10 Tumor-derived IL-27 reduces the frequencies of Treg cells in both DLNs and tumors 77 Figure 11 IL-27 deficiency impairs antitumor immune response and leads to faster tumor growth and metastasis ...... 79 Figure 12 IL-27 inhibits IL-2 production by both CD4 and CD8 T cells ...... 81 Figure 13 Treg depletion enhances antitumor CTL responses and tumor rejection in EBI3-/- mice ...... 84 Figure 14 Increased number and suppressive function of CD4+Foxp3+ cells in EBI3-/- mice ...... 86 Figure 15 EBI3-deficiency impairs tumor specific CTL responses in vaccinated mice ...... 89 Figure 16 Intra-tumoral injection of Ad-IL-27 enhances anti-tumor immunity ...... 91 Figure 17 The impacts of IL-27 signaling on CD8+ and CD4+ T cells in the tumor microenvironment ...... 108

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Abbreviations

cDNA Complementary DNA mRNA messenger RNA mAb Monoclonal Antibody

EBI3 Epstein Barr Virus-Induced Gene 3

Kb Kilo bases

TLR Toll-like receptor

ROS Reactive Oxygen Species

RNS Reactive Nitrogen Species

NOS Nitric Oxide Synthase

ITIM Immunoreceptor Tyrosine-based Inhibitory Motif

JAK Janus Kinase/Just another kinase

TYK Tyrosine kinase

ERK Extracellular signal regulated Kinase

STAT Signal Transducers and activators of Transcription

ICAM Intracellular Adhesion Molecule

DLN Draining Lymph node

MHC Major Histocompatibility Complex

CIA Collagen Induced Arthritis

RA Rheumatoid Arthritis

EAE Experimental Autoimmune Encephalomyelitis

B-CLL B cell Chronic Lymphoproliferative Leukemia

AML Acute Myeloid Leukemia

ALL Acute Lymphocytic Leukemia

PCM Plasma Cell Myeloma

BCLD B cell Lymphoproliferative Disease

HNSCC Head and Neck Squamous Cell Carcinoma

SCID Severe Combined Immunodeficiency

NK Natural Killer

TAMC Tumor Associated Myeloid Cells

MDSC Myeloid Derived Suppressor Cells

TAM Tumor Associated Macrophages

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IMC Immature Myeloid Cells

TIL Tumor Infiltrating Lymphocytes

CTL Cytotoxic T Lymphocyte

Tr1 Type I regulatory cells

CAF Cancer Associated Fibroblasts

TEC Tumor Endothelial Cells

DCs Dendritic Cells

TE Terminal Effector T cells

MPC Memory Precursor Cells

TCM Central memory T cells

TEM Effector memory T cells

TSCM T memory stem cells

BM-NSCs Bone marrow derived neural stem-like cells

GM-CSF Granulocyte Macrophage Colony Stimulating Factor

IL Interleukin

IFN Interferon

IRF IFN Regulatory Factor

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TNF-α Tumor Necrosis Factor- α

TGF-β Transforming Growth Factor beta

TCR T Cell Receptor

CNTFR Ciliary Neurotrophic Factor Receptor

ELISA Enzyme Linked Immunosorbent Assay

LPS Lipopolysaccheride

PEG Polyethylene Glycol poly(I:C) Polyinosinic:polycytidylic acid

ACT Adoptive Cell transfer c-Maf avian musculoaponeurotic fibrosarcoma v-maf

SOCS3 Suppressor of Cytokine Signaling 3

Sca-1 Stem Cell Antigen-1

Bcl B-Cell Lymphoma

Eomes Eomesodermin

T-bet Th1-specific T box transcription factor

Foxp3 Forkhead Box P3

ICOS Inducible Costimulatory Molecule

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RANKL Receptor Activator of Nuclear Factor Kappa-B Ligand

TIM3T T cell Immunoglobulin and Mucin domain-containing protein 3

LAG3 Lymphocyte Activation Gene 3 protein

PD-1 Programmed Cell Death Protein 1

CTLA-4 Cytotoxic T lymphocyte Antigen 4 (CTLA-4)

TRP-1 Tyrosine Related Protein-1

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Chapter 1 Introduction

1.1 Immunotherapy for the treatment of cancer

1.1.1 General principles of cancer immunotherapy

Besides radiation, chemotherapy, surgery, immunotherapy is another promising approach for treating cancers nowadays. Immunotherapy is the treatment that employs the power of immune system to attack cancer cells and interfere with their out-of-control growth. It includes a wide array of treatments which can be divided into two categories: active immunotherapy and passive immunotherapy. Active immunotherapies, such as cancer vaccines and adjuvant immunotherapies, aim to induce and stimulate the host immune system to work more efficient or longer to kill cancer cells. Passive immunotherapies, such as administration of monoclonal antibodies and adoptively transfer of spontaneous- occurring or genetic-engineered tumor antigen specific T cells, provide patients with autologous or man-made immune components to attack their cancer cells. Given active and passive immunotherapies respectively empower some parts of immune system, combination immunotherapies have shown to be more effective to activate multiple cellular constituents of immune surveillance which results in delayed tumor growth or even complete tumor rejection. Furthermore, immunotherapies are also utilized together with other types of treatments, such as radiation and chemotherapy, to acquire better efficacy in fighting cancers in current regime. Some treatments, such as Interferon-alfa

(IFN-α), interleukin-2, and monoclonal antibodies are now routinely used in cancer therapy. Immunotherapeutic drugs are now used to treat a variety of cancers, including cancers of the bladder, breast, colon, kidney, lung, and prostate, as well as leukemia, lymphoma, multiple myeloma, and melanoma.

1.1.2 Cytokine therapy of cancer

Established tumors are complex masses that contain heterogeneous cell populations including tumor cells, stromal fibroblasts, endothelial cells, and infiltrating leukocytes, such as dendritic cells, macrophages, natural killer cells, and T lymphocytes. During tumorigenesis, they produce a variety of soluble proteins or glycoproteins, which are known as cytokines. Cytokines act in an autocrine or paracrine fashion regulating the growth, differentiation and activation of immune cells, as well as modulating the crosstalk between tumor cells and cancer associated fibroblasts (CAFs), tumor endothelial cells (TECs) or tumor infiltration lymphocytes (TILs). The interactions between various cell types in the tumor microenvironment determine the effects of cytokines on tumor development and progression. Upon tumor initiation, both proinflammatory and anti-inflammatory cytokines are produced by activated myeloid cells.

Proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-17 often lead to the inhibition of antitumor immunity and the acceleration of tumor progression. Nevertheless, other pro-inflammatory cytokines, such as Interferon

(IFN)-γ, IL-2, IL-12, and IL-15, have been shown to enhance antitumor immunity and stop cancer cells from growing. Anti-inflammatory cytokines like IL-10 has been reported to act as a double-edged sword on tumor growth depending on the source and amounts of IL-10 produced. Although cytokines play an important role in tumor

2 pathogenesis, the extensive pleiotropism and apparent redundancy of cytokine actions, and dual functions of some cytokines in both immune stimulation and suppression, raise the difficulties to determine their exact functions in the tumor microenvironment.

Therefore, gaining a more complete understanding of cytokine networks is the current challenge regarding these molecules.

As the mixture of cytokines present in the tumor microenvironment plays an essential role in tumor development, therapeutic manipulation of cytokines constitutes one strategy to promote anti-tumor immunity. In general, there are four types of cytokine-based immunotherapies: systemic administration, direct tumor injection, cytokine-based tumor cell vaccine, and the use of cytokines as adjuvants in cancer vaccine and adoptive cell therapies.

1.1.2.1 Systemic administration of cytokines

The systemic administration of individual cytokines, such as IFN-α, IL-2, IL-7, IL-12,

IL-15, IL-18, and GM-CSF, have been employed in clinical trials for patients with advanced cancer. IFN-α has been considered as the most effective antitumor cytokine in treating melanoma and some hematological malignancies[1]. High dose, bolus IL-2 administration results in a defined objective clinical response in 15-20% of melanoma and renal cell carcinoma patients [2, 3]. Low doses of IL-2 are less toxic and increase the numbers of NK cells [4], while clinical benefits are limited. To date, IFN-α and IL-2 have achieved FDA approval as single agents for cancer treatment. The infusion of IL-12 provokes striking antitumor activities in murine models of colon carcinoma, melanoma, renal cell carcinoma (RCC), and mammary carcinoma and sarcoma [5-9], but its

3 objective response rate is less than 5% in a melanoma Phase I clinical trial [10]. Systemic administration of GM-CSF confers some extent of clinical advantages in melanoma, prostate cancer and pulmonary metastasis [11-13]. Overall, their high dose administrations are always associated with substantial toxicities, and the clinical benefits were not very ideal. Therefore, more research needs to be done to overcome these obstacles.

Table 1 Current cytokine therapies in clinical trials

Cytokine Drug Application Clinical trial phase IL-2 adenoviral-IL-2 renal cell carcinoma Phase I/II soft tissue carcinoma Phase I/II melanoma Phase I/II head and neck cancer Phase I/II

IL-2 MVA-MUC1-IL-2 kidney cancer Phase II prostate cancer Phase II lung cancer Phase II

IL-4 IL-4 coupled to exotoxin malignancy glioma Phase II kidney/prostate cancers Phase I

IL-6 humanized anti-IL-6R myeloma Phase I/II

IL-12 recombinant IL-12 lymphoma Phase II

IL-18 recombinant IL-18 melanoma Phase II renal cell carcinama Phase II lymphoma Phase I

IL-21 recombinant IL-21 metastic melanoma Phase I renal cell carcinama Phase I

IFN-α PEG recombinant IFN-α leukemia Phase III melanoma Phase III lymphoma Phase II

GM-CSF recombinant GM-CSF leukemia Phase II melanoma Phase II prostate cancer Phase II

1.1.2.2 Intratumoral injection of cytokines

In attempt to maximize the effects of cytokine in a physiological way, some researchers injected the cytokine, such as IL-2, directly into the tumor mass, which led to tumor rejection [14]. As the emergence of gene transfer techniques, researchers developed a variety of innovative strategies for delivery of therapeutic cytokines. They can be done in four ways: 1) cytokine-antibody fusion molecules; 2) chemical conjugation to polyethylene glycol (PEGylation) to improve the kinetics of the cytokine; 3) directly injecting recombinant viral vector encoding certain cytokine into the tumor; 4) ex vivo transfecting cancer cells with viral vectors encoding cytokine genes, such as GM-CSF and IL-12 [15, 16], and then inoculating them into syngeneic mice. Indeed, antitumor immune responses triggered against genetically modified tumor cells have been documented in a number of murine models. These models have generally focused on cytokine-mediated antitumor T cell responses, including IL-12[17], IL-18[17], IL-15[18],

IL-21[19], IL-23[20, 21], and IL-27[22]. In all, all mentioned approaches allow for the localization of cytokines to the tumor site, promoting the expansion and effector function of neighboring immune cells and resulting in tumor rejection.

1.1.2.3 Cytokine-based tumor cell vaccine

Autologous or allogeneic cytokine-releasing tumors have been used as cellular vaccines that augment systemic immunity against wild type tumors. Immunization with GM-

CSF/IL-12–overexpressing myeloma cells caused highly efficient and prolonged cross- protection of mice from challenge with myeloma cells [23].

1.1.2.4 The use of cytokines as adjuvants in cancer vaccine and adoptive cell therapies

Current cancer vaccine strategies can induce objective tumor regressions in only a small minority of cancer patients. Cytokines have been used as adjuvants to augment the immune responses triggered by the cancer vaccines. High dose of IL-2 was used together with HLA-A2-restricted gp-100 peptide in HLA-A2-positive patients with metastatic melanoma [24-26]. GM-CSF was fused with a prostate cancer antigen, which is then loaded into autologous peripheral blood monocytes, and lastly the treated monocytes were put back to the patients. This approach helps dendritic cell-based vaccine to elicit stronger immune responses against cancer cells.

In current adoptive cell therapy regimen, cytokines have also been widely used as adjuvant to optimize its efficacy. IL-1β, IL-4, IL-6, TNF-α, and GM-CSF are used in the development and activation of dendritic cells; IL-2, IL-7, IL-15, and IL-21 are used in the stimulation and expansion of T cells; IL-2 is also utilized to enhance the survival and maintain persistence of adoptively transferred T cells by systemic administration after T cell infusion [27]. To date, IL-2 treatment combined with T cell transfer has become a standard procedure of adoptive cell therapies to cancer patients.

Although cytokine-based immunotherapies for treating tumors have shown objective success in a variety of murine models and even in patients with certain tumors, severe toxicities are associated with these immunotherapeutic agents. Therefore, application of novel cytokines with potent antitumor activity but devoid of systemic toxicity is a promising alternative approach. Furthermore, characterizing cytokine pathways or networks in the tumor microenvironment is necessary to delineate how they affect tumor

6 growth and progression, and in turn to provide the rational for implementing new immunotherapies.

In this study, we examined the antitumor effects of a newly identified cytokine, IL-27, and studied the mechanisms by which IL-27 enhances antitumor CTL responses and tumor rejection. In addition, we demonstrated the delicate relationships among three cytokines (IL-27, IL-10 and IFN-γ) and how they affect different types of tumor initiation, progression and metastases. The study provides more insight and evidence of their functions in tumor immunity.

1.2 Interleukin (IL)-27

1.2.1 The history of IL-27

The EBI3 gene (Epstein Barr virus-induced gene 3) was identified by the induction of its expression in B lymphocytes by Epstein-Barr virus infection [28]. It encodes a 34-kDa soluble glycoprotein belonging to the hematopoietic receptor family related to the IL-12 p40 subunit and the ciliary neurotrophic factor receptor (CNTFR). Six years later, Pflanz et al. discovered the p28 subunit through searching sequence database [29]. EBI3 and p28 form a stable heterodimeric protein complex, which was designated as IL-27. Since IL-27 is structurally similar to IL-12, it was classified into the IL-12 cytokine family.

1.2.2 The structure and expression of IL-27

IL-27 is a heterodimeric cytokine composed of two subunits: EBI3 and p28 [29]. Human

EBI3 gene is localized on chromosome 19, band p13.3, and encodes a 229 amino acid polypeptide. Murine EBI gene encodes a 228 amino acid polypeptide, which is 96% identical to human EBI3. While EBI3 is only 27% identical to the IL-12 p40 subunit, it has conservative substitutions at many other residues. Similar to IL-12 p40 and in contrast to the other members of IL-12 cytokine family, EBI3 lacks a membrane- anchoring motif and is secreted readily by itself [28]. Human p28 gene is located on chromosome 16p11, and encodes a 243 amino acid polypeptide. In contrast, murine p28 cDNA sequence encodes a 234 amino acid polypeptide. Human and murine p28 are 73% identical. Unlike EBI3, p28 either is not or is inefficiently secreted on its own unless co- expressed with EBI3 [29]. EBI3 and p28 are linked by a covalent bond instead of a disulfide bond, which has been seen to link the subunits of IL-12 and IL-23. The structural difference allows for the production of the two subunits, EBI3 and p28, by different cells followed by extracellular association.

Similar to IL-12 and IL-23, coexpression of EBI3 and p28 within the same cell is required for IL-27 to exert its biological functions [29]. Early studies demonstrated that activated antigen-presenting cells are the main source of both human and murine IL-27.

The expression of human IL-27 has been detected in monocytes, DCs derived from monocytes, endothelial cells, and trophoblast cells [29, 30], while the expression of murine IL-27 has been detected in activated macrophages and microglia cells [29, 31].

Although the regulation of IL-27 expression has not been studied in great details, it has been shown that signaling via toll-like receptors (TLR) is a key inducer of IL-27.

Stimulation of macrophages with agonists [poly (I:C)], LPS, R848 for TLR3, TLR4, and

TLR7/8 correspondingly resulted in concurrent expression of EBI3 and p28 [32]. EBI3 expression in DCs was substantially reduced in the absence of TLR2, TLR4, TLR9, or

MyD88, suggesting that TLR stimulation is required for IL-27 expression [33]. In addition, EBI3 expression in DCs was also shown to be induced via activation of the transcription factors NF-B and PU-1 [33]. Human IL-27p28 mRNA was preferentially induced by Toll/IL-1R-containing adaptor inducing IFN-beta-coupled TLR ligands and following CMV infection, and IRF3 activation is a master switch for IL-27 synthesis [34].

Besides TLR ligands, a variety of host derived factors, such as CD40L, IL-1, IFN-,

IFN-and IFN-can upregulate the expression of IL-27 as well [32, 35]. In contrast, the nucleotide ATP negatively regulates the expression of IL-27 [36]. In all, IL-27 is a cytokine produced mainly by antigen presenting cells (APC) under immune-stimulating conditions.

However, EBI3 and p28 are not always expressed together in the same cell, and some factors preferentially induce one or the other component of IL-27 [29, 31, 37]. EBI3 is expressed at a high level in human B lymphoblast cell lines transformed in vitro by EBV;

Along with IL-12 p35 and p19, EBI3 were found to be expressed by human intestinal epithelial cells [38]; EBI3 was also found in the placenta, where IL-27p28 was barely detected [28]. In DCs, mRNA expression levels of EBI3 and IL-27p28 were induced at different pace upon E. Coli stimulation. p28 was up-regulated first peaking at 6-12 h, while EBI3 was induced more slowly peaking at 18-24 h [36]. Furthermore, EBI3 has been found to associate noncovantly with IL-12 p35 to form another IL-12 cytokine family member, IL-35 [39]. This finding increases the difficulties in studying the exact

9 role of IL-27 in tumor immunology, as well as inflammatory diseases. Indeed, the EBI3 deficiency mice display distinct phenotype to mice that lack IL-27 receptor (WXS-1).

Figure 1 The Structures of IL-12 family members and their receptors

1.2.3 The structure and expression of IL-27 Receptor

IL-27 receptor is also a heterodimeric molecule that consists of WSX-1 and gp130. By virtue of sequence databases and structure based alignment tools, a gp130-like protein,

WSX-1 (also called TCCR), was identified as a subunit of IL-27 receptor (IL-27R) [40].

