Elucidating the Roles of Il-15 in the Tumor Microenvironment

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The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences

12-2020

ELUCIDATING THE ROLES OF IL-15 IN THE TUMOR MICROENVIRONMENT

Rosa Maria Santana Carrero

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Recommended Citation Santana Carrero, Rosa Maria, "ELUCIDATING THE ROLES OF IL-15 IN THE TUMOR MICROENVIRONMENT" (2020). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 1057. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/1057

This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. ELUCIDATING THE ROLES OF IL-15 IN THE TUMOR MICROENVIRONMENT

Rosa M. Santana Carrero, B.S.

APPROVED:

Shao-Cong Sun , Ph.D. Advisory Professor

Kimberly S. Schlu~s, Ph.D.

~,Ph.D.

ia cAllister, M.D. WH Jagannadha Sastry, Ph.D.

Joya Chandra, Ph .D.

APPROVED:

Dean, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences ELUCIDATING THE ROLES OF IL-15 IN THE TUMOR MICROENVIRONMENT

DISSERTATION

Presented to the Faculty of

The University of Texas

MD Anderson Cancer Center UTHealth

Graduate School of Biomedical Sciences

in Partial Fulfillment

of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Rosa M. Santana Carrero, B.S. Houston, Texas

December, 2020 ACKNOWLEDGEMENTS

I would first like to thank my family and loved ones for the unconditional love and support that they have shown me throughout my life, but especially as I embarked on my graduate school journey. A special and most deserved mention to my husband,

Cristian, who has been my rock, protector and counselor through this incredible experience. Thank you, my love, for taking the risk of moving away from all our friends and family to take on the challenge of starting new careers and starting over in a new city. Your strength and kindness have helped me thrive and overcome all the obstacles that come with having a career in science and being an almost perpetual student. As I am closing this chapter as a graduate student, we are undertaking new challenges as a family to ensure that we grow in love and as professionals. I can’t imagine sharing this journey with anybody else.

I would also like to thank my parents for the support they have provided, including listening to me talk incessantly about my research and discussing difficult biological concepts. They have provided wisdom to overcome the obstacles and challenges that I have faced, and they have cheered me on when I achieved new milestones in my career. Thank you Mami and Papi for teaching me perseverance and to never give up on my dreams. Most importantly, thank you for teaching me that, as long as I give it my very best effort the outcomes don’t really make much of a difference, because the lessons learned will provide more than enough compensation.

I would also like to thank my mentor Dr. Kimberly Schluns for all the guidance and advice that she has provided me along my time in her lab. Kim, thank you for being supportive but firm as my advisor and my colleague. I have learned that true leadership is achieved by setting an example and executing every day to ensure we live up to that

iii standard. Moreover, thank you for your support as I dared to take on responsibilities outside of lab and thank you for keeping me in check when I was veering off track. You have been the best mentor that I could have asked for, and I wish you all the best in your new endeavors.

Other important people that I would like to thank are the current and past members of the Schluns’ lab for making my long days in lab more enjoyable. I would especially like to mention Shweta Hegde, because you have been an amazing co- worker and friend. Thank you, Shweta, for always lending a hand during the long days of tumor experiments and for always listening to me talk about food, my dogs, my family and all the happenings at the graduate school. Your loving and open spirit have made you one of my favorite people to be around. I will miss you so much, but I hope to maintain our friendship through the distance and through the years.

I would like to acknowledge my advisory committee members and the administrative staff at the GSBS for playing important roles as advisors and problem solvers during my time as a graduate student. Lastly, I would like to thank all my friends in Houston and at the GSBS for sharing amazing moments and memories throughout these past 5 years. You all made Houston my home, and I will always carry our memories together in my heart.

iv ELUCIDATING THE ROLES OF IL-15 IN THE TUMOR MICROENVIRONMENT

Rosa M. Santana Carrero, B.S.

Advisory Professors: Shao-Cong Sun, Ph.D. & Kimberly S. Schluns, Ph.D.

Interleukin-15 (IL-15) is a factor that promotes activation, proliferation, cytotoxicity, and survival of CD8 T cells and NK cells, and has been shown to have anti-tumor effects.

Moreover, loss of IL-15 expression in human colorectal tumors correlates with increased risk of relapse, diminished survival, decreased density and proliferation of T cells. All together these findings suggest that IL-15 expressed locally in the tumor microenvironment (TME) is an important mediator of anti-tumor responses by tumor infiltrating lymphocytes (TILs) representing a potentially powerful immunotherapeutic target. Unfortunately, the regulation and roles of IL-15 within the TME is unclear.

I hypothesized that IL-15 produced in the TME is an important factor promoting anti- tumor responses by cytotoxic lymphocytes stimulating their optimal numbers, enhancing their effector functions and metabolic fitness. In this dissertation I investigated the cellular sources and regulation of IL-15 in the TME and its roles in enhancing anti-tumor immunity. I showed, using several murine tumor models, that soluble IL-15/IL-

15R complexes (sIL-15 complexes) are present at high levels in the interstitial fluid of early tumors and decrease with tumor growth. Depletion of local IL -15 in tumors led to decreased numbers of TILs without directly affecting tumor growth, likely by promoting their infiltration into tumors. I also found that levels of sIL-15 complexes could be upregulated in advanced tumors by local activation of the Stimulator

v of Interferon Genes (STING) pathway. While treatment of tumors with STING agonists induces tumor regression, optimal STING-mediated systemic anti- tumor immunity required IL-15 expression. In addition, I examined the direct effect of IL-15 agonists in poorly T-cell infiltrated tumors and found that recombinant sIL-15/IL-

15R-Fc complexes (rsIL-15c) and a polymer-conjugated IL-15R agonist significantly slowed tumor growth and increased the total numbers of TILs. Moreover, intratumoral IL-

15 agonists in combination with anti-CTLA-4 immune checkpoint blockade synergistically induced better tumor growth control and enhanced mouse survival in a poorly infiltrated melanoma tumor model. These findings reveal the ability of IL-15 expressed within the TME to regulate TIL numbers and upon upregulation by inflammatory signals, contribute to enhanced anti-tumor responses.

vi TABLE OF CONTENTS

APPROVAL PAGE ...... I

TITLE PAGE ...... II

ACKNOWLEDGEMENTS ...... III

ABSTRACT ...... V

TABLE OF CONTENTS ...... VII

LIST OF FIGURES ...... X

CHAPTER 1: INTRODUCTION ...... 1-57

1.1 TUMOR MICROENVIRONMENT ...... 2

1.1.1 Detection of tumors by the immune system ...... 3

1.1.2 Composition of the tumor microenvironment ...... 5

1.1.3 Mechanisms of evasion and suppression of anti-tumor immunity ...... 15

1.2 TYPE I INTERFERONS IN CANCER ...... 19

1.2.1 IFN-I signaling ...... 19

1.2.2 IFN-I roles in cancer immunoediting ...... 21

1.2.3 Anti-metastatic and immunostimulatory effects of IFN-I ...... 23

1.2.4 IFN-I therapy in cancer ...... 25

1.2.5 STING pathway in spontaneous and therapeutic responses to cancer 26

1.3 INTERLEUKIN 15 (IL-15): A KEY CYTOKINE FOR CYTOTOXIC LYMPHOCYTES RESPONSES

...... 32

1.3.1 IL-15 discovery and functions ...... 32

1.3.2 Mechanisms of action of IL-15 ...... 34

vii

1.3.3 IL-15 functions during homeostasis and inflammation ...... 38

1.3.4 IL-15 regulation ...... 41

1.3.5 Models for IL-15 detection...... 42

1.4 THERAPEUTIC POTENTIAL OF IL-15 IN CANCER ...... 44

1.4.1 Recombinant IL-15 as cancer monotherapy ...... 44

1.4.2 IL-15/IL-15R agonists ...... 46

1.4.3 IL-15 agonists in combination with other immunotherapies ...... 51

1.5 SUMMARY ...... 55

CHAPTER 2: METHODOLOGY...... 58-63

CHAPTER 3: IL-15 IS AN IMPORTANT INFLAMMATORY SIGNAL IN THE TME THAT

ENHANCES ANTI-TUMOR RESPONSES ...... 64-110

3.1 INTRODUCTION ...... 64

3.2 RESULTS ...... 66

3.2.1 IL-15 expression and regulation in the tumor microenvironment ...... 66

3.2.2 Cellular sources of soluble IL-15/IL-15R complexes ...... 71

3.2.3 The tumor microenvironment is abundant in IL-15-expressing myeloid cells,

composed predominately of CD11b+Ly6ChiLy6G- cells ...... 77

3.2.4 Basal IL-15 in tumors regulates TIL numbers ...... 86

3.2.5 IL-15 expression can be upregulated in tumors by activating the STING

pathway ...... 94

3.2.6 Upregulation of IL-15 enhances CD8 T cell responses and

promotes anti-tumor immunity ...... 97

3.3 SUMMARY ...... 108 viii CHAPTER 4: EFFECT OF INTRATUMORAL IL-15 AGONISTS IN POORLY T-CELL

INFILTRATED TUMORS ...... 111-130

4.1 INTRODUCTION ...... 111

4.2 RESULTS ...... 113

4.2.1 Intratumor delivery of recombinant sIL-15/IL-15R-Fc complexes (rsIL-15c)

increases the immune cell infiltrate in melanoma tumors and enhances anti-tumor

responses ...... 113

4.2.2 Delivery of polymer-conjugated IL-15R agonist NKTR-255 directly into tumors

slows tumor growth and increases immune infiltrate in melanoma tumors . 120

4.2.3 Combination of intratumoral IL-15/IL-15R agonists and immune checkpoint

inhibitors delays tumor growth and enhances mice survival ...... 124

4.3 SUMMARY ...... 128

CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS ...... 131-139

BIBLIOGRAPHY ...... 140-209

VITA ...... 210

LIST OF FIGURES

FIGURE 1. IMMUNE CELLS AND TUMORS COOPERATE IN THE TME TO

SUPPRESS ANTI-TUMOR IMMUNITY ...... 14

FIGURE 2. SPONTANEOUS TUMOR RECOGNITION IS MEDIATED BY

RECOGNITION OF DSDNA BY THE STING PATHWAY IN INNATE IMMUNE CELLS

...... 27

FIGURE 3. MECHANISMS OF ACTION OF IL-15 ...... 36

FIGURE 4. SIL-15 COMPLEXES ARE PRODUCED IN THE TUMOR

MICROENVIRONMENT ...... 67

FIGURE 5. REGULATION OF SIL-15 COMPLEXES PRODUCTION IN TUMORS AND

TIL NUMBERS BY IFN-I ...... 69

FIGURE 6. STROMAL AND TUMOR CELLS ARE SOURCES OF SIL-15

COMPLEXES IN THE TUMOR MICROENVIRONMENT ...... 72

FIGURE 7. MULTIPLE MYELOID CELL SUBSETS AND NON-HEMATOPOIETIC

CELLS GENERATE SIL-15 COMPLEXES IN THE STROMAL COMPARTMENT OF

THE TME ...... 75

FIGURE 8. CD11B+LY6CHILY6G- CELLS ARE THE MAJOR SUBSET

EXPRESSING IL-15 IN TME ...... 79

FIGURE 9. IL-15 REPORTER EXPRESSION IS INCREASED IN TUMOR MYELOID

CELLS OVER SIMILAR MYELOID POPULATIONS IN THE SPLEEN ...... 82

FIGURE 10. IL-15R EXPRESSION BY TUMOR MYELOID CELLS IS STABLE IN

EARLY AND ESTABLISHED TUMORS ...... 85

FIGURE 11. IL-15 NEUTRALIZATION IN TME LEADS TO DECREASED TIL

NUMBERS ...... 87

FIGURE 12. IL-15 BLOCKADE DOES NOT SIGNIFICANTLY AFFECT TIL

PROLIFERATION, SURVIVAL AND GLUCOSE METABOLISM IN MELANOMA

TUMORS ...... 90

FIGURE 13. STIMULATION OF THE STING PATHWAY UPREGULATES SIL-15

COMPLEXES IN TUMORS ...... 95

FIGURE 14. STING ACTIVATION IN THE TME PROMOTES PROLIFERATION OF

CD8 T CELLS IN PERIPHERAL SECONDARY LYMPHOID ORGANS ...... 98

FIGURE 15. IL-15 PRODUCED IN RESPONSE TO LOCAL STING AGONIST

TREATMENT IN THE TME INDUCES IFN- PRODUCTION IN CD8 T CELLS IN THE

PERIPHERY ...... 100

FIGURE 16. STING ACTIVATION IN THE TME PROMOTES TUMOR REGRESSION

VIA IL-15 DEPENDENT MECHANISMS ...... 103

FIGURE 17. IL-15 IS ESSENTIAL FOR STING-MEDIATED REGRESSION OF

DISTANT SECONDARY TUMORS ...... 106

FIGURE 18. INTRATUMORAL DELIVERY OF RSIL-15C DECREASES GROWTH OF

MELANOMA TUMORS ...... 115

FIGURE 19. DIRECT DELIVERY OF RSIL-15C INTO TUMORS INCREASES

TUMOR INFILTRATING IMMUNE CELLS ...... 118

FIGURE 20. LOCALIZED DELIVERY OF NKTR-255 INTO TUMORS DECREASES

TUMOR GROWTH AND AUGMENTS THE IMMUNE CELLS FOUND IN MELANOMA

TUMORS ...... 122

FIGURE 21. COMBINATION THERAPY WITH SYSTEMIC ANTI-CTLA-4 AND

INTRATUMOR RSIL-15C CONTROLS TUMOR GROWTH AND INCREASES

SURVIVAL IN MICE WITH MELANOMA TUMORS ...... 126

FIGURE 22. INTRATUMORAL IL-15 AGONISTS SYNERGIZE WITH IMMUNE

CHECKPOINT BLOCKADE TO ENHANCE THERAPEUTIC RESPONSES IN POORLY

IMMUNOGENIC TUMORS ...... 130

xii

CHAPTER I: INTRODUCTION

CD8 T cells and natural killer (NK) cells are key mediators of anti-tumor immune responses due to their ability to kill tumor cells with their cytotoxic properties. CD8 T cells are adaptive lymphocytes that become activated when their T cell receptor (TCR) recognizes its cognate antigen bound to a major histocompatibility complex I (MHC-I) molecule on the cell surface of an antigen-presenting cell (APC). Conversely, NK cells are innate lymphocytes whose activation is not limited by antigen recognition and presentation by an MCH molecule. Instead their activation is tightly regulated by the balanced interactions of inhibitory and activating receptors on the cell surface of NK cells with MHC-I molecules on target cells. Understanding how to enhance the responses and functions of these cytotoxic lymphocytes is important to ensure that robust anti-tumor immune responses are achieved. Interleukin-15 (IL-15), a cytokine that preferentially stimulates CD8 T cells and NK cells, will be the focus of this dissertation. In recent years it has garnered attention as a potential immunotherapeutic cytokine, but very little was known of its endogenous expression and roles as an inflammatory protein in tumors. This dissertation will concentrate on uncovering its roles and effects on anti-tumor responses when it is expressed in the tumor microenvironment (TME). In the following chapter I will provide an overview of how the

TME impacts responses of cytotoxic lymphocytes, highlighting mechanisms of immunosuppression. I will also elaborate on the role of type I interferons (IFN-I) in anti- tumor immunity and I will discuss known functions of IL-15 as a promoter of CD8 T cells and NK cells responses and known mechanisms of regulation by other inflammatory signals.

1.1 Tumor microenvironment

Cancer as we understand it today is a group of diseases that is characterized by abnormal and uncontrolled cellular growth that has the potential to invade surrounding tissues and spread throughout the body. The first description of cancer was first reported back in 3000 BC in ancient Egypt in the Edwin Smith Papyrus, where masses of the breast tissue were surgically removed (1, 2). The Greek physicians Hippocrates and Galen and the Roman physician Celsus are credited with being the firsts to refer to ulcer-forming tumors are carcinomas, using the word oncos to describe tumors, and translating the Greek word carcinoma to the Latin cancer, respectively (2). Regardless of the origin of the word, cancers proved to be extremely difficult to treat and for many years were thought incurable (1). However, as we have developed more advanced investigative techniques and more targeted treatment options, we have a better understanding of the key events that must take place for cancer development and the characteristics that make certain cancers more difficult to treat and cure.

The development of cancer is a multi-step and multifactorial process that begins with alterations to the genetic material of a single cell, that can include the activation of cancer-promoting genes, or oncogenes, and/or the loss of function of tumor suppressor genes, that initiate the process of cellular transformation and proliferation of this mutated cell (3). Upon clonal expansion of this mutated cell, accumulation of more mutations in this population of cells can confer selective advantages such as enhanced growth capability to certain cells that become the dominant cell population. This process termed clonal selection, occurs throughout tumor development generating a heterogenous mass of tumor cells. As mutations continue to accumulate, these transformed cells acquire malignant characteristics that make them more efficient at

2 evading the attempts of the body to control their unmitigated cellular proliferation and invasion of surrounding tissues (4).

Our current understanding of how tumors develop and progress to invasive masses that can metastasize throughout the body led to the identification of very specific characteristics of tumors that were termed the hallmarks of cancer by Hanahan and Weinberg (5, 6). In 2000 they proposed six main hallmarks of cancer that included: self-sufficiency of growth signals, evading growth suppressors, evading apoptosis, limitless replicative potential, sustained angiogenesis and tissue invasion and metastasis (5). In their apt analysis of tumors they also pointed to potential contributions by other cell types that form the tumor mass and that now we come to appreciate are key components of the TME. In their 2011 revised hallmarks of cancer they included genomic instability, deregulation of cellular energetics, tumor promoting inflammation and avoiding immune destruction (6). Importantly, they demonstrated that the cells that are part of the TME are key contributors to these important processes and characteristics that promote the progression of tumor development and metastatic potential. In this section I will provide an overview of how tumors are recognized by the immune system, the cells that are part of the TME and their roles in either promoting or impeding robust anti-tumor responses, and specific mechanisms of immune evasion.

1.1.1 Detection of tumors by the immune system

The immune system is designed to protect humans and animals from invading pathogens by the detection of microbial and viral components. This feat is achieved because many innate immune cells express on their cell surface and the cytoplasm a variety of pattern recognition receptors (PRRs) that recognize pathogen associated

3 molecular patterns (PAMPs) from viruses and bacteria and distress signals from host cells, known as danger associated molecular patterns (DAMPs) (7). APCs such as dendritic cells (DCs) and macrophages can detect PAMPs and DAMPs that can directly ligate PRRs on the cell surface of APCs or can be engulfed by these innate immune cells and interact with cytoplasmic PRRs. The ligation of PRRs induces downstream signaling pathways that promote anti-viral and anti-bacterial inflammatory processes that aid in the destruction of the pathogen. Once APCs engulf these particles, they process them inside the cells to present on the cell surface bound to MHC-I and MHC-II molecules. APCs present antigens to cells of the adaptive arm of the immune system, including CD8 and CD4 T cells, that become activated and exert their functions to eliminate the foreign invaders.

Similarly, products of cell death including DNA, RNA and tumor-derived antigens are also sensed by PRRs on APCs that trigger the production of cytokines, including

IFN-I and chemokines, and priming of T cells by presentation of tumor antigens (8-10).

Furthermore, cellular stress and damage can increase the expression of ligands on tumor cells that bind activating receptors on NK cells and leads to tumor cell death. On the other hand, mutations in the genome that occur during the transformation process, induce the production of mutated proteins by cancer cells that are also presented on the cell surface on MHC-I molecules (11) and can be recognized as foreign by adaptive lymphocytes. These mutated proteins are known as neoantigens and are currently being targeted for enhancing the efficacy of immunotherapies including adoptive cell therapies and personalized cancer vaccines (12).

Consequently, although for many years it was thought that the immune system was not capable of distinguishing between normal and malignant self-tissues, we now

4 understand that immunosurveillance by innate and adaptive immune cells is important for protecting the host against not only foreign pathogens but also cancer and disease development (13) . Nonetheless, despite the many ways in which the immune system can effectively recognize cancer cells, tumors have developed several strategies to undermine both the innate and adaptive immune responses and are aided in part by the stromal cells that are part of the TME. In the next section I will describe the cells that form part of the stromal component of the TME and the mechanisms of immune evasion will be further discussed in section 1.3.3 of this chapter.

1.1.2 Composition of the tumor microenvironment

Tumors are masses composed of a heterogeneous population of transformed cells and several other cell types that are part of the TME. The other cellular components of the tumor mass include stromal cells like fibroblasts, vascular endothelial cells, blood vessels, lymphatic vessels and immune cells. Other components of the TME include inflammatory mediators and soluble factors such as cytokines, chemokines, growth factors, extracellular matrix (ECM) proteins and nutrients including oxygen, glucose, amino acids, fatty acids and their metabolites. In the next few paragraphs I will describe and discuss the functions and roles of some of these components in the TME.

The first cell type I will discuss are fibroblasts, which are the main generators of

ECM in connective tissues and produce transforming growth factor  (TGF-) during tissue repair and wound healing (14). Fibroblasts that reside in secondary lymphoid organs are known as fibroblastic reticular cells (FRCs) and in this context they produce

ECM that allows formation of lymphatic conduits for the transport of chemokines,

5 cytokines and antigens to present to T cells in lymphoid tissues (15). They also produce several chemokines like C-C motif ligand 19 (CCL19) and CCL21, that upon binding to C-C motif chemokine receptor 7 (CCR7) on DCs and naïve T cells promotes the DC and T cell interactions for their activation (16). Other chemokines and cytokines produced by FRCs include C-X-C motif ligand 13 (CXCL13) , CXCL12, IL-7 and B-cell

Activating Factor (BAFF) are important for recruitment of T cells, B cells, follicular helper T cells and DCs to secondary lymphoid organs and for survival of T cells and B cells (15). In the TME normal fibroblasts can become cancer associated fibroblasts

(CAFs) in response to several stimuli including direct contact with cancer cells and induction of Notch signaling, in response to inflammatory proteins like IL-1 and IL-6 and in response to physiological stress and DNA damage (17).

CAFs can promote tumor progression and suppress immune responses against tumors by several mechanisms including deposition and remodeling of ECM to increase metastatic spread of tumor cells, limiting CD8 T cell interaction with tumor cells and increasing the stiffness of tumor tissues making it harder for immune cells to infiltrate into tumors (18-20). They can also produce growth factors like TGF-, leukemia inhibitory factor (LIF) and fibroblast growth factor 5 (FGF5) that can promote proliferation and invasiveness in cancer cells (17, 21-23). CAFs can also promote angiogenesis through the production of vascular endothelial growth factor (VEGF) (24).

They can also produce immunosuppressive cytokines and chemokines including IL-6 and CXCL12, that coats cancer cells and decreases TIL infiltration (25-27). Lastly, they can cross-present antigens that induce specific deletion of CD8 T cells (28).

The next component of the TME that I will discuss are tumor endothelial cells

(TECs) and blood vessels. TECs line the inside of the blood vessels and in tumors are

6 involved in the process of angiogenesis. Angiogenesis is the formation of new blood vessels in tumors to supply oxygen and nutrients to sustain tumor cell growth (29). In response to hypoxia, an angiogenic switch is undergone in tumor cells that produce angiogenic factors, like VEGF-A, that interacts with VEGF receptor-2 (VEGFR-2) on nearby TECs initiating the formation of new blood vessels (29, 30). In this setting TECs use matrix metalloproteinases to remodel and breakdown the ECM allowing the migration of TECs to form new tumor blood vessels (31). The blood vessels in tumors present an immature structure that leads to irregularly shaped vessel networks that do not maintain their typical organization (32). These blood vessels have fewer smooth muscle cells and pericytes, which allow the transendothelial migration and dissemination of tumor cells to the circulation. TECs can also express Programmed

Death-1 (PD-1) ligands 1 (PD-L1) and PD-L2, ligands for the PD-1 inhibitory receptor, that can further inhibit T cell responses in tumors (33). Moreover, while normal endothelial cells express cell adhesion molecules like ICAM-1, in contrast TECs downregulate expression of these adhesion molecules in response to VEGF-A limiting

T cell extravasation into tumors (30, 34).

Lymphatic vessels are also important components of the TME that aid in the spread and dissemination of tumor cells to other parts of the body, however they are also essential for mounting anti-tumor immune responses. Lymphatic vessels in and around tumors can expand and generate new vessels in response to VEGF-C, produced by macrophages and tumor cells in a process termed lymphangiogenesis (35-37).

Lymphatic vessels surrounding tumors can expand and enlarge in response to VEGF-C and this increased surface area can provide more contact space between lymphatic vessels and tumor cells to allow for their entry into the lymph and metastatic spread to

7 nearby lymph nodes (38, 39). Moreover, lymphatic endothelial cells (LECs) can also cause immune suppression of T cell responses by crosspresenting antigens on MHC-I molecules in the absence of costimulatory signals leading to anergy of antigen specific

T cells (40). LECs in tumors can upregulate expression of PD-L1 leading to engagement with PD-1 on T cells that can contribute to their tolerization and dysfunction (41). LECs can also suppress T cell responses by their expression of indoleamine 2,3 deoxygenase (IDO) that leads to tryptophan depletion, a necessary amino acid for T cell proliferation and functions (42-44). However, lymphatics also play important roles in promoting anti-tumor responses. Lymphatic vessels can carry tumor antigens to lymph nodes where APCs can process them and present them to T cells to induce the activation of these cells (37). Interestingly, lymphangiogenesis and expression of VEGF-C are associated with increased TILs numbers and better responses to immunotherapies in melanoma (45). These findings are likely mediated by the upregulated expression of chemokines like CCL21, that promote homing of DCs to lymph nodes, in response to VEGF-C (45). The previously discussed cell types form part of the structural component of the tumor stroma, in the next paragraphs I will discuss the cells that compose the immune infiltrate in the TME.

In the TME we find a variety of myeloid cells that have both mature and immature phenotypes and that can have both stimulatory and suppressive roles for anti-tumor responses mediated by cytotoxic lymphocytes. The first subset of cells to be discussed are the DCs, which are the classical example of professional APCs. In the TME, immature dendritic cells can uptake and process tumor antigens, including products of cellular death, that induce their maturation and their ability to present them on the cell surface on MHC-I and MHC-II molecules. Mature DCs can home to tumor draining

8 lymph nodes where they interact with CD4 and CD8 T cells. The main function of DCs is to activate T cells by providing several signals required for effective T cell activation.

Signal 1 is achieved by the presentation of antigen on MHC molecules on the surface of DCs and the interaction with the TCR on the cell surface of T cells (46). The second signal is achieved by the interaction of costimulatory molecules expressed on the cell surface of DCs, such as CD80 or CD86, with the receptor CD28 on the cell surface of T cells (46, 47) . Costimulation signals are necessary for T cell proliferation, differentiation and even survival. Without costimulation T cells become anergic and eventually die (48).

DC subsets in mice can be divided into two main types: plasmacytoid DCs (pDCs) and conventional DCs (cDCs), which can be further classified into type 1 cDCs (cDC1) and type 2 cDCs (cDC2) subsets (49). While cDC1s have been identified as key promoters of CD8 T cell mediated anti-tumor immunity, the roles for the other two subsets of DCs in the TME are not clearly defined. The cDC1 subset of DCs, which rely on basic leucine zipper transcriptional factor ATF-like 3 (Batf3) transcription factor for their development and express the integrin CD103 (50), are of particular importance in anti-tumor responses. cDC1 DCs are also known as Batf3+ DCs. Several studies have shown that IFN-I signaling in Batf3+ DCs is crucial for tumor growth control (9, 51).

Moreover, loss of cDC1 cells leads to failure of adoptive cell therapies and checkpoint inhibitor therapies to reject immunogenic tumors (52-54). Importantly, Batf3+ DCs are extremely efficient in cross-presentation of exogenous antigens to CD8 T cells and in the TME they are required for spontaneous priming of CD8 T cells and loss of these cells decreased the infiltration of endogenous and effector CD8 T cells into tumors (53-

55). Moreover, cDC1s in the TME produce chemokines CXCL9 and CXCL10 to recruit effector CD8 T cells into tumors (54).

Regarding the roles of the other two subsets of DCs in anti-tumor immunity very little is known. pDCs are known for their important roles in anti-viral immune responses and they generate high levels of IFN-I upon activation of TLRs by binding to nucleic acids (56). In the context of cancer, pDCs found in tumors are usually associated as tumor-promoting immunosuppressive cells that, in breast and ovarian cancers, produce low levels of IFN-I and can even promote Treg differentiation (56-58). Lastly cDC2 cells are very adept at presenting antigens on MHCII molecules to CD4 T cells, and are capable of uptake of tumor antigens and inducing T cell proliferation in vitro (52).

As DCs in the TME play crucial roles in stimulating anti-tumor immunity, the tumor has developed mechanisms to suppress the functions of DCs. Several of these mechanisms include the production of soluble factors that inhibit DC functions including

IL-6, IL-10, VEGF, TGF- (59). These factors suppress DC functions by inhibiting their maturation, differentiation, migration and even turning DCs into tolerogenic DCs that can induce T cell anergy (60). For example, IL-6, TGF- and IL-10 can inhibit DC maturation and IL-10 can convert immature DCs into tolerogenic DCs (61-64).

Among the cells that compose the tumor stroma the tumor-associated macrophages

(TAMs) are one of several other myeloid cells types, that are particularly associated with sponsoring tumor progression. Monocytes in the circulation migrate to the TME in response to growth factors and once they reside there they differentiate into TAMs (65).

Two main types of TAMs can be identified based on their phenotype: M1, which have a more inflammatory and protective phenotype and M2, which have a more immunosuppressive phenotype. TAM differentiation into M1 or M2 phenotypes is

10 mediated by exposure to cytokines and growth factors in the TME (66, 67). M2 TAMs acquire immunosuppressive functions and can release many types of soluble factors that promote tumor progression including VEGF, TGF-, IL-10, prostaglandin E2

(PGE2) (66-70). They can also produce arginase that depletes arginine from the TME

(71), which in turn leads to decreased expression of TCR  and decreased T cell proliferation (72)

Other subsets of granulocytes found in the TME are tumor-associated neutrophils

(TANs) and their immature counterparts including monocytic and granulocytic myeloid derived suppressor cells (MDSCs). TANs, similar to TAMs, are classified as anti- tumorigenic N1 neutrophils and pro-tumorigenic N2 neutrophils. N1 neutrophils can induce TRAIL-mediated apoptosis of tumor cells, promote ECM degradation, produce reactive oxygen species (ROS) for lysis of cancer cells and mediate antibody dependent cell cytotoxicity (ADCC) of cancer cells (73). In contrast, N2 neutrophils in the TME can promote tumor progression and growth by promoting angiogenesis through production of VEGF and ECM remodeling by upregulation of matrix metalloproteinases (74). They can also recruit regulatory T cells (Tregs) and produce arginase to deplete arginine and suppress T cell function and proliferation (75-77). The second granulocytic population to be discussed here are MDSCs. MDSCs are a heterogenous population of immature myeloid cells that have similar characteristics to granulocytes and monocytes (78). MDSCs can be activated in the TME by several soluble factors including cytokines, chemokines and growth factors and they exert immunosuppressive functions (79). In the TME, MDSCs can suppress protective lymphocyte anti-tumor responses by upregulating enzymes such as arginase, NOS and

IDO that limit T cell and NK cell cytotoxic activities against tumor cells (78-81).

A very important subset of cells found in the TME are tumor-infiltrating lymphocytes, among which there are several types that have both pro- and anti-tumor roles. The lymphocyte subsets that will be discussed in the next several paragraphs are

CD8 T cells, NK cells and CD4 T cells including Tregs. First, CD8 T cells are one of the two subsets of lymphocytes that can directly mediate tumor cell killing and their presence in tumors correlates with better prognosis and therapeutic responses (82-85).

To be able to travel into the tumor, naïve CD8 T cells must become activated effector cells upon recognition of tumor antigens presented by DCs in tumor draining lymph nodes. These cytotoxic effector cells then migrate to the tumor site via lymphatic vessels and are guided by chemokine gradients, especially chemokines CCL5 and

CXCL9 produced by tumors cells and myeloid cells in the TME, respectively (86) .

Upon entry into the tumors they can recognize tumor cells via their TCR and mediate tumor cell death by secretion of perforin and Granzyme B or through direct induction of apoptosis via ligation of death receptors (87). Upon activation, cytotoxic CD8 T cells upregulate the expression of inhibitory receptors including cytotoxic T lymphocyte associated protein 4 (CTLA-4) and PD-1 which upon binding to their respective ligands suppresses T cell function (46). Among these, the PD-1/PD-L1 inhibitory checkpoint has been shown to be particularly important for inhibiting CD8 T cell responses in tumors (88). Moreover, once in the TME CD8 T cell functions can also be suppressed by anti-inflammatory and suppressive cytokines and metabolic barriers induced by catabolic metabolism of nutrients essential for T cell functions and survival.

The second subset of cytotoxic lymphocytes found in tumors are NK cells. These innate lymphocytes can also kill tumor cells using similar mechanisms as CD8 T cells however, their function is not restricted by antigen presentation on MHCI molecules.

Activation of NK cells is mediated by engagement of cell surface activating and inhibitory receptors with ligands in normal and transformed cells. In the TME, tumor cells can downregulate the cell surface ligands to the activating receptors on NK cells and avoid their cytotoxic actions (89). While NK cells are generally thought as being one of the key mediators of anti-tumor immunity, new evidence suggest that, while in the TME, NK cells can undergo polarization and even transformation into MDSCs in response to factors like VEGF and granulocyte-macrophage colony-stimulating factor

(GM-CSF) (90, 91).

The last subsets of immune cells in the TME to be discussed in this section are

CD4 T cells, which also include Tregs. CD4 T cells can be further subdivided into Th1,

Th2, Th9, Th17, Th22 and Tregs, but in in this dissertation I will only discuss the Th1,

Th17 and Treg subsets. Th1 cells are pro-inflammatory and secrete IL-2 and IFN- to aid CD8 T cell responses. Th17 cells have been shown to play dual roles including promoting tumor growth by releasing IL-17A or turning into Tregs and potentiating CD8

T cell functions by increasing their recruitment to the tumor (92, 93). Lastly, Tregs differentiate from naïve CD4 T cells, in response to IL-10 and TGF- and high numbers of these cells in tumors are associated with decreased patient survival (94).

They can also inhibit DC maturation by secreting TGF- and IL-10, and they can inhibit

T cell activation by depleting IL-2 by releasing soluble CD25 (95, 96).

In the TME, complex interactions between cancer cells and stromal cells can lead to the eventual escape of tumor cells from the attack of adaptive immune cells (Fig. 1).