Subsequently, gp130 was identified as the other IL-27R subunit [41].

WSX-1 is a type I cytokine receptor with four conserved cysteine residues and a

WSXWS protein motif in the carboxyl terminus [40]. WSX-1 is homologous to subunits of the IL-6 superfamily receptors, such as IL-12Rβ2 subunit, LIFR, and gp130. WSX-1 is expressed mainly in lymphocytes, as well as mast cells and monocytes [41]. Naïve CD4+

T cells and NK cells appear to express the highest levels of WSX-1, while differentiated

Th1 and Th2 cells show low expression of WSX-1[41]. gp130 is a common receptor for

IL-6 and other IL-6 family cytokines, including IL-11, CNTF, LIF, CLC, OSM, and CT-

1. It is widely expressed by both immune and non-immune cells [42].

WSX-1 and gp130 have been shown to co-express on a variety of immune cell types, including CD4+ T cells, CD8+ T cells, NK cells, monocytes, mast cells, neutrophils, and

B cells [41, 43]. Although WSX-1 has high affinity to IL-27 in the absence of gp130 [29], it is not sufficient to IL-27-mediated signal transduction [41]. In contrast, IL-27 is unlikely to bind gp130 in the absence of WSX-1, as soluble gp130 does not interfere with

IL-27 signaling [44]. Thus, co-expression of WSX-1 and gp130 on the same cell is pivotal for the biological activity of IL-27 and examination of either expression is not quite informative to assess actions of IL-27.

1.2.4 IL-27/IL-27R signaling

The ligation of IL-27 with IL-27R has been shown to activate Janus Kinase (JAK) 1-2,

Tyrosine kinase (TYK) 2, STAT 1-5 in naïve CD4+ T cells [45]. The JAK/STAT signaling pathway is essential for mediating biological responses induced by a variety of cytokines. Therefore, the function of a cytokine is dependent on which members of the

JAK and STAT families are activated by its ligation on the receptor. Co- immunoprecipitation studies and pull-down assays show that the intracellular domain of

WSX-1 is constitutively associated with JAK1 and contributes to tyrosine phosphorylation of STAT1 [46], while gp130 constitutively interacts with JAK1/TYK2 and elicits a strong activation of STAT3 [41, 47]. By using STAT1-/- or STAT3-/- murine

T cells, STAT3 was shown to be indispensable for IL-27-mediated proliferative effect on naïve CD4+ T cells, while STAT1 was found to be dispensable [47]. In contrast,

Yoshimura et al. reported that IL-27 preferentially induces STAT3 in fully activated

CD4+ T cells, while both STAT1 and STAT3 are activated in early activated T cells upon

IL-27 stimulation [48]. Since IFN-α/β/γ utilize JAK1/2, TYK1/2, and STAT1 [49, 50],

IL-6 has the ability to activate JAK1/2, TYK2, STAT1/3 [51, 52], IL-27 was considered to have similar biological actions to IFN-α/β/γ and IL-6.

Recently, another downstream signaling pathway mediated by IL-27 has been observed in CD4+ T cells. The addition of IL-27 in naïve CD4+ T cell culture results in upregulation of another transcriptional factor, c-Maf, as well as IL-21 receptor and inducible costimulatory molecule (ICOS). All of them are essential for the differentiation of IL-10-producing Treg (Tr1) cells [53-56].

Besides CD4+ T cells, IL-27 has been shown to play a pivotal role in CD8+ T cells. In naïve CD8+ T cells, IL-27 activates STAT1 and promotes expression of T-bet and Eomes, which are responsible for the generation of effector CTLs [57]. Another IL-27-mediated signaling pathway in CD8+ T cells was descried in an influenza infection model. IL-27 cooperates with cognate antigen and CD4+ T cell-derived IL-2 triggering some effector

CD8+ T cells to produce IL-10 in a Blimp-1-dependent manner [58].

1.3 The roles of IL-27 in immune regulation

1.3.1 The controversial roles of IL-27 in Th1 response

IL-27 was initially identified as a proinflammatory cytokine that induces Th1 immune response. In the in vitro culture system, IL-27 drives rapid clonal expansion of naïve

CD4+ T cells but not memory CD4+ T cells [29]. IL-27 also induce the expression of T- bet and T-bet downstream gene IL-12Rβ2, both molecules are essential for the differentiation of Th1 cells [37, 45, 46]. In addition, IL-27 synergizes with IL-12 to trigger IFN-γ production by both naïve CD4+ T cells and NK cells [29]. Correspondingly,

WSX-1-/- CD4+ T cells produce less IFN-γ than wild-type counterparts upon antigen or polyclonal stimulation [59, 60].

In the in vivo system, the IL-27R (WSX) and IL-27 (EBI3) deficient mice have been used to evaluate the effect of IL-27 in Th1 response. WSX-1-/- mice exhibited an impaired Th1 response measured by INF-γ production upon protein antigen stimulation. They were more susceptible to infection with an intracellular pathogen, Listeria monocytogenes and

Leishmania major [59, 60]. However, their Th1 response and IFN-γ production were restored at the later phase of the infection [60]. These observations suggest that WSX-1 is essential for the initial mounting of Th1 responses but dispensable for their maintenance.

Later, Zahn et al. utilized another deficiency model, EBI3-/- mice, and got the similar results. EBI3 deficiency resulted in less IFN-γ production at the early phase of infection and led to larger lesions 3-10 weeks post Leishmania major infection [61]. The above two murine models confirmed the previous in vitro findings that IL-27 plays a positive role in inducing Th1 response at the early phase of infection.

As for the molecular basis for IL-27 initiating Th1 responses, there are several molecular pathways have been documented. First, the WSX-1/STAT-1/T-bet/IL-12Rβ2 signaling pathway: The engagement of IL-27 to IL-27R triggers the activation of STAT1, and

STAT1 promotes expressions of T-bet and IL-12Rβ2, which are molecules essential for

Th1 generation [45, 47]. However, the other study showed that IL-27 seems to be able to induce T-bet and IL-12Rβ2 at mRNA levels in the absence of STAT1 [45]. Second, the

ICAM-1/LFA-1/ERK1/2 signaling pathway: IL-27 rapidly up-regulates the expression of

ICAM-1 on naive CD4+ T cells, which promotes Th1 differentiation; blocking Abs against ICAM-1 and LFA-1 inhibits the IL-27-induced Th1 differentiation; ERK1/2 is a gene downstream of ICAM-1/LFA-1, and the ERK1/2 inhibitor impairs ICAM-1/LFA-1- dependent Th1 differentiation. This molecular pathway is dependent on STAT1 but not

T-bet [62]. Third, the p38MARK/T-bet signaling pathway: p38MAPK is located upstream of T-bet, the p38MAPK inhibitor reduced the expression of T-bet and thus suppressed IL-27-induced Th1 differentiation [63].

Although considerable number of in vitro and in vivo studies has shown that IL-27 signaling is critical for inducing Th1 response, some studies found that IL-27 also has immunosuppressive effect in Th1 response. WSX-1 deficient CD4+ T cells produce more

IFN-γ than wild-type counterparts when stimulated with low doses of antigen in the presence of IL-12[64]; IL-27 suppresses IFN-γ and other pro-inflammatory cytokine production by activated CD4+ T cells in vitro [48]. Furthermore, several in vivo infection models also demonstrated this inhibitory effect of IL-27 in Th1 response, cytokine production and inflammation. When infecting WSX-1-/- mice with the intracellular pathogen Toxoplasma gondii, researchers observed increased level of inflammatory cytokines, which led to a lethal CD4+ T cell-dependent inflammatory disease after two weeks of infection. This pathology was characterized by the excessive production of IFN- gamma, persistence of highly activated T cells, and enhanced T cell proliferation in vivo

[64]. Infections with other Th1-dependent pathogens, such as Mycobacterium tuberculosis, Trypanosoma cruzi, and Leishmania donovani, subsequently confirmed that effective Th1 responses could occur in the absence of IL-27 signaling [65-67]. Similarly,

WSX-1-/- mice were more susceptible to Con A treatment than wild-type mice, showing elevated levels of IFN-γ, IL-4, IL-1, IL-6 and TNF-α production [68]. These studies indicate that IL-27 serves as a negative regulator in Th1-mediated inflammation that helps protect the host from excessive inflammation. However, the molecular basis for the negative effect of IL-27 on Th1 response remains unclear. IL-27 has been shown to suppress IL-2 expression in CD4+ T cells [69], Owaki et al. went on to show that this effect was mediated by SOCS3 [70]. As IL-2 is of importance in the growth and survival

15 of Th1 cells, the IL-27-mediated IL-2 suppression may contribute to one of the mechanisms by which IL-27 exerts a suppressive effect on Th1 response.

1.3.2 The inhibitory role of IL-27 in Th2 response

In contrast to the controversial effects of IL-27 on Th1 response, the inhibitory effect of

IL-27 on other helper T cells is clear. IL-27 suppresses Th2 response via down-regulating the expression of GATA3, a master regulator of Th2 cells, in a STAT1-dependent way

[45]. In vivo, the suppressive effect of IL-27 on Th2 response has been documented in both infection and autoimmune disease models. WSX-1-/- mice infected with the gastrointestinal helminth Trichuris muris, which could be removed by Th2-mediated immune response, cleared the pathogen more effectively than wild type controls due to increased production of Th2 cytokines [71]. IL-27 stimulation failed to mount Th2 response against Strongyloides venezuelensis infection [72]. Similarly in autoimmune diseases, WSX-1 deficiency resulted in impaired Th1 differentiation and normal Th2 skewing in a spontaneous glomerulonephritis model. WSX-1-/- mice displayed significantly reduced IFN-γ production along with elevated IL-4 expression [73]. More recently, iNKT-derived IL-27 has been reported to suppress Th2-associated allergic inflammation by alpha-GalCer [74]. Furthermore, IL-27 is capable of inhibiting already differentiated Th2 cells to produce IL-5 and IL-13 via the activation of both STAT1 and

STAT3 [72].

1.3.3 The inhibitory role of IL-27 in Th17 response

In addition to inhibiting Th1 and Th2 responses, IL-27 has been shown to prevent the development of Th17 cells in vitro and in vivo. In vitro, IL-27 is a potent suppressor of

IL-17 production by activated CD4+ T cells [48, 75]; IL-27 antagonizes the development of Th17 driven by TGF-β/IL-6 or IL-23 [48]. IL-27 has been shown to restrain the expressions of RORγt/RORα, the master transcriptional factors for Th17 differentiation, in human and mouse CD4+ T cells, respectively [76, 77]. The first in vivo evidence of IL-

27 inhibiting Th17 cells was shown in a murine model of Toxoplasma gondii infection.

Stumhofer et al. showed that WSX-1-/- mice chronically infected with Toxoplasma gondii developed severe CNS inflammation associated with augmented Th17 responses [69]. IL-

27 is also a potent suppressor of autoimmune diseases that are primarily mediated by

Th17. WSX-1-/- mice were highly susceptible to EAE and generated more Th17 cell accumulation in the draining lymph nodes and CSN, and neutralizing of IL-17 attenuated their disease phenotype[75]. Another group used recombinant IL-27 to treat EAE mice and showed that IL-27 decreased the disease incidence and severity via suppressing Th17 cells [78]. Amadi-Obi et al. also suggested that IL-27 inhibited Th17-mediated autoimmune pathology in experimental autoimmune uveoretinitis [79].

Above observations indicate that IL-27 is a potential therapeutic agent for treating inflammatory diseases and autoimmune diseases which are mediated by Th17. However, the mechanism for IL-27-mediated Th17 inhibition remains unclear. Two recent studies showed that IL-27 directly inhibited the productions of IL-17A and IL-17F in naive T cells by suppressing RORγt in a STAT1-dependent manner [76, 77]. In addition, IL-27 has been found to decrease the expression of GM-CSF and thereby dampens the

17 development of Th17 cells [80]. Since IL-6 is an important factor to polarize Th17 cells, and IL-6 and IL-27 share the common receptor subunit gp130, it was speculated that IL-

27 may compete with IL-6 for receptor binding, which may result in the inhibition of

Th17 cells. However, this hypothesis seems to be overthrown by the following studies. In the absence of IL-6, IL-27 still possesses the ability of inhibiting IL-17 production by

CD4+ T cells [69]. To date, there are still numerous questions centered on how IL-27 suppresses Th17 response.

1.3.4 The role of IL-27 in the development of T regulatory cells

Naïve CD4+ T cells cultured with TGF-β and IL-2 in vitro up-regulate the transcriptional factor Foxp3, conferring a differentiation to T regulatory cells. When these inducible T regulatory cells (iTreg) are exposed to IL-27 during differentiation, Foxp3 upregulation is inhibited[81, 82]. In addition, the expressions of another two Treg markers, CD25 and

CTLA-4, were also decreased by IL-27[82]. Recently, Cox et al. transferred WSX-1-/-

CD45Rbhi T cells into CB17-SCID recipient mice and observed diminished weight loss and reduced colonic inflammation. They noted that WSX-1-/- recipients contained approximately 2-3 times more Foxp3+ T cells compared to WT recipients, suggesting that

IL-27 functions via restraining the development of Treg cells [83]. Later on, Wojno et al. demonstrated that IL-27 transgenic mice developed systemic inflammation at 8-11 week of age, which was associated with their decreased levels of Treg cells in lymph organs

[84]. The above in vitro and in vivo studies clearly illustrated that IL-27 has a prominent function in regulating Treg cell development. As for the mechanism by which IL-27 inhibits Treg cell development, earlier studies showed that IL-27 down-regulates the expression of Foxp3 at least partially dependent of STAT3 signaling, but independent of

STAT1 signaling [81, 82]. Furthermore, IL-27 has been identified as a suppressor of IL-2 production in T cells [63, 85], and IL-2 is essential to the generation and persistence of

Treg cells, thus IL-27-mediated IL-2 suppression may contribute to the negative regulatory role of IL-27 on Treg cells. To prove this hypothesis, Wojno et al. showed that in IL-27 transgenic mice the ability of IL-27 to suppress IL-2 is associated with Treg deficit, which promotes inappropriate inflammation [84].

Type 1 regulatory (Tr1) cells are another type of induced Treg cells that have strong immunosuppressive properties, predominantly produce IL-10 with variable amounts of

IFN-γ, but do not express Foxp3. Tr1 cells were first described in severe combined immuno-deficient (SCID) patients who had developed long-term tolerance to stem cell allografts, suggesting that these cells suppressed immune responses in human [86]. The ability of Tr1 cells to cripple effector T cell responses has been ascribed to their high IL-

10 production [87]. Adoptive transfer of Tr1 cells has been shown to suppress autoimmunity, colitis, graft-vs-host disease, and tissue inflammation [88]. Although previous studies have demonstrated that IL-27 is capable of inhibiting TGF-β-driven

Foxp3+ Treg cells, later around the same time, three groups reported that IL-27 potently induced naïve murine CD4+ T cells into IL-10 producing Tr1 cells [53-55]. Batten et al. observed that IL-27 stimulated both CD4+ and CD8+ T cells to produce IL-10 [89]. In agreement with in vitro experiments, WSX-1-/- mice generate significantly less number of

IL-10+ T cells during listeria monocytogenes infection and experimental autoimmune encephalomyelitis [89]. Similarly, IL-27 plays a key role in human T cells by promoting

IL-10-secreting Tr1 cells and inhibiting Th17 cells, which provides a dual regulatory mechanism to control autoimmunity and tissue inflammation [90-92]. Not only inducing

IL-10 production by CD4+ T cells, IL-27 cooperates with IL-2 and cognate antigen and leads to more IL-10+ CTLs during acute viral infection [58]. Thus, IL-27 is an essential differentiation factor for the generation of Tr1 cells in both murine and human naïve

CD4+ T cells. The molecular mechanism underlying this action was initially reported to require both STAT1 and STAT3 [55]. Pot et al. revealed a new molecular pathway for this effect: IL-27 induces the transcriptional factor c-Maf, cytokine IL-21, and inducible costimulatory receptor ICOS, which coordinately act together to promote differentiation of IL-10-producing Tr1 cells [93]. They noted that each of these 3 elements is essential because loss of c-Maf, IL-21 signaling, or ICOS decreases the frequency of IL-27-nduced

IL-10-producing Tr1 cells.

1.3.5 The immunostimulatory role of IL-27 in CD8+ cell response

Besides affecting the differentiation and function of CD4+ T cell subsets, IL-27 plays a crucial role in CD8+ T cell response as well. IL-27 has been reported to stimulate CD8+ T cells and enhance their cytotoxic activity, resulting in effective protection against viral infection and cancer. Matsui et al. demonstrated that pre-injection of IL-27 expression plasmid before immunization resulted in great increases in the number of IFN-γ- producing, HCV-specific CD8+ cells [94]. At the same year, Hisada et al. and Salcedo et al. reported that IL-27 has potent antitumor activity, which is mediated mainly through

CD8+ T cells, against murine tumor models of colon carcinoma C26 [95] and neuroblastoma TBJ [96]. In WSX-/- mice, tumor-specific CTL generation was impaired compared to those in WT mice, which resulted in higher tumor growth rate [97]. In lines with the above in vivo findings, the proliferation and IFN-γ production of murine CD8+ T cells are remarkably enhanced in the presence of IL-27 in vitro [57]. IL-27 has also been

20 shown to increase the T-bet expression level, proliferation, and effector function of human CD8+ T cells [98]. Similar to CD4+ T cells, IL-27 activates STAT proteins and augments the expressions of T-bet, IL-12Rβ2, and cytotoxic molecules in naïve CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies [98, 99]. Meanwhile, IL-27 enhances SOCS3 expression to control excessive proliferation in CD8+ T cells [100]. It has been confirmed that IL-27 induces the generation and functional CTLs by activation of STAT1 and subsequent augment of two related T-box transcriptional factors: T-bet and EOMES. Since IL-27 plays a pivotal role in both early induction of Th1 response and generation of CTLs, leading to promotion of type 1 cell-mediated immunity, IL-27 represents an attractive therapeutical agent applicable to the immunotherapy against pathogens and cancers.