Several mechanisms of immune evasion have been established and they mostly rely on evading immune recognition, hindering immune cell activation and survival, and

13 impeding tumor-killing functions of cytotoxic lymphocytes. The specific mechanism that suppress tumor immunity will be discussed in the following section.

Figure 1. Immune cells and tumors cooperate in the TME to suppress anti-tumor immunity. In the TME, both tumor cells and suppressive stromal cells can produce factors such as TGF-, IL-6, IL-10, PGE2 and ROS that can inhibit the functions and proliferation of cytotoxic lymphocytes and Batf3+ DCs. Other enzymes like arginase and IDO produced in the TME can deplete important amino acids for cytotoxic lymphocyte function and metabolic activities. In support of tumor growth, endothelial cells and cancer associated fibroblasts and even immune cells produce growth factors and angiogenic factors that promote generation of new blood vessels, proliferation and invasive potential of tumor cells.

1.1.3 Mechanisms of immune evasion and suppression of anti-tumor immunity

To avoid and escape detection by innate and adaptive immune cells tumors have developed several strategies that result in progression and eventual metastatic spread of disease. For example, tumors downregulate MHC I molecules to avoid detection by

APCs and T cells that can recognize mutated tumor antigens on their cell surface (97).

What is more, downregulation of MHC I molecules correlates with worst clinical outcomes in patients (97). Moreover, tumor cells and stromal cells in the TME secrete soluble factors including IL-6, TGF- and VEGF that can inhibit DC maturation and downregulation of costimulatory molecules necessary for the successful activation of

CD8 T cells against tumors (98-100). If T cells fail to receive adequate costimulatory signals they become anergic or tolerogenic leading to their eventual demise (101).

Interestingly, when the immune cells initially recognize and eliminate malignant cells that express mutated antigens this means that less immunogenic cancer cells survive and these cells are, as a result of transformation, more adept at evading detection by immune cells. This process is known as immunoediting and it eventually leads to tumor progression (13, 102). Moreover, tumor cells can also decrease the expression of ligands for activating receptors on the cell surface of NK cells, thus avoiding recognition by these innate lymphocytes (103). Consequently, decreased expression of MHC-I molecules, immunoediting and inhibition of lymphocyte activation, by loss of costimulatory signals and ligands for activating receptors are a few of the intrinsic mechanisms that allow cancer cells to avoid immune detection.

There are other physical and chemical barriers in the TME that prevent the infiltration and activity of cytotoxic lymphocytes. Stromal cells including CAFs, TECs, and MDSCs in the TME contribute to the exclusion of CD8 T cells from tumors. CAFs

15 can remodel and deposit ECM to make tumor tissues more rigid and produce CXCL12, that coats cancer cells, to limit CD8 T cell infiltration (19, 25). TECs can induce CD8 T cell apoptosis due to their high expression of FasL (104). In addition, MDSCs can produce reactive nitrogen species (RNS) that can nitrate important chemokines for CD8

T cell infiltration (105).

In the TME, infiltrating cytotoxic lymphocytes face a very metabolically-challenging environment, especially as activated T cells rely on glucose metabolism, through glycolysis, for their proliferation and effector functions (106). Cancer cells have a very high rate of glucose uptake and glycolytic activity leads to production of high lactate levels (107). This process occurs even when oxygen is present in the system and tumor cells have normal mitochondria (108). Low levels of glucose in the TME can lead to T cell anergy, decreased proliferation, decreased secretion of cytokines and reduced anti-tumor activity (109, 110).

Cancer cells can also use amino acids and fatty acids to maintain their metabolic needs and proliferate (111). MDSCs and other immunosuppressive cells in the TME can produce arginase to deplete arginine, a critical amino acid for T cell effector functions (112) and produce ROS and RNS that are toxic to CD8 T cells in tumors

(113). Furthermore, high levels of lactate in the TME are associated with decreased

IFN- production by cytotoxic lymphocytes in tumors (114). Lastly, tumor and stromal cells in the TME produce IDO that depletes tryptophan and produces toxic metabolites that can suppress the anti-tumor responses by CD8 T cell and NK cells (115-117).

The last mechanisms of immune suppression that I will discuss is the engagement of inhibitory receptors on the cell surface of T cells that cause attenuation of their functions (118). Regulation of immune responses by adaptive lymphocytes is

16 necessary to ensure that autoimmunity is not developed. As part of the normal process of immune activation, immune checkpoints are engaged to limit the responses of lymphocytes and to ensure that once the threat has been eliminated no damage occurs to the host. The most widely known and studied immune checkpoints are CTLA-4 and

PD-1. These were discovered between the 1980’s and 1990’s and identified as mechanisms to attenuate T cell responses. T cells that have high expression of these inhibitory receptors show decreased production of cytokines, like IFN- and show an exhausted phenotype (119). These inhibitory receptors and their respective ligands are expressed by T cells, tumor cells, and other immune cells in the TME.

The ligands for CTLA-4 are CD80 and CD86 and for PD-1 they are PD-L1 and PD-

L2 and they can be upregulated on tumor cells in consequence of constitutive activation of oncogenic pathways (120). CTLA-4 mostly inhibits T cell activation by disrupting TCR signaling and decreasing the accessibility to costimulation by engaging with CD80/86 on the surface of APCs (121, 122). PD-1 inhibits T cell activity by decreasing the expression of survival factors and decreasing cytokine production (121,

123). While CTLA-4 can affect CD8 T cell functions, it mostly modulates CD4 T cells and, due to its important role in preventing autoimmunity it can also promote immunosuppressive functions of Tregs (124). Engagement of inhibitory immune checkpoints causes inhibition of T cell and NK cell effector functions and promotes

Tregs activity. Thus, activation of these inhibitory receptors is another mechanism that tumors use to suppress the immune response and currently therapies targeting these immune checkpoints, also known as immune checkpoint blockade, are being widely developed and studied for the treatment of many solid tumors and other malignancies.

The TME with its immunosuppressive actions in combination with intrinsic mechanisms of immune evasion by tumor cells prove that, although these mechanisms may seem redundant, as many cell types produce very similar tumor-promoting growth factors and cytokines, they are necessary for cancers to ensure their survival. This also underscores how capable these malignant cells are to adapt to the arsenal of anti- tumor actions of the immune system. Accordingly, we must ensure to consider these evasion mechanisms when designing new therapies or finding ways to improve current ones. However, as the mechanisms that tumors co-opt to ensure their continued development become better understood, new therapeutic interventions have been developed to alter the TME to become more permissive of immune cell functions.

1.2 Type I Interferons in cancer therapy

Interferons (IFN) were discovered in 1957 and received their name because they interfere with viral replication and mediate viral interference, a phenomenon in which a virus-infected cell becomes resistant to a secondary infection by another virus

(125). Three classes of IFNs have been identified including: type I IFN (IFN-I), type II

IFN (IFN-II), and type III IFN (IFN-III). The IFN-I family of cytokines is encoded by multiple genes that produce several IFN- proteins (13 in humans and 14 in mice), one

IFN- and other less studied IFNs including IFN-, IFN-, IFN-, IFN-, IFN- and IFN-

 (125). IFN- is the only member of the IFN-II family and its roles as an effector molecule, produced primarily by activated lymphocytes and to a lesser extent by APCs, are well documented (126). The IFN-III family is composed of four homologous proteins

IFN- 1-4 (127). Regardless of the type of IFN they promote anti-viral responses by both innate and adaptive immune cells. In this section, I will highlight the important functions of endogenous IFN-I during anti-tumor responses.

1.2.1 IFN-I signaling

IFN-I are produced by virtually all cell types upon stimulation of receptors known as PRRs. During infection with pathogens or in situations of cellular death, PRRs recognize microbial products, known as PAMPs, products of necrotic cell death, known as alarmins or DAMPS and cytosolic nucleic acids (7). Several types of cell surface and cytosolic PRRs are associated with IFN-I production including Toll-like receptors

(TLRs), retinoic acid-inducible gene 1 (RIG-1), melanoma differentiation-associated gene 5 (MDA-5) and NOD-containing protein-1 (NOD1) and NOD2 (128-131). Other

19 cytosolic receptors, that recognize cytosolic DNA, including cytosolic G-AMP synthase

(cGAS) and Z-DNA-binding protein 1 (ZBP1), can also induce IFN-I production (132,

133). The multitude of PRRs that can induce IFN-I expression underscores the importance of IFN-I expression during pathogenic infections and upon situations of cellular damage or death.

Upon stimulation of PRRs several downstream signaling pathways converge on the Interferon Regulatory Factor (IRF) family of transcription factors, particularly IRF3 and IRF7, which in turn upregulate the transcription of IFN-I genes. IFN-I induce signaling through the dimeric IFN-/ receptor (IFNAR) which can be composed of two

IFNAR1 subunits or a heterodimer of IFNAR1 and IFNAR2 (134). Downstream signaling of the IFNAR includes induction of the Janus activated kinase- signal transducer and activation of transcription (JAK/STAT) pathway, phosphoinositide 3- kinase (PI3K) pathway, mitogen-activated protein kinase (MAPK) pathway and the canonical and non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) signaling pathways (135-137). Successively, these various signaling pathways upregulate the transcription of many IFN-stimulated genes (ISGs) whose products including cytokines, chemokines and survival factors among others, are not only important for intrinsic anti-viral mechanisms but can enhance the maturation and functions of many immune cells such as DCs, macrophages, CD8 T cells and NK cells

(136). Thus, IFN-I are critical promoters of both innate and adaptive immune responses against viruses. Likewise, due to their stimulatory effects on many immune cell types they are now appreciated as important factors for intrinsic and therapeutic anti-tumor responses.

1.2.2 IFN-I roles in cancer immunoediting

For many years it was believed that our immune system was blind to transformed cells because they are derived from self-tissues. It wasn’t until the 1970’s and 1980’s, that the theory of cancer immunosurveillance, a process by which the immune system protects the host against oncogenesis, was independently proposed by

Burnett and Thomas (138-140). However, it took several decades to prove that the immune system can recognize malignant cells and eliminate them without overt or external stimulation. This delay was due in part by the lack of adequate animal models in which to test this theory. As proper animal models were developed, including

Recombination Activating Gene-1 (Rag1)-/- and Rag2-/- mice, more evidence was generated for cancer immunosurveillance by Robert Schrieber’s group. They proposed a more comprehensive theory for how the immune system protects against tumor development and how it manipulates the immunogenicity of tumors. This new theory was termed cancer immunoediting and it consists of three major steps: 1) recognition and elimination of malignant cells by the immune system (cancer immunosurveillance);

2) equilibrium between malignant cells and the immune system and 3) escape of neoplastic cells that have reduced immunogenicity (13, 102, 141). After the cancer immmunoediting theory was proposed several studies have generated strong evidence that supports the idea that IFN, in particular IFN-I and IFN-II, play important roles during the three phases of the cancer immunoediting process.

Studies using IFNAR1-deficient mice and antibody blockade of IFNAR1, showed that loss of IFN-I signaling by the host increased the formation of 3-Methylcolantrene

(MCA)-induced tumors and abrogated the rejection of highly immunogenic MCA- derived sarcomas in mice (142). Similar results were observed in mice deficient in IFN-

 signaling, indicating that both IFN-I and IFN-II play important, but not redundant roles, in the intrinsic immune responses against tumor development (13, 102, 141, 143).

Moreover, Dunn and colleagues showed that responsiveness to IFN-I by host hematopoietic cells, but not tumor cells, was necessary to induce tumor regression

(142). These results established a clear role for IFN-I during the cancer immunosurveillance or elimination phase of the cancer immunoediting process.

The second phase of the cancer immunoediting process, known as equilibrium, occurs when a balance between malignant cells and immune cells is maintained and no clinical signs of tumor development may be noticeable. Work from Schreiber’s group showed that this process can last for long periods of time and it is largely mediated by adaptive lymphocytes and IFN-II (144). At the interface of the equilibrium phase and the escape phase of cancer immunoediting is where the process of editing is thought to occur. In this process the immune system’s attack on transformed cells induces the development of less immunogenic transformed cells that can eventually escape immune recognition. Importantly, IFN-I are also significant mediators in this process.

Schrieber’s group showed that around 40% of MCA tumors derived from IFNAR1-/- mice were rejected when implanted into naive wild-type mice, suggesting that these tumors conserved a highly immunogenic or unedited phenotype (142). Whereas MCA tumors derived from wild-type mice grow rapidly when transferred into naïve wild-type mice, having a less immunogenic phenotype (142). These results exposed important functions of IFN-I to influence the immunogenicity of tumors.

1.2.3 Anti-metastatic and immunostimulatory effects of IFN-I IFN-I can induce an array of stimulatory effects on innate immune cells that support their central functions during spontaneous anti-neoplastic immune responses.

Diamond and colleagues showed that IFN-I signaling is required for early anti-tumor immune responses, as blockade of IFNAR1 during early tumor development failed to induce tumor regression of immunogenic MCA tumors (51). Moreover, Tom Gajewski’s group found that IFN-I production by hematopoietic cells, specifically CD11c+ DCs in tumor-draining lymph nodes, is observed prior to generating tumor-specific CD8 T cell responses (9). Moreover, mice deficient in IFNAR1 or STAT1 signaling fail to induce tumor-specific T cell priming (51). Schreiber’s group also showed that responsiveness to IFN-I signaling, specifically by innate immune cells, was critical for tumor rejection and for the generation of tumor-specific CD8 T cells (51). Notably, CD8+ DCs and

CD103+ DCs were shown to mediate cross-presentation of tumor antigens to CD8 T cells and loss of these specific DCs subsets abrogated rejection of tumors in mice (55,

145). In consequence, studies by several groups established the importance of IFN-I responsiveness in Batf3+ DCs for accumulation of these APCs in tumors, cross- presentation of tumor-derived antigens, priming of antigen-specific CD8 T cells and spontaneous rejection of tumors (9, 51, 146). Therefore, IFN-I production by innate immune cells and signaling on specific DC subsets are essential for spontaneous immune recognition and rejection of tumors.

IFN-I are considered a bridge between the innate and adaptive immune responses because of their immunostimulatory effects on cells of both types of immune responses. Importantly, IFN-I enhance maturation and antigen-presentation functions of DCs (147). They also promote migration of DCs to lymph nodes and enhance cross-

23 presentation to CD8 T cells (146, 148). Macrophages also respond to IFN-I stimulation by increasing inflammatory cytokines like IL-18 (149). Critical players that directly mediate tumor-cell death, including cytotoxic CD8 T cells and NK cells, also depend on

IFN-I for maturation, enhanced effector functions and survival. For CD8 T cells, IFN-I provide a third signal during activation to induce differentiation into cytolytic effectors

(150, 151). Furthermore, IFN-I signaling is necessary for CD8 T cell clonal expansion and memory generation in settings of infection with LCMV (152). In this process IFN-I promote survival of CD8 T cells during the antigen-driven proliferation phase. In addition, IFN-I can protect CD8 T cells from NK cell-mediated death by changing the expression of activating and inhibitory ligands for NK cells (153, 154).

It has long been appreciated that NK cells, the second subset of cytotoxic lymphocytes that mediate tumor cell death, are targets of IFN-I. First, IFN-I treatment can enhance NK cell cytotoxic activity in vitro (155, 156). Second, IFNAR1-/- and

IFNAR2-/- mice have decreased proportions of NK cells in the periphery and show decreased survival after implantation with NK-cell sensitive tumors (157). In addition,

IFN-I mediate NK cell priming indirectly through the induction of IL-15 production and trans-presentation by CD11c+ DCs (158). Lastly, NK cells from IFNAR1-/- and IFN--/- mice display impaired cytotoxic functions against tumor cells in vitro (142, 157, 159).

Thus IFN-I are critical during NK cell maturation, priming and anti-tumor responses.

IFN-I are also important in prevention of metastasis formation. It is now clear that both CD8 T cells and NK cells are important in preventing the dissemination of cancer from a primary tumor site to other tissues, illustrated by studies where depletion of CD8 T cells and NK cells causes increased metastatic dissemination to the bones in breast cancer tumor models (160). Conversely, increased metastasis formation was

24 also associated to loss of endogenous IFN-I signaling (161). Importantly, the control of metastatic dissemination in these models of breast cancer was dependent on IFN-I signaling and the presence of CD8 T cells and NK cells in the host. Thus IFN-s support the control of metastasis development, likely through their actions on innate and adaptive cytotoxic lymphocytes.

1.2.4 IFN-I therapy in cancer

IFN- was the first immunotherapeutic drug approved for the treatment of hairy cell leukemia (82) and it subsequently showed promise as a therapy for melanomas, myelomas, lymphomas and certain carcinomas inducing at least partial regression of the disease (162). Moreover, IFN- has been used as adjuvant therapy after surgical removal of melanomas (163) and due to their ability to inhibit angiogenesis, they have been used as antiangiogenic agents in several tumor types including Kaposi’s sarcomas (164). In bladder cancer IFN- is used in patients that fail to respond to BCG therapy (165). Although initial success was observed when using IFN- in the treatment of certain malignancies, the systemic side effects induced by this treatment at high doses were particularly problematic and included fever, chills, weight loss, anorexia and fatigue (166). Thus, with the generation of more specific targeted therapies the use of IFN- in clinical settings has declined. However, as activation of many other signaling pathways, including TLR stimulation and STING pathway activation induce IFN-I production, the focus has now shifted to target the activation of these pathways for the therapeutic benefits of IFN-I production and enhancement of anti-tumor immunity.

1.2.5 STING pathway in spontaneous and therapeutic responses to cancer

Recognition of pathogens and their products is an important function of innate immune cells to generate specific immunity and protect the host from disease. In response to PRR engagement, innate immune cells activate the transcription of important genes including IFN-I to activate other immune cells to the threat. Of particular importance to the work presented in this dissertation is the activation of a cytosolic DNA sensing pathway known as the STING pathway. This pathway was first studied in the context of immune recognition of DNA viruses and it was soon discovered to also be involved in immune sensing of certain bacterial infections (167). In brief, cytosolic DNA is recognized by the cyclic GMP-AMP synthase (cGAS) enzyme that generates a second messenger, cyclic

GMP-AMP (c-GAMP) (168, 169). c-GAMP activates the STING protein in the ER membrane which induces the activation of IRF3 and its translocation to the nucleus where it can upregulate transcription of IFN-I (132, 170, 171). Moreover, certain bacteria produce cyclic dinucleotides that can directly activate STING and induce IFN-I production (172).

While, STING pathway activation is important for anti-viral and anti-bacterial immune responses, recent findings shed light into its pivotal role in anti-tumor immune responses.

As discussed in the previous section spontaneous anti-tumor immune responses and priming of cytotoxic T cells require IFN-I expression and signaling (9). However, it wasn’t until recently that the specific mechanism by which innate immune cells sense tumors and induce IFN-I production was discovered (173). Studies by Tom Gajewski’s lab identified that recognition of tumor DNA, as a product of cellular death, in the cytosol of innate immune cells induced the activation of the STING pathway and induced IFN-I production which in turn promotes priming of CD8 T cells to tumor antigens (173) (Fig.2).

Moreover, activation of the STING pathway in tumor cells can also restrict tumor 26 development as it can induce the production of proinflammatory cytokines, chemokines and growth factors associated with decreased tumorigenesis and cellular senescence

(174-176). In addition, loss of STING signaling in tumor cells led to increased tumor growth in part by decreased infiltration of cytotoxic lymphocytes in tumors (177-179).

Figure 2. Spontaneous tumor recognition is mediated by recognition of dsDNA by the STING pathway in innate immune cells. Apoptotic tumor cells can release dsDNA that can be recognized by cGAS in the cytosol of both tumor cells and innate immune cells, like DCs. Recognition of dsDNA by cGAS leads to activation of

STING which in turn upregulates the transcription of IFN-I. In the TME, IFN-I can promote

DC maturation and enhance CD8 T cell crosspriming both in the TME and in the tumor draining lymph nodes. IFN-I can also promote cytolytic activity of CD8 T cells and NK cells to promote tumor cell killing.

STING signaling is important not only for the spontaneous anti-tumor responses but also enhances responses to other therapeutic modalities including radiation therapy, chemotherapy and immune checkpoint blockade (180-183). In brief, STING-deficient mice failed to control tumor growth in response to radiotherapy and STING activation in DCs induced IFN-I production that was required for antigen-specific CD8 T cell anti-tumor responses (180). Interestingly, while radiotherapy induces STING pathway activation and can promote anti-tumor responses, if used at high doses it can trigger down-regulation of c-GAS/STING pathway in tumors by induction of DNA exonuclease TREX-1 to degrade cytosolic DNA (184). In addition, activation of the STING pathway in cancer cells by several chemotherapy agents including Poly (ADP ribose) polymerase 1 (PARP1) and checkpoint kinase 1 (CHK1) inhibitors was essential for their therapeutic effects (181, 182).

Moreover, STING signaling was essential for the therapeutic response to anti-PD-L1 treatment in melanoma tumors (183). Altogether, these findings strongly suggest that

STING activation in TME plays essential roles in enhancing spontaneous and therapeutic anti-tumor responses and should be the focus of new therapeutic approaches.

Due to the strong rationale for inducing STING activity in the TME to improve anti- tumor responses several approaches were undertaken to induce intratumoral activation of the c-GAS/STING pathway (180, 185, 186). As cyclic dinucleotides produced by mammalian cells and bacterial dinucleotides, like 2’3’-c-GAMP and cyclic diguanylate monophosphate (c-di-GMP), could induce STING activation (168, 172, 187, 188), they were first used as intratumoral therapies. 2’3’-c-GAMP was used in combination with radiotherapy in the treatment of MC38 colon carcinomas and potentiated the anti-tumor effects of radiotherapy by enhancing CD8 T cell functions (180). Furthermore, c-GAMP delivery into melanoma and colon tumors in mice, alone and in combination with

29 checkpoint blockade for PD-1 and CTLA-4, induced tumor growth control and enhanced

CD8 T cell responses (189). c-di-GMP was also used alone and in combination with OVA- peptide vaccination and enhanced survival of glioma-bearing mice (185).

With the success of natural STING agonists in preclinical tumor models, the focus shifted to other compounds and designing novel synthetic dinucleotides that could act as

STING agonists and be safely delivered to the tumor. One of the first compounds studied was 5’-6’ dimethylxanthenone-4-acetic acid (DMXAA) that binds to mouse STING but not human STING (190). Tom Gajewski’s group showed that intratumoral delivery of DMXAA into melanoma tumors efficiently primes CD8 T cells for rejection of established tumors

(186). They also developed novel cyclic dinucleotides that bind to human STING and enhance local and systemic anti-tumor responses to treated and distant secondary tumors

(186). They designed a novel cyclic dinucleotide first termed ML RR-S2 CDA, now known as ADU-S100, which is currently being tested in clinical trials in combination with other immunotherapies (191-193). Intratumoral STING agonists have also been evaluated in preclinical models in combination with other immunotherapy strategies including immune checkpoint inhibitors and CAR T cell therapy (189, 194, 195).

Many other STING agonists have been developed and some of them are being studied as intratumoral therapeutics in combination with other cancer therapies (196, 197).

Nevertheless, recent reports suggest that considerations should be taken in regard to dosing strategies to ensure that combination strategies with immune checkpoint inhibitors and intratumoral STING agonists induce robust activation of CD8 T cells in tumors and long term maintenance of anti-tumor immunity (198).

STING activation is important for sensing tumors and activating spontaneous and therapeutic anti-tumor immune responses by the production of IFN-I. Importantly, we showed that STING activation induces production of sIL-15 complexes in the circulation in response to cyclic dinucleotides and after total body irradiation (TBI) (199, 200), thus in this dissertation I investigated if STING activation in the TME could enhance IL-15 production in tumors and the results of these experiments will be discussed in more detail in Chapter 3.

1.3 Interleukin-15 (IL-15): a key cytokine for cytotoxic lymphocyte responses

1.3.1 IL-15 discovery and functions

IL-15 is a 14-15 kDa cytokine discovered in 1994 as a T cell growth factor, similar in structure and function to IL-2 (201, 202). Although IL-2 and IL-15 share no sequence homology, they have similar protein structures and functions in vitro. Both cytokines can stimulate T cell proliferation, NK cell cytotoxicity and induce lymphokine- activated killer cell activity (201, 203-206). Initial studies using northern blots, indicated that IL-15 mRNA is widely expressed in many tissues including placenta, skeletal muscle, heart, liver, lungs, brains, spleens, kidneys and intestines but detection of IL-15 protein was very low (201, 206, 207). In fact, detection of IL-15 protein in serum or cell culture supernatants was minimal even when cells were stimulated with inflammatory mediators (208-210). These findings hinted to sophisticated mechanisms of IL-15 protein expression regulation.

Studies using antibodies against the IL-2 receptor (IL-2R) subunits demonstrated that IL-15 binds to the IL-2R and common gamma chain (c) complex but does not bind to the IL-2R subunit (206). On the other hand, IL-15 has its own IL-

15R chain that binds to IL-15 with very high affinity (Ka=1011 M-1) and its mRNA expression was detected in many tissues, largely overlapping with IL-15 mRNA expression (211). While IL-15 and IL-2 can form heterotrimeric receptor complexes with

IL-2R, c and their respective R chains, the IL-15R and IL-2R chains are not essential for mitogenic signaling through the IL-2R and c complex (208, 212).

Importantly, interaction of either cytokine with the IL-2R/c complex induces signaling through the JAK1/JAK3 STAT3/STAT5 pathway (213). Suggesting that IL-2 and IL-15 signaling is almost identical; however, the different expression patterns of mRNAs for 32 these cytokines and the difficulties in detecting IL-15 protein in tissues pointed to potentially different functions in vivo and different regulatory mechanisms limiting their expression.

While IL-2 protein is expressed primarily by activated T cells and is primarily regulated at the transcription level, the low amounts of IL-15 protein, that could be detected in tissues and in stimulated cells, hinted at more rigorous regulatory mechanisms for IL-15 protein expression. In fact, several studies confirmed that IL-15 expression is subject to transcriptional, translational and post-translational regulation.

This regulation is mediated in part by multiple start sites in the 5’ UTR region, two different signal peptides and regulatory elements found in the 3’UTR regions of the IL-

15 gene (210, 214, 215).

In terms of biological activity, studies using knockout mouse models provided evidence for distinct in vivo biological functions for IL-15 and IL-2. Mice that lack IL-2 or

IL-2R develop a fatal autoimmune disorder, while thymic development of adaptive lymphocytes remains largely unaffected, indicating that IL-2 plays an important role in regulation of autoreactive immune cell populations (216-218). In contrast, IL-15 -/- and

IL-15R-/- mice do not develop any autoimmunity but show defects in specific immune cell compartments (219, 220). Further differences in function between IL-15 and IL-2 include the major roles of IL-2 in Fas-mediated activation-induced cell death (AICD) of activated CD4 T cells and in the thymic development and long-term survival of Tregs in the periphery (221-224). In contrast, IL-15 can inhibit AICD mediated by IL-2 and

TRAIL-mediated apoptosis by inducing the expression of anti-apoptotic molecules like

Bcl-2 and Bcl-XL (225-227).

Studies using IL-15-/- and IL-15R-/- mice demonstrated that IL-15 plays important roles in the development of several subsets of lymphocytes including NK cells, NKT cells, CD8+ intra-epithelial lymphocytes (IELs) and memory CD8 T cells

(219, 220). IL-15-/- mice lack functional mature NK cells, and have a marked decrease in NKT cells in the thymus and periphery, CD8 T cells with a memory phenotype and

CD8+ IELs (219). IL-15R-/- mice are lymphopenic and show a similar phenotype to

IL-15-/- mice (220). Importantly, IL-15-/- mice are relatively healthy but when challenged with vaccinia virus they are not able to clear the infection and subsequently die (219), suggesting that IL-15 is crucial for host defense against viral infections.

1.3.2 Mechanisms of action of IL-15

The high binding affinity of IL-15R to IL-15, their similar mRNA expression patterns across many tissue types and immune cells and the similar phenotypes of IL-

15-/- and IL-15R-/- mice suggested a possible requirement for the interaction of IL-15 with IL-15R for its actions on IL-15 responsive cells. As IL-15R expression was detected in CD8 T cells and NK cells (228, 229), studies dissecting if co-expression of

IL-15R by IL-2R/c expressing cells was necessary for IL-15 responsiveness were next undertaken. Unexpectedly, IL-15R expression by either NK cells or CD8 T cells was not required for their survival or proliferation in response to IL-15 stimulation (230-

233). However, when transferred into wild-type hosts or bone marrow chimeras, IL-

15R-/- lymphocytes differentiated, proliferated and survived when IL-15R was expressed by radiation-sensitive hematopoietic cells (230, 231, 233). The results from the studies described above led to the hypothesis that expression of IL-15R on

34 immune cells different from lymphocytes, was necessary for IL-15-mediated effects in vivo. Accordingly, studies by Schluns et al subsequently showed that bone marrow

(BM)- derived and parenchymal cells expressing IL-15R were required for naïve CD8

T,  T cell, NK and NKT cell development (234). In contrast, expression of IL-15R by

BM-derived cells was required for memory CD8 T cell differentiation and maintenance through basal homeostatic proliferation (234).

As IL-15R expression by non-lymphocytic cells is required for IL-15 functions in vivo, this peculiar requirement pointed to a likely unique mechanism of action for endogenous IL-15. Importantly, in vitro studies investigating the role of IL-15R on IL-

15 signaling showed that membrane bound IL-15/IL-15R complexes could act in trans to stimulate proliferation of neighboring cells that express the IL-2R/c complex (235).

This new mechanism of action was termed “transpresentation” and provided a rationale for why IL-15R expression by cells, other than CD8 T cells and NK cells, was necessary for IL-15-mediated effects. Subsequently, it was shown using elegant bone marrow chimeras studies that expression of IL-15 and IL-15R by the same cell was necessary for development and basal proliferation of mature NK cells and memory CD8

T cells (236, 237). Thus, transpresentation is a unique mechanism of action, attributed to IL-15, that requires direct cell to cell contact of IL-15 producing cells with IL-15 responsive cells (Fig. 3A).

Figure 3. Mechanisms of action of IL-15. The IL-15/IL-15R complex is formed intracellularly in the endoplasmic reticulum and shuttled to the cell surface of IL-

15 expressing cells including myeloid cells, stromal and epithelial cells. IL-15 exerts its immunostimulatory effects mainly on CD8 T cells and NK cells. A) During steady-state, trans-presentation of IL-15 by IL-15R, requiring direct cell-to-cell contact with IL-2/IL-

15R and c expressing cells is the main signaling mechanism. B) During inflammation, the IL-15/IL-15R complex is cleaved from the cell surface by proteases and can send paracrine signals to nearby cytotoxic lymphocytes.

The findings discussed in the previous paragraphs pointed to transpresentation of IL-15 as an important mechanism regulating IL-15 effects on adaptive immune cells.

Still, other studies showed that in response to inflammatory signals the IL-15/IL-15R complex can be cleaved from the cell surface from the IL-15 producing cells and potentially induce paracrine signaling on cells that express the IL-2R/c complex (238-

240). Hence, pointing to a second possible mechanism of action in which IL-15 and IL-

15R form soluble complexes that act on nearby cells (Fig. 3B).

Cleavage of cell surface IL-15/IL-15R cell complexes is likely mediated by proteases and the specific metalloproteases that mediate the cleavage in vivo are still being studied. Several analyses pointed to the metalloprotease A Desintegrin and

Metalloprotease 17 (ADAM17) as a possible mediator of this cleavage. ADAM17 is known to mediate the cleavage of membrane bound TNF- and several other substrates, this in part because ADAM17 is expressed in many tissues (241-243).

Importantly, ADAM17 can cleave IL-15R from the cell surface of cells in vitro (239,

244). Thus, our group sought to study the role of ADAM17 in generating soluble IL-

15/IL-15R complexes (sIL-15 complexes). We found evidence that ADAM17 mediates the cleavage of IL-15/IL-15R complexes from the cell surface of bone marrow derived

DCs in response to IFN- stimulation (199). However, when ADAM17 expression was abrogated in a tamoxifen-inducible knockout mouse model we found that ADAM17 expression was only partially required for the generation of sIL-15 complexes in response to Poly I:C, but was not necessary for the IFNAR-independent generation of sIL-15 complexes in vivo (199). Consequently, the evidence suggests that other metalloproteases could also be mediating the generation of sIL-15 complexes in vivo.

Regardless of the nature of the IL-15 complexes, whether soluble or membrane- bound, it is clear that IL-15R expression by IL-15-producing cells could be important not only mediating the effects on IL-15-responsive cells but also in enhancing the stability of the cytokine signal. Thus, it was not surprising that studies revealed that co- expression of IL-15 and IL-15R by the same cell is necessary to stabilize the IL-15 protein intracellularly by forming an IL-15/IL-15R complex in the endoplasmic reticulum that is then shuttled to the cell surface (238, 245). Accordingly, cell surface expression of IL-15 is tightly regulated by coupling to IL-15R and once on the cell surface the IL-15/IL-15R complex can induce juxtracrine signaling or be cleaved and potentially induce paracrine signaling.

1.3.3 IL-15 functions during homeostasis and inflammation

Studies focusing on ablation or selective expression of IL-15R on specific cell subsets provided the first evidence of the roles of specific cell types transpresenting IL-

15 in lymphocyte development, maintenance and survival during steady state (246-

250). Studies using IL-15R-/- mice and cell specific IL-15R transgenic mice determined that DCs and macrophages transpresent IL-15 to support the development and maintenance of mature NK cells and homeostatic proliferation of memory CD8 T cells (247, 248, 251). Importantly, intestinal epithelial cells trans-present IL-15 to drive development, differentiation and survival of CD8+ IELs (250).

In addition to myeloid cells, other cells of non-hematopoietic origin including keratinocytes, liver, kidney, skeletal muscle cells, microglia and thymic epithelial cells express IL-15 mRNA (201, 206, 207, 246, 250, 252-254). In keratinocytes IL-15 acts a

38 factor that promotes survival and proliferation (254). In the kidney, IL-15 serves as a survival factor to protect tubular epithelial cells from transformation and cell death (255,

256). In skeletal muscle cells, IL-15 is thought to act as a pleiotropic myokine that regulates the oxidative metabolism of muscle fibers and reduces adipose tissue mass

(257). Studies where skeletal muscle-specific IL-15 was overexpressed showed increased mitochondrial mass and activity in the adipose tissue, pointing to a role for

IL-15 in the interaction between muscle and adipose cells (257). Consequently, IL-15 is not only important for development and survival of cells of the immune compartment but also plays critical roles promoting survival and metabolism of many other cell types.

After the discovery of IL-15 it was soon demonstrated that activation of monocytes, and macrophages with TLR agonists, including LPS, Poly:IC and IFN-I led to upregulation of IL-15 mRNA expression and protein production (203, 258-260).

Other studies also showed that in vivo delivery of these inflammatory mediators could induce IL-15 mRNA expression (156, 261). As these inflammatory proteins are produced by infectious pathogens it was important to use viral models of infection to investigate the roles of IL-15 during inflammation.

To this extent, studies in IL-15-/- mice showed that in absence of IL-15 the mice succumbed to infections with vaccinia virus and genital herpes simplex virus type 2

(HSV2). These poor responses were mostly due to the lack of mature NK cells and defects in NKT cells in these mice (219, 262). Studies using murine cytomegalovirus

(MCMV) showed that IFN-I and IL-15 were necessary for the activation, cytotoxic activity and survival of proliferating NK cells (156). Moreover, IL-15 expression was necessary for the generation of antigen-specific memory CD8 T cells in a vesicular stomatitis virus (VSV) model of infection (228). Importantly, in settings of infection with

VSV and lymphocytic choriomeningitis virus (LCMV) IL-15 was required for the generation and long-term maintenance of memory CD8 T cells (228, 232). Additionally,

IL-15 has been shown to have protective effects against bacterial infections like

Mycobacterium tuberculosis, Salmonella enterica and in sepsis models (263). Taken together, all these findings demonstrate that IL-15 plays essential roles in generation of effective anti-viral and anti-bacterial immune responses by CD8 T cells and NK cells.

IL-15 has also been shown to promote CD8 T cell and NK cell migration to sites of inflammation (264-266). IL-15 produced in the lung airways of influenza infected mice was important to recruit effector CD8 T cells to the site of infection and treatment with exogenous IL-15/IL-15R complexes was enough to restore the frequency of effector CD8 T cells in IL-15-/- mice after influenza infection (266). A follow-up study by the same group demonstrated that IL-15 mediated the recruitment of NK cells to the lung airways after influenza infection (265). These results support early in vitro studies that showed that IL-15 could act as a chemoattractant to T cells and NK cells and could induce the expression of chemokines and chemokine receptors on T cells (264, 267-

269).

As IL-15 stimulates NK cells and CD8 T cells, it is not surprising that IL-15 has also been associated with states of overt immune cell activation. Transgenic mice that overexpress IL-15 develop a form of T-cell leukemia and IL-15 expression is linked with pathology observed in several inflammatory and autoimmune diseases including rheumatoid arthritis, psoriasis, Celiac Disease, inflammatory bowel diseases, and multiple sclerosis (270-273). IL-15 is not only an important regulator of cytotoxic lymphocyte development and homeostasis but is an important inflammatory mediator

40 during overt states of inflammation including bacterial and viral infections and inflammatory and autoimmune diseases.

1.3.4 IL-15 regulation

IL-15 expression during homeostatic conditions is tightly regulated not only at the transcription and post-transcriptional level but also by its interaction with IL-15R.

These mechanisms ensure a low constitutive level of lL-15 is available for the development and survival of lL-15 responsive cells. During inflammation, IL-15 production is increased and generation of sIL-15 complexes is also observed. In this section I will discuss several mechanisms by which IL-15 responses are regulated in situations of immune activation and in response to several inflammatory stimuli.

As mentioned previously, soon after the discovery of IL-15 it was observed that in response to inflammatory mediators including TLR4 and TLR3 agonists, like LPS and Poly I:C, both IL-15 mRNA and protein expression is upregulated (203, 258-260).

In vitro stimulation with LPS and Poly I:C increase IL-15 mRNA expression by macrophages and DCs (240, 259, 261). In vivo stimulation with these TLR agonists induces production of IL-15/IL-15R complexes by DCs (240). Moreover, our group and others showed that VSV infection, CD40 stimulation and total body irradiation can also enhance IL-15 expression and production of sIL-15 complexes (158, 199, 274,

275).

During infections or because of TLR stimulation, IFN-I are generated and positively regulate IL-15 production. Both in vitro and in vivo treatment with IFN-I upregulates transcription and cell surface expression of IL-15 and IL-15R (259, 261,

276-278). Importantly, our group showed that in vivo treatment with IFN- induces the production of sIL-15 complexes found in the sera of mice (199). Importantly, we showed that the upregulation of sIL-15 complexes observed upon stimulation with IFN-

, Poly I:C, agonists of the STING pathway and total body irradiation was dependent on IFN-I signaling, as generation of sIL-15 complexes was impaired in mice deficient in the Interferon Alpha Receptor 1 (IFNAR-/-) (199, 200) . Although IFN-I signaling is required for cell-surface IL-15/IL-15R expression and upregulation during VSV infection and during TLR4 stimulation (158, 277), our study revealed that other signaling pathways other than IFN-I signaling may be responsible for generation of sIL-

15 complexes during VSV infection and upon CD40 stimulation (199). These findings suggest that IL-15 production in response to inflammation is regulated by IFN-I dependent and independent pathways. In Chapter 3 of this dissertation I will show how

IFN-I regulate the production of sIL-15 complexes early during tumor development and how this regulation correlates with tumor-infiltrating lymphocyte numbers.

1.3.5 Better models for IL-15 detection

Since detection of IL-15 protein in vivo proved difficult using conventional hybridization methods, the development of novel techniques and mouse models for detecting IL-15 protein were important to clarify IL-15 requirements and sources. To this extent, the generation of two different transgenic IL-15 reporter mouse models has aided in the quest to confirm the identity of IL-15 producing cells (277-279). The first IL-

15 GFP reporter mouse model was generated using a bacterial artificial chromosome

(BAC) in which GFP is expressed under the control of the IL-15 promoter (277). The second transgenic IL-15 reporter model was generated by introducing a modified BAC

42 that contains the IL-15 gene locus, where IL-15 is linked to GFP using a 2A peptide

(279). In the first transgenic model, GFP expression reflects the activity of the IL-15 promoter, modeling transcription of IL-15. The second transgenic reporter model serves as a model for IL-15 translation, where GFP expression is concurrent with IL-15 expression however, it does not mimic the trafficking and secretion of IL-15.

Studies using these transgenic IL-15 reporter mouse models confirmed that IL-

15 expression in cells of hematopoietic origin is largely confined to myeloid cells. IL-15 reporter expression was confirmed in basophils, mast cells, eosinophils, neutrophils, monocytes, macrophages and in several subsets of DCs including CD8+ DCs and

CD11c+ DCs, albeit at very low levels in the latter (280). Moreover, the expression of

IL-15 reporter was observed during steady state conditions and could be further enhanced in certain cell subsets after infection with vaccinia virus or after total body irradiation (200, 280). Moreover, the development of more sensitive enzyme-linked immunosorbent assays (ELISA), demonstrated that the physiological configuration of the IL-15 cytokine consists of either membrane-bound or sIL-15 complexes, as soluble

IL-15 is not found in the circulation (274). These new models provided an important avenue for our group to study endogenous IL-15 production, specific cellular sources and regulation of IL-15 expression in the setting of tumor development (281). In

Chapter 3 of this dissertation, I will discuss in detail the findings of our most recent published work (281) .

1.4 Therapeutic potential for IL-15 in cancer

The idea of using cytokines as therapies for cancer was solidified with the approval of IFN- as therapy for hairy cell leukemia by the FDA in 1986 (282).

Subsequently IFN- was approved for treatment of melanoma, Kaposi sarcoma and other hematological malignancies (283, 284). In 1992, IL-2 became the second cytokine to receive approval as a cancer immunotherapy for metastatic renal cell carcinoma, and in 1998 it was approved for treatment of advanced melanoma (285,

286). Unfortunately, both IFN- and IL-2 had to be administered at high doses due to their short in vivo half-life, had low response rates and provoked severe adverse effects

(283). Since then several cytokines including IL-12, GM-CSF, IL-21 and IL-15 have also been studied as monotherapies for cancer (283, 284). The focus of this section is to discuss the current therapeutic formulations of IL-15 studied as cancer therapeutics.

1.4.1 Recombinant IL-15 as cancer monotherapy

As mentioned previously, IL-15 shares similar functions to IL-2, including inducing proliferation of activated T cells and in differentiation of effector T cell subsets.

In contrast to IL-2, IL-15 acts as a survival factor for T cells at different stages of development, does not promote maintenance of Tregs and its actions in vivo preferentially stimulate CD8 T cells and NK cells, key cytotoxic mediators of tumor cell death. Moreover, in pre-clinical studies in mice IL-15 administration or overexpression induced potent anti-tumor responses by CD8 T cells in metastatic colon carcinomas and fibrosarcomas, rhabdomyosarcomas and melanomas (275, 287-289). These findings provided the first evidence of the potential of recombinant IL-15 as a cancer

44 therapeutic. These IL-15 attributes led to its consideration as one of the most promising agents for cancer immunotherapy by the National Cancer Institute in 2008.

Studies in non-human primates established the safety of recombinant human

(rh) IL-15 administration. Delivery of IL-15 at a dose of 20 g/kg/day for 10-12 days via intravenous (i.v.) bolus, continuous intravenous (c.i.) or subcutaneous (s.c.) infusions led to ~10 fold increase in circulating NK cells and between 10 to 100 fold increase in number of circulating effector memory CD8 T cells (290-292). While no capillary leak syndrome or hemodynamic instability were observed in these studies, grade 3 and 4 transient neutropenia was observed with i.v. bolus administration of rhIL-15 (290).

These findings in non-human primates provided the rationale to initiate clinical trials for safety of rhIL-15 in humans.

In the first phase-I clinical trial for rhIL-15 in adults with metastatic melanoma and renal cell carcinoma, daily i.v. administration of rhIL-15 led to a 10-fold expansion of NK cells in patients treated with a dose of 3g/kg of rhIL-15 (293). However, increases in other inflammatory cytokines including IL-6 and TNF- were found upon rhIL-15 administration at high doses. The increase in inflammatory cytokines was accompanied by acute toxicities including hypotension, fever and chills. Thus, it was concluded that the maximum tolerated dose for daily infusion of rhIL-15 was 0.3g/kg and with this dose the effects observed in NK cell increases were reduced to ~3-fold expansion.

Two other phase I clinical trials for safety and dose escalation of continuous infusion and s.c. administration of rhIL-15 were started. Recently, the phase-I dose escalating trial for daily s.c. administration of rhIL-15 in patients with advanced solid tumors was completed (294). It was determined that the maximum tolerated dose for

45 s.c. daily administration of rhIL-15 was 2 g/kg, demonstrating that s.c. delivery of rhIL-

15 allowed for significant dose escalation when compared to bolus i.v. administration.

Increases in circulating NK cells and CD8 T cells were observed to a similar extent as with bolus i.v. administration of rhIL-15. This trial was terminated early because one of the participants developed a severe adverse event associated with chest pain and hypotension. Importantly, in either of these trials significant clinical responses were observed, only maintenance of stable disease in a subset of patients. These trials shed light into some of the limitations that must be overcome to achieve functional anti-tumor responses by IL-15 monotherapy including its short half-life, the high-doses required for therapeutic efficacy and the increase in other inflammatory cytokines that could cause undesired side-effects.

1.4.2 IL-15/IL-15R agonists

To address the limitations of rhIL-15, several formulations of IL-15 agonists have been developed including mimics of the sIL-15 complexes. Initial studies suggested that a natural soluble form of the human IL-15R could act as an IL-15 antagonist by binding to membrane bound IL-15 (239). However, in vitro and in vivo studies in mice showed that treatment with IL-15 bound to IL-15R induces proliferation of memory- phenotype CD8 T cells and NK cells and enhances the anti-tumor activity of IL-15 (295-

298). What is more, these mimics of sIL-15 complexes are more potent than IL-15 treatment alone in inducing proliferation of NK cells and CD8 T cells in vivo (295-297,

299). Since IL-15/IL-15R heterodimer is the major form of IL-15 protein detected in the circulation of both humans and mice (274), it was important to generate

46 recombinant molecules that mimicked the natural configuration of the IL-15/IL-

15R complex.

Several different strategies have been used to mimic the endogenous IL-15/IL-

15R complex. The first strategy was developed by Pavlaki’s group, known as heterodimeric IL-15, which consists of recombinant IL-15/ IL-15R complexes that retain the native structure of the cytokine (300). To achieve this, human cell lines were engineered to overexpress the natural form of IL-15/IL-15R complexes. Studies in mice revealed heterodimeric IL-15 has an increased half-life of 4 hours when compared to less than 30 minutes for E. coli derived rhIL-15 (300). Heterodimeric IL-15 also induces potent CD8 T cell and NK cell proliferation, indicating superior bioactivity of heterodimeric IL-15 when compared to rhIL-15. Additionally, systemic treatment with heterodimeric IL-15 can induce activation, enhanced cytotoxicity and increased migration of antigen-specific CD8 T cells in a mouse model of adoptive cell therapy

(301). This particular IL-15 agonist is currently in clinical trials as a systemic monotherapy and in combination with anti-PD-1 checkpoint inhibitors for metastatic solid tumors (193).

A second strategy used mostly in pre-clinical models of cancer are recombinant soluble IL-15/IL-15R−Fc complexes (rsIL-15c). This is achieved by combining recombinant IL-15 with a fusion protein of soluble mouse IL-15R linked to the Fc portion of human IgG1 (296). Similar to heterodimeric IL-15, the in vivo half-life of rsIL-

15c was significantly increased to ~20 hours and systemic delivery of these complexes increased 50 to 100-fold the proliferation and expansion of memory CD8 T cells and

NK cells populations when compared to rhIL-15 (295-297, 299). Lastly, rsIL-15c show increased anti-tumor activity over IL-15, evidenced by their ability to limit tumor growth,

47 decrease metastatic spread and improving survival in metastatic melanoma and spontaneous pancreatic tumor models (295-298). The two types of soluble IL-15/IL-

15R mimics shed light on the effects of enhancing the stability and half-life of IL-15 by linking it to IL-15R, prompting others to focus on targeting specific domains of the IL-

15R to simplify the development of more stable and potent IL-15 agonists.

Studies dissecting the interaction of IL-15R to IL-15 determined that while the cytoplasmic domain of IL-15R is not necessary for transpresentation of IL-15 (302), the sushi domain of the IL-15R protein is however responsible for the high-affinity binding to IL-15 (303). The sushi domain of IL-15R is a 65 amino acid motif at the N- terminal domain of the IL-15R protein which mediates protein-protein interaction with

IL-15. A fusion protein called receptor-linker- IL-15 (RLI) was developed by combining human IL-15 to amino acid 1-77 of the sushi domain of IL-15R using a 20 amino acid linker peptide (304). This new agonist showed an increased half-life of ~3 hours and proved effective in enhancing anti-tumor immune responses by increasing mouse survival and limiting metastatic spread of melanoma tumors to the lungs and liver (305).

Although RLI enhanced anti-tumor functions it only partially inhibited tumor growth and in a model of colorectal cancer it was only effective delaying tumor growth when systemic monotherapy was started early during tumor development (306). Delayed treatment with RLI proved unsuccessful delaying tumor growth and this was correlated with an exhausted phenotype of CD8 TILs. Thus, RLI was used in combination with an anti-PD-1 checkpoint inhibitor and they acted synergistically to increase survival and improve complete response rates (306).

Taken together these findings show that conjugating IL-15 to the entire IL-

15R protein or just the sushi domain of IL-15R enhances the bioavailability of IL-15, 48 by stabilizing the protein and increasing its half-life. Although these IL-15/IL-

15R agonists prove potent inducers of CD8 T cell and NK cell anti-tumor responses that limit tumor growth and metastatic spread, monotherapy with IL-15 is not sufficient to completely eradicate tumors, suggesting that combination therapy with checkpoint inhibitors and other immunotherapies are the next logical steps to achieve complete remission of tumors. Currently, an ongoing phase I/Ib clinical trial is testing the safety and responsiveness of RLI as a monotherapy and in combination with an anti-PD-1 checkpoint inhibitor for advanced metastatic tumors (307).

Another approach to develop IL-15 agonists has also been explored and a new

IL-15R/c agonist is under development by Nektar Therapeutics (308). This new agonist, NKTR-255 is a recombinant human IL-15 protein that is conjugated to a polyethylene glycol polymer and it is currently being investigated in preclinical studies with non-human primates and mice and in combination with monoclonal antibodies in a phase I clinical trial (309, 310). I will discuss more about this agonist and its use as an intratumoral agent in Chapter 4 of this dissertation.

The last IL-15/IL-15R agonist that I will discuss is another fusion protein ALT-

803, which was recently renamed as N-803, that has shown great potential as a potent immunotherapy for cancer. This fusion protein was created by linking a mutated version of IL-15 (IL-15N72D) to a dimer of IL-15R sushi domain-IgG1 Fc fusion protein (311).

IL-15N72D, is a mutated IL-15 peptide in which an asparagine to aspartic acid amino acid substitution increases biological activity of this polypeptide over IL-15 (312). N-803 has an increased half-life of ~25 hours in mice and potently enhances CD8 T cells and

NK cells proliferation and activation (311, 313). Surprisingly, N-803 proved to have increased anti-tumor activity over IL-15 in a variety of liquid and solid tumors (313-318).

In models of multiple myeloma N-803 induced a reduction of 90% of myeloma cells in the bone marrow and prolonged survival of myeloma-bearing mice (313). It also protected mice from a second tumor challenge in a CD8 T cells-dependent manner.

Lastly, it promoted proliferation and expansion of memory-phenotype CD8 T cells and increased their in vivo secretion of IFN- (313). Potent anti-tumor responses against solid tumors by N-803 were observed in animal models of melanoma, colon carcinomas, bladder cancers, glioblastomas, breast and ovarian cancers (313-317,

319). In particular, N-803 increased the infiltration of CD8 T cells into brain and bladder tumors, supporting a role for IL-15 in promoting TIL infiltration (314, 316).

The surprising efficacy of N-803 across multiple tumor types increased the interest in developing this IL-15/IL-15R superagonist as a clinical reagent. Importantly, although systemic administration of N-803 could increase the circulating levels of other inflammatory cytokines, including INF- and TNF-, no systemic toxicities were observed (315). Moreover, systemic administration of N-803 in non-human primates showed that its half-life was increased to ~8 hours and no significant adverse events were observed with treatment (317). The results from the preclinical studies in mice and non-human primates propelled the study of N-803 in several clinical trials as a monotherapy or in combination with checkpoint inhibitors and adoptive cell therapies across a multitude of tumor types (Lists of recent clinical trials for N-803 can be found in (320, 321)). Importantly, due to the promising results from two clinical trials (322,

323) an intravesical combination treatment using Bacillus Calmette-Guerin (BCG) and

N-803 received the breakthrough therapy designation by the FDA for patients with

BCG-unresponsive non-muscle invasive bladder cancers (324).

1.4.3 IL-15 agonists in combination with other immunotherapies for cancer

Cancer immunotherapy strategies, whose goal is to activate the patient’s immune system to react against malignancies, have come a long way since William

Coley’s 1890’s first immunotherapeutic trial using live and attenuated bacteria injected into patients’ tumors (325). After Coley’s first trial, in which he observed spontaneous regression in hundreds of patients of several types of malignancies, several decades passed before interest in cancer immunotherapy was renewed and thus progress was somewhat slow. Nonetheless, several types of immune based therapies have become more widely developed and accepted including monoclonal antibodies, immune modulators, cancer vaccines, adoptive cell therapy and the immune checkpoint inhibitors. In this section I will discuss how IL-15 has been used in combination with several immunotherapy strategies including adoptive cell therapy and checkpoint inhibitors for treatment of different cancers.

Conventional adoptive cell therapy consists of isolating T lymphocytes from patient tumor samples or blood and expanding them to great numbers to reinfuse them back into the patient in the hopes that they will recognize specific antigens produced by the individual patient’s tumor and induce tumor cell death via cytotoxic mechanisms.

This first type of adoptive cell therapy is commonly referred to as Tumor-Infiltrating

Lymphocyte (TIL) therapy. However, other types of adoptive cell therapy require the genetic manipulation of these lymphocytes to engineer a transgenic TCR or a chimeric antigen receptor (CAR) on the surface of the lymphocytes that recognizes a specific antigen expressed primarily by tumor cells. These therapies are known as engineered

TCR therapy and CAR T cell therapy.

IL-15 has been used for the ex vivo expansion of T cells during TIL therapy (289,

326). Moreover, TILs expanded in the presence of IL-15 maintained a central memory

T cell phenotype and migrate better into tumors than IL-2 expanded TILs (289, 327). IL-

15 can also restore responsiveness to antigens in tolerant CD8 TILs and promote tumor clearance (328-330). Moreover, leukemia and glioma-specific CAR T cells engineered to express IL-15 have improved proliferation and survival and better anti- tumor responses than conventional CAR T cells (331-333).

NK cells are currently being studied for adoptive cell therapies using haploidentical NK cells, autologous NK cells and CAR NK cells for the treatment of liquid tumors including acute myeloid leukemia and multiple myeloma (334-336). To this extent combination therapy using rhIL-15 in combination with adoptive NK cell therapies including haploidentical and autologous NK cells have been tested in several clinical trials (NCT01875601, NCT02395822, NCT01385423, (336)). Moreover, cord blood derived CD19 CAR NK cells were engineered to express IL-15 and this addition enhanced their killing activity against leukemia cell lines and improved survival in a mouse model of lymphoma (335). These CD-19 CAR NK cells were tested in a phase

I/II clinical trial in patients with relapsed or refractory non-Hodgkin’s lymphoma and chronic lymphocytic leukemia with no adverse effects and responses in 8 out of 11 patients, including 7 complete remissions (337).

In addition to adoptive cell therapies, immune checkpoint blockade strategies have also been studied in combination with systemic treatments with IL-15 and its agonists in both preclinical and clinical studies. The rationale for using these combination strategies is based on the upregulation of immune checkpoints including

CTLA-4, TIGIT, TIM3 and PD-1 on T cells (338-340). In preclinical models of prostate

52 and colon cancers rhIL-15 was used in combination with anti-CTLA-4 and anti-PD-L1 mAb and the triple combination enhanced the anti-tumor activity of rhIL-15 and enhanced mouse survival (341, 342). Similarly, rsIL-15c and N-803 were evaluated in combination with anti-PD-1/PD-L1 mAbs in mouse tumor models of colon cancer and triple negative breast cancers and the combination enhanced responses to anti PD-

1/PD-L1 therapy (343, 344). Lastly, both rhIL-15 and N-803 have been tested in clinical trials in combination with immune checkpoint blockade against PD-1 and CTLA-4 in refractory and metastatic solid tumors and non-small cell lung cancer, respectively

(NCT03388632, (345)).

Proof of the potential immunotherapeutic value for IL-15 is abundant and with its potential synergistic effects with many other types of immune-based therapies it is not surprising that most studies have focused on its therapeutic effects against cancers.

However, as an endogenous promoter of natural and intrinsic responses by CD8 T cells and NK cells, it is important to investigate if this homeostatic and inflammatory cytokine plays important functions during unstimulated intrinsic anti-tumor responses.

Studies of human colorectal tumors highlighted an important role for IL-15 expression in tumors. Jerome Galon’s group performed genetic analysis of human colorectal tumors and found that a group of 13 cytokines were differentially expressed.

Importantly, IL-15 was deleted in a subset of tumors and the loss of IL-15 correlated with increased risk of tumor relapse, decreased patient survival and decreased proliferation of tumor-infiltrating T and B cells (346). Moreover, studies using IL-15 deficient mice discovered that loss of IL-15 expression in colitis-associated colon carcinomas leads to increased incidence and tumor burden, decreased overall survival, and correlates with upregulated expression of inflammatory mediators that promote

53 colon cancer progression in tumor tissues (347). Several other preclinical studies where cancer cells where engineered to express IL-15 showed that IL-15 in the TME can prevent tumor and metastasis development, control tumor growth and potentiate

NK cell killing of tumor cells (348-354). Accordingly, IL-15 expression in the TME was shown to augment anti-tumor immunity and taken together these findings pointed to an important role for endogenous IL-15 as an inflammatory mediator in the TME. While these findings suggested the importance of TME-derived IL-15, direct evidence as to how IL-15 production is regulated in tumors, the cellular sources and the mechanisms by which it enhances anti-tumor immunity was lacking and I aimed to address this gap in knowledge in this dissertation. These findings will be discussed in Chapters 3 and 4 of this dissertation.

1.5 Summary

The complex interplay between the immune system and cancer cells usually results in tumor cells escaping elimination by innate and adaptive lymphocytes and eventual spread of disease to nearby organs. Tumors are surrounded by stromal cells and infiltrating immune cells that can promote disease progression and spread of disease.

What is more, tumors have evolved intrinsic and extrinsic mechanisms to curtail immune responses leading to decreased infiltration of T cells and NK cells in tumors, and increased infiltration of immunosuppressive cells including Tregs and MDSCs. Although, rejection and elimination of tumors is rarely achieved naturally or in response to therapies, the immune system is still capable of mounting strong anti-tumor responses when the mechanisms of immunosuppression in tumors can be circumvented. This has been demonstrated with the sustained responses achieved with immune checkpoint inhibitors in solid tumors like melanomas and with adoptive CAR T cell therapies for leukemias and lymphomas (355, 356).

Understanding how the cells and inflammatory factors in the TME can be polarized to promote more robust anti-tumor responses by CD8 T cells and NK cells is crucial for achieving better and broader responses and positive clinical outcomes, including increased survival and decreased chances of relapsing, in patients with very aggressive and hard to treat cancers such as pancreatic, brain and colon cancers and in patients with disease that has already metastasized to other organs. To this extent, my studies focused on investigating the roles of locally-produced IL-15 in TME as a promoter of CD8 T cell and

NK cell responses. Based on the results from human colorectal tumors and other preclinical models of several types of cancers, there was strong evidence pointing to this inflammatory cytokine as an important part of the inflammatory milieu in the TME. Thus, in 55 this dissertation I investigated the hypothesis that IL-15 produced in the TME is an important promoter of anti-tumor responses by cytotoxic lymphocytes enhancing their optimal numbers, effector functions and metabolic fitness. To test this hypothesis, I pursued the following specific aims:

Aim 1. Determine the importance of IL-15 expressed within the tumor for anti-tumor responses by CD8 T cells and NK cells in melanoma and colon carcinoma tumors.

Aim 2. Investigate the mechanisms by which IL-15 regulates TIL numbers and induces anti-tumor responses.

Aim 3. Examine if upregulation of IL-15 in tumors, by local IL-15 therapy, promotes anti-tumor responses in poorly immunogenic melanoma tumors.

To test these aims I used several mouse tumor models including B16 melanomas and MC38 colon carcinomas. These tumor models were chosen because B16 melanomas are poorly infiltrated by T cells (357) and can represent the subset of non-T cell inflamed tumors, and MC38 tumors are more infiltrated and are more similar to T-cell inflamed tumors (358, 359). This classification of tumors as T-cell inflamed or non-T cell inflamed tumors has been used to describe subsets of tumors found in patients, especially in melanoma, that differ in their responses to immune checkpoint blockade and have distinct gene signatures (360). T-cell inflamed tumors have a gene signature indicative of T cell infiltration which includes expression of chemokines such as CCL2, CCL3, CCL4, CCL5 and CXCL9 &10 and IFN-I production (9, 361), while non-T cell inflamed tumors lack this signature. However immunosuppressive cells such as TAMs, TECs, CAFs are still present in these non-T cell inflamed tumors (19, 361-364). Importantly, non-T cell inflamed tumors are thought to evade or fail to sustain spontaneous anti-tumor responses likely due to the

56 loss of Batf3+ DCs and through activation of tumor intrinsic oncogenic pathways, such as the Wnt/-catenin pathway leading to exclusion of T cells from the TME and failure to respond to immunotherapies that boost T cell cytotoxic activities (53, 365). Consequently, exploring therapeutic avenues to promote spontaneous T cell priming and recruitment of these cytotoxic lymphocytes, by boosting the recognition of tumors by innate immune cells in the TME, should be the focus to circumvent the resistance mechanisms to immunotherapy employed by non-T cell inflamed tumors.

CHAPTER 2: METHODOLOGY

Mice

C57Bl/6 (WT) and CD45.1+ C57Bl/6 mice were purchased from NCI/Charles River

(Frederick, MD). All transgenic and gene-deficient mice used are on the C57Bl/6 background. IL-15Rfl/fl (251), CD11cCre (366), LysM-Cre (367), and Tmem173-/- mice

(368) were purchased from Jackson Laboratories (Bar Harbor, MN). IL-15R-/- knockout

(RKO) mice (220) were originally generated and obtained by Dr. Averil Ma through Leo

Lefrancois and backcrossed to the C57Bl/6 line. CCR2-DTR Tg mice (369) were generated by and provided by Dr. Eric G. Pamer (Memorial Sloan Kettering, New York,

NY). IFNAR1-/- mice were provided by Dr. Paul W. Dempsey (Department Of Microbiology and Molecular Genetics, University of California, Los Angeles and Dr. Tadatsugu

Taniguchi, Department of Immunology, Tokyo University, Japan) to W. Overwijk (Nektar

Therapeutics) and crossed to the C57Bl/6 background (370). IL-15 transcriptional reporter mice were generated by Dr. Leo Lefrancois (Department of Immunology, University of

Connecticut, Farmington, CT) (280). IL-15 translational reporter mice (IL-15 TE) were provided by Drs. Pippa Marrack and Ross Kedl (Integrated Department of Immunology,

University of Colorado, Denver, CO) (279). CCR2-RFP reporter mice (371) were originally obtained from Jackson Laboratories through Dr. Tomasz Zal (Department of Immunology,

UTMDACC) and bred to the IL-15 transcriptional reporter mice to generate GFP+/RFP+ reporter mice. All mice described were maintained under specific pathogen-free conditions at the institutional animal facility. The animal facility is fully accredited by the Association of

Assessment and Accreditation of Laboratory Animal Care International. All animal procedures were conducted on mice between 6-10 weeks of age, in accordance with the

58 animal care and use protocols (100409934) approved by the Institutional Animal Care and

Use Committee at the UT MD Anderson Cancer Center.

To generate BM chimeras, BM was collected from the tibia and femurs of IL-15R-/-

(CD45.2) and WT (CD45.2) mice and depleted of T cells as previously described (247) .

WT (CD45.1) recipients were irradiated with 1,000 RADs and injected i.v. with 5 x106 BM cells. BM reconstitution was confirmed 8-12 weeks later by analysis of BM derived cells

(CD45.2+) in the peripheral blood prior to tumor implantation.

Tumor implantation, treatment, and monitoring

B16-F10 melanoma cells (B16), B16-F10 cells expressing OVA (B16-OVA) and

MC-38 murine colon adenocarcinoma tumor cell lines were obtained from W. Overwijk and maintained in RPMI culture medium containing 10% FBS, 1% Hepes, 1% L-glutamine and

1% Penicillin/Streptomycin (P/S). MCA-205 fibrosarcoma were obtained from Dr. Tomasz

Zal, and maintained in IMDM culture medium containing 5% FBS, 1% P/S and 50μM 2-

ME. After trypsinizing and washing, 300,000 cells were injected s.c. into the flank of the indicated mice. Tumor growth was measured every other day using a caliper and tumor surface area (mm2) was calculated as length X width. Mice were sacrificed at various times post-implantation or when tumors reached 200mm2. For analysis of naive tumor- specific T cell responses, CD45.1+ OT-I TCR transgenic CD8 T cells (RAG-/-) were isolated from LNs and spleen, labeled with 2mM CFSE, and adoptively transferred to

CD45.2+ WT recipients.

Analysis of Cytokine Expression and Lymphocytes

For analysis of sIL-15 complexes, spleens and tumors were weighed prior to being homogenized in a constant volume of PBS and pelleted by centrifugation. Supernatants were collected and analyzed for levels of sIL-15 complexes using an ELISA specific for murine sIL-15 complexes (eBioscience, San Diego, CA) according to manufacturer’s recommendations. The amount of sIL-15 complexes present in the respective tissue was normalized to tissue weight and expressed as the amount of sIL-15 complexes/g of tissue.

For analysis of immune cells and IL-15 reporter expression in tumors, tumors were isolated, digested in RPMI media, 5% FCS and 100 U/ml collagenase, 1mM CaCl2 and

1mM MgCl2 for 30 min at 37°C with stirring and then subjected to a 44-67% Percoll centrifuge gradient. Cells in the interphase were harvested, washed, and then stained for flow cytometric analysis. Spleens were homogenized in HBSS containing HEPES, L- glutamine, gentamicin and pen-strep using frosted slides. RBC were lysed with Tris- ammonium chloride. All cells were filtered through a 70m nitex prior to staining. For flow cytometric analysis, cells were stained in 1x PBS containing 0.2% BSA and 0.1% NaN3 with appropriately diluted Ab at 4°C for at least 20 min. Ki-67 and Granzyme B staining were conducted after staining cell surface molecules and permeabilization using the

FoxP3/ Transcription Factor Staining Buffer Set according to manufacturer’s instructions

(eBioscience). For IFN- staining, isolated lymphocytes were stimulated in the presence of plate-bound CD3Ab for 5 hours in the presence of Golgi Plug (BD Bioscience). IFN- staining was conducted after staining for cell surface molecules and permeabilization using

Cytofix/Cytoperm buffer according to manufacturer’s instructions (BD Bioscience). The following mAbs were purchased from BD Biosciences (San Jose, CA), eBiosciences, or

BioLegend: CD45, CD45.1, CD45.2, CD19, CD3, TCRβ, CD11b, CD11c, Ly6G, Ly6C,

F4/80, CD8, NK1.1, CD44, Ki-67, IFN-, and Granzyme B. Lineage+ cells were identified as CD19+, TCRβ+, or NK1.1+. Rat IgG2a-APC (Biolegend) was used as an isotype control for F4/80-APC, while Rat IgG1-PE (BD Biosciences) was used as an isotype control for

IFN--PE and Granzyme B-PE. Flow cytometric data were acquired with a LSRII (BD

Biosciences) or LSR Fortessa (BD Biosciences) and analyzed with Flowjo software version

9.7.6 and 10 (Flowjo LLC, Ashland).

Cell depletions, STING agonist treatments, and IL-15 neutralization

To deplete mice of Ly6G+ cells, mice were treated i.p. with Ly6G mAb (clone 1A8,

400 μg, BioXcell) or Rat IgG (Jackson ImmunoResearch Laboratories) when tumors became palpable (day 8-9) and 3 days later with Ly6G mAb (100 μg i.p and 50 μg i.t.). To deplete CCR2+ cells, CCR2-DTR Tg and WT mice were treated with 250 ng of Diphtheria

Toxin (Sigma) every two days starting 4 days post tumor implantation. Efficiency in depletion of Ly6G+ cells and Ly6C+ monocytes in the respective models were confirmed by flow cytometry. Levels of sIL-15 complexes in B16 tumors and spleens were analyzed

2-3 days later by ELISA as described. For stimulation of the STING pathway, mice were administered i.t. c-di-GMP or 2’3’-c-GAMP (Invivogen, San Diego, CA) at the indicated doses. Neutralizing IL-15 mAb (clone M96) (372) was obtained from Amgen (Thousand

Oaks, CA). For systemic neutralization of IL-15, mice received one treatment (50g, i.p.) of IL-15 mAb at indicated time after tumor implantation. Mouse IgG2a (Jackson

ImmunoResearch Laboratories) was used as the isotype control. For local neutralization of IL-15, IL-15 Ab was delivered intratumorally at indicated doses when tumors became palpable. Efficient neutralization of IL-15 with antibody was confirmed by the absence of

NK cells.