1.3.6 The roles of IL-27 on other cells

Although T cells are the major targets of IL-27, other types of cells have also been shown to be affected by IL-27 ligation. As mentioned before, IL-27R is distributed on a variety of cell types, including NK cells, mast cells, B cells, macrophages, DCs, and neutrophiles.

Thus, there are also some studies documenting the effects of IL-27 on these cells.

Pflanz et al. showed that IL-27 acts synergistically with IL-2 and IL-12 to promote proliferation and IFN-γ production of NK cells in vitro [29]. Moreover, Matsui et al. demonstrated that IL-27 enhances viability and cytotoxic activity of NK cells [101].

However, NK cells from WSX-1-/- mice produce more IFN-γ and IL-4 compared to wild- type mice in response to con A-induced hepatitis [68], indicating that IL-27 signaling has a negative effect on NK effector functions in vivo. The apparent discrepancy between in

21 vitro and in vivo system suggests that IL-27 has a regulatory role in NK cell function.

Also, IL-27 exerts a negative effect on NKT cells [65, 68].

Similarly, the immunostimulatory effect of IL-27 was noted for mast cells in vitro, while analysis of WSX-/- mice generated opposite results. IL-27 treatment activates STAT3 and up-regulates its downstream cytokine gene expressions, such as IL-1α, IL-1β, IL-18, TNF,

OX40, RANKL, APRIL, and BRAFF [30]. However, enhanced mast cell responses were detected in WSX-/- mice compared to WT mice.

Reportedly, B cells express the functional IL-27R complex depending on the mode of activation. IL-27 promotes the proliferation of anti-immunoglobulin-activated naïve B cells and anti-CD40-activated naïve and germinal center B cells [102]. For lipopolysaccharide and anti-CD40-stimulated B cells, IL-27 directly induces IgG2a class switching in a STAT1 and T-bet dependent manner [103], in line with in vivo studies that

WSX-1-/- mice has less IgG2a in serum [59, 73]. Since IL-27 is able to induce both NK activation and IgG production, it is a feasible cytokine to provoke therapeutic ADCC to attack diseases in which appropriate monoclonal antibodies have not been practically available.

In addition to adaptive immune cells, IL-27 also regulates innate immune cells. IL-27 inhibits macrophages to produce cytokines, such as IL-12 and TNF-α [104]. Furthermore,

IL-27 suppresses reactive oxygen production by granulocytes and macrophages [105].

Moreover, IL-27 has been shown capable of crippling the function of DCs, which was demonstrated in enhanced antigen-presenting function of DCs in WSX-/- mice [106].

Adoptively transfer of WSX-/- DCs has higher efficacy in tumor eradication than that of

WT DCs, which indirectly indicates immunosuppressive effect of IL-27 on DCs [97].

IL-27 also induces STAT1 and STAT3 in osteoblasts, but its physiological relevance has not been identified. It may be involved in protecting host from bone destructive autoimmune diseases [107]. In summary, IL-27 has profound immunosuppressive effects in innate immune responses. However, the underlying mechanisms are poorly understood.

1.4 The antitumor activities of IL-27

In the past decade, IL-27 has attracted considerable interest as a potent antitumor cytokine due to its similarities to IL-12. IL-12 has been proven to be effective in controlling tumor growth and metastases. The mechanisms responsible for the antitumor effects of IL-12 include: promoting IFN-γ production, antiangiogenesis, triggering programmatic changes in suppressive cellular components within tumors, and enhancing lytic abilities of CTLs, NK cells, and NKT cells to eradicate tumors [108, 109]. However,

IL-12 treatment to either murine tumor-bearing mice or human cancer patients is always associated with severe side effects, which may due to its excessive pro-inflammatory effects. Since IL-27 was discovered in 2002, IL-27 gene has been transduced into a series of murine and human tumor cells lines. The potent antitumor effect of IL-27 has come into agreement in almost all literatures. Given its pleiotropic roles in different types of immune cells, several groups investigated how IL-27 impacts the immunological responses within tumors and results in tumor rejection.

1.4.1 Murine colon carcinoma

Hisada et al. transduced colon carcinoma C26 cells with the single-chain IL-27 cDNA and inoculated them or control tumor cells into immunocompetent mice. Mice challenged with C26-IL-27 tumor cells exhibited minimal tumor growth and survived longer with complete tumor remission compared to mice challenged with control tumor cells [95].

Tumor-derived IL-27 enhanced IFN-γ production and CTL responses against C26 tumors.

In sharp contrast, the antitumor activity of IL-27 was almost diminished in nude mice, which indicates that the adaptive immunity plays an important role in IL-27-mediated tumor rejection. Furthermore, C26-IL-27 tumors established in T-bet-deficient mice, WT mice experienced CD8+ T cell depletion and IFN-γ neutralization. These results suggest that IL-27 exerts its potent antitumor activity mediated mainly through CD8+ T cells, T- bet, and IFN-γ.

To further investigate the molecular mechanisms underlying the augmentation of CTL response by IL-27, Morishima et al. cultured murine CD8+ T cells in the presence of IL-

27 in vitro. IL-27 was shown to activate STAT-1, -2, -3, -4, and -5 and increased the expressions of T-bet, IL-12Rβ2, perforin, and granzyme B. IL-27 also promoted the proliferation of naïve CD8+ T cells [99].

In 2004, another group retrovirally transduced colon C26 cells with p28-linked EBI3 cDNA and showed that IL-27 secreted from C26 colon tumors exerted antitumor effects as well [110]. They compared the antitumor effects of cytokine combinations (IL-12, IL-

23, and IL-27) with negative controls in tumor development. They mixed populations of cytokine producers (C26/IL-12, C26/IL-23, and C26/IL-27) and inoculated them into one

24 flank of syngeneic mice, while the other flank was given parent tumors. All mice rejected the mixed cytokine producers, and the growth of parent tumors in the other flank was also significantly retarded compared with that of parent tumors developed in both flanks. This study confirmed the antitumor activity of IL-27 in murine colon carcinoma model.

Chiyo et al. published the second paper based on their C26/IL-27 model [22]. In disagreement with Hisada group, they observed that mice inoculated with C26-EBI3 or

C26-p28 developed tumors and their survival remained the same as that of the mice inoculated with parent C26 tumor cells. They also showed that the antitumor activity of

IL-27 was not completely abolished in syngeneic nude mice, while depletion of natural killer cells from nude mice diminished the growth retardation of colon 26/IL-27 tumors.

Thus, besides CD4+ and CD8+ T cells, natural killer (NK) cells are another type of effector cells by which IL-27 exerts its antitumor effects on colon tumors.

Zhu et al. employed an IL-27-based gene therapy to cure colon cancer. They administrated IL-12- and IL-27-encoding plasmid DNA sequentially into C26 tumor- bearing mice through i.m. electroporation and found that CT26 tumors were completely eradicated [111]. They showed T cells and NK cells were required for this sequential gene therapy-mediated tumor rejection. This study suggests that IL-27 can be used together with IL-12 to induce a stronger protective immune response against colon tumors.

1.4.2 Murine neuroblastoma TBJ

The antitumor activity of IL-27 has also been reported in murine neuroblastoma, the most common extracranial solid tumor in children. Salcedo et al. transduced TBJ

25 neuroblastoma cells with either p-IL-27/FLAG-CMV-1 (TBJ-IL-27) or the empty p-

FLAG-CMV-1 alone, and then injected them into immunocompetent mice [96]. TBJ-IL-

27 tumors showed obvious delayed tumor growth and complete durable tumor regression.

Most mice that experienced complete regression of TBJ-IL-27 tumors were resistant to tumor rechallenge, indicating that IL-27 is able to induce immunologic memory response against tumors. The underlying mechanisms that contribute to the potent antitumor activity of IL-27 have been revealed as below: IL-27 enhances the infiltration of CD8+ T cells in the tumor microenvironment, up-regulates gene expression levels of IFN-γ and

MHC class I within TBJ-IL-27 tumors, and provokes the generation of tumor-specific

CTL reactivity. Thus, IL-27-mediated tumor regression in murine neuroblastoma is critically dependent on CD8+ T cells, but not CD4+ T cells or NK cells.

Despite the strong inhibitory effects of IL-27 against s.c. and orthotopic primary neuroblastoma tumors, IL-27 alone is not able to control the neuroblastoma metastasis.

Therefore, Salcedo group delivered the combination of IL-27 and IL-2 into mice bearing disseminated neuroblastoma metastasis [112]. They showed that this combined treatment synergistically induce tumor regression and long term survival in mice bearing TBJ metastasis in liver and bone marrow. IL-27 plus IL-2 significantly enhanced the tumor- specific CTL response, not only at the initial sensitization of effector cells but also at the effector phase when primed effector cells were restimulated with tumor cells. Notably,

IL-27 induced potent memory CTL responses in mice cured of their disseminated disease.

This study suggests that IL-27 could be used together with IL-2 to treat disseminated neuroblastoma.

1.4.3 Murine melanoma B16F10

IL-27-mediated antitumor effect has been shown not only in immunogenic models such as C26 colon carcinoma and TBJ neuroblastoma but also in B16F10, a murine melanoma model that is poor immunogenic characteristic by low MHC class I expression. B16F10 tumors that were engineered to over-express single chain of IL-27 showed impaired tumor growth of primary tumors and pulmonary metastasis [113]. Interestingly, the antitumor and anti-metastatic activities were still observed in IFN-γ-/- mice and NOD-

SCID mice, suggesting that new mechanisms other than enhancing adaptive immune responses contribute to antitumor effects of IL-27 on melanoma. They demonstrated that

IL-27 up-regulated expressions of anti-angiogenic markers, such as IP-10 and MIG, and thus inhibited angiogenesis in vivo. Thus, antiangiogenesis appears to be a new mechanism for the antitumor and anti-metastatic effects of IL-27 on murine melanoma.

Another group did a similar investigation in 2006. Single chain IL-27-transfected B16F10

(B16/IL-27) exhibited apparent retardation of tumor growth at the early stage. However, the depletion of NK cells remarkably accelerated the tumor growth of B16/IL27 to almost the same rate as the tumor growth of B16/control [114]. This indicates that NK cells play an essential role in IL-27-mediated melanoma rejection. Of importance, they reported for the first time that IL-27 induced less adverse effects than IL-12, suggesting that the therapeutic usage of IL-27 might be more tolerated than that of IL-12 for cancer patients.

Besides inducing antiangiogenesis and enhancing NK cell function in the melanoma tumor microenvironment, IL-27 also possesses a direct antiproliferative activity on melanoma cells [115]. B16F10 cells transduced with WSX-1 cDNA became responsive

27 to IL-27 and grew much slower upon IL-27 sensitization in vitro. IL-27 suppressed the proliferation of B16F10/WSX-1 cells via the WSX-1/STAT1 signaling pathway and partially through IRF-1. To be noted, this study also examined several human melanoma cells and revealed that they expressed both IL-27 receptor subunits, and upon IL-27 stimulation, STAT1 and STAT3 were activated, and tumor growth was inhibited.

Collectively, IL-27 has a direct antiproliferative activity on melanomas through WSX-

1/STAT1 signaling.

Although above researchers ruled adaptive immune cells out as effectors for IL-27 to inhibit tumor growth of melanoma, Shinozaki et al. challenged WSX-/- mice with B16 melanoma cells and demonstrated that generation of tumor-specific CTL was substantially diminished in WSX-/- mice, which was in conjunction with higher tumor growth rate [97]. It suggests that IL-27 suppressed B16 melanoma tumor growth via enhancing tumor-specific CTL generation. In addition, they also discovered that IL-27 had suppressive effects on DC function. Thus, they recommended that the combination of

WT T cells and IL-27 signal-defective DCs might have therapeutic potential against tumors.

So far, we can conclude that IL-27-mediated antitumor activities in murine melanoma models are not only attributed to signaling in immune and tumor cells but also in vascular endothelial cells that around the tumor microenvironment. It employs various antitumor pathways depending on the tumor microenvironment that specific tumor creates.

1.4.4 Murine head and neck squamous cell carcinoma SCCVII

Head and neck squamous cell carcinoma (HNSCC) is one type of tumors that is relatively resistant to conventional immunotherapeutic methods because it is usually unsusceptible to natural killer cytolysis and CTLs specific for HNSCC are hard to induce. Although the in vivo depletion of NK cells significantly restored the growth of IL-27-producing

HNSCC [22, 114], it remains unclear whether IL-27 activates NK cells directly or indirectly through inducing IFN-γ. To address this question, Matsui et al. examined the regulatory activity of IL-27 on murine NK cells and evaluated the therapeutic potential of

IL-27 against s.c. HNSCC in mice [101]. First, they showed that IL-27 induced phosphorylation of STAT1/STAT3 and upregulated their downstream gene expression levels of T-bet and granzyme B in murine DX-5+ NK cells. Secondly, IL-27 positively regulated viability and cytotoxic activity of NK cells both in vitro and in vivo. Thirdly, therapeutic administration of IL-27 drastically suppressed the growth of SCCVII tumors, resulting in significant prolongation of the survival time. To further investigate the mechanism of how IL-27 utilizes NK cells to kill tumors, they performed NK cytotoxicity assay and ADCC assay. They found that SCCVII tumors were extremely unsusceptible to NK cytotoxicity, while IL-27 increased tumor-specific IgG in the sera as well as activated NK cells. These results suggest that the IL-27-induced antibody and activated NK cells cooperated to kill the NK-resistant HNSCC cells through the ADCC machinery.

1.4.5 Murine Lewis lung carcinoma LLC-1

Lung cancer is the leading cause of human cancer deaths worldwide. COX-2 has been widely considered as a target in lung cancer because it is over-expressed in both early and advanced lung cancer tissues [116]. Elevated levels of tumor COX-2 and its metabolite,

PGE2, contribute to modulation of apoptosis, stimulation of angiogenesis, promotion of cancer invasion, and suppression of antitumor immunity. IL-27 was shown to directly suppress lung tumorigenicity through downregulation of COX-2 and PGE2 in tumor cells

[117]. In this study, single-chain of IL-27 cDNA was transduced into murine Lewis lung carcinoma (LLC-1) cell line (LLC-1/scIL-27). In contrast with control LLC tumors,

LLC/scIL-27 tumors exhibited decreased expression of COX-2 and PGE2, resulting in lower vimentin expression, impaired cellular migration and invasion, and significant retardation of tumor growth. The antitumor activity of IL-27 on lung cancer cells was confirmed by the treatment with rIL-27 on the murine LLC-1 and human non-small cell lung carcinoma (NSCLC) cell lines. Thus, in addition to the regulatory effects on immune cells, IL-27 is capable of inhibiting expressions of tumor-promoting genes, such as COX-2, PGE2, and vimentin.

1.4.6 Murine Prostate Tumor

Prostate cancer is the most common cause of death from cancer in men over age 75.

There are various types of treatment for prostate cancer patients, such as surgery, radiation, hormone therapy, chemotherapy, and immunotherapy. The potential of IL-27 gene delivery by sonoporation to control prostate tumor growth was evaluated by

Zolochevska et al. in 2011 [118]. They used three models of immune-competent prostate

30 adenocarcinoma and characterized their tumor growth, gene-profile, and effector cellular profile after the IL-27 gene therapy. Transfection of prostate cancer cell lines TRAMP-

C2 and RM1 with a plasmid coding IL-27 resulted in less viable and migrating cells, suggesting that IL-27 could act on prostate cancer cells directly and inhibit their growth and metastasis. The sonoporation using pIL-27 was potent to halt tumor growth by ~50% and increase survival time. This therapy resulted in a dramatic up-regulation of p28, EBI3,

WSX-1, T-bet, IFN-γ, IL-12, IL-10, NF-κB, and IRF-1. Sono-IL-27 enhanced the percentage of CD8+ T cells by ~120% over Sono-Ctrl, while MDSCs and Treg cells were decreased in Sono-IL-27 treated tumors.

1.4.7 Human Multiple Myeloma

Multiple myeloma (MM) is cancer of plasma cells in bone marrow. It represents the second most common hematologic malignancy worldwide, and its prognosis remains poor. In multiple myeloma, transformed plasma cells establish tumors in solid bones, which hurdles bone marrow to make healthy blood cells and platelets. Pathogenesis of

MM is complex and dependent on the communications between tumor cells and microenvironment in the BM. In 2010, Cocco et al. investigated the activity of IL-27 on

MM cells in terms of their angiogenesis, proliferation, apoptosis, and chemotaxis [119].

They showed that human primary MM cells expressed IL-27R and IL-27 engagement inhibited angiogenesis, demonstrated by lower expression of VEGF-D and CCL-2 within

MM tumors. However, IL-27 did not show apparent effect in the proliferation, apoptosis, chemotaxis, or cytokine release of MM cells. Next, they extended to study the role of IL-

27 on osteoclasts, which interact with myeloma cells and promote tumor expansion and bone degradation. Osteoclast progenitors from MM patients and controls expressed IL-

27R as well, and IL-27 directly dampened osteoclast differentiation and their bone lytic activity. Moreover, IL-27 enhanced the expansion of mature osteoblast but with no impact on their function. Lastly, they evaluated the efficacy of hrIL-27 in eradicating human MM using immunodeficient SCID-NOD mice. Systemic IL-27 treatment remarkably inhibited tumor growth, and the antiangiogenesis effect of IL-27 contributed more than other mechanisms to IL-27-mediated tumor rejection.

1.4.8 Pediatric B-acute lymphoblastic leukemia

B-acute lymphoblastic leukemia (B-ALL) is the most common pediatric hematological cancer that derives from aberrant expansion of early B lymphocytes in the bone marrow.

Canale et al. used a preclinical model to test the antitumor effect of IL-27 on pediatric B-

ALL cells [120]. They injected B-ALL cells from pediatric patients into NOD/SCID/IL-

2rg-/- mice intravenously, and treated them with hrIL-27 or PBS. The number of human

B-ALL cells was significantly reduced by IL-27 treatment in PB, BM, and spleens. They assumed this could be ascribed to the suppression effect of IL-27 on cell proliferation and angiogenesis. In addition, they showed another two abilities of IL-27 in this model. First,

IL-27 was able to induce tumor cell apoptosis; Second, IL-27 downregulated MiR-155, a putative oncomiR accumulated in human B-cell lymphomas. All of them together contribute to the antitumor effect of IL-27 in B-ALL.

1.4.9 Pediatric acute myeloid leukemia

Acute myeloid leukemia (AML) is a type of fatal disease in children, which accounts for more than half of fatal cases in pediatric leukemia patients. In 2012, Zorzoli et al. gained

AML samples from 16 pediatric patients and injected them into NOD/SCID/IL-2rg-/-

32 mice intravenously followed by PBS or IL-27 treatment [121]. They showed that IL-27 treatment impaired the dissemination of AML in NOD/SCID/IL-2rg-/- mice. The mechanisms involved in this model have been demonstrated in 2 aspects: first, IL-27 down-regulated proangiogenic (ANGPT2, ANGPT3, IL-6, CXCL1, CXCL6, and VEGF-

C) and spreading (CXCR4, MMP2, MMP7 and MMP9) related genes in the tumor microenvironment; second, IL-27 inhibited the proliferation of AML cells directly.

1.4.10 Intracranial Gliomas GL-26

Bone marrow derived neural stem-like cells (BM-NSCs) transducing with an adenovirus vector carrying the single-chain IL-27 cDNA showed better efficacy in rejecting intracranial gliomas than those with an empty vector (Yuan et al., 2005). CD8+ T cells were shown to be the main mediators in this antitumor process. This study took advantages of the potent anti-tumor ability of IL-27 and the tumor tracking property of

BM-NSCs to treat brain tumors, which represents a novel promising gene therapy. It is consistent with other studies in IL-27-mediated tumor rejection.

Taken together, these studies have come to the agreement that IL-27 is a potent antitumor cytokine. The underlying mechanisms could be generally concluded in the following 4 aspects: 1) IL-27 directly acts on CD8+ T cells in the tumor environment, promotes its activation and killing ability against tumor cells; 2) IL-27 enhances the viability and cytotoxicity of NK cells, which are also protective immune cells against tumors; 3) IL-27 constrains tumor angiogenesis through inhibiting expressions of a variety of proangiogenic genes; 4) IL-27 activates the STAT1 signaling pathway in tumor cells directly, and subsequently suppresses their proliferation and migration. It’s worthy to note that the

33 first mechanism listed above seems to be the most important one contributing to the antitumor effect of IL-27. However, the exact underlying mechanisms remain unclear.

This study was set out to address how IL-27 enhances the antitumor CTL responses and leads to tumor rejection.

Table 2 List of studies of IL-27 in tumor immunity

Year Disease Method Function 2004 murine colone carcinoma C26-IL-27 antitumor 2004 murine neuroblastoma TBJ-IL-27 antitumor 2005 intracranial gliomas GL-26 Ad-IL-27 antitumor 2006 murine melanoma B16F10-IL-27 antiangiogenic 2008 murine melanoma B16F10-WSX-1 antiproliferation 2009 murine lewis lung carcinoma LLC/scIL-27 antitumor 2010 murine colone carcinoma IL-27-encoding DNA antitumor 2010 human multiple myeloma IL-27 treatment antitumor 2011 murine prostate IL-27 gene therapy antitumor 2011 pediatric B-acute leukemia hrIL-27 antitumor 2012 pediatric acute myeloid leukemia IL-27 treatment antitumor

1.5 The biological roles of interleukin-10 in tumor immunity

Interleukin-10 (IL-10) is a homodimeric cytokine. Through engaging with its heterodimeric receptor complex, IL-10 regulates biological activities of immune cells, keratinocytes, endothelial cells, and tumor cells. IL-10 can be released from macrophages, dendritic cells, epithelial cells, T cells and tumor cells. IL-10 is an anti-inflammatory cytokine which negatively regulates various inflammatory responses. The anti- inflammatory effects of IL-10 have been demonstrated in the following three aspects: first, IL-10 suppresses secretion of pro-inflammatory cytokines such as IFN-γ, TNF-α,

IL-2, IL-3, IL-6, IL-1β, and IL-12; second, IL-10 inhibits expressions of MHC and costimulatory molecules [122]; Third, IL-10 antagonizes several functions of antigen presenting cells through preventing their differentiation and maturation [122]. However, increasing number of research showed that IL-10 also exerts immunostimulatory effects on selected cell types. Early on, IL-10 was shown to exhibit growth factor activity for mouse mast cells and thymocytes [123, 124]. IL-10 enhances B cell survival, proliferation, and antibody production [125, 126]; Furthermore, IL-10 induces recruitment, proliferation and cytotoxic activity of CD8+ T cells in both mouse and human [127-131]; IL-10 increases expression of some genes in TLR-activated macrophages and DCs [132]. In all, IL-10 has demonstrated dual roles which highly depend on disease contexts. As for the role of IL-10 in tumor immunity, it must be said that much confusion has been raised in the past decade. It is debated whether it is a good component of immune system to eliminate the tumor.

1.5.1 The pro-tumor effects of IL-10

Several preclinical studies showed that IL-10 can promote tumor growth via blunting the immune responses against cancer. In vitro, tumors that produce IL-10 (human basal and squamous cell carcinoma) triggered poor tumor specific T cell responses, while neutralization of their IL-10 production by anti-IL-10 antibody could rescue their immune privilege [133]; tumor cells (e.g., melanoma, lymphoma) treated with rIL-10 or transfected with the IL-10 gene showed low MHC class I expression and low sensitivity to specific CTL-mediated lysis [134]; furthermore, IL-10-conditioned human dendritic cells not only exhibited impaired allostimulatory capacity, but also triggered CD8+ T cells anergy to melanoma-associated antigens [135]; autocrine IL-10 prevented both

35 spontaneous maturation of DCs and LPS-/CD40-mediated maturation of DCs [136];

Treg-derived IL-10 suppressed immune responses against tumor cells [137-139]. In vivo, lewis lung tumor grew faster in IL-10 transgenic mice than wild type control mice, yet administration of blocking IL-10 mAbs significantly slowed down the tumor growth

[140]; tumor-derived IL-10 prevented DCs from being recruited and accumulating at the tumor site [141]; administration of neutralizing IL-10 antibody reduced the sensitivity of

DCs to response to TLR ligands as well as CD40L [136]; IL-10 neutralization also resulted in enhancement of CTL responses and led to complete tumor eradication in mice

[142]. In humans, IL-10 has been found to be an independent prognostic factor in multivariate analysis for B-cell lymphoma patients. It inhibits the induction of the costimulation molecule CD86 on DCs and the ability of DCs to stimulate T cells [143].

As reported in above studies, IL-10 favors the development of tumors mainly through immunosuppressive mechanisms, such as down-regulating expressions of MHC class I and costimulatory molecules, inhibiting maturation and function of APCs, and suppressing recruitment and killing ability of cytotoxic CD8+ T cells. In addition to its immunosuppressive effects in regulating immune components in the tumor microenvironment, IL-10 has been shown to exert a positive or negative effect on tumor growth directly (e.g. B lymphoma, melanoma) [144-148]. Since IL-10 is one of the inducing cytokines for STAT3, which is constitutively activated in a large variety of solid tumors and cell lines, it may promote tumor progression through activation of STAT3 in tumor cells.

1.5.2 The antitumor effects of IL-10

Although the immunosuppressive effects of IL-10 in tumor immunity have received much attention in early studies, growing evidence now suggests that IL-10 is able to enhance the antitumor immune responses and leads to tumor rejection. Overexpressing

IL-10 in tumor cell lines, including Chinese hamster ovary cells [149], mammary adenocarcinoma [150], breast cancer [151], melanoma [152], prostate primary tumor

[153, 154], colon carcinoma [155], resulted in potent tumor rejection. These IL-10- producing tumor cells are highly immunogenic, triggering cytotoxic CD8+ T cells, Th2 cells, neutrophiles, NK cells and even the antibody responses in the tumor microenvironment. In addition, antitumor effects of IL-10 were examined and obtained by systemically administration of recombinant IL-10 [156, 157] or use rIL-10 as an adjuvant of vaccines [158, 159]. Intravenous administration of rIL-10 to humans elicited higher numbers of IFN-γ, IFN-inducible protein 10 (IP-10), TNF, IL-1, and granzyme B and induced activation of NK and CTL cells as well. Injection of IL-10 after immunized tumor bearing mice significantly enhances antitumor immune responses and vaccine efficacy. Some research groups, using IL-10 transgenic (IL-10TG) and deficiency mice, proved the antitumor effects of IL-10 in several tumor mouse models [160-162]. Mumm et al. subjected WT, IL-10-/-, and IL-10TG mice to skin tumors and found more tumors developed in IL-10-/- mice than WT control mice, while IL-10TG mice barely developed tumors [161]. They demonstrated that IL-10 induced several essential mechanisms to boost antitumor immune privilege: CD8+ T cells, IFN-γ, granzyme B, and intratumoral antigen presenting molecules. Tanikawa et al. showed that IL-10 deficiency resulted in an increased level of myeloid-derived suppressor cells (MDSC) and CD4+Foxp3+ regulatory

T (Treg) cells in both tumor microenvironment and tumor-draining lymph nodes [162].

Therefore, in some tumor circumstances, IL-10 exerts permissive effects in preventing tumorigenesis and tumor development. IL-10 may represent a new tool in current tumor immunotherapies.

1.6 Tumor infiltrated lymphocytes (TIL) in the tumor microenvironment

1.6.1 The anti-tumor effector T cells in the tumor microenvironment and adoptive cell therapies (ACT)

The anti-tumor effector T cells, including CTLs [163] and activated CD4+ T cells [164,

165], are usually generated at the initiation stage of tumor establishment. They are activated by encounters with tumor-associated antigens that are presented by specialized antigen presenting cells (APCs). Activated T cells are, theoretically, capable of directly recognizing antigens that presented on the surfaces of tumor cells and eradicating them through releasing cytotoxic molecules, such as IFN-γ, granzyme B, and perforin. Based on this theory, today physicians treat cancer patients with either their naturally occurring or gene-engineered T cells and have obtained substantial clinical benefits in several trials.

This treatment has been designed as adoptive cell therapy (ACT). They isolate and expand autologous T cells ex vivo to a sizable population [166], and then adoptively infuse them back into the cancer patient [167]. Those adoptively transferred T cells can traffic to the tumor bed and are capable of eradicating the tumor mass [168]. However, this type of immunotherapy has limitations. In practice, the success of adoptive T cell therapy is considerable (~50%), but not complete [169]. This is perhaps because they experience chronic activation within the tumors [170] and their functions are diminished

38 by tumor immunosuppressive molecules, such as T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), lymphocyte activation gene 3 protein (LAG3), programmed cell death protein 1 (PD-1), and cytotoxic T lymphocyte antigen 4 (CTLA-4)

[171]. In addition, tumor cells are smart. They educate immature macrophages or DCs to develop into immunosuppressive cell populations; they also promote the infiltration of

CD4+Foxp3+ T regulatory cells into the tumor microenvironment. These two cell populations together create an immunosuppressive microenvironment preventing adoptively transferred T cells from functioning and persisting in the patient body.

To overcome the above mentioned obstacles, several approaches have been developed and exploited in current ACT. Some researchers clone and insert a specific TCR gene into autologous T cells before infuse them back to the patients, which confers T cells with a high affinity and excellent specificity for target tumors antigens [172-174]. Most frequently, investigators use retroviruses or lentiviruses encoding T cell receptor specific for the target antigen to transfect T cells. To date, This approach has been used clinically in treating melanoma [174], neuroblastoma [175], synovial cell sarcoma [176], leukemia and lymphoma [177-179]. Another approach involves the transduction of autologous T cells to express chimeric antigen receptors (CARs) comprised of an extracellular scFv portion of a tumor specific antibody and an intracellular signaling domain capable of activating T cells [178]. Since CARs have the specificity of a monoclonal antibody, they are not HLA restricted and can be used to treat any patients as long as their tumor express the specific antigen. This approach has been demonstrated very effective in eliminating lymphoma and leukemia [178, 180, 181]. The invention of CARs is a tremendous breakthrough in cancer immunology history. In addition, some physicians combined

“preparative lymphodepletion” with ACT and demonstrated that it could raise the response rate to about 70% and prolong the persistence of transferred T cells. This lymphodepletion procedure provides an “expansion space” and meanwhile creates a Treg free environment for infiltrated effector T cells [182, 183]. Recently, investigators are working to reprogram T cells to become “stem-like” T cells that display superior antitumor activities [184-186]. A poorly explored possibility is the use of tumor antigen specific CD4+ T cells as the therapeutic agent alone or in conjunction with antigen specific CD8+ T cells. Few studies that have focused on adoptive transfer of CD4+ T cells suggest: 1) its addition to CD8+ T cell therapy augments clinical success, and 2) its efficacy is higher because CD4+ T cells can mount a broader antitumor response by direct and indirect recognition of tumor cells [187, 188].

1.6.2 Memory CD8+ T cells in tumor immunology and immunotherapy

A key feature of adaptive immunity is the ability to generate long-lived populations of self-renewing memory cells. In the tumor microenvironment, transformed cells trigger all kinds of innate cells to migrate to the tumor mass. They capture and present tumor antigens to naïve T cells in lymphoid organs, which are activated and progressively differentiate into effector T cells. When antigen supply ceases, primed T cells become quiescent and enter into the T memory stem cell (TSCM), central memory T cell (TCM), and effector memory T cell (TEM) pools [189]. The extent of T cell differentiation is dependent on various factors, mainly on the strength of the TCR signal and the cytokine environment. Several studies have shown that the differentiation state of the adoptive T cell populations is crucial to the success of ACT-based approaches. Berger et al. found

+ that CMV-specific effector CD8 T cells derived from TCM rather than TEM displayed

40 better survival in the circulation, bone marrow, and lymph nodes [190]. Naïve T cells have been shown to be more effective than memory T cells in eradicating tumor cells

[191]. Within the memory pools, TCM cells reject tumors more efficiently than TEM cells

[192]. These findings suggest that the differentiation state of CD8+ T cell is inversely correlated to antitumor efficacy (Figure 2: Nature Reviews, Volume 12, April 2012, page 276). In terms of T cells used for ACT, the culture conditions currently used may cause further differentiation of isolated TILs, which provides another reason for the low efficiency of current ACT procedure. Thus, manipulating T cell differentiation status is a new direction for physicians to perform ACT on cancer patients.

T memory stem cell (TSCM), mentioned above, is a newly identified population of memory CD8+ T cells [193] [186]. These cells express high levels of stem cell antigen 1

(Sca-1), B cell lymphoma 2 (Bcl-2) and CD122 (IL-2Rβ) [186]. Of importance, they have the stem cell-like properties of self-renewal and multipotency [186]. They survival better, persist longer after adoptive cell transfer, and have superior antitumor properties compared with TCM and TEM. In Gattinoni’s study, they also showed that TSCM cells have a genetic program that enables them to proliferate extensively and further differentiate into TCM and TEM cells (Figure 2). However, very few TSCM can be detected in the circulation of cancer patients. Nowadays, several groups are dedicating to develop methods to reprogram naïve T cells into a large number of TSCM cells in vitro for use in current ACT procedure.

Figure 2 Progressive T cell differentiation diminishes proliferative and antitumor capacities

T cells experience progressive changes in their phenotypes upon antigenic stimulation. Depending on the strength and duration of the signals, they are launched on a pathway of proliferation and differentiation. The process of T cell differentiation results in the loss of proliferative and self-renewal capacity.

1.6.3 T regulatory (Treg) cells in the tumor microenvironment

T regulatory (Treg) cells, representing a subpopulation of CD4+ T cells, play an essential role in sustaining self-tolerance and immune homeostasis through suppressing various physiological and pathological immune responses. In 1995 and 2003, CD25 (IL-2 receptor) and fork-head box P3 (Foxp3) were identified separately as markers for Treg cells [194, 195]. As the master transcriptional factor of Treg cells, Foxp3 controls expressions of multiple genes and regulates development, maintenance and function of

Treg cells [195]. Treg cells also express effector surface molecules such as CTLA4,

LAG3, CD39, or CD73 and co-stimulatory molecules such as CD28, CD80/86, CD40, or

OX40. They confer Treg cells to maintain and function in the peripheral [196]. In addition, some integrins and chemokine receptors such as α4β1 integrin, αEβ7 integrin,

α4β7 integrin, CCR1-2, CCR4-10, and CXCR3-6 have also been found to be expressed on the surface of Treg cells [197]. These molecules combine together function redundantly to regulate Treg cells homing and migration to lymph organs and disease occurrence sites. To date, Treg cells have been classified into 2 categories: naturally occurring Treg (nTreg) cells, which differentiates in the thymus, accounts for 5-10% of the CD4+ T cells; induced Treg (iTreg) cells, developing from naïve CD4+ T cells in the peripheral, obtains suppressive function upon stimulation in the presence of TGF-β [198].

They suppress effector cells via mechanisms as follows: first, they secret immunosuppressive cytokines, such as IL-10, TGF-β, IL-35, and thus inhibit immune responses [199]; second, they produce granzyme B to lyse target effector cells [200]; third, they compete with effector cells for IL-2, which disrupts the metabolism of effector cells [201]; last, CTLA4 on Treg cells engages with CD80/CD86 on DCs and delivers a negative signal preventing priming of adaptive immune responses [202].