Metabolic assays, Annexin V and BrdU staining

Tumor homogenates were pulsed with 20 µM 2-NBDG (Cayman Chemical) in 10%

FBS-containing media for 1hr at 37°C. Cells were surface stained and analyzed by flow cytometry. For Annexin V staining, tumors were dissociated and stained for 30 minutes at room temperature using the Annexin V staining kit (Invitrogen), according to manufacturer’s recommendations. For BrdU accumulation and flow cytometry analysis tumor bearing mice received i.p. injections of BrdU (2 mg; Sigma Aldrich, St. Louis, MO) every 2 days for 7 days. Tissues were harvested and cell surface stained in 1X PBS containing 0.2% BSA and 0.1% NaN3 at 4˚C for at least 20 min. Staining for BrdU was performed using BrdU Flow kit according to manufacturer’s instructions (BD Biosciences

Pharmingen, San Jose, CA). In brief, cells were fixed and permeabilized using BD cytofix/cytoperm buffer, followed by DNase (30 µg) treatment for 1 hour at 37˚C. Cells were then stained with anti-BrdU FITC for 20 min at room temperature.

Immune checkpoint inhibition and IL-15 agonists treatment

Anti-CTLA-4 mAb (clone 9H10, from BioXcell) was kindly provided by James Allison and Michael Curran (MD Anderson Cancer Center, Houston TX). It was administered to tumor-bearing mice i.p. at an initial dose of 200g/mouse on day 5 post-tumor implantation and then at a dose of 100g/mouse every three days for a total of 5 treatments. For intratumoral treatment with rsIL-15c, 50ng of recombinant murine IL-15 (PreproTech) were combined with 300ng of recombinant IL-15R-Fc (R& D Systems) in PBS and incubated at

37°C for 10 minutes. Mice received intratumoral injections on days 8, 11 and 14. A polymer-conjugated recombinant IL-15 (NKTR-255) was provided by Nektar Therapeutics

(San Francisco, CA). Mice received intratumoral injections of 60 ng of NKTR-255 on

62 indicated days after tumor implantation and treatments were repeated every other day until tumor regressed or reached 200 mm2.

Statistical Analysis

Statistical differences were determined by a two-tailed Students t test, one-way ANOVA or

Log-rank Test. *, **, ***, **** indicate p<0.05, p<01, p<0.001, and p<0.0001, respectively.

Analyses were performed using GraphPad Prism, version 8 (GraphPad Software, San

Diego, CA) or Microsoft Excel 2016 (Redmond, WA).

CHAPTER 3: IL-15 IS AN IMPORTANT INFLAMMATORY SIGNAL IN THE TME THAT

ENHANCES ANTI-TUMOR RESPONSES

This work is based upon “IL-15 is a component of the inflammatory milieu in the tumor microenvironment promoting anti-tumor responses” by Rosa M. Santana Carrero,

Figen Beceren-Braun, Sarai C. Rivas, Shweta M. Hegde, Achintyan Gangadharan, Devin

Plote, Gabriel Pham, Scott M. Anthony and Kimberly S. Schluns. 2019. Proceedings of the

Natural Academy of Sciences USA (PNAS); presented with permission from PNAS.

3.1: Introduction

Patients vary considerably in their responses to cancer therapies. The presence of tumor-infiltrating T cells strongly correlates with positive clinical outcomes in melanoma, colon, breast, cervical, and brain cancers (82-85). Specifically, the density, depth, and functional attributes of cytolytic CD8 T cells are associated with the greatest clinical outcomes (82). Altogether, these features are a better prognostic indicator than the tumor-node metastasis classification that is currently used. IL-15 is a cytokine that preferentially stimulates CD8 T cell and NK cell activation, proliferation, and cytolytic activity. Not surprisingly, these functional activities of IL-15 translate to enhanced anti- tumor responses in multiple tumor models (289, 341, 373). As such, systemic treatment with IL-15 or IL-15 analogs are currently being evaluated as potential cancer therapeutics. In addition to the ability of IL-15 to act systemically to promote anti-tumor responses, there is evidence that IL-15 expression within the tumor microenvironment

(TME) is crucial for optimal anti-tumor responses (346, 374). Jerome Galon’s group showed that loss of IL-15 expression within colorectal tumors correlated with lower T cell density, decreased T cell proliferation, higher risk of relapse, and decreased

64 survival (346). While these studies suggest IL-15 produced within the TME is important for effective anti-tumor responses by CD8 T cells, the mechanisms regulating IL-15 within tumors are unknown.

As little was known of the significance of locally produced IL-15 in tumors in adaptive anti-tumor immune responses this study focused on investigating if and how

IL-15 expression was regulated by factors such as other inflammatory mediators and increased tumor growth and development. Several models of IL-15 expression were evaluated to investigate specific cellular sources of IL-15 since IL-15 protein expression had been difficult to quantify due to the stringent regulation of IL-15 production at the transcriptional, translational and post-translational levels. Furthermore, while transpresentation of IL-15 is known to mediate IL-15 effects during homeostatic conditions (235, 246, 249), transient increases in IL-15 levels can be observed by the generation of sIL-15 complexes in response to many types of innate immune cell stimulation (238, 272). Moreover, as therapeutic sIL-15 complexes are 50-100X times more potent than rhIL-15 in promoting CD8 T cell and NK cell proliferation in vivo, it was important to examine how the production of these complexes in the TME could affect TIL responses.

In this chapter I will demonstrate that IL-15 is expressed in the TME as sIL-15 complexes and regulates TIL numbers. sIL-15 complexes are abundant in early tumors but low in established tumors, even though IL-15 expressing cells are abundant in established tumors. Nonetheless, sIL-15 complexes can be upregulated by stimulating the inflammatory STING pathway. More importantly, the induction of IL-15 expression by locally delivered inflammatory signals was critical for mediating anti-tumor responses. 65

3.2 Results

3.2.1: IL-15 expression and regulation in the tumor microenvironment

Inflammatory mediators, including cytokines, are initially produced in the TME to promote anti-tumor responses, however as tumors develop this microenvironment gradually becomes immunosuppressive and hostile to immune cells (9, 375). Our group’s findings that sIL-15 complexes are produced in response to numerous forms of immune stimulation compelled us to investigate whether sIL-15 complexes are produced in the TME (199). To determine if sIL-15 complexes are produced in B16 tumors, B16 tumors (B16-F10) were established in WT (wildtype) C57Bl/6 mice, removed along with spleens, measured in size and weight (tumor weight between 20-

150mg), dissociated, and suspended in PBS. Supernatants generated after pelleting cells were analyzed for sIL-15 complexes using ELISA. We found that sIL-15 complexes were present at relatively high levels in small tumors and at lower levels in larger tumors (Fig. 4A). In analysis of MC-38 and MCA-205 tumors, sIL-15 complexes are also produced during early tumor growth demonstrating that production of sIL-15 complexes in tumors is not unique to B16 melanoma (Fig. 4B & 4C). Similar levels of sIL-15 complexes were found in B16-ovalbumin (OVA) tumors as B16-F10 tumors (Fig.

4D). As such, the B16-OVA line was used for in vivo experiments from here on as it allows the analysis of tumor-specific CD8 T cell responses. We also examined the levels of sIL-15 complexes in B16 tumors at different times post-implantation and found that sIL-15 complexes are higher at earlier stages of tumor growth and their levels are reduced with tumor growth (Fig. 4E). Additionally, in both B16 and MC-38 tumors, the higher levels of sIL-15 complexes occur in the earliest tumors prior to the infiltration of

CD8 T cells and decline thereafter prior to the decline in numbers of CD8 tumor- 66 infiltrating lymphocytes (TILs) that occurs with tumor growth (Fig. 4F & 4G). These findings suggest IL-15 positively regulates CD8 T cell numbers in the TME.

Figure 4. sIL-15 complexes are produced in the tumor microenvironment. A) B16-

F10 tumor cells were injected (0.3x106 cells, s.c.) into WT mice. Tumors were dissociated and supernatants were analyzed for sIL-15 complexes using ELISA.

Levels of sIL-15 complexes in B16-F10 tumors of different masses isolated at the same time post-implantation. B, C) MCA-205 or MC-38 tumors were isolated from WT mice

(MCA between 5-10 days/MC-38-between 10 and 18 days post-implantation) and

67 levels of sIL-15 complexes in tumors were analyzed by ELISA. D) Levels of sIL-15 complexes in B16-F10 and B16-OVA implanted at the same time and isolated 10 days post-tumor implantation. Average tumor weight (mg): B16-F10= 58± 4; B16-OVA=

41±17. E) Levels of sIL-15 complexes in B16-F10 tumors isolated at 10, 14, and 17 days post tumor implantation. n=5 mice/group. F, G) Levels of sIL-15 complexes (light blue bar, left axis) and total CD8 T cells/tumor (purple bars, right axis) in B16-F10 and

MC-38 tumors, tumors grouped by tumor size. Error bars represent SEM, * p<0.05,

**p<0.01 by Student’s T-test. Data for Figure 4 A, B, C and E generated by and used with the permission of Sarai C. Rivas, Figen Beceren-Braun, Scott M. Anthony and

Gabriel Pham.

Since IFN-I expressed in the TME are important for spontaneous anti-tumor responses (9) and we have previously demonstrated IFN-I are potent inducers of sIL-

15 complexes in vivo (199), we asked if IFN-I signaling is important for the production of sIL-15 complexes in the TME. In B16 tumors transplanted into IFNAR-/- mice, sIL-

15 complexes were decreased in the early tumors compared to those in WT mice (Fig.

5A) indicating IFN-I are important for production of sIL-15 complexes in the TME. Since

IFN-I can be induced by the STING pathway, we asked if the STING pathway was important for endogenous production of sIL-15 complexes. B16 tumors implanted into

TMEM173-/- mice showed similar levels of sIL-15 complexes as WT mice (Fig. 5B) suggesting this IFN-I response was not dependent on the STING pathway.

Furthermore, total numbers of CD8 T cells, CD4 T cells, and NK cells among TILs were decreased in B16 tumors implanted into IFNAR-/- mice compared to WT mice (Fig.

5C). The increase in the CD4:CD8 T cell ratio in tumors of IFNAR-/- mice reflects the more dramatic loss in CD8 T cells, which is consistent with the loss of IL-15 as IL-15 preferentially stimulates CD8 T cells over CD4 T cells (259). Overall, IL-15 is expressed as sIL-15 complexes in the TME in an IFN-I-dependent manner that also regulates the number of CD8 and NK TILs.

Figure 5. Regulation of sIL-15 complexes production in tumors and TIL numbers by IFN-I. A) B16-OVA cells were injected (0.3x106 cells, s.c.) into WT or

IFNAR-/-mice; levels of sIL-15 complexes in tissues were analyzed 10 days post- implantation; n=4-8 mice/group. Tumor masses range from 10-55 mg (WT ave=34.7 ±

17.6, KO ave=41.1±16.6). B) Levels of sIL-15 complexes in B16-OVA tumors implanted in TMEM173-/- mice 9 days tumor post-implantation. n=5-8 mice/group. Average tumor weights were WT 21± 7.1; KO=19.8±3.3. C) The numbers of CD8 T cells, CD4 T cells, and NK cells in B16-OVA tumors implanted into WT or IFNAR-/- mice were analyzed by flow cytometry and normalized to tumor weight. One representative experiment of three performed is shown (n=5 mice/group). Error bars represent SEM, * p<0.05, **p<0.01 by

Student’s T-test. Data for Figure 5 A&B were generated by and used with permission of

Sarai C. Rivas and Scott M. Anthony.

3.2.2 Cellular sources of sIL-15/ IL-15R complexes

To address the extent to which sIL-15 complexes were derived from the tumor stroma, levels of sIL-15 complexes were measured in tumors implanted into WT and IL-

15R-/- mice. sIL-15 complexes were not detected in B16 tumors implanted into IL-

15R-/- mice (Fig. 6A) indicating the sIL-15 complexes were derived from the tumor stroma and not the tumor cells. However, when either MC-38 or MCA-205 tumor cells were implanted in IL-15R-/- mice, sIL-15 complexes in the interstitial fluid of these tumors were still abundant, suggesting the tumor cells themselves were producing sIL-

15 complexes (Fig. 6B). This was confirmed in analysis of culture supernatants obtained from these tumor cell lines (Fig. 6C). Tumor cell lines, such as BP-1

Melanoma cells, MB49 bladder carcinoma, and 4T1 mammary tumor cells also produced sIL-15 complexes while T3M4 pancreatic cancer, A20 Lymphoma, and CT26 colon carcinomas cell lines did not produce detectable levels of sIL-15 complexes (Fig.

6D & E). Thus, the ability of tumor cell lines to produce sIL-15 complexes is variable but is not necessarily dictated by the tissue of origin. Therefore, we have identified different scenarios of sIL-15 complex production: one where sIL-15 complexes are exclusively derived from the TME (i.e. B16) and the other where sIL-15 complexes can come from both the tumor and the tumor stroma (i.e. MCA-205, MC-38).

Figure 6. Stromal and tumor cells are sources of sIL-15 complexes in the tumor microenvironment. Tumor cells were injected (0.3x106 cells, s.c.) into WT or IL-15Rα-

/- mice. Tumors and spleens were dissociated and supernatants were analyzed for sIL-

15 complexes using ELISA. A) Levels of sIL-15 complexes in B16-OVA tumors implanted in WT and IL-15Rα-/- mice; n=2-3 mice/group. Error bars represent SD. B)

Levels of sIL-15 complexes in MCA-205 tumors (day 9-11) and MC-38 tumors (day between 9-14) isolated from WT and IL-15Rα-/- mice. Average tumor mass of MCA-

205 tumors were WT =21.6 ±17, Rko =24.8±6; average tumor mass of MC-38 tumors

WT =32.8±8.3, Rko =28.4±9.7. C) Levels of sIL-15 complexes present in tumor cell culture supernatants. D) The following tumor cell lines: BP-1 Melanoma cells, T3M4 pancreatic cancer, A20 Lymphoma, CT26 colon carcinomas cell lines, MB49 bladder carcinoma and 4T1 mammary tumor cells were grown to almost confluent levels in complete media and culture supernatants were collected. Culture supernatants and the respective complete media were analyzed for sIL-15/IL-15R complexes by ELISA (3 72 wells/cell line), *p<0.05. E) In a different experiment, levels of sIL-15 complexes in tumor culture supernatants were measured and normalized to the number of cells recovered. Error bars represent SEM. * p<0.05, with Student’s T-test. Data for this figure were generated by and used in this dissertation with permission from Figen

Beceren-Braun, Sarai C. Rivas, Shweta M. Hegde and Achintyan Gangadharan.

Since we demonstrated that sIL-15 complexes present in the B16 tumors are derived exclusively from the tumor stroma, we chose to use the B16 model to further investigate the non-tumor derived sources of sIL-15 complexes in the TME. We utilized various IL-15R conditional knockout mouse models: IL-15R floxed mice (IL-15Rfl/fl) crossed to CD11c-Cre Tg mice or LysM-Cre Tg mice to delete IL-15R primarily in DCs and phagocytic cells (macrophage and neutrophils), respectively as previously described (251). Loss of IL-15 expression from either DCs (Fig. 7A) or phagocytic cells

(Fig. 7B) led to a significant reduction in the levels of sIL-15 complexes in B16 tumors, suggesting both cell types are contributing to baseline sIL-15 complex levels in the

TME. To examine the contribution of monocytes, B16 cells were implanted into CCR2-

DTR Tg+ and Tg- littermates and treated with diphtheria toxin (DT) (200 ng, i.p. every 2 days) to deplete CCR2+ myeloid cells. Treatment with DT consistently decreased the levels of sIL-15 complexes in B16 tumors implanted into DTR-Tg+ mice compared to the tumors in DT-treated Tg- mice (Fig. 7C); however, these differences did not reach statistical significance (p<0.1). To examine the specific contribution from tumor associated neutrophils or granulocytic myeloid-derived suppressive cells (MDSC), sIL-

15 complexes were analyzed in tumors from mice treated with Ly6G depleting Ab. This treatment had no effect on levels of sIL-15 complexes suggesting neutrophils/MDSCs are not a significant source of sIL-15 complexes in the TME (Fig. 7D). In no model examined, were sIL-15 complexes reduced by more than 50% indicating that there are multiple myeloid sources of sIL-15 complexes in the TME, including CD11c+, LysM+ phagocytic cells, and monocytic cells.

Figure 7. Multiple myeloid cell subsets and non-hematopoietic cells generate sIL-

15 complexes in the stromal compartment of the TME. A,B) Levels of sIL-15 complexes in spleens and B16-OVA tumors isolated from control IL-15Rfl/fl (blue),

CD11c-Cre+/+ x IL-15Rfl/fl (red), and LysM-Cre+/+ x IL-15Rfl/fl (red) mice 10 days tumor post-implantation (n=3-5 mice/group), one representative experiment of three is shown. C) B16-OVA tumors were implanted in CCR2-DTR-Tg- (blue) and CCR2-DTR-

Tg+ (red) mice. Beginning 7 days post-implantation, mice were treated i.p. with either

PBS or DT every two days. Tumors and spleens were isolated 12-13 days post- implantation. Tumors ranging between 50-100 mg were analyzed and average tumor mass was not significantly different between groups (n=3 tumors/group, n=4-6 spleens/group). D) B16-OVA tumors were implanted in WT mice. When tumors became palpable (day 8-9), mice were treated either with Ly6G Ab (clone 1A8, 400

μg, i.p.) or Rat IgG and 3 days later with Ly6G Ab (100 μg, i.p. plus 50 μg i.t.). Levels of sIL-15 complexes in B16 tumors and spleens were analyzed 2-3 days later. n=5 mice/group. E) Levels of sIL-15 complexes in B16-OVA tumors and spleens isolated from IL-15R-/- BM chimeras (IL-15R-/- BM→ WT recipients) and WT control BM chimeras (WT BM→ WT recipients) 2 weeks after implantation, n=4-5 mice/group.

Error bars represent SEM. * p<0.05 with Student’s T-test. Data for this figure were generated by and used in this dissertation with permission from Figen Beceren-Braun,

Sarai C. Rivas and Devin Plote.

We also examined the contribution of non-hematopoietic cells as sources of sIL-

15 complexes using bone marrow (BM) chimeras. IL-15R-/- BM chimeras (IL-15R-/-

BM→ WT recipients) and WT control BM chimeras (WT BM→ WT recipients) were generated and reconstitution of the hematopoietic compartment was confirmed ~12 weeks later followed by s.c. implantation of B16 tumor cells. Tumors and spleens were analyzed for sIL-15 complexes two weeks later. B16 tumors isolated from IL-15R-/-

BM chimeras expressed lower levels of sIL-15 complexes than WT BM chimeras

(p<0.1) (Fig. 7E) indicating non-hematopoietic cells may be an additional source of sIL-

15 complexes in the TME. Overall, our analyses demonstrate that there are multiple sources of sIL-15 complexes in the TME including multiple myeloid cells, non- hematopoietic cells, and in some instances the tumor cells themselves.

3.2.3 The tumor microenvironment is abundant in IL-15-expressing myeloid cells, composed predominately of CD11b+Ly6ChiLy6G- cells.

Our next objective was to more specifically identify the cells expressing IL-15 within the TME and determine how IL-15 expression in the TME differs from that in the spleen. To address this, B16 tumor cells were implanted into WT, IL-15 transcriptional reporter, or IL-15 translational reporter mice, allowed to grow, and tissues were isolated for flow cytometric analysis of GFP+ cells. Between 12-18 days post-implantation, GFP expression was compared in dissociated tumors and splenocytes. Among splenocytes, the majority of TCRαβ CD19 NK1.1 (lineage)- cells are GFP+ (~75%) and consist of

CD11chi DCs, neutrophils (CD11b+Ly6G+), monocytes (CD11b+Ly6Chi), and macrophages (CD11b+Ly6C-/loLy6G-), similar to that described in previous studies 77

(376) (Fig. 8A). Similar to spleen, most myeloid cells in the tumor are GFP+(80-90%); however, the composition of the GFP+ cells was different than that in the spleen. In

B16 tumors, a larger portion of CD45+lineage- cells were CD11bhi CD11clo/int than in the spleen (Fig. 8A). Additionally, while GFP+ CD11chi cells and other CD11blo cells are found in the spleen, the proportion of CD11chi cells among GFP+ populations in the tumor are low in comparison to that observed in the spleen (Fig. 8A). Among the

GFP+CD11b+ cells in B16 tumors, there are three main populations: Ly6ChiLy6G-

(monocytic), Ly6C+Ly6G+ (neutrophils or granulocytic MDSC), and Ly6C-/loLy6G- cells

(Fig. 8A). Unlike the spleen, the GFP+CD11b+ cells in B16 tumors were composed predominantly of Ly6ChiLy6G- cells while Ly6C+Ly6G+ cells were minimally represented (Fig. 8A). Similar analyses were also conducted with the MC-38 colon carcinoma cell line and MCA-205 fibrosarcoma implanted into IL-15 reporter mice. In general, the composition of GFP+ cells in MCA-205 and MC-38 tumors was similar to that observed in B16 tumors, except that MC-38 tumors harbored a higher percentage of CD11chiGFP+ cells and slightly less CD11b+Ly6C-/loLy6G- cells (Fig. 8B & C).

MCA-205 tumors harbored slightly more CD11b+Ly6C-/loLy6G- cells among GFP+ cells compared to B16 and MC-38 tumors but this was not statistical significant (Fig.

8B). The composition of GFP+ cells between the two IL-15 reporter mouse lines was equivalent (Fig. 8D). Overall, across multiple implantable tumor cell lines, tumor myeloid cells expressing IL-15, composed predominantly of CD11b+Ly6ChiLy6G- cells, are abundant in established tumors.

Figure 8. CD11b+Ly6Chi Ly6G- cells are the major subset expressing IL-15 in

TME. (A) Flow cytometric analysis of IL-15-GFP expressing myeloid subsets in spleens and B16-OVA tumors (200-250 mg) isolated from WT (left panel) and IL-15 translational-GFP reporter (right panel) mice 14 days post-implantation. Spleens and tumors from WT mice were used to set the gate for GFP+ cells. Expression of CD45+

79 and Lineage (TCR-β, CD19, NK1.1) negative cells were used to discriminate myeloid cell populations. Myeloid cells were then gated on GFP+ cells and the composition of

GFP+ cells was examined by CD11c, CD11b, Ly6G and Ly6C expression.

Representative data from 1 of 6 independent experiments are shown. (B) Composition of IL-15-GFP expressing cells among CD45+lineage- cells present in B16-OVA, MCA-

205 and MC-38 tumors isolated from IL-15 reporter mice. Tumors analyzed ranged from 35-100mm2. Bars show mean ± SD from n = 7-10 mice per group. * represents a significant difference in frequency of CD11chi cells compared to B16-OVA and MCA-

205 tumors. C) Flow cytometric plots showing cells in spleens and MCA and MC-38 tumors isolated at the same time from IL-15 translational reporter mice after gating on

CD45+, lineage (TCR-β, CD19, NK1.1) negative, and GFP+ cells. Ly6C and Ly6G expression was examined in GFP+ CD11b+cells. D) GFP/IL-15 expression in IL-15 translational and transcriptional reporter mice. Flow cytometric plots showing composition of GFP+ myeloid subsets in similar size MCA tumors isolated from IL-15 translational and transcriptional reporter mice. Data for this figure were generated by and used in this dissertation with permission from Figen Beceren-Braun.

To further define these myeloid subsets, the expression of CCR2, which is associated with inflammatory monocytes (377), was examined in myeloid cells in B16 tumors implanted into translational IL-15-GFP/CCR2-RFP double reporter mice. Among the GFP+CD11b+ cells in the tumors, the Ly6Chi Ly6G- cells expressed high levels of

CCR2 reporter, the Ly6C-/loLy6G-cells were predominantly CCR2+, while the

Ly6C+Ly6G+ cells were uniformly CCR2- (Fig. 9A). In addition, we examined F4/80 expression by the GFP+CD11b+ populations in tumors and found that the Ly6C-

/loLy6G- cells expressed higher levels of F4/80 than the other CD11b+ subsets implicating these cells as part of the macrophage lineage (Fig. 9B & C). Unlike in the spleen where macrophages are F4/80hi CD11bint cells, an analogous population was not observed in the TME but instead, an F4/80loCD11bhi population was observed

(Fig. 9D).

Figure 9. IL-15 reporter expression is increased in tumor myeloid cells over similar myeloid populations in the spleen. A) CCR2 reporter expression in indicated cells isolated from B16-OVA tumors implanted into either CCR2-RFP+/IL-15 transcriptional GFP+ reporter or CCR2-RFP-/IL-15- transcriptional GFP+ reporter. (B)

F4/80 expression on indicated populations after gating on GFP+CD11b+ cells in B16 tumors isolated from IL-15-transcriptional GFP reporter mice. C&D) Tumor cells were implanted s.c. into IL-15 reporter mice. Tumors and spleens were isolated and stained

82 as described. C) Panels show tumor and spleen cells stained with F4/80-APC and Rat

IgG2a-APC after gating on CD45+ lineage- cells. D) Histograms show staining of

F4/80-APC and Rat IgG2a-APC after gating on CD45+lineageneg, GFP+ CD11b+ cells in spleens and B16 tumors implanted into IL-15 translational reporter mice. (E) The relative IL-15-GFP reporter expression levels among analogous cells in spleen and tumors. GFP expression by the specific CD45+lineage- CD11b+ cell populations was calculated by dividing the MFI of the indicated population isolated from the tumor over the MFI of the analogous population from the spleen of the same mouse. n=7-10 mice per group, error bars represent SEM. * p<0.05, **p<0.01, ***p<0.001 with One-way

ANOVA. Data for this figure were generated by and used in this dissertation with permission from Figen Beceren-Braun.

To address whether the IL-15 expression is different in specific myeloid cells in tumors compared to the spleen, we gated on specific myeloid populations and compared the GFP expression between the spleen and the tumor within the same mice. In the B16 tumors, GFP expression was increased in the CD11b+Ly6ChiLy6G- compared to the spleen (Fig. 9E). GFP expression by CD11chi cells,

CD11b+Ly6ChiLy6G- and Ly6C-/loLy6G- cells was also increased in MCA-205 tumors compared to spleen but decreased in the Ly6C+Ly6G+ population (Fig. 9E). In contrast, in MC-38 tumors, only the CD11chi cells showed increased GFP expression relative to that in the spleen (Fig. 9E). Overall, these findings indicate that IL-15 expression is altered among myeloid cells within the TME depending on the myeloid cell expressing IL-15 and the tumor type.

Since IL-15R is required for expression of cell surface IL-15 complexes and soluble IL-15 complexes (235, 240), cell surface IL-15R expression by tumor myeloid cells was examined. All myeloid cell subsets expressed surface IL-15R with the

CD11b+Ly6Chi cells expressing the highest levels (Fig. 10A). In analysis of IL-15R at different stages of tumor growth, levels of IL-15R by the three major myeloid subsets did not significantly change between day 9 and day 14 tumors (Fig. 10B). These data suggest that tumor myeloid cells are capable of both transpresenting IL-15 and producing sIL-15 complexes.

Figure 10. IL-15R expression by tumor myeloid cells is stable in early and established tumors. B16 tumor cells were implanted s.c. into WT and IL-15R-/-

(Rko) mice 9 and 14 days prior to analysis to allow simultaneous analysis of IL-15R of two time points. Tumors were isolated, incubated with BD mouse Fc block, and stained for IL-15R using anti-mouse IL-15R-biotin (R&D Systems) followed by Streptavidin-

APC (Jackson ImmunoResearch). A) Histograms show representative staining for IL-

15R after gating on CD45+ lineageneg CD11b+ cells isolated from tumors in WT

(grey filled) and IL-15R-/- (open histogram) mice. B) Graph shows average MFI of IL-

15R staining from indicated tumor myeloid cells from IL-15R-/- mice (white bars) and from WT mice 9 days (light grey bars) and 14 days (dark grey bars) post-tumor implantation. N=3-5 mice/group. Data for this figure were generated by and used in this dissertation with permission from Figen Beceren-Braun.

3.2.4 Local IL-15 in tumors regulates TIL numbers

To directly examine the role of IL-15 in regulating TIL numbers, established palpable B16 tumors in WT mice were treated intratumorally with neutralizing IL-15 Ab or control Ig. Blocking IL-15 activity in the tumor led to a significant decrease in the total number of CD8 T cells and NK cells in the tumors (p<0.001) but did not significantly affect the total numbers of CD4 T cells (Fig. 11A). Decreases in TIL numbers were also observed with IL-15 blockade of MC-38 tumors (Fig. 11B). To determine if endogenous

IL-15 regulates proliferation of TILs, Ki-67 expression was examined in TILs in B16 tumors of mice treated with neutralizing IL-15 Ab. Ki-67 expression in CD8, CD4, or

NK1.1+ TILs was similar with IL-15 neutralization and control Ig treatment (Fig. 11C) suggesting IL-15 regulates TIL numbers independent of proliferation, possibly by promoting infiltration. TILs were also examined phenotypically and found to have similar levels of Tim-3+ and PD-1+Tim3+ among both groups (Fig. 11D). Surprisingly,

IL-15 blockade did not affect B16 or MC-38 tumor growth (Fig. 11E & F) suggesting the amount of IL-15 present in the TME is sufficient to regulate TILs but is not sufficient to break tolerance against the tumor.

Figure 11. IL-15 neutralization in TME leads to decreased TIL numbers. A) B16-

OVA tumors implanted into WT mice were treated intratumorally with IL-15Ab (50 g) or Rat IgG 9 days after tumor implantation. Tumor lymphocytes were analyzed three days later by flow cytometry and total cell numbers were normalized to tumor weight. B)

MC-38 tumors implanted into WT mice were treated intratumorally with IL-15Ab (50

g) 8 days post implantation. One week later, tumor lymphocytes were analyzed by flow cytometry and total cell numbers were normalized to tumor weight. C) Frequency of Ki67+ cells among indicated TILs isolated from B16-OVA tumors treated with IL-15

Ab or IgG. D) Frequency of TIM3+ and TIM3+PD-1+ cells among CD8 TILs isolated from B16-OVA tumors treated with IL-15 Ab or IgG. E, F) Tumor growth of B16-OVA and MC-38 tumors in WT mice treated with intratumoral IL-15 Ab or PBS 8 days post

B16 tumor implantation or 14 and 22 days post-MC-38 tumor implantation. n= 5 mice/group. Error bars represent SEM, *p<0.05, **p<0.01 by Student’s T-test.

As local IL-15 blockade regulated the numbers of CD8 TILs in tumors (Fig. 11A

&B) we next investigated several mechanisms by which IL-15 could influence the total numbers of CD8 TILs these in tumors. The more studied and established roles for IL-15 during steady-state conditions involve its functions in maintaining homeostatic levels of proliferation of CD8 T cells and serving as a survival signal for CD8 T cells during the contraction phase of an immune response (226, 378-380). In terms of enhancing proliferation we showed that Ki67 expression by CD4, CD8 and NK TILs was not affected by IL-15 neutralization in tumors (Fig. 11C). However, Ki67 expression by cells only represents the proliferation during a very short time period, thus we measured

BrdU incorporation as an indicator of proliferation over a longer time period. To this extent, mice bearing established B16-OVA tumors received i.p. injections of BrdU and tumors were treated with i.t. IL-15 ab and harvested for flow cytometry analysis.

Similar to the results for Ki67 expression, IL-15 blockade in tumors did not significantly impact the levels of BrdU incorporation by CD4 and CD8 TILs in tumors (Fig. 12A).

These results point to other mechanisms involved in the IL-15 regulation of TIL numbers in tumors.

Figure 12. IL-15 blockade does not significantly affect TIL proliferation, survival and glucose metabolism in melanoma tumors. A) B16-OVA tumors were implanted into WT mice and treated with 50g of IL-15 Ab i.t. or Rat IgG on day 7 post-implantation. Mice received i.p. injection of BrdU on days 4, 6, 8 and 10 after tumor implantation. On day 13 post-implantation tumors were harvested for flow cytometry analysis. Left panels- Flow cytometry plots of BrdU+ CD4 and CD8 TILs.

Right panels- frequency of BrdU+ cells among CD4 and CD8 TILs. B) B16-OVA tumors were implanted into WT mice and treated with 20g of IL-15 Ab i.t. or Rat IgG on day

8 post-implantation. On day 11 post-implantation tumors were harvested for flow cytometry analysis. Flow cytometry plots show frequency of Annexin V+ cells among

CD4 and CD8 TILs. C) B16-OVA tumors were implanted into WT mice and treated with

20g of IL-15 Ab i.t. or Rat IgG on day 12 post-implantation. On day 14 post- 90 implantation tumors were harvested and incubated with 20uM solution of 2-NBDG, a fluorescent glucose analog. Graph represents average 2-NBDG fluorescence in CD4,

CD8 and NK TILs. D) B16-OVA tumors were implanted into WT mice and treated with

20g of IL-15 Ab i.t. or Rat IgG on day 10 post-implantation. On day 12 post- implantation tumors were harvested for flow cytometry analysis. Graphs show average frequency of IFN- + cells among CD8 TILs. n=5 mice/group. Error bars indicate SEM.

The next mechanism we investigated was changes in survival. To examine if IL-

15 influences survival of TILs we treated established B16-OVA tumors with i.t -IL-15 Ab and analyzed of expression of apoptotic marker Annexin V in CD4 and CD8 TILs.

Annexin V expression was similar in TILs from IL-15 depleted tumors and control treated tumors (Fig. 12B). While IL-15 depletion from the TME did not induce changes in apoptotic markers, IL-15 has been shown to promote survival of CD8 T cell and NK cells by inducing the expression of anti-apoptotic proteins including BCL-2, BCL-XL, Mcl-1 and downregulating the expression of pro-apoptotic proteins including Bim (225-227,

381, 382). Hence, it would be important to look at the expression of these anti-apoptotic and pro-apoptotic molecules in more detail in future experiments.

Other aspects that could affect the survival of TILs could be decreased metabolic functions. Recently, studies focusing on T cell metabolism have shed light on the metabolic reprogramming that occurs during normal T cell development and under inflammatory conditions (383, 384). During homeostatic conditions naïve T cells rely mostly on oxidative phosphorylation (OXPHOS) and catabolic metabolic pathways, like fatty acid oxidation (FAO) (385). Once these cells become activated they must undergo a metabolic switch, using the glycolytic pathway to proliferate and exert their effector functions (386). Memory T cells, on the other hand, rely on the use of FAO and OXPHOS during homeostatic proliferation (387). Although IL-15 has clearly been shown to enhance proliferation, survival and cytotoxicity of effector CD8 T cells, very few studies have investigated the ability of IL-15 to promote their metabolic functions (388). These studies showed that in vitro stimulation of CD8 T cells with IL-15 promotes mitochondrial biogenesis to generate memory-like CD8 T cells that have increased spare respiratory capacity (388). To my knowledge, to date no in vivo studies had been performed to define

92 if IL-15 also induces these changes on CD8 T cells in the immunosuppressive TME.

Therefore, we investigated if IL-15 neutralization in the TME could affect glucose metabolism in TILs from B16-OVA tumors treated with an IL-15 blocking antibody. No significant difference in uptake of a fluorescent glucose analog, 2-NBDG, was detected in CD4, CD8 or NK TILs from tumors depleted of IL-15 and control treated tumors (Fig.

12C). Lastly, since IL-15 can enhance effector functions of CD8 T cells, we looked at expression of effector proteins, specifically IFN-, by CD8 TILs in tumors depleted of IL-

15. We did not detect significant differences in the frequency of IFN-+ cells among CD8

TILs between treatment groups (Fig. 12D). Consequently, these results combined with the lack of overt changes in apoptotic and proliferation markers in TILs from tumors where IL-15 was neutralized, suggest that the decreased numbers of CD8 TILs observed with IL-15 blockade could likely be due to regulation of trafficking and infiltration of these lymphocytes into tumors. Investigating if IL-15 produced in the TME regulates TIL numbers by promoting the trafficking and infiltration of these cells into tumors was of particular interest for me and due to time constraints and difficulties establishing an adequate model for these experiments I was not able to fully investigate this mechanism.

Future experiments could be performed looking at direct and indirect mechanisms by which IL-15 could regulate the infiltration of TILs into tumors. Some possible mechanisms that could be studied are increases in the expression of chemokines and chemokine receptors by tumor infiltrating myeloid cells and TILs, respectively.

3.2.5 IL-15 expression can be upregulated in tumors by activating the STING pathway

Although IL-15 reporter+ cells are numerous in established tumors (Fig. 8A), production of sIL-15 complexes is significantly reduced in the latter (Fig. 4A).

Therefore, we sought to determine if administrating exogenous STING agonists was capable of upregulating production of sIL-15 complexes in the TME. Despite STING signaling not being involved in regulation of sIL-15 complexes in early tumor development, intratumoral (i.t.) injection of the STING agonist, c-di-GMP (25 g) in large tumors (range 170-260mg) led to an impressive upregulation of sIL-15 complexes in the tumor (Fig. 13A). Importantly, these data demonstrate the IL-15 expressing cells present in the TME are capable of producing sIL-15 complexes in the tumor at this later stage of development. To address which cell types respond to STING activation by increasing IL-15 expression, B16 tumors established in IL-15 translational reporter mice were treated i.t. with c-di-GMP, and GFP expression by myeloid cells in the tumor was examined 24 hours later. In response to STING pathway activation in the TME, we observed significant increases in GFP expression on the CD11chi, CD11b+Ly6Chi, and

CD11b+Ly6C-/loLy6G- tumor-infiltrating myeloid cell subsets compared to PBS treatment (Fig. 13B & C). We also wanted to address whether stimulation of the STING pathway is capable of upregulating sIL-15 complexes directly in tumor cells. Hence,

B16, MCA-205, and MC-38 tumor cells were treated with STING agonist in vitro. As shown earlier, sIL-15 complexes were not produced by either B16-OVA or B16-F10 tumor cells, even after treatment with STING agonists (Fig. 13D). In contrast, STING activation increased production of sIL-15 complexes in the tumor cell lines with baseline detectable sIL-15 complexes, including MCA-205 and MC-38 cells (Fig. 13D) indicating that in these tumor models, both myeloid cells and tumor cells are capable of 94 responding to STING agonists. These results demonstrate that intratumoral activation of the STING pathway upregulates the translation of IL-15 and the production of sIL-15 complexes in tumors, even at later stages of tumor development.

Figure 13. Stimulation of the STING Pathway upregulates sIL-15 complexes in tumors. A) STING agonist, c-di-GMP (25g) or PBS was injected i.t. into large, well- established B16-OVA tumors and levels of sIL-15 complexes within tumors were analyzed 1 day later. Average tumor mass was 259 mg±53 and 173 mg±59 in untreated and c-di-GMP treated tumors respectively. B) IL-15 reporter expression in

B16-OVA tumors 24 hours after i.t. 2’3’-c-GAMP (25g) or PBS. Histograms show the

GFP levels on indicated cell populations in B16-OVA tumors treated with i.t. 2’3’-c-

GAMP (purple histogram) or PBS (blue histogram) after gating on CD45+lineage-

CD11b+ cells. Tumors were treated 14 days after implantation into IL-15 translational reporter mice. C) Graphs represent the averaged GFP levels of the respective

CD11b+ populations isolated from 2’3’-c-GAMP or PBS-treated tumors in IL-15 translational reporter mice. n= 3 mice/group. D) Sub-confluent B16-OVA, B16-F10,

MCA-205, and MC-38 cells were either left untreated or treated with c-di-GMP (6.6

g/ml) and 48 hours later culture supernatants were analyzed for levels of sIL-15 complexes and normalized to the number of cells recovered. n=3 wells per group. Error bars represent SEM, *p<0.05, **p<0.01 with Student’s T-test.

3.2.6 Upregulation of IL-15 in the tumor enhances CD8 T cell responses and promotes anti-tumor immunity

STING agonists have been shown to enhance anti-tumor responses when given intratumorally (173, 180, 186, 189, 195). As such, we asked whether tumor specific

CD8 T cell responses were increased by the STING agonist treatment. To examine this, naïve OVA-specific TCR Transgenic T cells (OT-I) were CFSE-labeled and injected into mice bearing B16-OVA tumors, followed by i.t. treatment with STING agonist. The frequency of OT-I T cells in tumor-draining lymph node (dLN) and spleens was increased in mice treated with c-di-GMP (Fig. 14A). Additionally, these OT-I T cells divided more (Fig. 14A). However, when similar experiments were performed to analyze OT-I T cells responses to STING agonists in WT mice treated with IL-15 Ab, the extent of OT-I proliferation was similar (Fig. 14B) suggesting IL-15 was not critical for STING-enhanced proliferation of OT-I T cells. This was surprising considering our previous studies showed STING-mediated bystander proliferation of memory CD8 T cells was IL-15 dependent (200). Nonetheless, we found that i.t. treatment with STING agonists increased the percentage of Ki67+ CD8 T cells in the dLN and the spleen but not in the tumor where frequency of Ki-67+ cells was already high (Fig. 14C). STING agonists also increased Ki67+ NK cells in the spleen, while STING agonist had only a minor effect on Ki67+ CD4 T cells in spleen, but not in dLN, and tumor (Fig. 14C).

Furthermore, the increase in the frequency of Ki-67+ among CD8 T cells was abrogated with treatment with neutralizing IL-15 Ab (Fig. 14C). Ki-67 expression by NK cells with IL-15 Ab treatment is difficult to analyze as this antibody treatment leads to the disappearance of NK cells (Fig. 14D) (372).

Figure 14. STING activation in the TME promotes proliferation of CD8 T cells in peripheral secondary lymphoid organs. A) Flow cytometry analysis of CFSE dilution in tumor-specific T cells. WT mice bearing B16-OVA tumors were injected with 0.5x106

CFSE-labelled OT-I cells on day 7 post-implantation and received one i.t. treatment with c-di-GMP (25g) once tumors became palpable. Three days post-treatment, the mice were sacrificed and spleen and draining lymph nodes were analyzed by flow cytometry. B) Flow cytometry analysis of CFSE dilution in OT-I T cells in Spleen and tumor-draining LNs (dLN) of WT mice bearing B16-OVA tumors. CFSE-labelled OT-I cells (0.5x106 cells) were injected on day 7 post-implantation and received one i.t. treatment with c-di-GMP (25g) once tumors became palpable. IL-15 Ab (50g) was delivered i.p. one day prior to and on the same day as treatment with c-di-GMP. Three days post-treatment, the mice were sacrificed and spleen and draining lymph nodes were analyzed by flow cytometry. Frequency of OT-I (CD45.1+) among CD8 T cells was determined. C) B16-OVA tumors were implanted into WT mice and treated with c- di-GMP on day 12 post-implantation. IL-15 Ab (50g) was delivered i.p. one day prior to and on the same day as treatment with c-di-GMP. dLN, spleens, and tumors were isolated three days later and Ki-67 staining by CD8 T cells, CD4 T cells and NK cells was measured by flow cytometry. D) Representative flow cytometry plots of NK1.1 and

CD4 staining in spleens and B16 tumors treated with IL-15 Ab or IgG (i.p.). Error bars indicate SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, with One-way ANOVA.

The effects of i.t. STING agonist treatment on CD8 T cell effector functions were also investigated. Similar to Ki-67 expression, IFN- and Granzyme B expression by

CD8 T cells were already high in untreated tumors and treatment with c-di-GMP did not further increase this (Fig. 15 A-C). However, i.t. c-di-GMP led to an increase in IFN-

 expression by CD8 T cells in the spleen that was abrogated by blocking IL-15 (Fig.

15B). A similar effect on IFN- expression by CD8 T cells was observed in dLN but was not statistically significant (Fig. 15B). Altogether, STING activation in the TME leads to increased proliferation of CD8 T cells and NK cells and an increased frequency of IFN-+ CD8 T cells in secondary lymphoid tissues in an IL-15 dependent manner.

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Figure 15. IL-15 produced in response to local STING agonist treatment in the TME induces IFN- production in CD8 T cells in the periphery. B16-OVA tumors were implanted into WT mice and treated with 25g of c-di-GMP on day 12 post-implantation. IL-15 Ab (50g) was delivered i.p. one day prior to and on the same day as treatment with c-di-GMP. dLN, spleens, and tumors were isolated three days later and Ki-67 staining by CD8 T cells, CD4 T cells and NK cells was measured by flow cytometry. A) Representative IFN- staining by CD8 T cells from B16 tumors. B)

Graphs show average frequency of IFN- + cells among CD8 T cells isolated from B16 tumors, dLN, and spleens. C) Frequency of Granzyme B+ CD8 T cells from B16 tumors. Error bars indicate SEM, **p<0.01, with One-way ANOVA.

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We next asked whether IL-15 expression induced by STING stimulation was important for STING-mediated anti-tumor responses. WT and IL-15R-/- mice bearing palpable B16 tumors were treated i.t. with STING agonist and tumor growth was measured over time. In the absence of STING stimulation, tumor growth progressed faster in IL-15R-/- mice than in WT mice providing evidence that IL-15 expression impacts baseline anti-tumor responses (Fig. 16A & C). While STING agonist induced potent anti-tumor immunity and tumor regression in WT mice, it failed to induce tumor regression in IL-15R-/- mice (Fig. 16B). These results indicate that IL-15 is a critical mediator driving STING-induced tumor regression. A similar dependence on IL-15 was also observed with STING agonist treatment of MCA-205 tumors in WT and IL-15R-/- mice (Fig. 16C). Since IL-15R-/- mice have inherent deficiencies in NK cells and CD8

T cells (389), we used an IL-15 neutralizing Ab to block IL-15 in tumor-bearing WT mice treated with i.t. STING agonists (Fig. 16B). In the presence of IL-15 neutralizing Ab,

STING-mediated tumor regression was impaired therefore recapitulating the results observed in IL-15R-/- mice.

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Figure 16. STING activation in the TME promotes tumor regression via IL-15 dependent mechanisms. A) B16-OVA tumor growth in WT and IL-15R-/- mice treated with i.t. injections of c-di-GMP (25g) or PBS on days 7, 10, and 13 post- implantation (indicated by arrows). Left side panel- grouped tumor growth results. Right side panels- Spider plot indicating individual tumor growth. n=4-5 mice per group. B)

Tumor growth of B16-OVA tumors in WT mice treated systemically with neutralizing IL-

15 Ab or PBS (7 days post tumor implantation) followed by three i.t. treatments with c- di-GMP (25g). Tumor growth was significantly different between groups at days 9-15 and days 20-21, p<0.05. C) WT and IL-15R-/- mice bearing MCA-205 tumors on the right flank received three i.t. injections of STING agonist, c-di-GMP (25g) on days 8,

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11, and 14 post-implantation. (n=4-5 per group). Error bars represent SEM, *p<0.05,

**p<0.01, with Student’s T-test.

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Since we observed IL-15 reporter expression increased upon treatment with

STING agonist, we asked whether the expression of IL-15 by CD11c+ cells was important for STING-mediated tumor regression. To this end, B16 tumors were implanted into both CD11c-Cre Tg X IL-15Rfl/fl mice followed by intratumoral treatment with c-di-GMP or PBS. The ability of STING agonist treatment to induce regression of tumors was not impaired in CD11c-Cre Tg X IL-15Rfl/fl mice (Fig. 17A

& B) suggesting the tumor regression was not dependent on upregulation of IL-15 by

CD11c+ cells in response to STING stimulation. Additionally, tumor growth was not increased in untreated CD11c-Cre Tg X IL-15Rfl/fl mice (Fig. 17C).

Since STING agonists have been shown to induce potent systemic anti-tumor responses resulting in regression of distant tumors (186, 189), we investigated whether this abscopal effect of STING agonist against a secondary tumor required the cooperation of IL-15 (Fig. 17D). We also examined whether tumor regression could also be induced by other STING agonists, such as 2’3’c-GAMP that represents a type of STING agonist produced by mammalian cells. We found intratumoral 2’3’c-GAMP induced tumor regression similar to c-di-GMP in an IL-15-dependent manner (Fig.

17D). Furthermore, the STING-mediated tumor regression of a secondary tumor was also dependent on IL-15. These results show the important role of the IL-15 produced in the TME in endogenous and STING-agonist induced anti-tumor immunity.

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Figure 17. IL-15 is essential for STING-mediated regression of distant secondary tumors. A, B) Growth of B16-OVA tumors in CD11c-Cre Tg X IL-15Rfl/fl and WT mice treated with i.t. injections of c-di-GMP (25g) or PBS on days 7, 10, and 13 post- implantation (indicated by arrows). WT mice were treated alongside the CD11c-Cre Tg

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X IL-15Rfl/fl mice to confirm the effectiveness of c-di-GMP treatment. (n=4-5 per group). Error bars represent SEM, *p<0.05, ***p<0.001. C) In a separate experiment, tumor growth of B16-OVA cells was compared in CD11c-Cre Tg X IL-15Rfl/fl and IL-

15Rfl/fl mice (n= 5-7 mice/group). D) WT and IL-15R-/- mice bearing B16-OVA tumors on the right flank were implanted with a secondary B16-OVA tumor on the left flank 72 hours later. The primary tumors were treated with three i.t. injections of 2’3’-c-

GAMP (15g) or PBS on days 7, 10, and 13. Right panels show tumor growth of primary tumors while left panels show tumor growth of secondary tumors. n=5 per group, error bars represent SEM, *p<0.05, **p<0.01, ****p<0.0001 with Student’s T- test.

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3.3 Summary

IL-15 is a cytokine that is constitutively expressed in multiple tissues for homeostatic maintenance of several subsets of CD8 T cells and NK cells. Moreover, IL-

15 can be upregulated when innate immune cells are stimulated and during inflammation, where it can stimulate cytolytic lymphocyte responses (200, 389). Recent findings indicate that IL-15 is also produced in human tumors and its absence is correlated with worst patient outcomes and decreased T cell proliferation in tumors

(346). These findings hinted at the possibility that IL-15 can act as a pro-inflammatory mediator in the TME. Thus, in this chapter I demonstrated that IL-15 expression is common in different tumor types, its expression is regulated in the TME, in part due to

IFN-I and upregulation of IL-15 by other inflammatory signals can enhance local and systemic anti-tumor immune responses.

In this study, the specific cell types that produce IL-15 in the TME using IL-15

GFP reporter mice and conditional knockout mouse models were identified. IL-15 expressing cells in tumors are limited to mainly tumor cells and three myeloid cell populations including a monocyte-like subset (CD11b+Ly6ChiLy6G-), a granulocytic subset (CD11b+Ly6G+), and a macrophage-like subset. Although these reporter systems provide some insight into IL-15 mRNA and protein levels produced in tumors, they have some limitations that are important to consider. Using these reporter systems we cannot distinguish between cell surface IL-15/IL-15R complexes and the cleaved sIL-15 complexes. Importantly, similar to our previous studies we were not able to detect cell surface IL-15 in tumor myeloid cells (248). However, we were able to examine the production of sIL-15 complexes using ELISA. We found that sIL-15 complexes in tumors are abundant during early tumor development but decrease as 108 tumors become larger. These findings were similar in three different murine tumor types including melanoma, colon carcinoma and fibrosarcoma. We also discovered that the high level of sIL-15 complexes found in early tumors is regulated by IFN-I. We also demonstrated the production of sIL-15 complexes can be increased in more advanced tumors by direct activation of the STING pathway in tumors. Demonstrating that while

IL-15 expressing cells are present and capable of producing sIL-15 complexes in large established tumors, they are not receiving potent inflammatory signals to maintain high production of these IL-15 complexes. Since we were not able to detect transpresented or cell surface bound IL-15/IL-15R complexes we cannot rule out the possibility that the regulation and the effects observed in these different tumor types could also be mediated by transpresented IL-15.

The results described herein also provide evidence that IL-15 produced in tumors plays important roles in promoting cytotoxic lymphocyte recruitment to tumors. Local inhibition of IL-15 in tumors led to decreased total numbers of CD8 and NK TILs, without directly affecting tumor growth. While this was unexpected, as other studies indicate that presence of cytotoxic TILs is a very important parameter that dictates anti-tumor responses (82, 390), we interpreted our findings to indicate that the low levels of IL-15 present in tumors, without overt stimulation, are not enough to break the tolerance established by other immunosuppressive cells and factors in the TME.

While IL-15 levels produced during inflammatory conditions, like viral infections, can enhance the effector functions and enhance the survival of activated CD8 T cells we noted no major changes in apoptotic markers or production of effector proteins in

CD8 TILs upon IL-15 neutralization in tumors. One caveat of these experiments is that although we used smaller doses of IL-15 neutralizing antibody, we cannot disregard the 109 possibility that the antibody is leaking into the systemic circulation and some of the observed effects may be due in part by the systemic loss of IL-15. Nonetheless, while physiological levels of IL-15 in tumors may not be enough to break tolerance they are still enough to regulate the number of TILs in tumors.

Conversely, by upregulating IL-15 to higher levels, through activation of the

STING pathway in tumors, we were able to break tolerance and enhance local and systemic anti-tumor responses. Direct activation of the STING pathway locally or in response to cell death has shown potent induction of anti-tumor immunity mediated by

CD8 T cells. These responses lead to regression of local and distant tumors (173, 180,

186, 189, 195). Our results demonstrate that STING-mediated primary and secondary tumor regression depends on IL-15. Specifically, IL-15 was necessary for the observed

STING-induced proliferation and enhanced effector functions of CD8 T cells and NK cells in secondary lymphoid tissues.

To conclude, I demonstrated that IL-15 is produced early during tumor development as sIL-15 complexes by cells in the TME, including stromal and tumor cells. As tumors continue to develop the levels of sIL-15 complexes decrease, likely due to increases in the tumor to stroma ratio and the immunosuppressive nature of more established tumors. However, stimulation of IL-15 expressing cells in tumors, with inflammatory mediators, can upregulate the production of sIL-15 complexes regardless of tumor size. Notably, the IL-15 produced in response to local inflammatory signals is critical for mediating systemic anti-tumor responses and tumor regression. Overall, these findings shed light into previously unknown roles for IL-15 as part of the inflammatory environment of tumors that potentiates anti-tumor immunity.

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CHAPTER 4: EFFECT OF INTRATUMORAL IL-15 AGONISTS IN POORLY T-CELL

INFILTRATED TUMORS

4.1 Introduction

Systemic therapy with recombinant IL-15 as a single agent has been difficult to use in clinical settings due to its poor half-life and its need for continuous infusion to maintain therapeutic levels of the cytokine in circulation. Thus, as discussed in Chapter

1, it became necessary to create agonists of the physiological version of the cytokine, which mimic its native conformation bound to IL-15R. While significant success in preclinical models has been achieved using systemic approaches with these IL-15 agonists and currently several agonists are being evaluated for safety in clinical trials

(391) there are still considerable limitations to their use, including potential off-target adverse effects and toxicities.

On the other hand, recent preclinical and clinical studies using localized therapies delivered directly to tumors have shown that intratumoral delivery of immune stimulating agents is effective at inducing sustained regression of local and distant secondary tumors (392). The strategies used in these studies include a variety of agents that aim to use the tumor itself as a type of vaccine to generate specific local and systemic activation of immune cells. Some of the currently studied agents include a variety of oncolytic viruses, immune cell receptor stimulators including TLR agonists, and STING agonists, other chemical agents and direct delivery of DCs into lesions.

For example, studies using intratumoral oncolytic viruses, which can infect and kill cancer cells, have shown positive clinical outcomes in several types of cancers including melanomas and liver cancers (393-396). These viruses were genetically

111 modified to express the immunostimulatory cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). In melanoma patients, intratumoral delivery of

Talimogene laherparepvec (T-VEC), a herpes-simplex 1 virus (HSV-1) that expresses

GM-CSF, led to increased durable response rate and increased overall survival, over

GM-CSF alone and in patients with metastatic disease (394, 395).

Another class of intratumoral therapeutic agents that has shown positive clinical outcomes are small molecule immune modulators including TLR and STING agonists.

In particular preliminary results of a phase I/II clinical trial revealed that intratumoral administration NKTR-262, a TLR7/8 agonist, in patients with advanced and metastatic solid tumors, led to a disease control rate of 45% out of a total of 11 patients (397,

398). Our group also showed that intratumoral delivery of poly I:C led to tumor control in a mouse model of non-muscle invasive bladder carcinoma (399). Furthermore, intratumoral delivery of cyclic dinucleotides that directly activate the STING pathway have shown potent local and abscopal effects in mouse models of melanomas and head and neck cancers (186, 189, 400). Currently a synthetic STING agonist, ADU-

S100, is being tested in a phase I clinical trial for patients with advanced/metastatic solid tumors and lymphomas (192).

Intratumoral immunotherapy delivery is particularly attractive to use with drugs that have dose-limiting toxicities as they allow high levels of available compound to remain in the tumor environment without the risk of systemic distribution (392). These strategies are also being used as neoadjuvant therapies for solid tumors with the idea that they can induce local regression of tumors that might otherwise be very difficult to resect or treat by conventional therapies (401). There are many ongoing clinical trials that are using intratumoral therapies in combination with conventional therapies such 112 as radiotherapy, chemotherapy and surgical resection (401). Moreover, combination studies using intratumoral therapies including oncolytic viruses and small molecule immune modulators and checkpoint inhibitors are currently under way (191, 192, 398,

402-410). Importantly, the majority of the trials that are using intratumoral agents have been conducted in patients with advanced disease and the main focus has been to test safety rather than efficacy of the treatments, thus further studies must be performed once the adequate dosing limits have been established (392, 401, 411).

Based on the results from the studies using other immunostimulatory agents, and the possible systemic off-target effects using systemic IL-15 therapies, I sought to deliver IL-15 agonists directly into poorly T-cell infiltrated tumors to assess their effects on anti-tumor immunity. In this chapter I will demonstrate that local delivery of IL-15 agonists alone can enhance anti-tumor responses and slow tumor growth. Moreover, it can increase the number of TILs in poorly immune infiltrated tumors and when used in combination with systemic checkpoint inhibitors can act synergistically to induce tumor regression and enhance survival.

4.2 Results

4.2.1 Intratumor delivery of recombinant sIL-15/IL-15R-Fc complexes (rsIL-15c) increases the immune cell infiltrate in melanoma tumors and enhances anti-tumor responses

Systemic delivery of recombinant soluble IL-15/IL-15R-Fc complexes (rsIL-15c) induces anti-tumor responses in a variety of tumor models (295-297). Interestingly, the anti-tumor immune responses induced by systemic rsIL-15c were specifically mediated by tumor-resident CD8 T cells (295), thus we sought to investigate if local delivery of

113 rsIL-15c could promote CD8 T cell responses and induce changes in tumor growth. To this extent mice bearing palpable B16-OVA tumors were treated with rsIL-15c directly into tumors and tumor growth and mouse survival were measured (Fig. 18A).

Intratumoral treatment with three doses of rsIL-15c led to slower tumor growth of B16 tumors when compared with control treated mice (Fig. 18B). However, treatment with rsIL-15c did not induce tumor regression or significantly increase mouse survival (Fig.

18C). Although tumor regression was not observed with treatment, the slower rate of tumor growth in rslL-15c treated tumors suggested that even at significantly smaller doses, localized treatment with rsIL-15c could enhance anti-tumor responses.

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Figure 18. Intratumoral delivery of rsIL-15c decreases growth of melanoma tumors. A) WT mice bearing s.c. B16-OVA tumors were treated with i.t. rsIL-15c or

PBS on days 8, 11 and 14 post implantation and tumor growth and mice survival were

115 measured. B) Growth curves for tumors treated with rsIL-15c (red) or PBS (black). Left panel- grouped tumor growth results. Right side panels- Spider plot indicating individual tumor growth. C) Kaplan-Meier analysis showing survival of mice from B. n=5, per group. Error bars indicate SEM, *p<0.05 with Student’s T-test.

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Based in the slower tumor growth observed with intratumoral rsIL-15c treatment we investigated if the localized delivery of rsIL-15c was enough to increase the amount of immune infiltrate in tumors. In a similar setup as our tumor growth experiment, palpable B16-OVA tumors were treated with three doses of rsIL-15c i.t. and tumors were harvested for flow cytometry analysis (Fig. 19A). We found that tumors treated with rsIL-15c had increased numbers of infiltrating CD45+ immune cells and CD3+ T cells (Fig. 19B). Moreover, treated tumors had statistically significant increases in the numbers of CD4, CD8 and NK TILs (Fig. 19C). The increases in total TIL numbers in rsIL-15c treated tumors are consistent with others’ findings that increased IL-15 levels can promote the infiltration of CD8 T cells and other lymphocytes into the inflamed tissues (265, 266, 412). Moreover, the frequency of CD8 TILs among all T cells in tumors was significantly increased (Fig. 19D). Consistently, the ratio of CD4:CD8 T cells was significantly decreased in tumors treated with rsIL-15c (Fig. 19E), suggesting that treatment preferentially increased the number of CD8 TILs. Taken together these findings indicate that local increases in IL-15 in tumors can increase the immune cell infiltrate and potentially convert a non-T cell inflamed tumor into a T-cell inflamed tumor.

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Figure 19. Direct delivery of rsIL-15c into tumors increases tumor infiltrating immune cells. A) WT mice bearing s.c. B16-OVA tumors were treated with i.t. rsIL-15c on days 8, 11 and 14 after implantation. One day after last treatment tumors were resected and analyzed by flow cytometry. B) Total number of CD45+ and

CD3+ cells in tumors after i.t. treatment with rsIL-15c or PBS. C) Total number of CD4,

CD8 and NK TILs in tumor treated as in A. D) Frequencies of CD4 and CD8 T cells

118 among total CD3+ TILs in tumors treated as in A. E) Ratio of CD4 to CD8 TILs in tumors treated as in A. n=5, per group. Bars represent SEM, *p<0.05, **p<0.01, with

Student’s T-test.

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4.2.2 Delivery of polymer-conjugated IL-15R agonist NKTR-255 directly into tumors slows tumor growth and increases immune infiltrate in melanoma tumors

Several versions of IL-15/IL-15R agonists have been developed and were discussed in Chapter 1. Another novel IL-15R (IL-2/IL-15Rc) agonist, NKTR-255, was developed by Nektar Therapeutics and is currently being pursued as an immuno- oncology drug in pre-clinical and clinical studies. NKTR-255 is a polymer-conjugated human IL-15 protein that has increased in vivo half-life when compared to recombinant

IL-15 and has shown promise in the treatment of solid and hematological malignancies

(308, 310). Previous pre-clinical studies in mice and non-human primates have used

NKTR-255 as a systemic therapy for several cancer types and found that it preferentially stimulates CD8 T cells and NK cell proliferation and responses (309).

Moreover, in lung-metastasis models of colon cancer they found that treatment with

NKTR-255 led to 85% decrease of metastasis in an NK cell-dependent manner (309).

Currently, NKTR-255 is being evaluated in a phase I dose-escalation clinical trial for refractory multiple myeloma and non-Hodgkin lymphoma as a monotherapy and in combination with targeted antibodies including anti-CD38 monoclonal antibody daratumumab and rituximab, an anti-CD20 monoclonal antibody (310).

Since most pre-clinical studies to date using NKTR-255 have used a systemic therapeutic approach, we asked if local delivery of NKTR-255 into tumors could promote anti-tumor responses by tumor infiltrating immune cells. To investigate this, we treated mice bearing palpable melanoma tumors with intratumoral injections of NKTR-

255 and measured tumor growth and analyzed tumor infiltrating immune cells by flow cytometry (Fig. 20A). Intratumoral treatment with NKTR-255 significantly slowed the tumor growth of melanoma tumors when compared to untreated control tumors (Fig. 120

20B). TIL numbers were increased including CD4, CD8 and NK cells (Fig. 20C), although results did not achieve statistical significance. Interestingly, the number of

CD11b+ tumor infiltrating myeloid cells was also increased with treatment, and among

CD11b+ cells only an increase in Ly6Chi cells was observed, while no increase in

Ly6G+ cells was detected (Fig. 20C). These results suggest increased IL-15 levels in the TME, achieved by local treatment with NKTR-255, is enough to increase the levels of tumor-infiltrating immune cells including myeloid and lymphoid cells to slow tumor growth progression in poorly infiltrated melanoma tumors.

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Figure 20. Localized delivery of NKTR-255 into tumors decreases tumor growth and augments the immune cells found in melanoma tumors. A) WT mice bearing

B16-OVA tumors were treated with 60ng of NKTR-255 i.t. or no treatment on days 5, 7,

9, 11, 14, 16 and 18 post-tumor implantation. On day 19 tumors were harvested for flow cytometry analysis. B) Growth curves for tumors treated with NKTR-255 (red) or

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PBS (black). Left panel- grouped tumor growth results. Right panels- Spider plots indicating individual tumor growth. C) Numbers of CD8 T cells, CD4 T cells, NK cells and CD11b+ cells in B16-OVA tumors treated as in A, normalized to tumor weight. D)

Numbers of Ly6Chi and Ly6G+ positive myeloid cells in B16-OVA tumors treated as in

A, normalized to tumor weight. n=3-5 mice/group. Error bars represent SEM, *p<0.05,

**p<0.01, with Student’s T-test.

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4.2.3 Combination therapy with intratumoral IL-15/IL-15R agonists and immune checkpoint inhibitors delays tumor growth and enhances mice survival

The new era for immunotherapeutic drugs began with the discovery of immune checkpoints, receptors on the cell surface of lymphocytes, that serve as natural regulatory mechanisms to ensure that immune responses are controlled and do not cause autoimmunity. While the CTLA-4 and PD-1 proteins were discovered in 1987 and 1992, respectively, their roles as immune checkpoints were not appreciated at the time (413, 414). In 1995 Jim Allison showed that engagement of CTLA-4, on the surface of T cells, with B7 on other cells negatively regulated T-cell activation and the use of an anti-CTLA-4 antibody enhanced T cell proliferation (415). Consequently,

Allison’s group also showed that systemic delivery of an anti-CTLA-4 antibody could induce tumor regression and protection against a secondary tumor challenge (416).

Similar findings in multiple tumors models including melanomas and prostate cancers, using anti-CTLA-4 monotherapy and combination therapy with other immunotherapy strategies (417-424) provided strong preclinical evidence to move forward with testing anti-CTLA-4 checkpoint blockade in human and non-human primates (425-430).

Positive clinical outcomes from clinical trials in patients with advanced melanoma led to

FDA approval of ipilimumab, a monoclonal antibody against CTLA-4, as an immunotherapy drug for patients with inoperable and metastatic melanoma in 2011.

Since then tens of combination approaches using several immune checkpoint inhibitors and therapies including targeted antibodies, radiotherapy, chemotherapy and other immune based therapies have been tested and many approved for a variety of solid tumors (356).

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As our understanding of the challenges faced by cytotoxic lymphocytes in the

TME becomes more thorough, it has become quite evident that combination therapy to combat different mechanisms of immunosuppression in the TME will be necessary to achieve long lasting and curative responses in most malignancies. Consequently, we focused on investigating if combination therapy with immune checkpoint inhibitors and localized IL-15 agonists could act in synergy to enhance anti-tumor immunity in a poorly immunogenic melanoma tumor model. Hence, mice bearing palpable B16-OVA tumors received systemic treatment with anti-CTLA-4 Ab in combination with intratumoral rsIL-15c (Fig. 21A). Monotherapy with anti-CTLA-4 Ab did not control tumor growth (Fig. 21B, blue lines), similar to previous findings by Allison’s group, where this treatment was not capable of overcoming the tolerance mechanisms in these poorly infiltrated and poorly immunogenic tumors (422). Nonetheless, combination treatment with anti-CTLA-4Ab and rsIL-15c induced significant tumor growth control of melanoma tumors (Fig. 21B, red lines). Moreover, the effects of combination therapy extended to significantly improving mouse survival (Fig. 21C).

Taken together, these results suggest that local delivery of IL-15 agonists can act synergistically with systemic immune checkpoint blockade to induce better tumor growth control and increase survival in mice bearing poorly immunogenic tumors.

Future experiments should be performed to look specifically at the amount and activation status of immune cells of tumors treated with combination therapy of anti-

CTLA-4 and rsIL-15c, because it is possible that by treating with rsIL-15c directly into tumors we have increased the infiltration of cytotoxic lymphocytes, such as CD8 T cells, into tumors, thus turning these poorly-infiltrated tumors into T-cell-inflamed tumors.