Although Treg cells are critical for prevention of autoimmune diseases and chronic inflammatory diseases, they help immune escape within the tumor microenvironment

[203]. CD4+CD25+Foxp3+ Treg cells have been reported to infiltrate into tumor sites in both mouse tumor models and cancer patients, and generally favor tumor progression by restricting antitumor T cell responses. CD25+ T cell-depletion significantly improved antitumor immunity in different mouse models [204]. In the tumor microenvironment,

43 tumor cells and myeloid cells produce some chemokines, such as CCL22 and CCL2, which chemoattracts CCR4-expressing Treg cells to migrate to the tumor sites [205, 206]; hypoxia also triggers Treg cell recruitment by up-regulating CCL28, and subsequently promotes the angiogenesis process [207]. Tumor cells also stimulate immature DCs to secret TGF-β and induce the conversion of naïve T cells to iTreg cells [198]. The in situ

Treg cells secret TGF-β, IL-10, IL-35 and helps create an immunosuppressive environment that blunts the anti-tumor effector responses by CD4+, CD8+, NK, and NKT cells [208].

Given the immunosuppressive role of Treg cells in tumor microenvironment, depleting or blocking Treg cells represents one of the promising immunotherapies for cancer patients.

Recently, a monoclonal antibody target CTLA-4 (ipilimumab, BMS) received approval from the U.S. FDA for the treatment of patients with metastatic melanoma [209].

Furthermore, Anti-PD-1 has been tested in early-phase trails for patients with metastatic melanoma and colon cancer [210]. However, Treg cells and activated T cells share several common surface markers. Some strategies currently used to deplete or block Treg cells, such as administration of CD25 antibody, CTLA-4 antibody, and OX40 antibody, also resulted in the reduction of effector cells. New strategies are needed to restrict Treg cell function rather than eliminate Treg cells. In this regard, IL-27 is a novel regulator of

Treg generation, maintenance and function (reference). It provides a new possibility to treat patients with cancer.

1.7 Hypothesis and goals of the study

Tumor antigen specific CD8+ T cells play a pivotal role in the inhibition of tumor initiation, establishment, progression and metastases. IL-27 has been shown to exert its potent antitumor activity via multiple mechanisms, most notably enhancing antitumor

CTL responses. However, the mechanisms by which IL-27 enhances the antitumor CTL responses remain unclear. We hypothesized that IL-27 affects both tumor antigen specific

CD8+ T cells and CD4+Foxp3+ T regulatory cells in the tumor microenvironment, which together contributes to the IL-27-mediated tumor rejection. To determine the molecular and cellular mechanisms that IL-27 boosts the antitumor immune responses and leads to tumor rejection, we set up goals as follows:

1) Examine the impacts of IL-27 on tumor antigen-specific CD8+ T cells

For this part of our study, we examined the impacts of IL-27 on tumor antigen specific

CD8+ T cells using both in vitro and in vivo tumor models. WT and IL-10-/- tumor antigen specific CD8+ T cells (P1CTLs) were stimulated in vitro with cognate peptide

P1A in the presence and absence of rmIL-27. Their differentiation, proliferation, activation, and survival were compared using multiple biochemical and cell-based assays.

IL-27-overexpressing tumor cells (J558-IL-27) were generated and inoculated into various genetically modified mice to study the impacts of tumor-derived IL-27 on adoptively transferred tumor antigen specific CD8+ T cells.

2) Investigate the role of IL-27 in the response and function of Treg cells in

the tumor microenvironment

For this aim we used the B16.OVA.IL-27 and B16.OVA.Ctrl tumor models to explore the impact of tumor-derived IL-27 on T regulatory cells in the tumor microenvironment.

We also utilized IL-27 deficiency (EBI3-/-) mice to further study how IL-27 affects the accumulation and function of Treg cells. We performed both Treg depletion and adoptive transfer of Treg cells on melanoma-bearing mice to determine the contribution of Treg cells in IL-27-mediated tumor rejection.

3) Determine whether using IL-27 is a promising strategy for Cancer

Immunotherapy

In this section of our work, we designed two experiments to determine the potential of using IL-27 to treat tumors. First, we immunized WT and IL-27 deficiency (EBI3-/-) mice with tumor specific antigen and evaluated the CTL responses upon immunization;

Second, we examined the implications of using an adenovirus encoding IL-27 (Ad-IL-27) as a therapeutic tool in cancer treatment.

Chapter 2 Experimental Materials and Methods

2.1 Mice

The following mice were used in the present work

BALB/c, C57BL/6, IL-10-/-C57BL/6, RAG1-/-C57BL/6, and IL-10-/-BALB/c mice were originally purchased from The Jackson Laboratories.

RAG2-/-BALB/c mice were purchased from Taconic Farms (Germantown, New York,

USA).

P1CTL Transgenic mice expressing a TCR specific for the tumor antigen P1A

(P1CTL), whose TCR recognizes H-2Ld:P1A35-43 complex, have been described [211].

P1CTL TCR transgenic mice were backcrossed with BALB/c mice for at least 15 generations before they were used for this study.

IL-10-/-P1CTL mice were generated by breeding IL-10-/-BALB/c mice with P1CTL mice.

EBI3-deficient (EBI3-/-) mice in the C57BL6 background have been described

(reference). EBI3-/-RAG1-/- mice were generated through breeding EBI3-/- mice with

RAG1-/- mice for two generations.

EBI3-/-IL-10-/- mice were generated through breeding EBI3-/- mice with IL-10-/- mice for two generations.

PCR was used for identification of mice genotypes. The primers used were: EBI3

(forward) 5’-CTG ATG GGT CAC TAA CTC GGA TCC-3’ and EBI3 (reverse) 5’-ACG

ACA TCA GGG TCT GAT ATC AAG-3’, and the primers used were mIL-10.G: 5’-

ATA GAC TTG CTC TTG CAC TAC CAA AG-3’ (forward) and 5’-CTC ATG GCT

TTC CCT AGG ACT CTC TA-3’ (reverse). RAG1 deficiency was identified by analyzing peripheral blood cells for lack of B220+ cells. All mice were maintained in the animal facilities of The Ohio State University that are fully accredited by American

Association for Accreditation of Laboratory Animal Care.

2.2 Real time RT-PCR

Quantitative real-time PCR was performed using an ABI 7900-HT sequence system (PE

Applied Biosystems) with the QuantiTect SYBR Green PCR kit (Qiagen) in accordance with the manufacturer’s instructions. PCR was done using previously determined conditions [212]. One microliter of first-strand cDNA product was amplified with platinum Taq polymerase (Invitrogen Life Technologies) and gene-specific primer pairs.

Each sample was assayed in triplicate and the experiments were repeated twice. The relative amount of mRNA was calculated by plotting the Ct (cycle number) and the average relative expression for each group was determined using the comparative method

(2-∆∆Ct). The following primers were used for amplifying specific genes: SOCS3: 5’-GAG

ATT TCG CTT CGG GAC TA-3’(forward) and 5’-ACT TGC TGT GGG TGA CCA T -

3’(reverse); Bcl6: 5’- GTG AGC CGT GAG CAG TTT AG-3’ (forward) and 5’-CTC

AGG GCT GAT TTC AGG AT -3’ (reverse); Sca-1: 5’-TGG ATT CTC AAA CAA

GGA AAG TAA AGA-3’ (forward) and 5’-ACC CAG GAT CTC CAT ACT TTC AAT

A-3’ (reverse); Eomes: 5’- AAG CGG ACA ATA ACA TGC AG-3’ (forward) and 5’-

TGT TGT TGT TTG CAC CTT TG-3’ (reverse); T-bet: 5’- CTG CCT GCA GTG CTT

CTA AC-3’ (forward) and 5’-AAG TTC TCC CGG AAT CCT TT-3’ (reverse); Blimp1:

5’- CAA GCC GAG GCA TCC TTA-3’ (forward) and 5’- CGT GTT CCC TTC GGT

ATG TA-3’ (reverse); Perforin: 5’-GAA GAC CTA TCA GGA CCA GTA CAA CTT-3’

(forward) and 5’-CAA GGT GGA GTG GAG GTT TTT G-3’ (reverse); Granzyme B:

5’-CGA TCA AGG ATC AGC AGC C-3’ (forward) and 5’-CTG GGT CTT CTC CTG

TTC T-3’ (reverse); Bcl-2: 5’-TGA GTA CCT GAA CCG GCA TCT-3’ (forward) and

5’-GCA TCC CAG CCT CCG TTAT-3’ (reverse); Bcl-xl: 5’- GAC AAG GAG ATG

CAG GTA TTG G -3’ (forward) and 5’- TCC CGT AGA GAT CCA CAA AAG T -3’

(reverse); IL-10: 5’-ACA GCC GGG AAG ACA ATA AC-3’ (forward) and 5’-CAG

CTG GTC CTT TGT TTG AA-3’ (reverse); IFN-: 5’-AGC TCT TCC TCA TGG CTG

TT-3’ (forward) and 5’-TTT GCC AGT TCC TCC AGA TA-3’ (reverse). The HPRT gene was simultaneously amplified as endogenous control. The primers were 5’-AGC

CTA AGA TGA GCG CAA GT-3’ (forward) and 5’-TTA CTA GGC AGA TGG CCA

CA-3’ (reverse).

2.3 Antibodies and flow cytometry

FITC-, PE-, APC- or PerCP- labeled antibodies to CD4, CD8, FoxP3, pSTAT3, V8.3,

Bcl-2, Bcl-6, Sca-1 (D7), Eomes (Dan11mag), Perforin (eBioOMAK-D), Granzyme B

(NGZB), IL-10 (JES5-16E3), IFN- (4S.B3), IL-2 (JES6-5H4) and isotype-matched

49 control antibodies were purchased from BD Biosciences (San Diego, CA) or eBiosciences (San Diego, CA). For staining of cell surface markers, cells (cultured lymphocytes and single cell suspensions of tumors) were stained with various antibodies in staining buffer (PBS with 1% FCS) and incubated on ice for 30 min. After washing with staining buffer, cells were fixed in 1% Paraformaldehyde in PBS. For intracellular cytokine staining, cells were stimulated in culture medium for 4 h with 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) and 300 ng/ml of ionomycin in the presence of

Golgistop (1:1500; BD Biosciences). Viable cells were then fixed in IC fixation buffer

(eBioscience), permeabilized with 1×permeabilization buffer (eBiosciece) and stained with respective antibodies. Staining for FoxP3, Bcl-2, Bcl-6 and AnnexinV/7-AAD were performed according to manufacturer’s protocol (BD Biosciences). Stained cells were analyzed on a FACSCalibur flow cytometer and data were analyzed using the flowjo software (Tree Star, Inc., OR).

2.4 ELISA

ELISA kits for the detection of IL-10, IL-2 and IFN- were purchased from eBiosciences.

Standard procedures were followed to detect releases of cytokines in culture supernatants in a variety of settings.

2.5 Western Blot

For detection of pSTAT3, Bcl-2, Bcl-xl, Bcl-6, Socs3, cleaved caspase-3 and -actin by

Western blotting, P1CTL cells were stimulated with P1A35-43 in the presence or absence

50 of IL-27 for 4 days. Whole-cell lysates from 3×106 cells were run on SDS-PAGE gel, transferred to a nitrocellulose membrane and blotted using rabbit anti-pSTAT3 (sc-8001-

R, Santa Cruz), rabbit anti-Bcl-2 (#2876, Cell Signaling), rabbit anti-Bcl-xl (#2764, cell signaling), rabbit anti-Bcl-6 (# 5650s, cell signaling), rabbit anti-Socs3 (#2932s, cell signaling) and rabbit anti-cleaved caspase-3 Ab (#9661, Cell Signaling), and detected using an HRP-conjugated anti-rabbit IgG (Santa Cruz).

2.6 Cancer cell lines and tumor establishment in mice

B16F10 melanoma cells expressing the full length chicken ovalbumin (referred to as

B16-OVA) has been described (reference). Mouse plasmocytoma J558 cells (H-2Ld) has been described [213]. To generate B16-IL-27 and J558-IL-27 cells, an IL-27 expression vector (Invivogen) was used together with a pcDNA3 (Invitrogen) expression vector containing hygromycin-resistant gene to transfect B16-OVA and J558 cells, respectively.

The resulting hygromycin-resistant cells were selected for IL-27 secretion by ELISA. pcDNA3 expression vector containing hygromycin-resistant gene alone was used to transfect B16-OVA and J558 cells to generate B16-Ctrl and J558-Ctrl cells simultaneously. The generated tumor cells were maintained in RPMI 1640 medium

(Gibco) supplemented with 100 g/ml penicillin, 100 g/mL streptomycin, and 5% FBS.

To establish subcutaneous tumors in mice, 1×105 B16-IL-27 or B16-Ctrl cells were subcutaneously (s.c.) inoculated into recipient mice at the flank; 5×106 J558-IL-27 or

J558-Ctrl cells were inoculated into recipient mice s.c. at the flank. The length (a) and width (b) of each tumor were measured using a digital caliper every 2 or 3 days. The

51 tumor volume was calculated according to the formula V = ab2/2, as described [214, 215].

To establish tumor lung metastasis, each mouse was injected with 1×105 B16-IL-27 or

B16-Ctrl cells via the tail vein. Mice were monitored up to 3–4 weeks depending on symptoms and treatments received. At the end of the experiments, mice were sacrificed and lungs were collected and weighted.

2.7 T cell culture

0.3 x 106/ml of spleen and lymph node cells from P1CTL or IL-10-/-P1CTL mice were cultured in click’s EHAA medium (Invitrogen) containing 100 g/ml penicillin and 100

g/ml streptomycin, 1 mM 2-ME, 5% fetal bovine serum (FBS), and 0.2 g/ml P1A35-

43 peptide in the absence or presence of 50 ng/ml recombinant mouse IL-27 (Biolegend) for up to 5 days. The culture supernatants and activated P1CTL cells were analyzed during the course of culture.

2.8 T cell adoptive transfer

To treat mice with established J558 tumors, 1×107 splenocytes from P1CTL-transgenic mice or 5×106 in vitro-activated P1CTLs were injected into tumor-bearing mice i.v.

Tumor growth was monitored as described above.

2.9 In vivo T cell proliferation assay

CD8+ T cells from P1CTL mice were labeled with CFSE and were injected into RAG-2-/- mice i.v. at a dose of 5 x 106/mouse. Immediately after T cell injection, each recipient mouse also received 10 x 106 J558-IL-27 or J558-Ctrl cells. 65 h later, mice were sacrificed and splenocytes were stained for CD8/V8.3 and analyzed by flow cytometry.

2.10 CD8+ T cell and NK cell depletion

To deplete CD8+ T cells in vivo, mice received i.p. injection of 250 μg rat anti-mouse

CD8 IgG (53-6.72, BioXcell) diluted in 200 μl PBS. Control animals received i.p. injection of 250 μg IgG2a isotype control mAb (2A3, BioXcell). Injection of antibody was repeated every 4 days for a total of 3 times to maintain the depletion. To deplete NK cells in Rag1-/-ebi3-/- mice, 250 μg anti-mouse NK1.1 IgG (PK136, BioXcell) diluted in

200 μl PBS were injected into each mouse i.p. weekly.

2.11 CD25+ Treg cell depletion

To deplete CD25+ Treg cells in vivo, mice received i.p. injection of 400μg rat anti-mouse

CD25 IgG (PC-61.5.3, BioXcell) diluted in 200μl PBS. Control animals received i.p. injection of 400μg IgG1 isotype control mAb (HRPN, BioXcell). Injection of antibody was repeated every 4 days for a total of 3 times to maintain the depletion.

2.12 Isolation of CD4+CD25+ Treg cells from spleens

Mononuclear cells were prepared from spleens as described (references). CD4+cells were isolated from splenocytes by first incubating cell suspensions with CD4 soup

(TIB210:GK1.4 at 1:1 ratio) on ice for 0.5 h, followed by negative isolation using

Dynabeads Sheep anti-Rat IgG (Invitrogen). CD25+ cells were isolated from CD4+ cells by first staining them with PE-anti-CD25 mAb (BD biosciences), followed by magnetic antibody cell separation using anti-PE microbeads (Miltenyi Biotec). The isolated cells were >90% pure.

2.13 Treg-mediated suppression assay

For Treg-mediated suppression assay, 1×106/ml purified CD4+CD25- T cells from EBI3-/- mice were cocultured with graded numbers of CD4+CD25+ Treg cells from EBI3-/- or

EBI3-/-IL-10-/- mice in the presence of irradiated splenocytes (2×106 /ml) from EBI3-/-

RAG1-/- mice and 0.1 mg/ml anti-CD3 mAb (2C11). After 48 h, 1 mCi/well [3H]-Tritium was pulsed into the cultures, and incorporation of [3H]-Tritium was measured in a liquid scintillation plate counter 12 h later.

2.14 Flow cytometry-based cytotoxicity assay (FloKA)

A flow cytometry-based cytotoxicity assay was used to measure in vitro cellular cytotoxicity of IL-27-stimulated WT and IL-10-/- P1CTL cells to P815 target cells. Target cells (P815) were washed with PBS, resuspended at 1×106 cell/ml, and then labeled at

370C degree for 15 min with 10 nM CFSE (Molecular Probes, Junction City, OR).

Labeling reactions were stopped with RPMI medium containing 5% of FBS. Labeled target cells (1×105) were added to 96-well plates along with graded numbers of effector cells, IL-27-stimulated WT and IL-10-/- P1CTL cells. The effector : target ratio was 4:1,

2:1, and 1:1 as indicated. Four hours after incubation, 1µg/ml of 7-AAD (eBioscience,

CA) was added to each well and its incorporation was analyzed by flow cytometry. 7-

AAD positive cell were considered as late cell apoptosis since it intercalates with DNA in cells that have lost membrane integrity.