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Figure 21. Combination therapy with systemic anti-CTLA-4 and intratumor rsIL-

15c controls tumor growth and increases survival in mice with melanoma tumors. A) Mice bearing B16-OVA tumors were treated with anti-CTLA-4 Ab or polyclonal hamster IgG i.p. on days 5,8,11,14 and 18 post-tumor implantation. Tumors were treated with i.t rsIL-15c complexes or PBS on days 8, 11 and 14 post-

126 implantation. Tumor growth and mouse survival were measured. B) Tumor growth curves for mice treated as in A. Left panel- Grouped tumor growth results. Right panels- Spider-plots showing individual tumor growth. C) Kaplan-Meier analysis showing survival of mice from B. n=5 mice/group. Error bars represent SEM, *p<0.05,

**p<0.01, with Student’s T-test or Log-rank Test.

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4.3 Summary

The immunosuppressive nature of the TME and the physical and biochemical barriers cytotoxic lymphocytes encounter to infiltrate, survive and execute their anti- tumor functions present big challenges for immune based therapeutics. Clinical evidence points to a positive relationship between the amount of infiltrating cytotoxic lymphocytes found in patient tumors and their responses to immune checkpoint blockade agents (360, 431, 432). Tumor classification based on T cell infiltration can stratify tumors in two main types: T-cell inflamed and non-T cell inflamed tumors. On one end of the spectrum, non-T cell inflamed tumors can have low levels or completely lack the presence of T cells and these “cold” tumors are less likely to respond to therapy with immune checkpoint inhibitors. On the other hand, there are “hot” tumors or

T cell inflamed tumors that have abundant tumor infiltrating T cells and patients with these types of tumors have better clinical outcomes using immune checkpoint inhibitors. As the number of TILs correlates with better responses to immunotherapy agents, an important strategy to improve the efficacy and applicability of immune checkpoint blockade therapy and other immune based therapies is to find avenues to increase the infiltration and total numbers of lymphocytes in tumors.

In chapter 3 of this dissertation I demonstrated that expression of IL-15 in the

TME can regulate the number of CD8 and NK TILs found in tumors likely due to its ability to promote infiltration of these lymphocytes into tumors. Based on those findings

I investigated if local upregulation of IL-15 in tumors, by intratumoral delivery of IL-15 agonists, could positively affect anti-tumor responses by CD8 T cells and other TILs. As systemic treatment with IL-15 and its agonists could enhance anti-tumor responses in multiple solid tumor models, I used an intratumoral approach to deliver IL-15 agonists 128 to tumors. I also studied the effects of combination therapy with intratumoral IL-15 agonists and immune checkpoint inhibitors.

Delivery of rsIL-15c into tumors increased the amount of CD4, CD8 and NK TILs found in tumors and induced tumor growth control on melanoma tumors. Moreover, intratumoral delivery of a polymer-conjugated human IL-15 protein, NKTR-255, that shows improved bioavailability and half-life over recombinant IL-15, also induced significant tumor growth control and increased the immune cell infiltrates in melanoma tumors. These findings suggest that local increases in IL-15 levels in tumors are sufficient to improve the amount of TILs found in poorly infiltrated melanoma tumors, and that localized strategies to deliver IL-15 agonists or other agents that upregulate IL-

15 production into tumors should be further explored for their potential benefits.

. Typically, preclinical studies using checkpoint inhibitors in poorly immunogenic tumors, like B16 melanomas, rely on the concurrent administration of cancer cell vaccines that express GM-CSF or Flt3-ligand to enhance the efficacy of checkpoint inhibitors (422, 433-436). These combination approaches using multiple checkpoint inhibitors and cancer vaccines are thought to increase the anti-tumor T cell immunity by enhancing T cell infiltration, increasing the inflammatory state of the tumors by upregulating cytokines like IFN-, and enhancing the activation of CD8 T cells in tumors

(434). Taking a similar approach to enhance T cell immunity, I showed that combination treatment with anti-CTLA-4 and local rsIL-15c induced significant tumor growth control when compared to either monotherapy. Furthermore, the combination approach significantly improved survival of mice bearing B16 tumors. The results from these experiments support my hypothesis that increasing levels of IL-15 locally in the TME can potentially convert poorly infiltrated tumors into T cell inflamed tumors that can 129 benefit from systemic immune checkpoint inhibitor therapy or other combinations of immune based therapeutics (Fig. 22).

Figure 22. Intratumoral IL-15 agonists synergize with immune checkpoint blockade to enhance therapeutic responses in poorly immunogenic tumors.

Poorly immunogenic and tumors that have low numbers of tumor infiltrating T cells are also known as cold tumors. In our model, cold tumors produce low levels of IL-15 and using intratumoral delivery of IL-15 agonists the immune cell infiltrate and T cell infiltrate in these cold tumors increases, turning these tumors into hot tumors.

Combination therapy with immune checkpoint inhibitors, specifically anti-CTLA-4 therapy, and IL-15 agonists led to better tumor growth control and increased survival in mice.

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CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS

The limited long-term benefits of cancer immune-based therapies, including immune checkpoint blockade, to a small subset of patients and tumor types has shed light into the importance of determining which attributes of tumors and their microenvironment make them more permissive to mount potent anti-tumor immunity.

Immune cell infiltration, particularly of tumor-infiltrating T cells, has been associated with positive clinical outcomes in a variety of cancers, including melanoma and colorectal tumors (82-85). Specifically, the density, location in the tumor, and cytotoxic activity of CD8 T cells are associated with better clinical outcomes (82, 437).

Consequently, understanding what factors in the TME promote better T cell infiltration can lead to improved clinical responses to current immunotherapies and to develop novel immunotherapy strategies that provide more clinical benefits to a broader population of patients and tumor types.

IL-15 is a pleiotropic cytokine that is important for the survival and maintenance of specific subsets of CD8 T cells and NK cells and has also been shown to enhance anti-tumor immunity as an immunotherapy agent. In human tumors, IL-15 expression correlated with better clinical outcomes and increased T cell proliferation in colorectal tumors (346). In this dissertation I set out to investigate the hypothesis that IL-15 produced in the TME is an important promoter of cytotoxic lymphocytes anti-tumor responses by stimulating optimal numbers in the TME and enhancing their cytotoxic and metabolic functions. As IL-15 in the TME is important for regulating numbers of

TILs, these findings suggest that IL-15 produced in tumors is an important inflammatory cytokine produced by tumor cells and stromal cells that can influence how adaptive lymphocytes access and infiltrate tumor masses and survive in this environment. 131

Moreover, IL-15 produced in response to intratumoral innate immune cell agonists enhances both local and systemic anti-tumor immune responses. These findings suggest that IL-15 production by innate immune cells in the TME can be boosted to stimulate responses by other lymphocytes against tumor cells.

My findings that sIL-15 complexes are produced at high levels early on during tumor development followed by infiltration of CD8 TILs suggest that upregulation of IL-

15 in the tumor could be an important inflammatory reaction likely promoting priming, activation and recruitment of cytotoxic lymphocytes to the tumor site. Moreover, the variety of cellular sources of sIL-15 complexes in the TME, including tumor and stromal cells, show that upon recognition of tumor-derived antigens or products of cellular death spontaneous anti-tumor immune responses are generated that upregulate IL-15 production. Importantly, IFN-I generation, as a result of activation of the STING pathway, is necessary for spontaneous priming of tumor-specific CD8 T cells (173) and

I showed that IFN-I signaling regulated production of sIL-15 complexes and TIL numbers early during tumor development. Thus, IFN-I regulate TIL numbers in tumors in part due to their upregulation of IL-15 in the TME. Furthermore, these findings suggest that any other agents that could promote IFN-I production could also stimulate and positively regulate IL-15 production and could be studied in the context of tumor immunology.

In this dissertation I also showed that many myeloid cell types and even non- hematopoietic cells in the TME can express IL-15 in the TME. It was interesting that while I looked at several tumor types, the composition of myeloid cells that express IL-

15 in the TME in these different tumor types was very similar. Regardless of the tumor type analyzed, the majority of the cells that expressed IL-15 in the TME were CD11b+ 132

Ly6ChiLy6G- cells, and due to their expression levels of CCR2 and the fact that they express IL-15, these cells likely represent classical inflammatory monocytes rather than monocytic MDSCs. However, more in-depth functional studies would have to be performed to ascertain the exact nature of these cells.

Importantly, while I detected low levels of sIL-15 complexes in advanced tumors, upon delivery of a STING agonist into the tumor the IL-15 producing cells in the TME responded by upregulating IL-15 expression and sIL-15 complexes production.

Consequently, while IL-15 expression in the TME is negatively regulated as tumors become more advanced IL-15-producing cells are capable of upregulating IL-15 expression if they receive strong inflammatory signals. These findings could provide evidence to use other inflammatory mediators to promote IL-15 production in the TME.

Since numerous inflammatory stimuli upregulate IL-15 transcription and production of sIL-15 complexes (199, 200, 240, 274), I suspect that other inflammatory pathways could have a similar effect. Thus, currently used innate immune stimulators could potentially exert their effects in part by increasing IL-15 production. It would be thought- provoking to use other agonists of PRRs like TLR agonists such as Poly I:C, that when used peritumorally induce IFN-I production in poorly immune infiltrated melanoma tumors (438), to investigate if the anti-tumor effects observed are also mediated by IL-

15 upregulation.

An important physiological process that affects the cytotoxic functions of CD8 and NK TILs is the competition for nutrients with cancer cells and the challenges they face to maintain their metabolic fitness in the suppressive TME. Activated CD8 T cells rely on glucose metabolism and activated NK cells rely on both glycolysis and FAO to generate the energy sources needed to exert and maintain their effector functions (439- 133

441). Moreover, mitochondrial content and function is also crucial for the survival and metabolic function of both memory CD8 T cells and activated NK cells (439-441). In these studies IL-15 blockade in the TME did not significantly impact the glucose metabolism of CD8 TILs. Furthermore, I was not able to examine NK cell glucose metabolism because the NK cell population is dependent on IL-15 for survival, and depletion of IL-15 from the TME led to complete loss of this population of lymphocytes.

However, I think that further research should be performed to specifically examine if IL-

15 in the tumor, whether endogenous or delivered as a therapeutic could be inducing changes in the metabolic state of T cells and NK cells in tumors.

It is important to look at possible effects on metabolism mediated by IL-15 because IL-15 was found to increase mitochondrial biogenesis, promote OXPHOS and

FAO in activated CD8 T cells in vitro (388, 442) and induce the PI3K/AKT/mTOR pathway in memory CD8 T cells (389). Furthermore, treatment with rsIL-15c led to accumulation of effector CD8 T cells in mucosal tissues in an mTOR dependent manner (412). For NK cells, IL-15 activates the PI3K/AKT/mTOR pathway for the survival, proliferation and IFN- production (443, 444). An experimental approach that could be used to examine the effect of IL-15 on TIL metabolism is performing extracellular flux analysis to analyze both glycolytic and FAO engagement and to obtain information about mitochondrial fitness, including mitochondrial mass and membrane potential. Moreover, if changes in metabolic activity are observed with IL-15 treatment or blockade then it would be important to examine the contribution of mTOR to these metabolic changes by using mTOR inhibitors.

While investigating the specific mechanisms by which basal levels of IL-15 in tumors regulate TIL numbers, my findings strongly suggest that IL-15 could be 134 promoting trafficking and infiltration of TILs into tumors. IL-15 has been shown to play a direct role in promotion of T cell infiltration into inflamed tissues. In vitro IL-15 can act as a chemoattractant of T cells and NK cells (267, 268) and in vivo can promote CD8 T cell infiltration to sites of infection and inflammation (266, 272, 412). Although IL-15 can induce infiltration of CD8 T cells to inflamed tissues, it remains to be clearly defined whether IL-15 is critical for their migration and infiltration into the TME. In pilot experiments, not shown in this dissertation, using naïve and activated antigen-specific

CD8 T cells I tried examining antigen-specific CD8 T cell infiltration into tumors upon IL-

15 blockade; however, several experimental difficulties with this model led me to conclude that other approaches should be used to measure the potential role of TME- derived IL-15 on TIL trafficking.

Some suggestions to directly measure the role of TME-derived IL-15 in promoting TIL infiltration include the use of intravital time-lapse two-photon imaging to directly observe the trafficking of transferred antigen-specific cytotoxic lymphocytes expressing recombinant fluorescent proteins. For example, intravital two photon imaging has been used to visualize GFP expressing OT-I T cells trafficking into OVA- expressing EL4 thymoma tumors in a model of adoptive cell therapy (445). Another approach would be to directly look at TIL infiltration using immunohistochemistry or immunofluorescence of tumor samples treated with IL-15 antibody blockade or in tumors where IL-15 could be conditionally knocked-out using Cre-recombinase inducible systems. Futhermore, IL-15 has been shown to enhance the expression of

LFA-1 on T cells and NK cells and promotes transendothelial migration of T cells in mouse models of rheumatoid arthritis (264, 268). Moreover the interaction of LFA-1 on cytotoxic lymphocytes and its ligand ICAM-1 on the cell surface of tumor cells is

135 required for tumor-cell killing by these lymphocytes (446). Thus examining the expression of this integrin on TILs from IL-15 depleted tumors would serve as another strategy to investigate if IL-15 directly enhances TIL infiltration into tumors.

IL-15 can also promote migration and infiltration of T cells via other indirect mechanisms that include the upregulation of chemokines and chemokine receptors in myeloid cells and cytotoxic lymphocytes, respectively (447). Of interest are the IFN-- inducible chemokines CXCL9 and CXCL10 produced by APCs. These chemokines, produced by Batf3+ DCs are key in mediating infiltration and recruitment of CXCR3+

CD8 T cells into tumors (53, 54, 448-451). Recently, a study using heterodimeric IL-15 as a systemic therapy in MC-38 tumors showed that this IL-15 agonist can upregulate the expression of chemokines CXCL9 and CXCL10 by myeloid cells in the TME, including DCs and macrophages, in part due to increased IFN- production by TILs

(452-454). Furthermore, IL-15 can increase the expression of the receptor for these chemokines, CXCR3, on CD8 T cells (455).

Accordingly, investigating if intratumoral treatment with IL-15 agonists can also enhance the infiltration of TILs into poorly infiltrated tumors would be an important next step to further elucidate how IL-15 therapeutics directly or indirectly enhance anti-tumor immune responses. Several experimental approaches could be used including measuring the levels of chemokines in the tumor using multiplex or ELISA for specific chemokines. Furthermore, expression of chemokine receptors on TILs could be measured using flow cytometry and gene expression profiling techniques could be used to examine upregulated and downregulated genes. Demonstrating that increased

IL-15 levels in tumors could enhance infiltration of T cells into tumors could provide strong and direct evidence that IL-15 can potentially convert cold tumors into T-cell 136 inflamed tumors. This in turn could lead to better combination strategies with other immune-based therapies to ensure that more patients with different tumor types can obtain long term benefits from immunotherapy. As an example, several IL-15 agonists are already undergoing clinical trials in combination with immune checkpoint blockade for PD-1 (NCT03520686, (345)), as IL-15 can induce the upregulation of PD-1 and PD-

L1 on T cells and (340, 344). Therefore, as I showed that the use of IL-15 and its agonists as intratumoral agents increased the immune infiltrate in tumors and can act cooperatively when used in combination with checkpoint inhibitors against CTLA-4, then the next possible step could be to combine intratumoral IL-15 agonists with other checkpoint inhibitors and possibly with other therapies including adoptive cell therapies.

Based on the findings in this dissertation, I propose the use of intratumoral treatment strategies to increase the levels of IL-15 in the TME to aid in the conversion of non-T cell inflamed or cold tumors into T-cell inflamed or hot tumors. While intratumoral delivery of immune based therapies circumvents the issues faced with systemic delivery in regard to dose-limiting toxicities, this delivery system has several disadvantages that must be considered when designing combination therapies. The first obvious disadvantage would be the difficulty of accessing many types of solid tumors as most clinical trials performed to date using intratumoral therapies have been conducted in easily accessible lesions like skin and breast cancers. However, recent developments in radiologic and diagnostic imaging techniques are now thought to bypass the issue of accessibility as long as primary lesions or metastatic lesions can be localized using any of these new imaging and diagnostic modalities (401, 456, 457).

Another practical disadvantage for intratumoral agents includes the possibility of requiring multiple doses delivered to the tumor site for generation of sustained systemic 137 immunity (401). Therefore, developing strategies that deliver these therapeutic agents into the tumors and maintain them localized to the tumor site, limiting their diffusion to the systemic circulation, are necessary to reduce the need for repetitive dosing and the potential to generate systemic toxicities (456). Several approaches have been developed to deliver immunotherapeutic agents like cytokines, such as IL-2 and IL-15, and immune checkpoint inhibitors to tumors and ensure slow release of the drug and limit the dissemination to the circulation. These approaches are based on conjugating these drugs to nanoparticles, liposomes and biodegradable polymers (456, 458-460). In short, intratumoral delivery of IL-2 encapsulated in microspheres of gelatin and chondroitin sulfate had a slower continuous in vivo release for 21 days and induced protective immunity in mouse models of brain and liver metastasis (458). IL-15 agonists have been delivered to the peritumoral space using alginate matrices and this led to tumor growth control of B16 melanoma tumors and infiltration of leukocytes into the tumor (461).

Immune checkpoint blockade antibodies against CTLA-4 have been injected into tumors in water and oil emulsions leading to slower release of the antibody while maintaining tumor eradication without autoimmune adverse effects observed with systemic therapy

(460). Lastly, Wang et al developed degradable microneedle patches, which can be applied to the skin, that release dextran nanoparticles conjugated to anti-PD-1 mAb that release the drug upon encounter of an acidic environment and this enhanced survival of mice in a melanoma tumor model (462). Consequently, the use of one or more of these approaches could be applied when considering the use of IL-15 agonists or other agents that can upregulate IL-15 in the TME as intratumoral therapies.

One last aspect to consider in the studies performed in this dissertation is the fact that we only used transplantable subcutaneous mouse tumor models. Subcutaneous

138 mouse tumor models provide important advantages including highly reproducible and rapid growth of tumors and easy tumor growth monitoring by visual inspection and manual measurements (463). However, since they arise from a clonal population of cells that have been cultivated, they may lack the heterogeneity observed in naturally developing tumors and since they are implanted in tissues different from their tissues of origin they fail to receive the adequate cues that can lead to development of a different inflammatory tumor microenvironment (464). Nevertheless, these mouse models have been widely used for the study of cancer immunotherapy drugs, including immune checkpoint inhibitors, and their results have been validated in clinical trials and thus continue to serve as important models for immuno-oncology research. A different approach that could have been taken was using orthotopic tumor models where syngeneic mouse tumor cells are implanted into the organ or tissues of the same histologic type as the tumor cells (463). While these orthotopic tumors develop in the organs or tissues of origin, they still present some disadvantages because they are derived from cultured cell lines that lack the genetic heterogeneity that is found in human tumors. Other mouse tumor models that could be used to circumvent the limitations of transplantable tumor models are genetically engineered mouse models in which tissue- specific overexpression of oncogenes or deletion of tumor suppressor genes can lead to spontaneous development of tumors. It would be interesting to perform studies using these different types of mouse tumor models in experiments using local IL-15 therapies to elucidate how the tumor microenvironment and the development of mutations over time affect the response to these therapeutics.

139

CHAPTER 6: REFERENCES

1. Health NIo Cancer: A Historic Perspective.

https://training.seer.cancer.gov/disease/history/ Date Accessed: 08/17/2020.

2. Society AC (2014) Early History of Cancer.

https://www.cancer.org/cancer/cancer-basics/history-of-cancer/what-is-

cancer.html Date Accessed: 08/16/2020.

3. (US) NIoH (2007) Understanding Cancer. Biological Sciences Curriciulum Study.

NIH Curriculum Supplement Series [Internet], (National Institutes of Health

(US), Bethesda (MD)).

4. Cooper GM (2000) The Development and Causes of Cancer. The Cell: A

Molecular Approach, (Sinauer Associates, Sunderland (MA)), 2nd edition Ed.

5. Hanahan D & Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57-70.

6. Hanahan D & Weinberg RA (2011) Hallmarks of cancer: the next generation.

Cell 144(5):646-674.

7. Kawai T & Akira S (2010) The role of pattern-recognition receptors in innate

immunity: update on Toll-like receptors. Nat Immunol 11(5):373-384.

8. Rakoff-Nahoum S & Medzhitov R (2009) Toll-like receptors and cancer. Nat Rev

Cancer 9(1):57-63.

9. Fuertes MB, Kacha AK, Kline J, Woo SR, Kranz DM, Murphy KM, & Gajewski

TF (2011) Host type I IFN signals are required for antitumor CD8+ T cell

responses through CD8{alpha}+ dendritic cells. J Exp Med 208(10):2005-2016.

10. Fuertes MB, Woo SR, Burnett B, Fu YX, & Gajewski TF (2013) Type I interferon

response and innate immune sensing of cancer. Trends Immunol 34(2):67-73.

140

11. Segal NH, Parsons DW, Peggs KS, Velculescu V, Kinzler KW, Vogelstein B, &

Allison JP (2008) Epitope landscape in breast and colorectal cancer. Cancer

Res 68(3):889-892.

12. Yarchoan M, Johnson BA, 3rd, Lutz ER, Laheru DA, & Jaffee EM (2017)

Targeting neoantigens to augment antitumour immunity. Nat Rev Cancer

17(4):209-222.

13. Dunn GP, Bruce AT, Ikeda H, Old LJ, & Schreiber RD (2002) Cancer

immunoediting: from immunosurveillance to tumor escape. Nat Immunol

3(11):991-998.

14. Gabbiani G (2003) The myofibroblast in wound healing and fibrocontractive

diseases. J Pathol 200(4):500-503.

15. Buechler MB & Turley SJ (2018) A short field guide to fibroblast function in

immunity. Semin Immunol 35:48-58.

16. Förster R, Davalos-Misslitz AC, & Rot A (2008) CCR7 and its ligands: balancing

immunity and tolerance. Nat Rev Immunol 8(5):362-371.

17. Sahai E, Astsaturov I, Cukierman E, DeNardo DG, Egeblad M, Evans RM,

Fearon D, Greten FR, Hingorani SR, Hunter T, Hynes RO, Jain RK, Janowitz T,

Jorgensen C, Kimmelman AC, Kolonin MG, Maki RG, Powers RS, Puré E,

Ramirez DC, Scherz-Shouval R, Sherman MH, Stewart S, Tlsty TD, Tuveson

DA, Watt FM, Weaver V, Weeraratna AT, & Werb Z (2020) A framework for

advancing our understanding of cancer-associated fibroblasts. Nature Reviews

Cancer 20(3):174-186.

18. Nguyen EV, Pereira BA, Lawrence MG, Ma X, Rebello RJ, Chan H, Niranjan B,

Wu Y, Ellem S, Guan X, Wu J, Skhinas JN, Cox TR, Risbridger GP, Taylor RA,

141

Lister NL, & Daly RJ (2019) Proteomic Profiling of Human Prostate Cancer-

associated Fibroblasts (CAF) Reveals LOXL2-dependent Regulation of the

Tumor Microenvironment. Mol Cell Proteomics 18(7):1410-1427.

19. Salmon H, Franciszkiewicz K, Damotte D, Dieu-Nosjean MC, Validire P,

Trautmann A, Mami-Chouaib F, & Donnadieu E (2012) Matrix architecture

defines the preferential localization and migration of T cells into the stroma of

human lung tumors. J Clin Invest 122(3):899-910.

20. Dumont N, Liu B, Defilippis RA, Chang H, Rabban JT, Karnezis AN, Tjoe JA,

Marx J, Parvin B, & Tlsty TD (2013) Breast fibroblasts modulate early

dissemination, tumorigenesis, and metastasis through alteration of extracellular

matrix characteristics. Neoplasia 15(3):249-262.

21. Shi Y, Gao W, Lytle NK, Huang P, Yuan X, Dann AM, Ridinger-Saison M,

DelGiorno KE, Antal CE, Liang G, Atkins AR, Erikson G, Sun H, Meisenhelder J,

Terenziani E, Woo G, Fang L, Santisakultarm TP, Manor U, Xu R, Becerra CR,

Borazanci E, Von Hoff DD, Grandgenett PM, Hollingsworth MA, Leblanc M,

Umetsu SE, Collisson EA, Scadeng M, Lowy AM, Donahue TR, Reya T,

Downes M, Evans RM, Wahl GM, Pawson T, Tian R, & Hunter T (2019)

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and

monitoring. Nature 569(7754):131-135.

22. Cazet AS, Hui MN, Elsworth BL, Wu SZ, Roden D, Chan CL, Skhinas JN, Collot

R, Yang J, Harvey K, Johan MZ, Cooper C, Nair R, Herrmann D, McFarland A,

Deng N, Ruiz-Borrego M, Rojo F, Trigo JM, Bezares S, Caballero R, Lim E,

Timpson P, O'Toole S, Watkins DN, Cox TR, Samuel MS, Martín M, & Swarbrick

A (2018) Targeting stromal remodeling and cancer stem cell plasticity

142

overcomes chemoresistance in triple negative breast cancer. Nat Commun

9(1):2897.

23. De Wever O, Nguyen QD, Van Hoorde L, Bracke M, Bruyneel E, Gespach C, &

Mareel M (2004) Tenascin-C and SF/HGF produced by myofibroblasts in vitro

provide convergent pro-invasive signals to human colon cancer cells through

RhoA and Rac. Faseb j 18(9):1016-1018.

24. Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC, Lu N, Selig M, Nielsen G,

Taksir T, Jain RK, & Seed B (1998) Tumor induction of VEGF promoter activity

in stromal cells. Cell 94(6):715-725.

25. Feig C, Jones JO, Kraman M, Wells RJ, Deonarine A, Chan DS, Connell CM,

Roberts EW, Zhao Q, Caballero OL, Teichmann SA, Janowitz T, Jodrell DI,

Tuveson DA, & Fearon DT (2013) Targeting CXCL12 from FAP-expressing

carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in

pancreatic cancer. Proc Natl Acad Sci U S A 110(50):20212-20217.

26. Monteran L & Erez N (2019) The Dark Side of Fibroblasts: Cancer-Associated

Fibroblasts as Mediators of Immunosuppression in the Tumor Microenvironment.

Front Immunol 10:1835.

27. Fearon DT (2014) The carcinoma-associated fibroblast expressing fibroblast

activation protein and escape from immune surveillance. Cancer Immunol Res

2(3):187-193.

28. Lakins MA, Ghorani E, Munir H, Martins CP, & Shields JD (2018) Cancer-

associated fibroblasts induce antigen-specific deletion of CD8 (+) T Cells to

protect tumour cells. Nat Commun 9(1):948.

143

29. Bergers G & Benjamin LE (2003) Tumorigenesis and the angiogenic switch. Nat

Rev Cancer 3(6):401-410.

30. De Palma M, Biziato D, & Petrova TV (2017) Microenvironmental regulation of

tumour angiogenesis. Nat Rev Cancer 17(8):457-474.

31. Hida K, Maishi N, Annan DA, & Hida Y (2018) Contribution of Tumor Endothelial

Cells in Cancer Progression. Int J Mol Sci 19(5).

32. Forster JC, Harriss-Phillips WM, Douglass MJ, & Bezak E (2017) A review of the

development of tumor vasculature and its effects on the tumor

microenvironment. Hypoxia (Auckl) 5:21-32.

33. Rodig N, Ryan T, Allen JA, Pang H, Grabie N, Chernova T, Greenfield EA, Liang

SC, Sharpe AH, Lichtman AH, & Freeman GJ (2003) Endothelial expression of

PD-L1 and PD-L2 down-regulates CD8+ T cell activation and cytolysis. Eur J

Immunol 33(11):3117-3126.

34. Dirkx AE, Oude Egbrink MG, Kuijpers MJ, van der Niet ST, Heijnen VV, Bouma-

ter Steege JC, Wagstaff J, & Griffioen AW (2003) Tumor angiogenesis

modulates leukocyte-vessel wall interactions in vivo by reducing endothelial

adhesion molecule expression. Cancer Res 63(9):2322-2329.

35. Mandriota SJ, Jussila L, Jeltsch M, Compagni A, Baetens D, Prevo R, Banerji S,

Huarte J, Montesano R, Jackson DG, Orci L, Alitalo K, Christofori G, & Pepper

MS (2001) Vascular endothelial growth factor-C-mediated lymphangiogenesis

promotes tumour metastasis. Embo j 20(4):672-682.

36. Schoppmann SF, Fenzl A, Nagy K, Unger S, Bayer G, Geleff S, Gnant M,

Horvat R, Jakesz R, & Birner P (2006) VEGF-C expressing tumor-associated

144

macrophages in lymph node positive breast cancer: impact on

lymphangiogenesis and survival. Surgery 139(6):839-846.

37. Farnsworth RH, Achen MG, & Stacker SA (2018) The evolving role of lymphatics

in cancer metastasis. Curr Opin Immunol 53:64-73.

38. Leu AJ, Berk DA, Lymboussaki A, Alitalo K, & Jain RK (2000) Absence of

functional lymphatics within a murine sarcoma: a molecular and functional

evaluation. Cancer Res 60(16):4324-4327.

39. Hoshida T, Isaka N, Hagendoorn J, di Tomaso E, Chen YL, Pytowski B,

Fukumura D, Padera TP, & Jain RK (2006) Imaging steps of lymphatic

metastasis reveals that vascular endothelial growth factor-C increases

metastasis by increasing delivery of cancer cells to lymph nodes: therapeutic

implications. Cancer Res 66(16):8065-8075.

40. Lund AW, Duraes FV, Hirosue S, Raghavan VR, Nembrini C, Thomas SN, Issa

A, Hugues S, & Swartz MA (2012) VEGF-C promotes immune tolerance in B16

melanomas and cross-presentation of tumor antigen by lymph node lymphatics.

Cell Rep 1(3):191-199.

41. Dieterich LC, Ikenberg K, Cetintas T, Kapaklikaya K, Hutmacher C, & Detmar M

(2017) Tumor-Associated Lymphatic Vessels Upregulate PDL1 to Inhibit T-Cell

Activation. Front Immunol 8:66.

42. Nörder M, Gutierrez MG, Zicari S, Cervi E, Caruso A, & Guzmán CA (2012)

Lymph node-derived lymphatic endothelial cells express functional costimulatory

molecules and impair dendritic cell-induced allogenic T-cell proliferation. Faseb j

26(7):2835-2846.

145

43. Tokumoto M, Tanaka H, Tauchi Y, Tamura T, Toyokawa T, Kimura K,

Muguruma K, Yashiro M, Maeda K, Hirakawa K, & Ohira M (2017)

Immunoregulatory Function of Lymphatic Endothelial Cells in Tumor-draining

Lymph Nodes of Human Gastric Cancer. Anticancer Res 37(6):2875-2883.

44. Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, & Mellor AL (1999)

Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp

Med 189(9):1363-1372.

45. Fankhauser M, Broggi MAS, Potin L, Bordry N, Jeanbart L, Lund AW, Da Costa

E, Hauert S, Rincon-Restrepo M, Tremblay C, Cabello E, Homicsko K, Michielin

O, Hanahan D, Speiser DE, & Swartz MA (2017) Tumor lymphangiogenesis

promotes T cell infiltration and potentiates immunotherapy in melanoma. Sci

Transl Med 9(407).

46. Chen L & Flies DB (2013) Molecular mechanisms of T cell co-stimulation and

co-inhibition. Nat Rev Immunol 13(4):227-242.

47. June CH, Ledbetter JA, Gillespie MM, Lindsten T, & Thompson CB (1987) T-cell

proliferation involving the CD28 pathway is associated with cyclosporine-

resistant interleukin 2 gene expression. Mol Cell Biol 7(12):4472-4481.

48. Mueller DL, Jenkins MK, & Schwartz RH (1989) Clonal expansion versus

functional clonal inactivation: a costimulatory signalling pathway determines the

outcome of T cell antigen receptor occupancy. Annu Rev Immunol 7:445-480.

49. Merad M, Sathe P, Helft J, Miller J, & Mortha A (2013) The dendritic cell lineage:

ontogeny and function of dendritic cells and their subsets in the steady state and

the inflamed setting. Annu Rev Immunol 31:563-604.

146

50. Murphy TL, Grajales-Reyes GE, Wu X, Tussiwand R, Briseño CG, Iwata A,

Kretzer NM, Durai V, & Murphy KM (2016) Transcriptional Control of Dendritic

Cell Development. Annu Rev Immunol 34:93-119.

51. Diamond MS, Kinder M, Matsushita H, Mashayekhi M, Dunn GP, Archambault

JM, Lee H, Arthur CD, White JM, Kalinke U, Murphy KM, & Schreiber RD (2011)

Type I interferon is selectively required by dendritic cells for immune rejection of

tumors. J Exp Med 208(10):1989-2003.

52. Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ, Barczak

A, Rosenblum MD, Daud A, Barber DL, Amigorena S, Van't Veer LJ, Sperling

AI, Wolf DM, & Krummel MF (2014) Dissecting the Tumor Myeloid Compartment

Reveals Rare Activating Antigen-Presenting Cells Critical for T Cell Immunity.

Cancer Cell 26(6):938.

53. Spranger S, Bao R, & Gajewski TF (2015) Melanoma-intrinsic β-catenin

signalling prevents anti-tumour immunity. Nature 523(7559):231-235.

54. Spranger S, Dai D, Horton B, & Gajewski TF (2017) Tumor-Residing Batf3

Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell

Therapy. Cancer Cell 31(5):711-723.e714.

55. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama M,

Calderon B, Schraml BU, Unanue ER, Diamond MS, Schreiber RD, Murphy TL,

& Murphy KM (2008) Batf3 deficiency reveals a critical role for CD8alpha+

dendritic cells in cytotoxic T cell immunity. Science 322(5904):1097-1100.

56. Reizis B (2019) Plasmacytoid Dendritic Cells: Development, Regulation, and

Function. Immunity 50(1):37-50.

147

57. Conrad C, Gregorio J, Wang YH, Ito T, Meller S, Hanabuchi S, Anderson S,

Atkinson N, Ramirez PT, Liu YJ, Freedman R, & Gilliet M (2012) Plasmacytoid

dendritic cells promote immunosuppression in ovarian cancer via ICOS

costimulation of Foxp3(+) T-regulatory cells. Cancer Res 72(20):5240-5249.

58. Sisirak V, Faget J, Gobert M, Goutagny N, Vey N, Treilleux I, Renaudineau S,

Poyet G, Labidi-Galy SI, Goddard-Leon S, Durand I, Le Mercier I, Bajard A,

Bachelot T, Puisieux A, Puisieux I, Blay JY, Ménétrier-Caux C, Caux C, &

Bendriss-Vermare N (2012) Impaired IFN-α production by plasmacytoid dendritic

cells favors regulatory T-cell expansion that may contribute to breast cancer

progression. Cancer Res 72(20):5188-5197.

59. Zong J, Keskinov AA, Shurin GV, & Shurin MR (2016) Tumor-derived factors

modulating dendritic cell function. Cancer Immunol Immunother 65(7):821-833.

60. Fu C & Jiang A (2018) Dendritic Cells and CD8 T Cell Immunity in Tumor

Microenvironment. Front Immunol 9:3059.

61. Brown RD, Pope B, Murray A, Esdale W, Sze DM, Gibson J, Ho PJ, Hart D, &

Joshua D (2001) Dendritic cells from patients with myeloma are numerically

normal but functionally defective as they fail to up-regulate CD80 (B7-1)

expression after huCD40LT stimulation because of inhibition by transforming

growth factor-beta1 and interleukin-10. Blood 98(10):2992-2998.

62. Steinbrink K, Jonuleit H, Müller G, Schuler G, Knop J, & Enk AH (1999)

Interleukin-10-treated human dendritic cells induce a melanoma-antigen-specific

anergy in CD8(+) T cells resulting in a failure to lyse tumor cells. Blood

93(5):1634-1642.

148

63. Steinbrink K, Wölfl M, Jonuleit H, Knop J, & Enk AH (1997) Induction of

tolerance by IL-10-treated dendritic cells. J Immunol 159(10):4772-4780.

64. Park SJ, Nakagawa T, Kitamura H, Atsumi T, Kamon H, Sawa S, Kamimura D,

Ueda N, Iwakura Y, Ishihara K, Murakami M, & Hirano T (2004) IL-6 regulates in

vivo dendritic cell differentiation through STAT3 activation. J Immunol

173(6):3844-3854.

65. Duluc D, Delneste Y, Tan F, Moles MP, Grimaud L, Lenoir J, Preisser L, Anegon

I, Catala L, Ifrah N, Descamps P, Gamelin E, Gascan H, Hebbar M, & Jeannin P

(2007) Tumor-associated leukemia inhibitory factor and IL-6 skew monocyte

differentiation into tumor-associated macrophage-like cells. Blood 110(13):4319-

4330.

66. Mantovani A, Sozzani S, Locati M, Allavena P, & Sica A (2002) Macrophage

polarization: tumor-associated macrophages as a paradigm for polarized M2

mononuclear phagocytes. Trends Immunol 23(11):549-555.

67. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, & Locati M (2004) The

chemokine system in diverse forms of macrophage activation and polarization.

Trends Immunol 25(12):677-686.

68. Pollard JW (2004) Tumour-educated macrophages promote tumour progression

and metastasis. Nat Rev Cancer 4(1):71-78.

69. Riabov V, Gudima A, Wang N, Mickley A, Orekhov A, & Kzhyshkowska J (2014)

Role of tumor associated macrophages in tumor angiogenesis and

lymphangiogenesis. Front Physiol 5:75.

149

70. Hao NB, Lü MH, Fan YH, Cao YL, Zhang ZR, & Yang SM (2012) Macrophages

in tumor microenvironments and the progression of tumors. Clin Dev Immunol

2012:948098.

71. Sharda DR, Yu S, Ray M, Squadrito ML, De Palma M, Wynn TA, Morris SM, Jr.,

& Hankey PA (2011) Regulation of macrophage arginase expression and tumor

growth by the Ron receptor tyrosine kinase. J Immunol 187(5):2181-2192.

72. Rodriguez PC, Zea AH, DeSalvo J, Culotta KS, Zabaleta J, Quiceno DG, Ochoa

JB, & Ochoa AC (2003) L-arginine consumption by macrophages modulates the

expression of CD3 zeta chain in T lymphocytes. J Immunol 171(3):1232-1239.

73. Masucci MT, Minopoli M, & Carriero MV (2019) Tumor Associated Neutrophils.

Their Role in Tumorigenesis, Metastasis, Prognosis and Therapy. Front Oncol

9:1146.

74. Piccard H, Muschel RJ, & Opdenakker G (2012) On the dual roles and polarized

phenotypes of neutrophils in tumor development and progression. Crit Rev

Oncol Hematol 82(3):296-309.

75. Zhou SL, Zhou ZJ, Hu ZQ, Huang XW, Wang Z, Chen EB, Fan J, Cao Y, Dai Z,

& Zhou J (2016) Tumor-Associated Neutrophils Recruit Macrophages and T-

Regulatory Cells to Promote Progression of Hepatocellular Carcinoma and

Resistance to Sorafenib. Gastroenterology 150(7):1646-1658.e1617.

76. Rotondo R, Bertolotto M, Barisione G, Astigiano S, Mandruzzato S, Ottonello L,

Dallegri F, Bronte V, Ferrini S, & Barbieri O (2011) Exocytosis of azurophil and

arginase 1-containing granules by activated polymorphonuclear neutrophils is

required to inhibit T lymphocyte proliferation. J Leukoc Biol 89(5):721-727.

150

77. Singel KL & Segal BH (2016) Neutrophils in the tumor microenvironment: trying

to heal the wound that cannot heal. Immunol Rev 273(1):329-343.

78. Gabrilovich DI (2017) Myeloid-Derived Suppressor Cells. Cancer Immunol Res

5(1):3-8.

79. Gabrilovich DI & Nagaraj S (2009) Myeloid-derived suppressor cells as

regulators of the immune system. Nat Rev Immunol 9(3):162-174.

80. Yu J, Du W, Yan F, Wang Y, Li H, Cao S, Yu W, Shen C, Liu J, & Ren X (2013)

Myeloid-derived suppressor cells suppress antitumor immune responses

through IDO expression and correlate with lymph node metastasis in patients

with breast cancer. J Immunol 190(7):3783-3797.

81. Mundy-Bosse BL, Lesinski GB, Jaime-Ramirez AC, Benninger K, Khan M,

Kuppusamy P, Guenterberg K, Kondadasula SV, Chaudhury AR, La Perle KM,

Kreiner M, Young G, Guttridge DC, & Carson WE, 3rd (2011) Myeloid-derived

suppressor cell inhibition of the IFN response in tumor-bearing mice. Cancer

Res 71(15):5101-5110.

82. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pagès C,

Tosolini M, Camus M, Berger A, Wind P, Zinzindohoué F, Bruneval P, Cugnenc

PH, Trajanoski Z, Fridman WH, & Pagès F (2006) Type, density, and location of

immune cells within human colorectal tumors predict clinical outcome. Science

313(5795):1960-1964.

83. Kim ST, Jeong H, Woo OH, Seo JH, Kim A, Lee ES, Shin SW, Kim YH, Kim JS,

& Park KH (2013) Tumor-infiltrating lymphocytes, tumor characteristics, and

recurrence in patients with early breast cancer. Am J Clin Oncol 36(3):224-231.

151

84. Piersma SJ, Jordanova ES, van Poelgeest MI, Kwappenberg KM, van der Hulst

JM, Drijfhout JW, Melief CJ, Kenter GG, Fleuren GJ, Offringa R, & van der Burg

SH (2007) High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is

associated with the absence of lymph node metastases in patients with large

early-stage cervical cancer. Cancer Res 67(1):354-361.

85. Kmiecik J, Poli A, Brons NH, Waha A, Eide GE, Enger P, Zimmer J, & Chekenya

M (2013) Elevated CD3+ and CD8+ tumor-infiltrating immune cells correlate with

prolonged survival in glioblastoma patients despite integrated

immunosuppressive mechanisms in the tumor microenvironment and at the

systemic level. J Neuroimmunol 264(1-2):71-83.

86. Dangaj D, Bruand M, Grimm AJ, Ronet C, Barras D, Duttagupta PA, Lanitis E,

Duraiswamy J, Tanyi JL, Benencia F, Conejo-Garcia J, Ramay HR, Montone

KT, Powell DJ, Jr., Gimotty PA, Facciabene A, Jackson DG, Weber JS, Rodig

SJ, Hodi SF, Kandalaft LE, Irving M, Zhang L, Foukas P, Rusakiewicz S,

Delorenzi M, & Coukos G (2019) Cooperation between Constitutive and

Inducible Chemokines Enables T Cell Engraftment and Immune Attack in Solid

Tumors. Cancer Cell 35(6):885-900.e810.

87. Martínez-Lostao L, Anel A, & Pardo J (2015) How Do Cytotoxic Lymphocytes Kill

Cancer Cells? Clin Cancer Res 21(22):5047-5056.

88. Juneja VR, McGuire KA, Manguso RT, LaFleur MW, Collins N, Haining WN,

Freeman GJ, & Sharpe AH (2017) PD-L1 on tumor cells is sufficient for immune

evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med

214(4):895-904.

152

89. Salih HR, Rammensee HG, & Steinle A (2002) Cutting edge: down-regulation of

MICA on human tumors by proteolytic shedding. J Immunol 169(8):4098-4102.

90. Bruno A, Ferlazzo G, Albini A, & Noonan DM (2014) A think tank of

TINK/TANKs: tumor-infiltrating/tumor-associated natural killer cells in tumor

progression and angiogenesis. J Natl Cancer Inst 106(8):dju200.

91. Park YJ, Song B, Kim YS, Kim EK, Lee JM, Lee GE, Kim JO, Kim YJ, Chang

WS, & Kang CY (2013) Tumor microenvironmental conversion of natural killer

cells into myeloid-derived suppressor cells. Cancer Res 73(18):5669-5681.

92. Bailey SR, Nelson MH, Himes RA, Li Z, Mehrotra S, & Paulos CM (2014) Th17

cells in cancer: the ultimate identity crisis. Front Immunol 5:276.

93. Martin-Orozco N, Muranski P, Chung Y, Yang XO, Yamazaki T, Lu S, Hwu P,

Restifo NP, Overwijk WW, & Dong C (2009) T helper 17 cells promote cytotoxic

T cell activation in tumor immunity. Immunity 31(5):787-798.

94. Kobayashi N, Hiraoka N, Yamagami W, Ojima H, Kanai Y, Kosuge T, Nakajima

A, & Hirohashi S (2007) FOXP3+ regulatory T cells affect the development and

progression of hepatocarcinogenesis. Clin Cancer Res 13(3):902-911.

95. Jang JE, Hajdu CH, Liot C, Miller G, Dustin ML, & Bar-Sagi D (2017) Crosstalk

between Regulatory T Cells and Tumor-Associated Dendritic Cells Negates Anti-

tumor Immunity in Pancreatic Cancer. Cell Rep 20(3):558-571.

96. Lindqvist CA, Christiansson LH, Simonsson B, Enblad G, Olsson-Strömberg U,

& Loskog AS (2010) T regulatory cells control T-cell proliferation partly by the

release of soluble CD25 in patients with B-cell malignancies. Immunology

131(3):371-376.

153

97. Marincola FM, Jaffee EM, Hicklin DJ, & Ferrone S (2000) Escape of human solid

tumors from T-cell recognition: molecular mechanisms and functional

significance. Adv Immunol 74:181-273.

98. Gabrilovich DI, Chen HL, Girgis KR, Cunningham HT, Meny GM, Nadaf S,

Kavanaugh D, & Carbone DP (1996) Production of vascular endothelial growth

factor by human tumors inhibits the functional maturation of dendritic cells. Nat

Med 2(10):1096-1103.

99. Chaux P, Favre N, Martin M, & Martin F (1997) Tumor-infiltrating dendritic cells

are defective in their antigen-presenting function and inducible B7 expression in

rats. Int J Cancer 72(4):619-624.

100. Gabrilovich DI, Ciernik IF, & Carbone DP (1996) Dendritic cells in antitumor

immune responses. I. Defective antigen presentation in tumor-bearing hosts.

Cell Immunol 170(1):101-110.

101. Harding FA, McArthur JG, Gross JA, Raulet DH, & Allison JP (1992) CD28-

mediated signalling co-stimulates murine T cells and prevents induction of

anergy in T-cell clones. Nature 356(6370):607-609.

102. Dunn GP, Old LJ, & Schreiber RD (2004) The immunobiology of cancer

immunosurveillance and immunoediting. Immunity 21(2):137-148.

103. Schmiedel D & Mandelboim O (2018) NKG2D Ligands-Critical Targets for

Cancer Immune Escape and Therapy. Front Immunol 9:2040.

104. Motz GT, Santoro SP, Wang LP, Garrabrant T, Lastra RR, Hagemann IS, Lal P,

Feldman MD, Benencia F, & Coukos G (2014) Tumor endothelium FasL

establishes a selective immune barrier promoting tolerance in tumors. Nat Med

20(6):607-615.

154

105. Molon B, Ugel S, Del Pozzo F, Soldani C, Zilio S, Avella D, De Palma A, Mauri

P, Monegal A, Rescigno M, Savino B, Colombo P, Jonjic N, Pecanic S,

Lazzarato L, Fruttero R, Gasco A, Bronte V, & Viola A (2011) Chemokine

nitration prevents intratumoral infiltration of antigen-specific T cells. J Exp Med

208(10):1949-1962.

106. Sugiura A & Rathmell JC (2018) Metabolic Barriers to T Cell Function in Tumors.

J Immunol 200(2):400-407.

107. Ward PS & Thompson CB (2012) Metabolic reprogramming: a cancer hallmark

even warburg did not anticipate. Cancer Cell 21(3):297-308.

108. Liberti MV & Locasale JW (2016) The Warburg Effect: How Does it Benefit

Cancer Cells? Trends Biochem Sci 41(3):211-218.

109. Jacobs SR, Herman CE, Maciver NJ, Wofford JA, Wieman HL, Hammen JJ, &

Rathmell JC (2008) Glucose uptake is limiting in T cell activation and requires

CD28-mediated Akt-dependent and independent pathways. J Immunol

180(7):4476-4486.

110. Cham CM, Driessens G, O'Keefe JP, & Gajewski TF (2008) Glucose deprivation

inhibits multiple key gene expression events and effector functions in CD8+ T

cells. Eur J Immunol 38(9):2438-2450.

111. Hensley CT, Wasti AT, & DeBerardinis RJ (2013) Glutamine and cancer: cell

biology, physiology, and clinical opportunities. J Clin Invest 123(9):3678-3684.

112. Geiger R, Rieckmann JC, Wolf T, Basso C, Feng Y, Fuhrer T, Kogadeeva M,

Picotti P, Meissner F, Mann M, Zamboni N, Sallusto F, & Lanzavecchia A (2016)

L-Arginine Modulates T Cell Metabolism and Enhances Survival and Anti-tumor

Activity. Cell 167(3):829-842.e813.

155

113. Mocellin S, Bronte V, & Nitti D (2007) Nitric oxide, a double edged sword in

cancer biology: searching for therapeutic opportunities. Med Res Rev 27(3):317-

352.

114. Pilon-Thomas S, Kodumudi KN, El-Kenawi AE, Russell S, Weber AM, Luddy K,

Damaghi M, Wojtkowiak JW, Mulé JJ, Ibrahim-Hashim A, & Gillies RJ (2016)

Neutralization of Tumor Acidity Improves Antitumor Responses to

Immunotherapy. Cancer Res 76(6):1381-1390.

115. Tanizaki Y, Kobayashi A, Toujima S, Shiro M, Mizoguchi M, Mabuchi Y, Yagi S,

Minami S, Takikawa O, & Ino K (2014) Indoleamine 2,3-dioxygenase promotes

peritoneal metastasis of ovarian cancer by inducing an immunosuppressive

environment. Cancer Sci 105(8):966-973.

116. Wang D, Saga Y, Mizukami H, Sato N, Nonaka H, Fujiwara H, Takei Y, Machida

S, Takikawa O, Ozawa K, & Suzuki M (2012) Indoleamine-2,3-dioxygenase, an

immunosuppressive enzyme that inhibits natural killer cell function, as a useful

target for ovarian cancer therapy. Int J Oncol 40(4):929-934.

117. Platten M, Wick W, & Van den Eynde BJ (2012) Tryptophan catabolism in

cancer: beyond IDO and tryptophan depletion. Cancer Res 72(21):5435-5440.

118. Sharma P & Allison JP (2015) The future of immune checkpoint therapy.

Science 348(6230):56-61.

119. Wherry EJ & Kurachi M (2015) Molecular and cellular insights into T cell

exhaustion. Nat Rev Immunol 15(8):486-499.

120. Parsa AT, Waldron JS, Panner A, Crane CA, Parney IF, Barry JJ, Cachola KE,

Murray JC, Tihan T, Jensen MC, Mischel PS, Stokoe D, & Pieper RO (2007)

156

Loss of tumor suppressor PTEN function increases B7-H1 expression and

immunoresistance in glioma. Nat Med 13(1):84-88.

121. Parry RV, Chemnitz JM, Frauwirth KA, Lanfranco AR, Braunstein I, Kobayashi

SV, Linsley PS, Thompson CB, & Riley JL (2005) CTLA-4 and PD-1 receptors

inhibit T-cell activation by distinct mechanisms. Mol Cell Biol 25(21):9543-9553.

122. Qureshi OS, Zheng Y, Nakamura K, Attridge K, Manzotti C, Schmidt EM, Baker

J, Jeffery LE, Kaur S, Briggs Z, Hou TZ, Futter CE, Anderson G, Walker LS, &

Sansom DM (2011) Trans-endocytosis of CD80 and CD86: a molecular basis for

the cell-extrinsic function of CTLA-4. Science 332(6029):600-603.

123. Keir ME, Liang SC, Guleria I, Latchman YE, Qipo A, Albacker LA, Koulmanda M,

Freeman GJ, Sayegh MH, & Sharpe AH (2006) Tissue expression of PD-L1

mediates peripheral T cell tolerance. J Exp Med 203(4):883-895.

124. Peggs KS, Quezada SA, Chambers CA, Korman AJ, & Allison JP (2009)

Blockade of CTLA-4 on both effector and regulatory T cell compartments

contributes to the antitumor activity of anti-CTLA-4 antibodies. J Exp Med

206(8):1717-1725.

125. Isaacs A & Lindenmann J (2015) Pillars Article: Virus Interference. I. The

Interferon. Proc R Soc Lond B Biol Sci. 1957. 147: 258-267. J Immunol

195(5):1911-1920.

126. Castro F, Cardoso AP, Gonçalves RM, Serre K, & Oliveira MJ (2018) Interferon-

Gamma at the Crossroads of Tumor Immune Surveillance or Evasion. Front

Immunol 9:847.

127. Stanifer ML, Pervolaraki K, & Boulant S (2019) Differential Regulation of Type I

and Type III Interferon Signaling. Int J Mol Sci 20(6).

157

128. Kawai T & Akira S (2006) TLR signaling. Cell Death Differ 13(5):816-825.

129. Watanabe T, Asano N, Fichtner-Feigl S, Gorelick PL, Tsuji Y, Matsumoto Y,

Chiba T, Fuss IJ, Kitani A, & Strober W (2010) NOD1 contributes to mouse host

defense against Helicobacter pylori via induction of type I IFN and activation of

the ISGF3 signaling pathway. J Clin Invest 120(5):1645-1662.

130. Loo YM & Gale M, Jr. (2011) Immune signaling by RIG-I-like receptors.

Immunity 34(5):680-692.

131. Gitlin L, Barchet W, Gilfillan S, Cella M, Beutler B, Flavell RA, Diamond MS, &

Colonna M (2006) Essential role of mda-5 in type I IFN responses to

polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus.

Proc Natl Acad Sci U S A 103(22):8459-8464.

132. Wu J, Sun L, Chen X, Du F, Shi H, Chen C, & Chen ZJ (2013) Cyclic GMP-AMP

is an endogenous second messenger in innate immune signaling by cytosolic

DNA. Science 339(6121):826-830.

133. Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, Lu Y, Miyagishi M,

Kodama T, Honda K, Ohba Y, & Taniguchi T (2007) DAI (DLM-1/ZBP1) is a

cytosolic DNA sensor and an activator of innate immune response. Nature

448(7152):501-505.

134. Ivashkiv LB & Donlin LT (2014) Regulation of type I interferon responses. Nat

Rev Immunol 14(1):36-49.

135. Platanias LC (2005) Mechanisms of type-I- and type-II-interferon-mediated

signalling. Nat Rev Immunol 5(5):375-386.

158

136. Hervas-Stubbs S, Perez-Gracia JL, Rouzaut A, Sanmamed MF, Le Bon A, &

Melero I (2011) Direct effects of type I interferons on cells of the immune

system. Clin Cancer Res 17(9):2619-2627.

137. Du Z, Wei L, Murti A, Pfeffer SR, Fan M, Yang CH, & Pfeffer LM (2007) Non-

conventional signal transduction by type 1 interferons: the NF-kappaB pathway.

J Cell Biochem 102(5):1087-1094.

138. Burnet FM (1970) The concept of immunological surveillance. Prog Exp Tumor

Res 13:1-27.

139. Burnet FM (1971) Immunological surveillance in neoplasia. Transplant Rev 7:3-

25.

140. Thomas L (1982) On immunosurveillance in human cancer. Yale J Biol Med

55(3-4):329-333.

141. Dunn GP, Old LJ, & Schreiber RD (2004) The three Es of cancer immunoediting.

Annu Rev Immunol 22:329-360.

142. Dunn GP, Bruce AT, Sheehan KC, Shankaran V, Uppaluri R, Bui JD, Diamond

MS, Koebel CM, Arthur C, White JM, & Schreiber RD (2005) A critical function

for type I interferons in cancer immunoediting. Nat Immunol 6(7):722-729.

143. Shankaran V, Ikeda H, Bruce AT, White JM, Swanson PE, Old LJ, & Schreiber

RD (2001) IFNgamma and lymphocytes prevent primary tumour development

and shape tumour immunogenicity. Nature 410(6832):1107-1111.

144. Koebel CM, Vermi W, Swann JB, Zerafa N, Rodig SJ, Old LJ, Smyth MJ, &

Schreiber RD (2007) Adaptive immunity maintains occult cancer in an

equilibrium state. Nature 450(7171):903-907.

159

145. Edelson BT, Kc W, Juang R, Kohyama M, Benoit LA, Klekotka PA, Moon C,

Albring JC, Ise W, Michael DG, Bhattacharya D, Stappenbeck TS, Holtzman MJ,

Sung SS, Murphy TL, Hildner K, & Murphy KM (2010) Peripheral CD103+

dendritic cells form a unified subset developmentally related to CD8alpha+

conventional dendritic cells. J Exp Med 207(4):823-836.

146. Lorenzi S, Mattei F, Sistigu A, Bracci L, Spadaro F, Sanchez M, Spada M,

Belardelli F, Gabriele L, & Schiavoni G (2011) Type I IFNs control antigen

retention and survival of CD8α(+) dendritic cells after uptake of tumor apoptotic

cells leading to cross-priming. J Immunol 186(9):5142-5150.

147. Montoya M, Schiavoni G, Mattei F, Gresser I, Belardelli F, Borrow P, & Tough

DF (2002) Type I interferons produced by dendritic cells promote their

phenotypic and functional activation. Blood 99(9):3263-3271.

148. Papewalis C, Jacobs B, Wuttke M, Ullrich E, Baehring T, Fenk R, Willenberg

HS, Schinner S, Cohnen M, Seissler J, Zacharowski K, Scherbaum WA, &

Schott M (2008) IFN-alpha skews monocytes into CD56+-expressing dendritic

cells with potent functional activities in vitro and in vivo. J Immunol 180(3):1462-

1470.

149. Zhu Q & Kanneganti TD (2017) Cutting Edge: Distinct Regulatory Mechanisms

Control Proinflammatory Cytokines IL-18 and IL-1β. J Immunol 198(11):4210-

4215.

150. Curtsinger JM, Valenzuela JO, Agarwal P, Lins D, & Mescher MF (2005) Type I

IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and

differentiation. J Immunol 174(8):4465-4469.

160

151. Hervas-Stubbs S, Riezu-Boj JI, Gonzalez I, Mancheño U, Dubrot J, Azpilicueta

A, Gabari I, Palazon A, Aranguren A, Ruiz J, Prieto J, Larrea E, & Melero I

(2010) Effects of IFN-α as a signal-3 cytokine on human naïve and antigen-

experienced CD8(+) T cells. Eur J Immunol 40(12):3389-3402.

152. Kolumam GA, Thomas S, Thompson LJ, Sprent J, & Murali-Krishna K (2005)

Type I interferons act directly on CD8 T cells to allow clonal expansion and

memory formation in response to viral infection. J Exp Med 202(5):637-650.

153. Crouse J, Bedenikovic G, Wiesel M, Ibberson M, Xenarios I, Von Laer D,

Kalinke U, Vivier E, Jonjic S, & Oxenius A (2014) Type I interferons protect T

cells against NK cell attack mediated by the activating receptor NCR1. Immunity

40(6):961-973.

154. Xu HC, Grusdat M, Pandyra AA, Polz R, Huang J, Sharma P, Deenen R, Köhrer

K, Rahbar R, Diefenbach A, Gibbert K, Löhning M, Höcker L, Waibler Z,

Häussinger D, Mak TW, Ohashi PS, Lang KS, & Lang PA (2014) Type I

interferon protects antiviral CD8+ T cells from NK cell cytotoxicity. Immunity

40(6):949-960.

155. Lee CK, Rao DT, Gertner R, Gimeno R, Frey AB, & Levy DE (2000) Distinct

requirements for IFNs and STAT1 in NK cell function. J Immunol 165(7):3571-

3577.

156. Nguyen KB, Salazar-Mather TP, Dalod MY, Van Deusen JB, Wei XQ, Liew FY,

Caligiuri MA, Durbin JE, & Biron CA (2002) Coordinated and distinct roles for

IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral

infection. J Immunol 169(8):4279-4287.

161

157. Swann JB, Hayakawa Y, Zerafa N, Sheehan KC, Scott B, Schreiber RD,

Hertzog P, & Smyth MJ (2007) Type I IFN contributes to NK cell homeostasis,

activation, and antitumor function. J Immunol 178(12):7540-7549.

158. Lucas M, Schachterle W, Oberle K, Aichele P, & Diefenbach A (2007) Dendritic

cells prime natural killer cells by trans-presenting interleukin 15. Immunity

26(4):503-517.

159. Mizutani T, Neugebauer N, Putz EM, Moritz N, Simma O, Zebedin-Brandl E,

Gotthardt D, Warsch W, Eckelhart E, Kantner HP, Kalinke U, Lienenklaus S,

Weiss S, Strobl B, Müller M, Sexl V, & Stoiber D (2012) Conditional IFNAR1

ablation reveals distinct requirements of Type I IFN signaling for NK cell

maturation and tumor surveillance. Oncoimmunology 1(7):1027-1037.

160. Bidwell BN, Slaney CY, Withana NP, Forster S, Cao Y, Loi S, Andrews D,

Mikeska T, Mangan NE, Samarajiwa SA, de Weerd NA, Gould J, Argani P,

Möller A, Smyth MJ, Anderson RL, Hertzog PJ, & Parker BS (2012) Silencing of

Irf7 pathways in breast cancer cells promotes bone metastasis through immune

escape. Nat Med 18(8):1224-1231.

161. Rautela J, Baschuk N, Slaney CY, Jayatilleke KM, Xiao K, Bidwell BN, Lucas

EC, Hawkins ED, Lock P, Wong CS, Chen W, Anderson RL, Hertzog PJ,

Andrews DM, Möller A, & Parker BS (2015) Loss of Host Type-I IFN Signaling

Accelerates Metastasis and Impairs NK-cell Antitumor Function in Multiple

Models of Breast Cancer. Cancer Immunol Res 3(11):1207-1217.

162. Borden EC, Sen GC, Uze G, Silverman RH, Ransohoff RM, Foster GR, & Stark

GR (2007) Interferons at age 50: past, current and future impact on biomedicine.

Nat Rev Drug Discov 6(12):975-990.

162

163. Wheatley K, Ives N, Hancock B, Gore M, Eggermont A, & Suciu S (2003) Does

adjuvant interferon-alpha for high-risk melanoma provide a worthwhile benefit? A

meta-analysis of the randomised trials. Cancer Treat Rev 29(4):241-252.

164. Lindner DJ (2002) Interferons as antiangiogenic agents. Curr Oncol Rep

4(6):510-514.

165. Lamm D, Brausi M, O'Donnell MA, & Witjes JA (2014) Interferon alfa in the

treatment paradigm for non-muscle-invasive bladder cancer. Urol Oncol

32(1):35.e21-30.

166. Weiss K (1998) Safety profile of interferon-alpha therapy. Semin Oncol 25(1

Suppl 1):9-13.

167. Barber GN (2014) STING-dependent cytosolic DNA sensing pathways. Trends

Immunol 35(2):88-93.

168. Gao P, Ascano M, Wu Y, Barchet W, Gaffney BL, Zillinger T, Serganov AA, Liu

Y, Jones RA, Hartmann G, Tuschl T, & Patel DJ (2013) Cyclic [G(2',5')pA(3',5')p]

is the metazoan second messenger produced by DNA-activated cyclic GMP-

AMP synthase. Cell 153(5):1094-1107.

169. Sun L, Wu J, Du F, Chen X, & Chen ZJ (2013) Cyclic GMP-AMP synthase is a

cytosolic DNA sensor that activates the type I interferon pathway. Science

339(6121):786-791.

170. Ishikawa H & Barber GN (2008) STING is an endoplasmic reticulum adaptor that

facilitates innate immune signalling. Nature 455(7213):674-678.

171. Ishikawa H, Ma Z, & Barber GN (2009) STING regulates intracellular DNA-

mediated, type I interferon-dependent innate immunity. Nature 461(7265):788-

792.

163

172. Burdette DL, Monroe KM, Sotelo-Troha K, Iwig JS, Eckert B, Hyodo M,

Hayakawa Y, & Vance RE (2011) STING is a direct innate immune sensor of

cyclic di-GMP. Nature 478(7370):515-518.

173. Woo SR, Fuertes MB, Corrales L, Spranger S, Furdyna MJ, Leung MY, Duggan

R, Wang Y, Barber GN, Fitzgerald KA, Alegre ML, & Gajewski TF (2014)

STING-dependent cytosolic DNA sensing mediates innate immune recognition

of immunogenic tumors. Immunity 41(5):830-842.

174. Dou Z, Ghosh K, Vizioli MG, Zhu J, Sen P, Wangensteen KJ, Simithy J, Lan Y,

Lin Y, Zhou Z, Capell BC, Xu C, Xu M, Kieckhaefer JE, Jiang T, Shoshkes-

Carmel M, Tanim K, Barber GN, Seykora JT, Millar SE, Kaestner KH, Garcia

BA, Adams PD, & Berger SL (2017) Cytoplasmic chromatin triggers

inflammation in senescence and cancer. Nature 550(7676):402-406.

175. Glück S, Guey B, Gulen MF, Wolter K, Kang TW, Schmacke NA, Bridgeman A,

Rehwinkel J, Zender L, & Ablasser A (2017) Innate immune sensing of cytosolic

chromatin fragments through cGAS promotes senescence. Nat Cell Biol

19(9):1061-1070.

176. Yang H, Wang H, Ren J, Chen Q, & Chen ZJ (2017) cGAS is essential for

cellular senescence. Proc Natl Acad Sci U S A 114(23):E4612-e4620.

177. Ranoa DRE, Widau RC, Mallon S, Parekh AD, Nicolae CM, Huang X, Bolt MJ,

Arina A, Parry R, Kron SJ, Moldovan GL, Khodarev NN, & Weichselbaum RR

(2019) STING Promotes Homeostasis via Regulation of Cell Proliferation and

Chromosomal Stability. Cancer Res 79(7):1465-1479.

178. Takashima K, Takeda Y, Oshiumi H, Shime H, Okabe M, Ikawa M, Matsumoto

M, & Seya T (2016) STING in tumor and host cells cooperatively work for NK

164

cell-mediated tumor growth retardation. Biochem Biophys Res Commun

478(4):1764-1771.

179. Ho SS, Zhang WY, Tan NY, Khatoo M, Suter MA, Tripathi S, Cheung FS, Lim

WK, Tan PH, Ngeow J, & Gasser S (2016) The DNA Structure-Specific

Endonuclease MUS81 Mediates DNA Sensor STING-Dependent Host Rejection

of Prostate Cancer Cells. Immunity 44(5):1177-1189.

180. Deng L, Liang H, Xu M, Yang X, Burnette B, Arina A, Li XD, Mauceri H, Beckett

M, Darga T, Huang X, Gajewski TF, Chen ZJ, Fu YX, & Weichselbaum RR

(2014) STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced

Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors.

Immunity 41(5):843-852.

181. Sen T, Rodriguez BL, Chen L, Corte CMD, Morikawa N, Fujimoto J, Cristea S,

Nguyen T, Diao L, Li L, Fan Y, Yang Y, Wang J, Glisson BS, Wistuba, II, Sage J,

Heymach JV, Gibbons DL, & Byers LA (2019) Targeting DNA Damage

Response Promotes Antitumor Immunity through STING-Mediated T-cell

Activation in Small Cell Lung Cancer. Cancer Discov 9(5):646-661.

182. Pantelidou C, Sonzogni O, De Oliveria Taveira M, Mehta AK, Kothari A, Wang

D, Visal T, Li MK, Pinto J, Castrillon JA, Cheney EM, Bouwman P, Jonkers J,

Rottenberg S, Guerriero JL, Wulf GM, & Shapiro GI (2019) PARP Inhibitor

Efficacy Depends on CD8(+) T-cell Recruitment via Intratumoral STING

Pathway Activation in BRCA-Deficient Models of Triple-Negative Breast Cancer.

Cancer Discov 9(6):722-737.

165

183. Wang H, Hu S, Chen X, Shi H, Chen C, Sun L, & Chen ZJ (2017) cGAS is

essential for the antitumor effect of immune checkpoint blockade. Proc Natl

Acad Sci U S A 114(7):1637-1642.

184. Vanpouille-Box C, Alard A, Aryankalayil MJ, Sarfraz Y, Diamond JM, Schneider

RJ, Inghirami G, Coleman CN, Formenti SC, & Demaria S (2017) DNA

exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity. Nat

Commun 8:15618.

185. Ohkuri T, Ghosh A, Kosaka A, Zhu J, Ikeura M, David M, Watkins SC, Sarkar

SN, & Okada H (2014) STING contributes to antiglioma immunity via triggering

type I IFN signals in the tumor microenvironment. Cancer Immunol Res

2(12):1199-1208.

186. Corrales L, Glickman LH, McWhirter SM, Kanne DB, Sivick KE, Katibah GE,

Woo SR, Lemmens E, Banda T, Leong JJ, Metchette K, Dubensky TW, Jr., &

Gajewski TF (2015) Direct Activation of STING in the Tumor Microenvironment

Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep

11(7):1018-1030.

187. Ablasser A, Goldeck M, Cavlar T, Deimling T, Witte G, Röhl I, Hopfner KP,

Ludwig J, & Hornung V (2013) cGAS produces a 2'-5'-linked cyclic dinucleotide

second messenger that activates STING. Nature 498(7454):380-384.