2.15 Lentivector immunization and Adenovector treatment

Lentivirus expressing TRP-1 gene (TRP1-lv) was provided by Dr. YuKai He, Georgia

Health Science University. For immunization, 2.5 ×107 transduction units of TRP1-Lv were injected into footpad of WT and EBI3-/- mice. Volumes of 50 µl were used for each mouse. On day 12 and 30 after immunization, about 50 µl of blood was taken and put into 1ml of PBS. Red blood cells were lysed by using 1 ml of lysis buffer. Cells were stimulated in 0.5 ml of RPMI media containing 1 ug/ml of TRP1-455 peptide in the presence of GolgiStop (1:1500) for 4 hrs at 370C. Intracellular staining of IFN-γ was performed to test CD8+ T cell response upon immunization.

Adenovirus expressing IL-27 (Ad-IL-27) was provided by Dr. Shulin Li, MD Anderson

Cancer Center. 1 ×109 PFU of Ad-IL-27 in 50 µl of PBS was injected directly into each

J558 tumor which was established in Balb/c mice. Control mice were treated with 50 µl of PBS per mouse. The treatment was repeated every 4 days for a total of 3 times.

2.16 Statistical analysis

Data are expressed as mean ± SD/SEM. Two-tailed Student’s t-test was used for statistical analysis. p<0.05 was considered significant.

Chapter 3 IL-27 enhances tumor antigen specific CD8+ T cell responses

and leads to tumor rejection via multiple mechanisms

3.1 IL-27 enhances survival of tumor antigen specific CD8+ T cells and programs them into IL-10 producing memory precursor-like effector cells

3.1.1 IL-27 enhances survival of tumor antigen specific CD8+ T cells

To determine the direct effects of IL-27 on the activation and differentiation of tumor antigen specific CD8+ T cells, spleen and lymph node cells from P1CTL mice, whose

CD8+ T cells bear a TCR transgene specific for tumor rejection antigen P1A, were stimulated with P1A35-43 in the presence or absence of recombinant IL-27. Cell numbers were counted every 24 hours for a period of 5 days. As shown in Fig.3A,

P1CTL cells stimulated with IL-27 yielded significantly higher numbers of viable cells compared to cells stimulated with P1A peptide alone. To determine if IL-27 affects the survival of activated P1CTL cells, the cultured P1CTL cells were stained for Annexin V and 7-AAD to quantify cell apoptosis over time. The addition of IL-27 resulted in substantially reduced cell apoptosis of activated P1CTL cells (Fig.3B). Similar results were obtained when purified CD8+ T cells from P1CTL mice were activated with plate- bound anti-CD3/anti-CD28 and IL-27 (not shown). Thus, IL-27 directly conveys a survival advantage to activated P1CTLs.

Figure 3 The role of IL-27 in the survival of activated P1CTLs A. Spleen and lymph node cells from P1CTL mice were stimulated with 0.2 µg/ml of

P1A peptide in the presence or absence of 50 ng/ml of rmIL-27. Live cells in

cultures were counted under microscope by Trypan blue exclusion every 24 hours

for a period of 5 days. Error bars indicate SD of 3 samples in each group. Data

shown represent five experiments with similar results.

B. Cultured P1CTL cells (as described in A) were stained for Annexin V and 7-AAD

on day 3-5, and analyzed by flow cytometry. Annexin V+7-AAD+ P1CTL cells

were plotted using day 4 data. Error bars represent SD of 4 samples in each group.

C. 5 x 106 J558-IL-27 or J558-Ctrl cells were injected into each Rag-2-/-BALB/c

mouse s.c. (n=5/group). The tumor volume was measured over time. Error bars

indicate SD of 5 mice in each group. Data shown represents three experiments

with similar results.

D. 5 x 106 J558-IL-27 or J558-Ctrl cells were injected into each Rag-2-/-BALB/c

mouse s.c. (n=4/group). When tumor reached 5 mm in diameter, 10 x 106

splenocytes from P1CTL mice were injected into each mouse i.v. Four days later,

mononuclear cells were prepared from spleens and tumors and were stained for

CD8 and Vα8.3. Flow cytometry analysis (left) shows data from one pair of

representative mice. Error bars in the right panel represent SD of samples from 4

mice in each group.

E. Mononuclear cells prepared from spleens and tumors described in D were stained for

CD8, V8.3 and Annexin V and analyzed by flow cytometry. Data shown are gated on

CD8+V8.3+ cells and represent two experiments with similar results.

F. CD8+ T cells from P1CTL mice were labeled with CFSE and were injected into

Rag-2-/- mice i.v. at a dose of 5 x 106/mouse. Immediately after T cell injection,

each recipient mouse also received 10 x 106 J558-IL-27 or J558-Ctrl cells. 65 h

later, mice were sacrificed and splenocytes were stained for CD8/Vα8.3 and

analyzed by flow cytometry. Data shown are gated on CD8+Vα8.3+ cells and

represent two experiments with similar results.

To test if IL-27 enhances P1CTL survival in vivo, mouse plasmacytoma J558 cells were transfected with an IL-27 expression vector or a control expression vector, and J558-IL-

27 and J558-Ctrl cells were generated. J558 cells express tumor antigen P1A that can be specifically recognized by P1CTL cells [211, 216]. S.c. injection of 5×106 J558-IL-27 or

J558-Ctrl tumor cells into Rag-2-/- mice resulted in similar tumor growth (Fig.3C).

P1CTL cells were injected i.v. into Rag-2-/- mice bearing established tumors of similar size. Four days later, more P1CTL cells were detected in the spleens and tumors from

59 mice bearing J558-IL-27 tumors compared to mice bearing J558-Ctrl tumors (Fig.3D).

Tumor infiltrating P1CTL cells from J558-IL-27 tumors also underwent less apoptosis compared to cells from J558-ctrl tumors (Fig.3E). An in vivo proliferation assay suggested that the dividing rate of P1CTL cells was similar in the two different hosts

(Fig.3F). Thus, IL-27 also enhances the survival of activated P1CTLs in the tumor- bearing host and tumor microenvironment.

3.1.2 IL-27 stimulates a large amount of IL-10 production by tumor antigen specific CD8+ T cells

One of the known roles of IL-27 is to stimulate IL-10 production by CD8+ T cells [55,

89]. To determine if IL-27 could induce IL-10 production in P1CTL cells in vivo, we injected P1CTL cells into Rag-2-/- mice bearing established J558-IL-27 or J558-Ctrl tumors. Four days after P1CTL transfer, we sacrificed mice and prepared single cell suspensions from both spleens and tumors, and stained cells for IL-10 and IFN-. As demonstrated in Fig.4A and quantified in Fig.4B, the number of IL-10+ P1CTLs infiltrating into J558-IL-27 tumors was substantially increased compared to those in

J558-Ctrl tumors. The number of IFN- and IL-10 positive P1CTLs were low in spleens from both J558-IL-27 and J558-Ctrl tumor-bearing mice.

Figure 4 The role of IL-27 in P1CTL IL-10 production in tumor microenvironment

A. Flow cytometry analyses of IL-10 and IFN- production in ex vivo P1CTL cells.

Each Rag-2-/-BALB/c mouse with established J558 tumor (5 mm in diameter)

received 10 x 106 splenocytes from P1CTL mice i.v. Four days later, mononuclear

cells were prepared from spleens and tumors, and stained for CD8, V8.3, IL-10

and IFN-Data shown were gated on CD8+V8.3+ cells.

B. Quantification of IL-10+, IL-10+ IFN-+ and IFN-+ CD8+ cells in A. Error bars

indicate SD of 5 samples in each group.

To determine if IL-27 induction of IL-10 affects P1CTL cell effector functions, spleen and lymph node cells from P1CTL and IL-10-/-P1CTL mice were stimulated with P1A peptide in the presence or absence of IL-27 for up to five days in vitro. As shown in

Fig.5A and quantified in Fig.5B, starting on day 3, high numbers of IL-10+IFN-- and IL-

10+IFN-+ cells were detected in IL-27-stimulated WT P1CTL cells (Fig.5A, first row), while IL-10+ cells were undetectable in WT P1CTL cells in the absence of IL-27 (Fig.5A, second row), or in activated IL-10-/-P1CTLs in the presence or absence of IL-27 (Fig.5A, third and fourth rows). Consistent with this observation, IL-10 mRNA expression was elevated more than 15 times in IL-27-stimulated WT P1CTLs relative to other cultures

(Fig.5C). Increased concentration of IL-10 protein in the culture supernatant from IL-27- stimulated WT P1CTLs was also detected (Fig.5D). IL-27 stimulation resulted in slightly reduced numbers of IFN-+ cells (Fig.5A, Fig.5B), lower IFN-γ mRNA expression

(Fig.5C), and reduced amounts of IFN- secretion into the culture supernatants (Fig.5D) in both WT and IL-10-/-P1CTLs. Since IL-27 equally inhibited IFN- production in WT and IL-10-/-P1CTL cells, it is likely that the down-regulation of IFN- by IL-27 is IL-10 independent. However, IL-27-differentiated P1CTL and IL-10-/-P1CTL cells showed similar cytotoxicity to P1A antigen positive P815 cells (Fig.5E).

Figure 5 The roles of IL-27 in CTL IL-10 production and effector functions Spleen and lymph node cells from WT and IL-10-/- P1CTL BALB/c mice were stimulated with 0.2g/ml P1A peptide in the presence or absence of 50 ng/ml of rmIL-27.

A. Flow cytometry analyses of intracellular IL-10 and IFN- production by P1CTL

cells. The cultured P1CTL cells were harvested every 24 hours for a period of 5

days and were stained for CD8, IL-10, and IFN-. Data shown were gated on

CD8+ cells. Data shown represent five experiments with similar results.

B. Quantification of IL-10 and IFN- producing cells. Cultured P1CTL cells were

harvested on day 4. IL-10 and IFN- production by P1CTL cells were measured

by intracellular staining as in A. Error bars indicate SD of 5 samples in each

group.

C. P1CTL Cells were harvested on day 4. Expression of IL-10 and IFNgenes was

analyzed by qRT-PCR. Error bars indicate SD of 3 samples in each group. Data

shown represent three experiments with similar results.

D. Concentrations of IL-10 and IFN- in the culture supernatants were measured by

ELISA. Error bars indicate SD of triplicates. Data shown represents three

experiments with similar results.

E. Cytotoxicity of IL-27-stimulated P1CTL and IL-10-/-P1CTL cells to P815 target

cells. Data shown represents three experiments with similar results.

3.1.3 IL-27-induced CTL IL-10 production is not essential for its pro-survival effect

Since IL-27 stimulates high amounts of IL-10 production by P1CTL cells, we tested if the survival enhancing effect of IL-27 was due to IL-10 production by P1CTL cells, as

IL-10 has been shown to enhance CTL expansion [159, 217]. Spleen and lymph node cells from IL-10-/-P1CTL mice were stimulated with P1A peptide in the presence or absence of IL-27, and numbers of viable cells were counted once a day for a period of 5 days. As shown in Fig.6A, IL-10-/-P1CTL cells cultured with IL-27 resulted in significantly higher numbers of viable cells compared to cells stimulated with P1A peptide alone. To determine if IL-27 affects the survival of activated IL-10-/-P1CTL cells, cultured IL-10-/-P1CTL cells were stained for Annexin V and 7-AAD to quantify cell death over time. The addition of IL-27 resulted in significantly reduced cell death of

64 activated IL-10-/-P1CTL cells (Fig.6B). Similar results were obtained when purified

CD8+ T cells from IL-10-/-P1CTL mice were activated with plate-bound anti-CD3/anti-

CD28 and IL-27 (not shown). These results suggest that IL-27 provides a survival advantage to activated IL-10-/-P1CTL cells. To test if IL-27 stimulates IL-10-/-P1CTL cell expansion in vivo, we injected IL-10-/-P1CTL cells into Rag-2-/- mice bearing established tumors of similar size. Four days later, more IL-10-/-P1CTL cells were detected in the tumors and spleens from mice bearing J558-IL-27 tumors (Fig.6C). Thus, IL-27 induced

IL-10 production does not affect IL-27 induced CTL survival in vitro and in vivo.

Figure 6 The role of IL-27 in the survival of activated IL-10-/- P1CTL cells

A. Spleen and lymph node cells from IL-10-/-P1CTL mice were stimulated with 0.2

g/ml of P1A peptide in the presence or absence of 50 ng/ml of rmIL-27. Live cells

in cultures were counted under a microscope by trypan blue exclusion every 24 hours

for a period of 5 days. Error bars indicate SD of 3 samples in each group. Data shown

represent five experiments with similar results.

B. Cultured IL-10-/-P1CTL cells were stained for Annexin V and 7-AAD on day 3-5, and

analyzed by flow cytometry. Annexin V+7-AAD+ P1CTL cells were plotted using day

4 data. Error bars represent SD of 4 samples in each group.

C. 5 x 106 of J558-IL-27 or J558-Ctrl cells were injected into each Rag-2-/-BALB/c

mouse s.c. (n=4/group). When tumor size reached 5 mm in diameter, 10 x 106

splenocytes from IL-10-/-P1CTL mice were injected into each mouse i.v. Four days

later, mononuclear cells were prepared from spleens and tumors and were stained for

CD8 and V8.3. Flow cytometry analysis (left) shows data from one pair of

representative mice. Error bars (right) represent SD of 4 samples in each group.

3.1.4 IL-27-induced CTL IL-10 production contributes to tumor rejection

To determine the effects of IL-27 induced CTL IL-10 production in tumor rejection, we injected IL-27-stimulated WT P1CTLs, IL-27-stimulated IL-10-/- P1CTLs, or P1A peptide alone-activated WT P1CTLs into J558 tumor-bearing Rag-2-/- mice and monitored their tumor growth. As shown in Fig.7A, IL-27-stimulated P1CTL had the best efficacy in rejecting established J558 tumors. In the second set of experiments, un- stimulated WT P1CTL or IL-10-/- P1CTL cells were injected into Rag-2-/- (Fig.7B) or

Rag-2-/-IL-10-/- (Fig.7C) mice with established J558-IL-27 tumors. Enhanced tumor

66 rejection was observed in P1CTL cell-treated mice than IL-10-/- P1CTL cell-treated mice in both models. Collectively, these data suggest that IL-27-induced IL-10 production by

CTLs contributes to tumor rejection.

Subcutaneous injection of J558-IL-27 cells into BALB/c mice barely established tumors, while J558-Ctrl cells established tumors that reached a size of ~800 mm3 after 20 days

(Fig.7D). Since J558-IL-27 tumors could be established in Rag-2-/- and showed similar growth kinetics to J558-Ctrl tumors, adaptive immunity must play a vital role in rejecting

J558-IL-27 tumors. Indeed, the depletion of CD8+ T cells in BALB/c mice resulted in progressive growth of J558-IL-27 tumors (Fig.7E), suggesting that IL-27-mediated tumor rejection in this model is CD8+ T cell-dependent. To determine the role of IL-10 production in CTL-mediated tumor rejection, IL-10-/-BALB/c and BALB/c mice were inoculated with J558-IL-27 tumor cells s.c. No tumors grew in WT mice, but J558-IL-27 tumors established and grew in about 50% of IL-10-/- mice (Fig.7F). Thus, CTL response rejected J558-IL-27 tumors in WT but not IL-10-/- mice.

Figure 7 The role of IL-27-induced CTL IL-10 production in tumor rejection

A. 5 x 106 J558-Ctrl cells were injected into each Rag-2-/-BALB/c mouse s.c. When

tumor size reached 5 mm in diameter, mice were randomly divided into three

groups and received: 1) 5 x 106 IL-27 + P1A peptide-stimulated P1CTL cells

(n=5); 2) 5 x 106 IL-27 + P1A peptide-stimulated IL-10-/-P1CTL cells (n=5) and 3)

5 x 106 P1A peptide alone-stimulated P1CTL cells (n=5). The tumor growth was

observed over time. *: p<0.05.

B. J558-IL-27 cells were injected into Rag-2-/-BALB/c s.c. at a dose of 5 x 106

cells/mouse. When tumor size reached 5 mm in diameter, mice were treated with

10 x 106/mouse IL-10-/-P1CTL or P1CTL splenocytes (n=5/group). **: p<0.01 by

student’s t test.

C. J558-IL-27 cells were injected into Rag-2-/-IL-10-/- mice s.c. at a dose of 5 x 106

cells/mouse. When tumor size reached 5 mm in diameter, mice were treated with

10 x 106 cells/mouse IL-10-/-P1CTL or P1CTL splenocytes (n=5/group). *:

p<0.05; **: p<0.01 by student’s t test.

D. 5 x 106 J558-IL-27 or J558-Ctrl cells were injected into each BALB/c mouse s.c. The

tumor growth was observed over time. Error bars indicate SD of 5 mice in each group.

Data shown represent three experiments with similar results.

E. 5 x 106 J558-IL-27 cells were injected into each BALB/c mouse s.c. On days 0, 5

and 9 after tumor cell injection, each mouse received either 250 g anti-CD8

mAb (53-6.72, BioXcell) or control IgG i.p. The tumor growth was observed over

time. Bars indicate SD of 3 mice in each group.

F. 5 x 106 J558-IL-27 cells were injected into each BALB/c or IL-10-/-BALB/c

mouse s.c. The tumor growth was observed over time. Bars indicate SD of 5 mice

in each group. Data shown represent three experiments with similar results.

3.1.5 IL-27/IL-10 axis induces a memory precursor/effector phenotype in tumor antigen specific CD8+ T cells

To further understand the mechanisms of IL-27-mediated effects on tumor antigen specific CD8+ T cells, we examined the expression of a variety of survival and differentiation genes associated with CD8+ T cells [218, 219] in cultured WT P1CTL and

IL-10-deficient P1CTL cells. As shown in Fig.8A, IL-27 significantly up-regulated the expression of SOCS3, Bcl-6, Sca-1, and Bcl-2 genes in both WT P1CTL and IL-10- deficient P1CTL cells. However, more significant induction of these genes was observed

69 in WT P1CTL cells, expressions of Bcl-xl and T-bet genes were up-regulated by IL-27 to a lesser extent in both WT P1CTL and IL-10-deficient P1CTL cells, Blimp-1 and

Perforin genes were up-regulated slightly by IL-27 in WT but not IL-10-deficient P1CTL cells. In contrast, expressions of Eomes and Granzyme B genes were down-regulated in both WT P1CTL and IL-10-deficient P1CTL cells. IL-27 has been shown to activate

STAT3 [37, 55], and STAT3 activation is known to induce expression of SOCS3 [219] and the Bcl family of proteins [219-221]. Western blot (Fig.8B) and flow cytometry

(Fig.8C) analyses verified that IL-27 activated STAT3 and up-regulated Bcl2, Bcl-6,

SOCS3, Sca-1, and perforin while reduced the expression of Eomes and Granzyme B in activated WT P1CTL cells; in IL-10-deficient P1CTL cells, IL-27 up-regulated p-Stat3,

Bcl-6, Sca-1 and down-regulated Eomes. However, IL-27 did not alter the expression of

Bcl-2, SOCS3, Bcl-xl, Granzyme B and Perforin at the protein level in IL-10-/-P1CTL cells. IL-27 stimulation also reduced the expression of cleaved caspase 3, a key executer of apoptosis [222], in activated WT and IL-10-deficient P1CTL cells (Fig.8B). Thus, IL-

27 induces a memory precursor cell (MPC) phenotype [219] in tumor antigen specific

CD8+ T cells characterized by strong expression of Bcl-6, SOCS3 and Sca-1. However, in the absence of IL-10, the induction of MPC phenotype and survival molecules by IL-

27 is diminished.

Figure 8 Phenotypes of IL-27 stimulated tumor antigen specific CD8+ T cells A. Expression of T cell differentiation genes in P1A-peptide activated P1CTL and

IL-10-/-P1CTL cells. P1CTL cells were stimulated for 4 days with P1A peptide in

the presence or absence of IL-27. Error bars indicate SD of 3 samples in each

group. Data shown represent two experiments with similar results. *: p<0.05; **:

p<0.01; ***: p<0.001.

B. Protein lysates were prepared from P1CTL and IL-10-/-P1CTL cells stimulated

under the same condition as in A and subjected to Western blot analysis using

various antibodies as indicated. Data shown represents three experiments with

similar results.

3.3 Using IL-27 is a promising strategy for cancer immunotherapy

3.3.1 EBI3-deficiency impairs the efficacy of tumor antigen vaccination

Since we have shown that IL-27 enhances the survival and induces a memory precursor cell (MPC) phenotype of tumor antigen specific CD8+ T cells, we hypothesize that using

IL-27 could be beneficial for cancer vaccine therapies. To test this hypothesis, WT and

EBI3-/- C57BL6 mice were immunized with 2.5×107 TU of lentivector expressing melanoma antigen TRP1 (TRP1-lv), On day 12 and day 32 after immunization, blood from immunized mice was collected and intracellular stained for IFN-γ after 4 hours of ex vivo stimulation with cognate peptide TRP1-455. As shown in Figure 15A, this tumor antigen vaccination stimulated more CD8+ T cells to produce IFN-γ in WT mice compared to EBI3-/- mice on both day 7 and 32. Next, we investigated if lentivector- stimulated CD8+ T cells could recognize and kill B16 tumor, each immunized mouse was inoculated with 1×105 B16 tumor cells subcutaneously. Tumor grew much slower in immunized WT mice than EBI3-/- mice during the following 21 days (Figure 15B). At the end of experiment, both groups of mice were sacrificed and tumors were isolated out and weighted. Consistent with tumor growth curve, immunized WT mice had significant smaller and lighter tumor burden than EBI3-/- mice (Figure 15C). Furthermore, we examined the tumor infiltrating CD8+ T cells and their IFN-γ production. Substantially higher number of CD8+ T cells was detected in tumors from immunized WT mice compared to those from EBI3-/- mice, and these tumor infiltrated CD8+ T cells produced comparatively more IFN-γ in WT mice than EBI3-/- mice (Figure 15D). Collectively,

EBI3-decifiency resulted in less effective CD8+ T cell response against tumor in a cancer

88 vaccine therapy, indicating that endogenous IL-27 is important for eliciting potent CD8+

T cell response upon tumor antigen vaccination.

A B WT EBI3-/- 2.5 4000 EBI3-/- EBI3-/-

) 3500

3 B6 2 WT

Day12 3000

in in blood + 1.5 2500 **

* 2000 in in CD8

+ 1 1500

γ - Day30 1000

0.5 VolumeTumor (mm

500

% of% IFN γ - 0 0 12 30 IFN 9 11 13 15 17 19 21 CD8 Days post vaccination Days post tumor injection

C D WT EBI3-/- 4.5 * CD8+ cells IFN-γ+CD8+ cells 4 6 0.45 3.5 WT 5 * 0.4 * 3 -/- 0.35 EBI 2.5 4

SSC 0.3 2 0.25

1.5 3 milliong / milliong / 0.2 1 Tumor weightTumor (g) 2 0.15 0.5 0.1 0 1

-/- γ EBI3 B6 - 0.05

0 0 IFN Vaccinated Non-Vaccinated Vaccinated Non-Vaccinated CD8 Figure 15 EBI3-deficiency impairs tumor specific CTL responses in vaccinated mice 2.5×107 TU of TRP1-Lv were injected into one footpad of WT or EBI3-/- mice

(n=5/group)

A. On day 12 and 32 after immunization, peripheral blood cells were stimulated with

peptide TRP1-455 for 4 hours, stained for CD8 and IFN-γ, and analyzed by flow

cytometry. The percentage of IFN-γ-producing CD8+ T cells of total CD8+ T cells

were calculated and presented as means ±SD.

B. On day 35 after immunization, 1×105 B16 tumor cells were injected at the right

flank of each mouse. Tumor growth was monitored by measuring the

perpendicular diameters of the tumor every 2-3 days.

C. On day 21 after tumor injection, mice were sacrificed and tumors were isolated

and weighted. Figure shows the average weigh of each group.

D. Single cell suspensions were prepared from tumor mass, stimulated with peptide

TRP1-455 for 4 hours, stained for CD8 and IFN-γ, and analyzed by flow

cytometry. The percentages of CD8+ T cells of total tumor cells and IFN-γ-

producing CD8+ T cells of total CD8+ T cells were calculated and presented as

means ±SD.

3.3.2 Intra-tumoral injection of IL-27 producing adenovirus enhances anti-tumor immunity

To further test if IL-27 can be practically used to enhance anti-tumor immunity, we injected J558-Ctrl tumor cells into BALB/c mice subcutaneously. When tumors were fully established, we injected adenovirus expressing IL-27 (Ad-IL-27) or vehicle only directly into established tumors. After 2 rounds of treatment, tumor growth was obviously slower in Ad-IL-27 treated mice compared to those treated with vehicle only (Figure

16A). To investigate if intra-tumoral injection of Ad-IL-27 induced anti-tumor immunity, we analyzed T cell responses in the tumors. We found higher numbers of CD8+ T cells in the Ad-IL-27-treated tumors than control tumors (Figure 16B). Moreover, CD8+ T cells from Ad-IL-27 treated tumors produced more IFN- and IL-10 (Figure 16C). As expected, we observed reduced numbers of CD4+Foxp3+ Treg cells in Ad-IL-27-treated tumors compared to those treated with vehicle only (Figure 16D). Our results indicated that IL-27 gene therapy does have biological relevance. Therefore, we suggest the use of

IL-27 can be applied to current tumor treatment protocols, in order to boost CTL response and enhance tumor rejection.

A B 1800 15 * Ad-IL-27 PBS Ad-IL-27

) 1600

3 PBS 1400 10

1200 (%)

1000 P=0.02 + SSC

800 CD8 5 600 400 Tumor volume (mm volume Tumor 200 0 CD8 0 PBS Ad-IL-27 8 10 12 15 18 22 Days post tumor injection

D C 50 40 PBS Ad-IL-27 PBS PBS Ad-IL-27 ** 40 ** Ad-IL-27 30

30 (%)

+ 20 10

- 20

Foxp3

IL Foxp3 % in CD8+in cells % 10 * 10

0 0 CD4 IFN-γ IFN-γ IL-10 PBS Ad-IL-27

Figure 16 Intra-tumoral injection of Ad-IL-27 enhances anti-tumor immunity A. 5 x 106 of J558 cells were injected into each BALB/c mouse s.c. (n=4/group).

When tumor reached a size of ~5-6mm diameter, mice were randomly assigned to

treatment groups. Adenovirus (1×109 pfu diluted in 50 l PBS) or 50 l PBS was

injected intra-tumorally. Treatments were repeated twice at the interval of 4 days.

The tumor growth was observed over time. Bars represent SD for 4 mice in each

group.

B. Flow cytometry analysis and quantification of CD8+ T cells in tumors with or

without Ad-IL-27 treatment. Data shown in left panel are from one pair of

representative mice. Data shown in the right panel are mean + SD of four mice in

each group.

C. Flow cytometry analysis and quantification of IFN- and IL-10 producing CD8+ T

cells in tumors with or without Ad-IL-27 treatment. Data shown in left panel are

from one pair of representative mice. Data shown in the right panel are mean +

SD of four mice in each group.

D. Flow cytometry analysis and quantification of CD4+FoxP3+ cells in tumors with

or without Ad-IL-27 treatment. Data shown in left panel are from one pair of

representative mice. Data shown in the right panel are mean + SD of four mice in

each group.

Chapter 4 Discussion

Since Hisada et al. first reported the anti-tumor efficacy of IL-27 in 2004, researchers from different groups transfected the IL-27 gene into a variety of mouse and human tumor cell lines, evaluated their tumor establishments on mouse models, and found that tumor-derived IL-27 triggered potent antitumor immune responses and led to almost complete tumor rejection. For instance, highly immunogenic murine colon carcinoma

Colon 26 cells transfected with the IL-27 expression vector exhibited minimal tumor growth, which is mediated mainly by CD8+ T cells, IFN-γ and T-bet [95, 96, 110-112].

While in poorly immunogenic tumors such as B16F10 melanoma, IL-27 exerts its antitumor effects using various mechanisms, including enhancing NK cell responses

[114], inhibiting angiogenesis [113], and directly suppressing tumor cell proliferation

[115]. IL-27 also activates NK cells and suppresses NK-resistant head and neck squamous cell carcinoma through inducing antibody-dependent cellular cytotoxicity

[101]. In mice bearing TBJ neuroblastoma tumors, IL-27 mediates overall tumor regression by up-regulating MHC class I expression on tumor cells, and enhancing the generation of both tumor-specific immune responsiveness and immunologic memory responses [112]. Furthermore, both over-expression of IL-27 and treatment with rIL-27 can directly inhibit expression of vimentin, COX-2, and its metabolite (PGE2) on lung cancer cells [117]. These effects result in the reduction of cancer migration and invasion.

IL-27 is also capable of inhibiting human multiple myeloma cell growth as well as osteoclast differentiation and activity [119]. Collectively, both IL-27-mediated immune and non-immune effects contribute to its potent antitumor activities. The mechanisms used by IL-27 to regulate tumor growth vary among different tumors depending on the characteristics of tumors.

It’s worthy to note that the T cell-dependent mechanism seems to be the most important one contributing to the antitumor effect of IL-27. While expression of IL-27 in cancer cells or IL-27-based vaccination/gene therapy resulted in robust CTL responses and tumor rejection, the enhancing roles of endogenous IL-27 in the generation of anti-tumor

CTL responses were also demonstrated by using IL-27R-deficient mice. However, the exact underlying mechanisms remain unclear. One possibility is that IL-27 directly enhances CTL differentiation and effector functions, since IL-27 has been shown to promote CD8+ T cells to express T-bet, IL-12Rβ2, and granzyme B.

In this study, we sought to determine this issue by analyzing the direct and indirect effects of IL-27 on tumor antigen specific CTLs using our well-established tumor models

[225]. We have made the following four major observations that could explain why IL-27 enhances anti-tumor CTL responses and tumor rejection. First, IL-27-stimulated CTL cells have survival advantage and produce high amount of IL-10. IL-27 conferred a survival advantage for CTL cells which is independent of CTL IL-10 production. Second,

IL-27-induced IL-10 production by tumor antigen specific CTL cells contributes to CTL- mediated tumor rejection. Third, IL-27 induces a unique memory precursor cell (MPC) phenotype in activated CD8+ T cells, characterized by up-regulation of SOCS3, Bcl-6 and

Sca-1, and give rise to memory T cells. The induction of MPC phenotype and T cell memory are largely dependent on IL-27-induced CTL IL-10 production. Fourth, IL-27 suppresses the development and function of Treg cells in the tumor microenvironment, which indirectly enhances antitumor CTL responses and leads to tumor rejection.

4.1 IL-27 enhances survival of tumor specific CD8+ T cells both in vitro and in vivo

After encountering with antigens, naïve T cells proliferate and differentiate into effector cells, which actually would die by apoptosis driven by the engagement of death receptors on their surface or “cytokine withdrawn”. The death of activated T cells can be prevented by cytokines both in vitro and in vivo. Several cytokines, such as IL-2, IL-4, IL-7, IL-9,

IL-15 and IL-21, have been broadly reported to promote the survival of T cells. Moreover, each of them has a distinct role depending on the characteristics of T cells. For example,

IL-7 is crucial for development and survival of naïve T cells; IL-2 regulates both expansion and contraction of effector T cells; while IL-15 is required for the maintenance and activation of memory T cells. Bcl-2 family members, downstream of cytokine signaling, have either anti-apoptotic or pro-apoptotic activities within T cells. Bcl-2 levels within activated T cells are decreased compared with resting cells.

A notable observation of this study is that IL-27 confers a survival advantage to activated

CTL cells both in vitro and in vivo. The pro-survival effect was observed in both IL-27- stimulated WT and IL-10-/- P1CTL cells, suggesting that the anti-apoptotic effect of IL-27 is independent of IL-27-induced IL-10 production. IL-27 and IL-10 both activate Stat3

[37, 55, 219], and activation of Stat3 is known to up-regulate Bcl family of molecules

[219-221]. Indeed, in this study we found that IL-27-stimulated CTL cells down- regulated production of cleaved caspase-3 and up-regulated a number of Bcl family genes, including Bcl-2, Bcl-6 and Bcl-xl. Thus, the IL-27-mediated pro-survival effect is associated with activation of Stat3-Bcl anti-apoptosis pathways. Although IL-27 promotes survival of IL-10-/-P1CTL cells, western blot analysis revealed a notable reduction of IL-27-induced Bcl-2 protein compared to IL-27 stimulated WT P1CTL cells.

This result suggests that IL-10-deficiency may affect CTL survival in more physiological conditions where high levels of IL-27 are not available. This point is supported by our finding that the long term survival of IL-27-stimulated IL-10-/-P1CTL cells was impaired in vivo (Fig.9). The strong anti-apoptosis effect of IL-27 could explain why, in tumor models, more CTLs accumulate in the IL-27-positive tumor microenvironment.

4.2 IL-27 programs activated CD8+ T cells to become memory precursor-like effector cells

CD8+ T cell memory is the ability of CD8+ T cells to respond faster and more strongly to reencounter of the same antigen. They are critical for long-term immunity and are the basis of vaccination. Most of our current knowledge on the differentiation, maintenance, reactivation, and function of memory T cells is based on the study of acute and chronic viral infections. However, little is known about the generation and characteristics of

CD8+ T cell memory subsets in the tumor microenvironment. To better investigate memory CD8+ T cells in the tumor bed, it is instructive to know several key aspects of memory T cell response upon virus infection.

After acute viral or bacterial infection, a subset of memory precursor cells (MPC) could be descent from a heterogeneous effector cell pool, and these cells progressively differentiated into mature memory T cells [226, 227]. Several factors have been reported to be required for this effector-to-memory transition. IL-2 signaling, mTOR activity, inflammation, prolonged antigenic stimulation were shown to favor the formation of terminal effector T cells (TE) [219]. A recent study from a Yale immunology group demonstrated that the IL-21-IL-10-STAT3 signaling pathway plays an important role in the differentiation and functional maturation of memory CD8+ T cells during LCMV infection. In their study, activation of STAT3 resulted in upregulation of several pro- memory transcriptional factors and associated proteins, such as Bcl-6, Eomes and SOCS3;

IL-10 and IL-21, two STAT3 upstream cytokine stimulators, cooperated to induce memory CD8+ T cells and promoted their maturation during LCMV infection [219].

In our study, we found that IL-27-stimulated CD8+ T cells had a mixed memory precursor/effector phenotype. Similar to the above cited study, IL-27 significantly up- regulated STAT3 responsive molecules SOCS3, Bcl-6, Sca-1, and IL-10 in P1CTLs.

SOCS3 and Bcl-6 have recently been shown to be critical in establishing CD8+ T cell memory [219]. Sca-1 is a cell membrane molecule usually expressed on self-renewing stem cells and is a marker of central memory CD8+ T cells [186, 193]. IL-10 has also been shown to play a vital role in CD8+ T cell memory [228], presumably via induction of SOCS3 [219]. Noteably, in the absence of IL-10, the capacity of IL-27 to induce MPC phenotype in CD8+ T cells is greatly diminished, which is mainly reflected by reduced expression of SOCS3, Bcl-2, and Bcl-6. Adoptive transfer experiments suggest that IL-

10-deficient CD8+ T cells do not give rise to good memory response compared to IL-27-

97 stimulated WT P1CTL cells. Thus, IL-27 induced CTL IL-10 production contributes to

MPC induction and CTL memory.

The expression of SOCS3, Bcl-6, Sca-1, and IL-10 in CTLs support a role for IL-27 in induction of the MPC phenotype in CTLs. However, we also found that IL-27 up- regulated the expression of T-bet, Blimp-1, and Perforin, down-regulated the expression of Eomes, IFN- and slightly down-regulated Granzyme B. Up-regulation of T-bet and down-regulation of Eomes have been demonstrated in IL-12-stimulated CD8+ T cells previously [108], and are associated with CD8+ T cell differentiation into effector but not memory T cells [108, 219]. The controversy of up-regulation of T-bet with down- regulation of IFN- in P1CTL can be explained by IL-27 induction of Bcl-6, which has been shown to inhibit T-bet induced IFN- production [229]. Collectively, these results suggest that IL-27 programs tumor antigen specific CD8+ T cells into MPC-like effector cells. This phenotype can potentially increase CTL “stemness”, without affecting their effector functions such as cytotoxicity. Interestingly, recent studies in humans [230] and mice [185] demonstrate that other effector T cells, such as Th17 cells, also have stem cell characteristics with prolonged survival, suggesting that effector cell “stemness” may be a universal mechanism for maintaining long term survival of effector T cells.

Although vaccination can often elicit tumor-reactive CD8+ T cell responses and even develop tumor-specific CD8+ T cell memory responses, current vaccinations against authentic tumor antigens only induce objective cancer regression in a small minority of cancer patients and have not consistently prevented tumor recurrence [231, 232]. Several obstacles to develop effective tumor vaccination have been identified to date, including: 1) in order to proliferate and metastasize, tumor cells convert immature myeloid dendritic

98 cells into TGF-β-secreting APCs, and the production of TGF-β induces an immunosuppressive population, Treg cells, which constrain the number and functionality of memory CD8+ T cells; 2) immune tolerance to tumor or tumor antigens, which are essentially non-mutated self-peptides capable of activating the immune system weakly; 3)

It has been shown that chronic antigen stimulation induces corrupted memory CD8+ T cells. However, most cancer vaccines were designed to amplify rather than correct the corrupted CD8+ T cell memory population [233]. Given IL-27 is capable of inducing

MPC phenotype in activated tumor antigen specific CD8+ T cells, we suggest that IL-27 can be used as an adjuvant to current cancer vaccination strategies. Indeed, we have shown that endogenous IL-27 is very important to induce potent antitumor CTL response upon tumor antigen vaccination (Figure 15).

4.3 IL-27 induces a large amount of IL-10 production by activated tumor antigen specific CD8+ T cells, which contributes to CTL-mediated tumor rejection

In this study, we found that tumor antigen specific CTLs do not normally produce IL-10.

However, upon stimulation with IL-27 in vitro and in vivo, CD8+ T cells produced high amounts of IL-10. These results are consistent with previous reports that IL-27 has a potent effect in induction of IL-10 production by CD8+ T cells [89]. A critical issue is whether the high amount of IL-10 production by CTLs enhances or diminishes the antitumor efficacy. Although IL-10-producing CD4+ T regulatory cells were shown to promote tumor growth [234], Over-expressing IL-10 in tumor cell lines, including

Chinese hamster ovary cells [149], mammary adenocarcinoma [150], breast cancer [151], melanoma [152], prostate primary tumor [153, 154], colon carcinoma [155], resulted in

99 significant tumor rejection. These IL-10-producing tumor cells are highly immunogenic, triggering cytotoxic CD8+ T cells, Th2 cells, neutrophiles, NK cells and even the antibody responses in the tumor microenvironment. In addition, antitumor effects of IL-

10 were examined and obtained by systemically administration of recombinant IL-10

[156, 157] or use rIL-10 as an adjuvant of vaccines [158, 159]. Intravenous administration of rIL-10 to humans elicited higher numbers of IFN-γ, IFN-inducible protein 10 (IP-10), TNF, IL-1, and granzyme B and induced activation of NK and CTL cells. Injection of IL-10 after immunized tumor bearing mice significantly enhances antitumor immune responses and vaccine efficacy. Some research groups took advantage of IL-10 transgenic and deficient mice and proved the antitumor effects of IL-10 in several tumor mouse models [160-162]. Mumm et al subjected WT, IL-10-/-, and IL-

10TG mice to skin tumors and found higher numbers of tumors developed in IL-10-/- mice than WT control mice, while IL-10TG mice barely developed tumors [161]. They demonstrated that IL-10 induced several essential mechanisms to boost antitumor immune privilege: CD8+ T cells, IFN-γ, granzyme B, and intratumoral antigen presenting molecules. Tanikawa T et al. showed that IL-10 deficiency resulted in an increased level of myeloid-derived suppressor cells (MDSC) and CD4+Foxp3+ regulatory T (Treg) cells in both tumor microenvironment and tumor-draining lymph nodes [162].

In this study, we demonstrate that IL-27-induced IL-10 production by CD8+ T cells contributes to their anti-tumor efficacy. This conclusion is supported by three lines of evidence. First, adoptive transfer of IL-27-stimulated P1CTL cells, which produce high amounts of IL-10, had better therapeutic efficacy than IL-27-stimulated IL-10-deficient

100

P1CTL. Second, adoptive transfer of P1CTL or IL-10-/-P1CTL cells into Rag2-/- or Rag2-

/-IL-10-/- mice with established J558-IL-27 tumors shows that P1CTL cells can reject tumors better than IL-10-/-P1CTL cells. Third, J558-IL-27 tumor cells, which failed to grow into tumors in BALB/c mice due to a potent anti-tumor CTL response, could grow into tumors in IL-10-/-BALB/c mice. IL-10 producing CD8+ T cells are usually considered as suppressor cells that down-regulate T cell responses [235]. However, recent studies showed that IL-10 producing CTLs were more highly activated and cytolytic than IL-10-deficient CTLs [161, 236]. In current study, we found that IL-27- stimulated IL-10-deficient CTLs expressed similar levels of IFN-, Granzyme B and perforin, and exhibited similar levels of cytotoxicity to target cells compared to their WT counterparts. However, IL-10-deficiency significantly reduced the expression of IL-27- induced survival molecules, such as SOCS3, Bcl-2, and Bcl-6 in CTLs. Thus, IL-27 induced CTL IL-10 production increases their survival potential, which can lead to stronger CTL responses. Our finding that IL-27 induced CTL IL-10 production contributes to their efficacy in tumor rejection suggests that IL-10 producing CTLs are better effectors rather than suppressor cells.

4.4 IL-27 suppresses T regulatory cell response in the tumor microenvironment

The data presented here provide direct evidence that IL-27-mediated Treg suppression contributes to its antitumor ability. IL-27 derived from tumor cells reduces the frequencies of Foxp3+CD4+ Treg cells in the draining lymph nodes and tumors in two preclinical models: B16 melanoma and J558 plasmacytoma. In line with these results,

IL-2-producing capacities of both CD4+ and CD8+ T cells are significantly attenuated in

101

IL-27-enriched tumor microenvironment. Consequently, the antitumor CD8+ T cell responses are remarkably enhanced by IL-27. In contrast with IL-27-overexpressing tumors, tumors implanted into IL-27 deficiency (EBI3-/-) mice contain more Foxp3+CD4+

Treg cells and IL-2 compared to tumors in WT mice. EBI3-/- mice not only develop s.c. melanoma tumors faster but also formed more melanoma foci in their lungs than WT mice. Treg depletion in EBI3-/- mice reverses their tumor-promoting property. In addition, we investigate the role of IL-10 in IL-27-mediated Treg suppression. It seems that IL-10-

/-EBI3-/- Treg cells are less suppressive than EBI3-/- Treg cells, indicating that IL-10 plays an enhancing role for the suppressive ability of EBI3-/- Treg cells (data not shown).

Collectively, IL-27 constrains Treg response in the tumor microenvironment through inhibiting IL-2 production by T cells, which in turn boosts antitumor CD8+ T cell response and leads to substantial reduced tumor growth rate and metastatic colonies.

Based on the past decade of intensive studies on IL-27 in various disease models, IL-27 is mainly an inhibitory cytokine but also have some stimulating effects. It has been shown to inhibit differentiation of helper T cells in general. Recent studies demonstrate that IL-

27 plays a crucial role in Treg conversion. First, IL-27 antagonizes the TGF-β-driven

Foxp3+CD4+ Treg conversion from naïve CD4+ T cells in vitro [81]; in addition, splenic

Treg cells express high level of IL-27 receptor, WSX-1[84]. Second, in a T cell transfer model, absence of IL-27R (WSX-1) on transferred T cells increases the frequencies of

Foxp3+CD4+ Treg cells in gut and secondary lymph organs, and results in less colitis symptoms, indicating that IL-27 signaling influences the conversion of Foxp3+CD4+ Treg cells in a pathogenic condition [83]. Third, IL-27 transgenic mice are almost completely lack of Treg cells in lymph organs and susceptible to systemic inflammation, further

102 comfirming the suppressive effect of IL-27 on Treg cells [84]. These studies together suggest that IL-27 modulates the conversion of naïve CD4+ T cells into Treg cells rather than the expansion of natural Treg cells. Since most Treg cells in the tumor microenvironment are tumor antigen-induced Treg cells, our results derived from three different models are consistent with their findings. The frequencies of Foxp3+CD4+ Treg cells are significantly decreased by IL-27 produced by B16-OVA melanoma or J558 plasmacytoma, while this reduction disappears in EBI3-/- mice. Of importance, our study shows that IL-27 also affects the function of Treg cells, demonstrated by which Treg cells isolated from EBI3-/- lymph organs suppress T cell proliferation more efficiently than

Treg cells isolated from WT mice. Thus, despite IL-27 has no effect on the expansion of natural Treg cells, absence of IL-27 on nature Treg cells strengthens its regulating function. Further studies should focus on the mechanisms by which IL-27 weakens the

Treg suppressive activity.

The engagement of IL-27 to IL-27 receptor on T cells recruits several Jak family kinases and subsequently activates mainly STAT1 and STAT3. One early in vitro study shows that STAT1 is required for inhibition of IL-2 production by IL-27. Due to the important role of IL-2 in the generation and maintenance of Treg cells, IL-27-STAT1-IL-2 signaling pathway may represent one of the mechanisms by which IL-27 constrains Treg response. In contrast, later studies show that IL-27 activates STAT3, but not STAT1, and down-regulates the expression of Treg-related markers, such as Foxp3, CD25, and

CTLA-4. To date, which one is the main transcriptional factor responsible for IL-27 to downregulate Foxp3 expression has not been elucidated. Regardless of STAT1 or STAT3 activation, our study confirms that the presence of IL-27 in the tumor microenvironment

103 strikingly reduced IL-2 production, by both CD4+ and CD8+ T cells, while IL-2 production is restored in IL-27 deficiency mice. These are consistent with the previous study using IL-27 transgenic mice, in which IL-2 production is highly impaired. Thus,

IL-2 is an essential mediator involved in IL-27-mediated Treg suppression.

IL-27 is well-known as a potent antitumor cytokine to date. It has been shown to mediate marked antitumor efficacy in a variety of preclinical tumor models, such as colon carcinoma, neuroblastoma, melanoma, breast cancer, and lung cancer. In previous studies, several mechanisms have been reported accounting for IL-27-mediated tumor rejection.

IL-27 promotes the generation and activation of cytotoxic CD8+ T cells and NK cells by enhancing their production of IFN-γ and granzyme B. In a melanoma study, IL-27 is shown to directly interact with melanoma tumor cells and inhibits their proliferation.

Furthermore, another melanoma study demonstrates that IL-27 controls angiogenesis which in turn suppresses tumor growth and metastases. Although various immune or non- immune mechanisms have been discussed, the inhibitory effect of IL-27 on Treg cells, a tumor promoting population, has not been well investigated yet. To determine whether this proinflammatory effect of IL-27 contributes to IL-27-mediated tumor rejection, we generated two IL-27-overexpression tumor cells lines (B16-IL-27 and J558-IL-27) and took advantage of EBI3-/- mice, which can be considered as IL-27 deficiency mice. Both sides of tumor models confirmed that IL-27 plays a role in constraining Treg development and results in slower establishment of local and metastatic tumors. Treg depletion conducted in EBI3-/- mice diminishes its negative impact on tumor progression.

Furthermore, Treg transfer assay confirms that EBI3-/- Treg cells are more effective than

WT Treg cells in suppressing T cell proliferation, which leads to faster tumor growth rate

104

and more metastatic foci formed on lungs. Taken together, IL-27-mediated Treg

suppression could be considered as one of the mechanisms responsible for its antitumor

effects.

4.5 Concluding remarks and future directions

This study was designed and performed to determine the molecular and cellular

mechanisms by which IL-27 enhances the antitumor CTL response and leads to tumor

rejection. The goals of this dissertation were: a) To examine the direct impacts of IL-27

on tumor antigen specific CD8+ T cells; b) To determine the role of IL-27 in T regulatory

cell response in the tumor microenvironment; c) To determine if using IL-27 is a feasible

approach for cancer immunotherapy. Our research has resulted in the following

observations and conclusions.

1) We demonstrate that the addition of recombinant IL-27 in tumor antigen specific

CD8+ T cell culture significantly reduces their apoptosis rate, indicating that IL-27 provides a survival advantage to these tumor antigen specific CD8+ T cells. Based on our finding, physicians can use recombinant IL-27 as an agent to improve their current ACT procedure, particularly use IL-27 to ex vivo expand tumor antigen specific T cells, which confers them better survival and thus more efficacy in killing tumor cells.

2) Tumor-derived IL-27 increases the infiltration of adoptively transferred tumor antigen specific CD8+ T cells into both spleens and tumors. The mechanism underlying this phenomenon is that IL-27 enhances the expressions of several anti-apoptotic genes in infiltrated T cells and prevents them from dying before executing effector functions. Thus, we believe that a high dose of IL-27 in the tumor microenvironment could boost the

105 efficacy of current adoptive T cell therapy. This can be done either by injecting adenoviral vectors for the transfer of IL-27 gene directly into solid tumors or using IL-27- overexpressing tumor cells as a vaccine.

3) IL-27 induces a unique gene expression program in the activated tumor antigen specific CD8+ T cells, which is characterized by upregulation of several memory- associated gene expressions (SOCS3, Bcl-6, Sca-1 and IL-10). In contrast, IL-27 does not seem to affect the effector function of tumor antigen specific CD8+ T cells as much as we expect. Therefore, we think that IL-27 programs naïve CD8+ T cells more likely to become memory precursor cells rather than terminal effector cells. Moreover, this pro- memory effect of IL-27 has been confirmed in our in vivo T memory development assay.

In the future, we can use IL-27 as an adjuvant to the vaccine strategies, which would give rise to more and stronger memory T cells against tumor cells.

4) In this study, we show that IL-27 is able to trigger activated tumor antigen specific CD8+ T cells to produce high amount of IL-10 and this induction contributes to its antitumor activity. This is in consistent with several recent studies regarding to the antitumor effect of IL-10. Therefore, it would be interesting and worthwhile to compare the efficacy of IL-27-induced IL-10 production by CTLs and IL-10 itself in tumor eradication in the future.

5) We also show that the IL-27-induced CTL IL-10 production is required for the formation of tumor specific memory CD8+ T cells. However, the exact molecular signaling pathway that mediates this effect has not been elucidated yet. There are several experiments we plan to do: a) Blocking or knockdown several transcriptional factors, such as STAT3, c-Maf, or Blimp-1, in tumor antigen specific CD8+ T cells and determine

106 which one is responsible for the IL-27/IL-10-induced MPC phenotypes; b) We also can use STAT3 conditional knockout mice to examine if IL-27 increases the memory potential of CD8+ T cells through activating STAT3; c) To further confirm the role of IL-27/IL-10 axis on effector and memory CD8+ T cell differentiation during tumorigenesis, we can deprive cells of these cytokines using a combination of genetic and mAb blockade approaches.

6) Besides directly working on tumor specific CD8+ T cells, IL-27 has also been shown to interfere with the infiltration and function of CD4+Foxp3+ Treg cells in the tumor microenvironment in current study. Although we have demonstrated that IL-2 inhibition by IL-27 serves as one of the mechanisms by which IL-27 suppresses Treg cell responses, other molecular pathways remain to be addressed and deciphered.

Given the potent effects of IL-27 in enhancing antitumor CTL response and in inhibiting

pro-tumor Treg cells, using IL-27 or IL-27-based strategies should provide an option for

the immunotherapies of human cancer. Our successful treatment of plasmacytoma using

adeno-IL-27 proves that this approach is feasible.

Taken together, our investigation on the role of IL-27 in tumor immunity has resulted in

the following working mode (Figure 17). First, IL-27 directly engage IL-27R on CD8+ T

cells and activates STAT3, leading to the induction of survival molecules, MPC

phenotype, and IL-10 production in CD8+ T cells, which will greatly benefit the survival

of CTL effector cells and contribute to T cell memory formation. IL-27-induced CTL IL-

10 production will further strengthen this process. In addition, IL-27 can directly inhibit

Treg response via suppressing IL-2 production.

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Figure 17 The impacts of IL-27 signaling on CD8+ and CD4+ T cells in the tumor microenvironment IL-27 exerts three STAT3 activation-mediated effects on tumor antigen specific CD8+ T cells, which provides an explanation of why IL-27 enhances anti-tumor CTL responses. First, IL-27 up-regulates anti-apoptotic molecules such as Bcl-2 and inhibits activation of Caspase 3 in activated CD8+ T cells, which leads to improved survival of activated CD8+ T cells. Second, IL-27 induces a unique stem cell/memory precursor cell (MPC) phenotype in activated CD8+ T cells, characterized by up-regulation of SOCS3, Bcl-6 and Sca-1. Third, IL-27 induces high amount of IL-10 production by CTL. IL-27-induced CTL IL-10 production contributes to MPC phenotype, T cell memory and tumor rejection. Besides stimulating CD8+ T cells, IL-27 interacts with IL-27R expressed on CD4+ T cells and inhibits their IL-2 production, which subsequently impairs the generation and function of Treg cells in the tumor microenvironment. Four lines of mechanisms work together and lead to reduced tumor incidence, growth, and metastasis.

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