188. Zhang X, Shi H, Wu J, Zhang X, Sun L, Chen C, & Chen ZJ (2013) Cyclic GMP-

AMP containing mixed phosphodiester linkages is an endogenous high-affinity

ligand for STING. Mol Cell 51(2):226-235.

189. Demaria O, De Gassart A, Coso S, Gestermann N, Di Domizio J, Flatz L, Gaide

O, Michielin O, Hwu P, Petrova TV, Martinon F, Modlin RL, Speiser DE, & Gilliet

166

M (2015) STING activation of tumor endothelial cells initiates spontaneous and

therapeutic antitumor immunity. Proc Natl Acad Sci U S A 112(50):15408-15413.

190. Conlon J, Burdette DL, Sharma S, Bhat N, Thompson M, Jiang Z, Rathinam VA,

Monks B, Jin T, Xiao TS, Vogel SN, Vance RE, & Fitzgerald KA (2013) Mouse,

but not human STING, binds and signals in response to the vascular disrupting

agent 5,6-dimethylxanthenone-4-acetic acid. J Immunol 190(10):5216-5225.

191. Anonymous Efficacy and Safety Trial of ADU-S100 and Pembrolizumab in Head

and Neck Cancer. https://ClinicalTrials.gov/show/NCT03937141.

192. Anonymous Safety and Efficacy of MIW815 (ADU-S100) +/- Ipilimumab in

Patients With Advanced/Metastatic Solid Tumors or Lymphomas.

https://ClinicalTrials.gov/show/NCT02675439.

193. Anonymous A Phase I/Ib Study of NIZ985 in Combination With PDR001 in

Adults With Metastatic Cancers. https://ClinicalTrials.gov/show/NCT02452268.

194. Smith TT, Moffett HF, Stephan SB, Opel CF, Dumigan AG, Jiang X, Pillarisetty

VG, Pillai SPS, Wittrup KD, & Stephan MT (2017) Biopolymers codelivering

engineered T cells and STING agonists can eliminate heterogeneous tumors. J

Clin Invest 127(6):2176-2191.

195. Ager CR, Reilley MJ, Nicholas C, Bartkowiak T, Jaiswal AR, & Curran MA

(2017) Intratumoral STING Activation with T-cell Checkpoint Modulation

Generates Systemic Antitumor Immunity. Cancer Immunol Res 5(8):676-684.

196. Le Naour J, Zitvogel L, Galluzzi L, Vacchelli E, & Kroemer G (2020) Trial watch:

STING agonists in cancer therapy. OncoImmunology 9(1):1777624.

197. Ager CR, Zhang H, Wei Z, Jones P, Curran MA, & Di Francesco ME (2019)

Discovery of IACS-8803 and IACS-8779, potent agonists of stimulator of

167

interferon genes (STING) with robust systemic antitumor efficacy. Bioorg Med

Chem Lett 29(20):126640.

198. Sivick KE, Desbien AL, Glickman LH, Reiner GL, Corrales L, Surh NH, Hudson

TE, Vu UT, Francica BJ, Banda T, Katibah GE, Kanne DB, Leong JJ, Metchette

K, Bruml JR, Ndubaku CO, McKenna JM, Feng Y, Zheng L, Bender SL, Cho CY,

Leong ML, van Elsas A, Dubensky TW, Jr., & McWhirter SM (2018) Magnitude

of Therapeutic STING Activation Determines CD8(+) T Cell-Mediated Anti-tumor

Immunity. Cell Rep 25(11):3074-3085.e3075.

199. Anthony SM, Howard ME, Hailemichael Y, Overwijk WW, & Schluns KS (2015)

Soluble interleukin-15 complexes are generated in vivo by type I interferon

dependent and independent pathways. PLoS One 10(3):e0120274.

200. Anthony SM, Rivas SC, Colpitts SL, Howard ME, Stonier SW, & Schluns KS

(2016) Inflammatory Signals Regulate IL-15 in Response to Lymphodepletion. J

Immunol 196(11):4544-4552.

201. Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V,

Beers C, Richardson J, Schoenborn MA, Ahdieh M, & et al. (1994) Cloning of a

T cell growth factor that interacts with the beta chain of the interleukin-2

receptor. Science 264(5161):965-968.

202. Burton JD, Bamford RN, Peters C, Grant AJ, Kurys G, Goldman CK, Brennan J,

Roessler E, & Waldmann TA (1994) A lymphokine, provisionally designated

interleukin T and produced by a human adult T-cell leukemia line, stimulates T-

cell proliferation and the induction of lymphokine-activated killer cells. Proc Natl

Acad Sci U S A 91(11):4935-4939.

168

203. Carson WE, Ross ME, Baiocchi RA, Marien MJ, Boiani N, Grabstein K, &

Caligiuri MA (1995) Endogenous production of interleukin 15 by activated human

monocytes is critical for optimal production of interferon-gamma by natural killer

cells in vitro. J Clin Invest 96(6):2578-2582.

204. Henney CS, Kuribayashi K, Kern DE, & Gillis S (1981) Interleukin-2 augments

natural killer cell activity. Nature 291(5813):335-338.

205. Bamford RN, Grant AJ, Burton JD, Peters C, Kurys G, Goldman CK, Brennan J,

Roessler E, & Waldmann TA (1994) The interleukin (IL) 2 receptor beta chain is

shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-

cell proliferation and the induction of lymphokine-activated killer cells. Proc Natl

Acad Sci U S A 91(11):4940-4944.

206. Giri JG, Ahdieh M, Eisenman J, Shanebeck K, Grabstein K, Kumaki S, Namen

A, Park LS, Cosman D, & Anderson D (1994) Utilization of the beta and gamma

chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J 13(12):2822-

2830.

207. Reinecker HC, MacDermott RP, Mirau S, Dignass A, & Podolsky DK (1996)

Intestinal epithelial cells both express and respond to interleukin 15.

Gastroenterology 111(6):1706-1713.

208. Tagaya Y, Bamford RN, DeFilippis AP, & Waldmann TA (1996) IL-15: a

pleiotropic cytokine with diverse receptor/signaling pathways whose expression

is controlled at multiple levels. Immunity 4(4):329-336.

209. Waldmann TA & Tagaya Y (1999) The multifaceted regulation of interleukin-15

expression and the role of this cytokine in NK cell differentiation and host

response to intracellular pathogens. Annu Rev Immunol 17:19-49.

169

210. Bamford RN, Battiata AP, & Waldmann TA (1996) IL-15: the role of translational

regulation in their expression. J Leukoc Biol 59(4):476-480.

211. Giri JG, Kumaki S, Ahdieh M, Friend DJ, Loomis A, Shanebeck K, DuBose R,

Cosman D, Park LS, & Anderson DM (1995) Identification and cloning of a novel

IL-15 binding protein that is structurally related to the alpha chain of the IL-2

receptor. EMBO J 14(15):3654-3663.

212. Anderson DM, Kumaki S, Ahdieh M, Bertles J, Tometsko M, Loomis A, Giri J,

Copeland NG, Gilbert DJ, Jenkins NA, & et al. (1995) Functional

characterization of the human interleukin-15 receptor alpha chain and close

linkage of IL15RA and IL2RA genes. J Biol Chem 270(50):29862-29869.

213. Johnston JA, Bacon CM, Finbloom DS, Rees RC, Kaplan D, Shibuya K, Ortaldo

JR, Gupta S, Chen YQ, Giri JD, & et al. (1995) Tyrosine phosphorylation and

activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15. Proc

Natl Acad Sci U S A 92(19):8705-8709.

214. Bamford RN, DeFilippis AP, Azimi N, Kurys G, & Waldmann TA (1998) The 5'

untranslated region, signal peptide, and the coding sequence of the carboxyl

terminus of IL-15 participate in its multifaceted translational control. J Immunol

160(9):4418-4426.

215. Onu A, Pohl T, Krause H, & Bulfone-Paus S (1997) Regulation of IL-15 secretion

via the leader peptide of two IL-15 isoforms. J Immunol 158(1):255-262.

216. Schorle H, Holtschke T, Hünig T, Schimpl A, & Horak I (1991) Development and

function of T cells in mice rendered interleukin-2 deficient by gene targeting.

Nature 352(6336):621-624.

170

217. Kundig TM, Schorle H, Bachmann MF, Hengartner H, Zinkernagel RM, & Horak

I (1993) Immune responses in interleukin-2-deficient mice. Science

262(5136):1059-1061.

218. Willerford DM, Chen J, Ferry JA, Davidson L, Ma A, & Alt FW (1995) Interleukin-

2 receptor alpha chain regulates the size and content of the peripheral lymphoid

compartment. Immunity 3(4):521-530.

219. Kennedy MK, Glaccum M, Brown SN, Butz EA, Viney JL, Embers M, Matsuki N,

Charrier K, Sedger L, Willis CR, Brasel K, Morrissey PJ, Stocking K, Schuh JC,

Joyce S, & Peschon JJ (2000) Reversible defects in natural killer and memory

CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191(5):771-780.

220. Lodolce JP, Boone DL, Chai S, Swain RE, Dassopoulos T, Trettin S, & Ma A

(1998) IL-15 receptor maintains lymphoid homeostasis by supporting

lymphocyte homing and proliferation. Immunity 9(5):669-676.

221. Lenardo MJ (1991) Interleukin-2 programs mouse alpha beta T lymphocytes for

apoptosis. Nature 353(6347):858-861.

222. Refaeli Y, Van Parijs L, London CA, Tschopp J, & Abbas AK (1998) Biochemical

mechanisms of IL-2-regulated Fas-mediated T cell apoptosis. Immunity

8(5):615-623.

223. Lio CW & Hsieh CS (2008) A two-step process for thymic regulatory T cell

development. Immunity 28(1):100-111.

224. Fan MY, Low JS, Tanimine N, Finn KK, Priyadharshini B, Germana SK, Kaech

SM, & Turka LA (2018) Differential Roles of IL-2 Signaling in Developing versus

Mature Tregs. Cell Rep 25(5):1204-1213 e1204.

171

225. Marks-Konczalik J, Dubois S, Losi JM, Sabzevari H, Yamada N, Feigenbaum L,

Waldmann TA, & Tagaya Y (2000) IL-2-induced activation-induced cell death is

inhibited in IL-15 transgenic mice. Proc Natl Acad Sci U S A 97(21):11445-

11450.

226. Wu TS, Lee JM, Lai YG, Hsu JC, Tsai CY, Lee YH, & Liao NS (2002) Reduced

expression of Bcl-2 in CD8+ T cells deficient in the IL-15 receptor alpha-chain. J

Immunol 168(2):705-712.

227. Berard M, Brandt K, Bulfone-Paus S, & Tough DF (2003) IL-15 promotes the

survival of naive and memory phenotype CD8+ T cells. J Immunol

170(10):5018-5026.

228. Schluns KS, Williams K, Ma A, Zheng XX, & Lefrancois L (2002) Cutting edge:

requirement for IL-15 in the generation of primary and memory antigen-specific

CD8 T cells. J Immunol 168(10):4827-4831.

229. Carson WE, Fehniger TA, Haldar S, Eckhert K, Lindemann MJ, Lai CF, Croce

CM, Baumann H, & Caligiuri MA (1997) A potential role for interleukin-15 in the

regulation of human natural killer cell survival. J Clin Invest 99(5):937-943.

230. Koka R, Burkett PR, Chien M, Chai S, Chan F, Lodolce JP, Boone DL, & Ma A

(2003) Interleukin (IL)-15R[alpha]-deficient natural killer cells survive in normal

but not IL-15R[alpha]-deficient mice. J Exp Med 197(8):977-984.

231. Lodolce JP, Burkett PR, Boone DL, Chien M, & Ma A (2001) T cell-independent

interleukin 15Ralpha signals are required for bystander proliferation. J Exp Med

194(8):1187-1194.

172

232. Becker TC, Wherry EJ, Boone D, Murali-Krishna K, Antia R, Ma A, & Ahmed R

(2002) Interleukin 15 is required for proliferative renewal of virus-specific

memory CD8 T cells. J Exp Med 195(12):1541-1548.

233. Burkett PR, Koka R, Chien M, Chai S, Chan F, Ma A, & Boone DL (2003) IL-15R

alpha expression on CD8+ T cells is dispensable for T cell memory. Proc Natl

Acad Sci U S A 100(8):4724-4729.

234. Schluns KS, Nowak EC, Cabrera-Hernandez A, Puddington L, Lefrançois L, &

Aguila HL (2004) Distinct cell types control lymphoid subset development by

means of IL-15 and IL-15 receptor alpha expression. Proc Natl Acad Sci U S A

101(15):5616-5621.

235. Dubois S, Mariner J, Waldmann TA, & Tagaya Y (2002) IL-15Ralpha recycles

and presents IL-15 In trans to neighboring cells. Immunity 17(5):537-547.

236. Sandau MM, Schluns KS, Lefrancois L, & Jameson SC (2004) Cutting edge:

transpresentation of IL-15 by bone marrow-derived cells necessitates expression

of IL-15 and IL-15R alpha by the same cells. J Immunol 173(11):6537-6541.

237. Schluns KS, Klonowski KD, & Lefrancois L (2004) Transregulation of memory

CD8 T-cell proliferation by IL-15Ralpha+ bone marrow-derived cells. Blood

103(3):988-994.

238. Bergamaschi C, Rosati M, Jalah R, Valentin A, Kulkarni V, Alicea C, Zhang GM,

Patel V, Felber BK, & Pavlakis GN (2008) Intracellular interaction of interleukin-

15 with its receptor alpha during production leads to mutual stabilization and

increased bioactivity. J Biol Chem 283(7):4189-4199.

173

239. Mortier E, Bernard J, Plet A, & Jacques Y (2004) Natural, proteolytic release of a

soluble form of human IL-15 receptor alpha-chain that behaves as a specific,

high affinity IL-15 antagonist. J Immunol 173(3):1681-1688.

240. Mortier E, Woo T, Advincula R, Gozalo S, & Ma A (2008) IL-15Ralpha

chaperones IL-15 to stable dendritic cell membrane complexes that activate NK

cells via trans presentation. J Exp Med 205(5):1213-1225.

241. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner

BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA,

Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, & Cerretti

DP (1997) A metalloproteinase disintegrin that releases tumour-necrosis factor-

alpha from cells. Nature 385(6618):729-733.

242. Peschon JJ, Slack JL, Reddy P, Stocking KL, Sunnarborg SW, Lee DC, Russell

WE, Castner BJ, Johnson RS, Fitzner JN, Boyce RW, Nelson N, Kozlosky CJ,

Wolfson MF, Rauch CT, Cerretti DP, Paxton RJ, March CJ, & Black RA (1998)

An essential role for ectodomain shedding in mammalian development. Science

282(5392):1281-1284.

243. Edwards DR, Handsley MM, & Pennington CJ (2008) The ADAM

metalloproteinases. Mol Aspects Med 29(5):258-289.

244. Khawam K, Giron-Michel J, Gu Y, Perier A, Giuliani M, Caignard A, Devocelle A,

Ferrini S, Fabbi M, Charpentier B, Ludwig A, Chouaib S, Azzarone B, & Eid P

(2009) Human renal cancer cells express a novel membrane-bound interleukin-

15 that induces, in response to the soluble interleukin-15 receptor alpha chain,

epithelial-to-mesenchymal transition. Cancer Res 69(4):1561-1569.

174

245. Duitman EH, Orinska Z, Bulanova E, Paus R, & Bulfone-Paus S (2008) How a

cytokine is chaperoned through the secretory pathway by complexing with its

own receptor: lessons from interleukin-15 (IL-15)/IL-15 receptor alpha. Mol Cell

Biol 28(15):4851-4861.

246. Castillo EF, Acero LF, Stonier SW, Zhou D, & Schluns KS (2010) Thymic and

peripheral microenvironments differentially mediate development and maturation

of iNKT cells by IL-15 transpresentation. Blood 116(14):2494-2503.

247. Castillo EF, Stonier SW, Frasca L, & Schluns KS (2009) Dendritic cells support

the in vivo development and maintenance of NK cells via IL-15 trans-

presentation. J Immunol 183(8):4948-4956.

248. Stonier SW, Ma LJ, Castillo EF, & Schluns KS (2008) Dendritic cells drive

memory CD8 T-cell homeostasis via IL-15 transpresentation. Blood

112(12):4546-4554.

249. Stonier SW & Schluns KS (2010) Trans-presentation: a novel mechanism

regulating IL-15 delivery and responses. Immunol Lett 127(2):85-92.

250. Ma LJ, Acero LF, Zal T, & Schluns KS (2009) Trans-presentation of IL-15 by

intestinal epithelial cells drives development of CD8alphaalpha IELs. J Immunol

183(2):1044-1054.

251. Mortier E, Advincula R, Kim L, Chmura S, Barrera J, Reizis B, Malynn BA, & Ma

A (2009) Macrophage- and dendritic-cell-derived interleukin-15 receptor alpha

supports homeostasis of distinct CD8+ T cell subsets. Immunity 31(5):811-822.

252. Blauvelt A, Asada H, Klaus-Kovtun V, Altman DJ, Lucey DR, & Katz SI (1996)

Interleukin-15 mRNA is expressed by human keratinocytes Langerhans cells,

175

and blood-derived dendritic cells and is downregulated by ultraviolet B radiation.

J Invest Dermatol 106(5):1047-1052.

253. Weiler M, Rogashev B, Einbinder T, Hausmann MJ, Kaneti J, Chaimovitz C, &

Douvdevani A (1998) Interleukin-15, a leukocyte activator and growth factor, is

produced by cortical tubular epithelial cells. J Am Soc Nephrol 9(7):1194-1201.

254. Ruckert R, Asadullah K, Seifert M, Budagian VM, Arnold R, Trombotto C, Paus

R, & Bulfone-Paus S (2000) Inhibition of keratinocyte apoptosis by IL-15: a new

parameter in the pathogenesis of psoriasis? J Immunol 165(4):2240-2250.

255. Giron-Michel J, Azzi S, Khawam K, Mortier E, Caignard A, Devocelle A, Ferrini

S, Croce M, Francois H, Lecru L, Charpentier B, Chouaib S, Azzarone B, & Eid

P (2012) Interleukin-15 plays a central role in human kidney physiology and

cancer through the gammac signaling pathway. PLoS One 7(2):e31624.

256. Shinozaki M, Hirahashi J, Lebedeva T, Liew FY, Salant DJ, Maron R, & Kelley

VR (2002) IL-15, a survival factor for kidney epithelial cells, counteracts

apoptosis and inflammation during nephritis. Journal of Clinical Investigation

109(7):951-960.

257. Barra NG, Palanivel R, Denou E, Chew MV, Gillgrass A, Walker TD, Kong J,

Richards CD, Jordana M, Collins SM, Trigatti BL, Holloway AC, Raha S,

Steinberg GR, & Ashkar AA (2014) Interleukin-15 modulates adipose tissue by

altering mitochondrial mass and activity. PLoS One 9(12):e114799.

258. Doherty TM, Seder RA, & Sher A (1996) Induction and regulation of IL-15

expression in murine macrophages. J Immunol 156(2):735-741.

176

259. Zhang X, Sun S, Hwang I, Tough DF, & Sprent J (1998) Potent and selective

stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity

8(5):591-599.

260. Neely GG, Robbins SM, Amankwah EK, Epelman S, Wong H, Spurrell JC,

Jandu KK, Zhu W, Fogg DK, Brown CB, & Mody CH (2001) Lipopolysaccharide-

stimulated or granulocyte-macrophage colony-stimulating factor-stimulated

monocytes rapidly express biologically active IL-15 on their cell surface

independent of new protein synthesis. J Immunol 167(9):5011-5017.

261. Mattei F, Schiavoni G, Belardelli F, & Tough DF (2001) IL-15 is expressed by

dendritic cells in response to type I IFN, double-stranded RNA, or

lipopolysaccharide and promotes dendritic cell activation. J Immunol

167(3):1179-1187.

262. Ashkar AA & Rosenthal KL (2003) Interleukin-15 and natural killer and NKT cells

play a critical role in innate protection against genital herpes simplex virus type 2

infection. J Virol 77(18):10168-10171.

263. Perera PY, Lichy JH, Waldmann TA, & Perera LP (2012) The role of interleukin-

15 in inflammation and immune responses to infection: implications for its

therapeutic use. Microbes Infect 14(3):247-261.

264. Oppenheimer-Marks N, Brezinschek RI, Mohamadzadeh M, Vita R, & Lipsky PE

(1998) Interleukin 15 is produced by endothelial cells and increases the

transendothelial migration of T cells In vitro and in the SCID mouse-human

rheumatoid arthritis model In vivo. J Clin Invest 101(6):1261-1272.

177

265. Verbist KC, Rose DL, Cole CJ, Field MB, & Klonowski KD (2012) IL-15

participates in the respiratory innate immune response to influenza virus

infection. PLoS One 7(5):e37539.

266. Verbist KC, Cole CJ, Field MB, & Klonowski KD (2011) A role for IL-15 in the

migration of effector CD8 T cells to the lung airways following influenza infection.

J Immunol 186(1):174-182.

267. Wilkinson PC & Liew FY (1995) Chemoattraction of human blood T lymphocytes

by interleukin-15. J Exp Med 181(3):1255-1259.

268. Allavena P, Giardina G, Bianchi G, & Mantovani A (1997) IL-15 is chemotactic

for natural killer cells and stimulates their adhesion to vascular endothelium. J

Leukoc Biol 61(6):729-735.

269. Perera LP, Goldman CK, & Waldmann TA (1999) IL-15 induces the expression

of chemokines and their receptors in T lymphocytes. J Immunol 162(5):2606-

2612.

270. Fehniger TA & Caligiuri MA (2001) Interleukin 15: biology and relevance to

human disease. Blood 97(1):14-32.

271. Fehniger TA, Suzuki K, Ponnappan A, VanDeusen JB, Cooper MA, Florea SM,

Freud AG, Robinson ML, Durbin J, & Caligiuri MA (2001) Fatal leukemia in

interleukin 15 transgenic mice follows early expansions in natural killer and

memory phenotype CD8+ T cells. J Exp Med 193(2):219-231.

272. McInnes IB, al-Mughales J, Field M, Leung BP, Huang FP, Dixon R, Sturrock

RD, Wilkinson PC, & Liew FY (1996) The role of interleukin-15 in T-cell

migration and activation in rheumatoid arthritis. Nat Med 2(2):175-182.

178

273. Di Sabatino A, Calarota SA, Vidali F, Macdonald TT, & Corazza GR (2011) Role

of IL-15 in immune-mediated and infectious diseases. Cytokine Growth Factor

Rev 22(1):19-33.

274. Bergamaschi C, Bear J, Rosati M, Beach RK, Alicea C, Sowder R, Chertova E,

Rosenberg SA, Felber BK, & Pavlakis GN (2012) Circulating IL-15 exists as

heterodimeric complex with soluble IL-15Ralpha in human and mouse serum.

Blood 120(1):e1-8.

275. Zhang M, Yao Z, Dubois S, Ju W, Müller JR, & Waldmann TA (2009) Interleukin-

15 combined with an anti-CD40 antibody provides enhanced therapeutic efficacy

for murine models of colon cancer. Proc Natl Acad Sci U S A 106(18):7513-

7518.

276. Sikora AG, Jaffarzad N, Hailemichael Y, Gelbard A, Stonier SW, Schluns KS,

Frasca L, Lou Y, Liu C, Andersson HA, Hwu P, & Overwijk WW (2009) IFN-

alpha enhances peptide vaccine-induced CD8+ T cell numbers, effector

function, and antitumor activity. J Immunol 182(12):7398-7407.

277. Colpitts SL, Stoklasek TA, Plumlee CR, Obar JJ, Guo C, & Lefrançois L (2012)

Cutting edge: the role of IFN-α receptor and MyD88 signaling in induction of IL-

15 expression in vivo. J Immunol 188(6):2483-2487.

278. Colpitts SL, Stonier SW, Stoklasek TA, Root SH, Aguila HL, Schluns KS, &

Lefrançois L (2013) Transcriptional regulation of IL-15 expression during

hematopoiesis. J Immunol 191(6):3017-3024.

279. Sosinowski T, White JT, Cross EW, Haluszczak C, Marrack P, Gapin L, & Kedl

RM (2013) CD8alpha+ dendritic cell trans presentation of IL-15 to naive CD8+ T

179

cells produces antigen-inexperienced T cells in the periphery with memory

phenotype and function. J Immunol 190(5):1936-1947.

280. Colpitts SL, Stoklasek TA, Plumlee CR, Obar JJ, Guo C, & Lefrancois L (2012)

Cutting edge: the role of IFN-alpha receptor and MyD88 signaling in induction of

IL-15 expression in vivo. J Immunol 188(6):2483-2487.

281. Santana Carrero RM, Beceren-Braun F, Rivas SC, Hegde SM, Gangadharan A,

Plote D, Pham G, Anthony SM, & Schluns KS (2019) IL-15 is a component of

the inflammatory milieu in the tumor microenvironment promoting antitumor

responses. Proc Natl Acad Sci U S A 116(2):599-608.

282. Golomb HM, Jacobs A, Fefer A, Ozer H, Thompson J, Portlock C, Ratain M,

Golde D, Vardiman J, Burke JS, & et al. (1986) Alpha-2 interferon therapy of

hairy-cell leukemia: a multicenter study of 64 patients. J Clin Oncol 4(6):900-

905.

283. Waldmann TA (2018) Cytokines in Cancer Immunotherapy. Cold Spring Harb

Perspect Biol 10(12).

284. Berraondo P, Sanmamed MF, Ochoa MC, Etxeberria I, Aznar MA, Pérez-Gracia

JL, Rodríguez-Ruiz ME, Ponz-Sarvise M, Castañón E, & Melero I (2019)

Cytokines in clinical cancer immunotherapy. Br J Cancer 120(1):6-15.

285. Fyfe G, Fisher RI, Rosenberg SA, Sznol M, Parkinson DR, & Louie AC (1995)

Results of treatment of 255 patients with metastatic renal cell carcinoma who

received high-dose recombinant interleukin-2 therapy. J Clin Oncol 13(3):688-

696.

286. Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, Abrams J,

Sznol M, Parkinson D, Hawkins M, Paradise C, Kunkel L, & Rosenberg SA

180

(1999) High-dose recombinant interleukin 2 therapy for patients with metastatic

melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin

Oncol 17(7):2105-2116.

287. Munger W, DeJoy SQ, Jeyaseelan R, Sr., Torley LW, Grabstein KH, Eisenmann

J, Paxton R, Cox T, Wick MM, & Kerwar SS (1995) Studies evaluating the

antitumor activity and toxicity of interleukin-15, a new T cell growth factor:

comparison with interleukin-2. Cell Immunol 165(2):289-293.

288. Evans R, Fuller JA, Christianson G, Krupke DM, & Troutt AB (1997) IL-15

mediates anti-tumor effects after cyclophosphamide injection of tumor-bearing

mice and enhances adoptive immunotherapy: the potential role of NK cell

subpopulations. Cell Immunol 179(1):66-73.

289. Klebanoff CA, Finkelstein SE, Surman DR, Lichtman MK, Gattinoni L, Theoret

MR, Grewal N, Spiess PJ, Antony PA, Palmer DC, Tagaya Y, Rosenberg SA,

Waldmann TA, & Restifo NP (2004) IL-15 enhances the in vivo antitumor activity

of tumor-reactive CD8+ T cells. Proc Natl Acad Sci U S A 101(7):1969-1974.

290. Lugli E, Goldman CK, Perera LP, Smedley J, Pung R, Yovandich JL, Creekmore

SP, Waldmann TA, & Roederer M (2010) Transient and persistent effects of IL-

15 on lymphocyte homeostasis in nonhuman primates. Blood 116(17):3238-

3248.

291. Waldmann TA, Lugli E, Roederer M, Perera LP, Smedley JV, Macallister RP,

Goldman CK, Bryant BR, Decker JM, Fleisher TA, Lane HC, Sneller MC,

Kurlander RJ, Kleiner DE, Pletcher JM, Figg WD, Yovandich JL, & Creekmore

SP (2011) Safety (toxicity), pharmacokinetics, immunogenicity, and impact on

181

elements of the normal immune system of recombinant human IL-15 in rhesus

macaques. Blood 117(18):4787-4795.

292. Sneller MC, Kopp WC, Engelke KJ, Yovandich JL, Creekmore SP, Waldmann

TA, & Lane HC (2011) IL-15 administered by continuous infusion to rhesus

macaques induces massive expansion of CD8+ T effector memory population in

peripheral blood. Blood 118(26):6845-6848.

293. Conlon KC, Lugli E, Welles HC, Rosenberg SA, Fojo AT, Morris JC, Fleisher TA,

Dubois SP, Perera LP, Stewart DM, Goldman CK, Bryant BR, Decker JM, Chen

J, Worthy TA, Figg WD, Sr., Peer CJ, Sneller MC, Lane HC, Yovandich JL,

Creekmore SP, Roederer M, & Waldmann TA (2015) Redistribution,

hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine

production during first-in-human clinical trial of recombinant human interleukin-

15 in patients with cancer. J Clin Oncol 33(1):74-82.

294. Miller JS, Morishima C, McNeel DG, Patel MR, Kohrt HEK, Thompson JA,

Sondel PM, Wakelee HA, Disis ML, Kaiser JC, Cheever MA, Streicher H,

Creekmore SP, Waldmann TA, & Conlon KC (2018) A First-in-Human Phase I

Study of Subcutaneous Outpatient Recombinant Human IL15 (rhIL15) in Adults

with Advanced Solid Tumors. Clin Cancer Res 24(7):1525-1535.

295. Epardaud M, Elpek KG, Rubinstein MP, Yonekura AR, Bellemare-Pelletier A,

Bronson R, Hamerman JA, Goldrath AW, & Turley SJ (2008) Interleukin-

15/interleukin-15R alpha complexes promote destruction of established tumors

by reviving tumor-resident CD8+ T cells. Cancer Res 68(8):2972-2983.

182

296. Rubinstein MP, Kovar M, Purton JF, Cho JH, Boyman O, Surh CD, & Sprent J

(2006) Converting IL-15 to a superagonist by binding to soluble IL-15R{alpha}.

Proc Natl Acad Sci U S A 103(24):9166-9171.

297. Stoklasek TA, Schluns KS, & Lefrancois L (2006) Combined IL-15/IL-15Ralpha

immunotherapy maximizes IL-15 activity in vivo. J Immunol 177(9):6072-6080.

298. Dubois S, Patel HJ, Zhang M, Waldmann TA, & Muller JR (2008) Preassociation

of IL-15 with IL-15R alpha-IgG1-Fc enhances its activity on proliferation of NK

and CD8+/CD44high T cells and its antitumor action. J Immunol 180(4):2099-

2106.

299. Elpek KG, Rubinstein MP, Bellemare-Pelletier A, Goldrath AW, & Turley SJ

(2010) Mature natural killer cells with phenotypic and functional alterations

accumulate upon sustained stimulation with IL-15/IL-15Ralpha complexes. Proc

Natl Acad Sci U S A 107(50):21647-21652.

300. Chertova E, Bergamaschi C, Chertov O, Sowder R, Bear J, Roser JD, Beach

RK, Lifson JD, Felber BK, & Pavlakis GN (2013) Characterization and favorable

in vivo properties of heterodimeric soluble IL-15.IL-15Ralpha cytokine compared

to IL-15 monomer. J Biol Chem 288(25):18093-18103.

301. Ng SSM, Nagy BA, Jensen SM, Hu X, Alicea C, Fox BA, Felber BK,

Bergamaschi C, & Pavlakis GN (2017) Heterodimeric IL15 Treatment Enhances

Tumor Infiltration, Persistence, and Effector Functions of Adoptively Transferred

Tumor-specific T Cells in the Absence of Lymphodepletion. Clin Cancer Res

23(11):2817-2830.

302. Wu Z, Xue HH, Bernard J, Zeng R, Issakov D, Bollenbacher-Reilley J, Belyakov

IM, Oh S, Berzofsky JA, & Leonard WJ (2008) The IL-15 receptor {alpha} chain

183

cytoplasmic domain is critical for normal IL-15Ralpha function but is not required

for trans-presentation. Blood 112(12):4411-4419.

303. Wei X, Orchardson M, Gracie JA, Leung BP, Gao B, Guan H, Niedbala W,

Paterson GK, McInnes IB, & Liew FY (2001) The Sushi domain of soluble IL-15

receptor alpha is essential for binding IL-15 and inhibiting inflammatory and

allogenic responses in vitro and in vivo. J Immunol 167(1):277-282.

304. Mortier E, Quemener A, Vusio P, Lorenzen I, Boublik Y, Grotzinger J, Plet A, &

Jacques Y (2006) Soluble interleukin-15 receptor alpha (IL-15R alpha)-sushi as

a selective and potent agonist of IL-15 action through IL-15R beta/gamma.

Hyperagonist IL-15 x IL-15R alpha fusion proteins. J Biol Chem 281(3):1612-

1619.

305. Bessard A, Sole V, Bouchaud G, Quemener A, & Jacques Y (2009) High

antitumor activity of RLI, an interleukin-15 (IL-15)-IL-15 receptor alpha fusion

protein, in metastatic melanoma and colorectal cancer. Mol Cancer Ther

8(9):2736-2745.

306. Desbois M, Le Vu P, Coutzac C, Marcheteau E, Béal C, Terme M, Gey A,

Morisseau S, Teppaz G, Boselli L, Jacques Y, Béchard D, Tartour E, Cassard L,

& Chaput N (2016) IL-15 Trans-Signaling with the Superagonist RLI Promotes

Effector/Memory CD8+ T Cell Responses and Enhances Antitumor Activity of

PD-1 Antagonists. J Immunol 197(1):168-178.

307. Anonymous Study of SO-C101 and SO-C101in Combination With Pembro in

Adult Patients With Advanced/Metastatic Solid Tumors.

https://ClinicalTrials.gov/show/NCT04234113.

184

308. Peter Kirk MA, Thomas Chang, Ping Zhang, Marina Konakova, Katsunobu

Hagihara, Steven Pai, Laurie VanderVeen, Palakshi Obalapur, Peiwen Kuo, Phi

Quach, Lawrence Fong, Deborah H Charych, Jonathan Zalevsky ( 2016 )

NKTR-255: an IL-15-based therapeutic with optimized biological activity and

anti-tumor efficacy. Journal for ImmunoTherapy of Cancer 4:(Suppl 1).

309. Peter Kirk PK, Murali Addepalli, Takahiro Miyazaki, Jane Gunther, Laurie

VanderVeen, Ping Zhang, Palakshi Obalapur, Mekhala Maiti, Amol Murkar,

Arunasree Lanka, Werner Rubas, Jonathan Zalevsky (2017) Pre-clinical efficacy

and tolerability of NKTR-255, a polymer-conjugated IL-15 for immuno-oncology.

Journal for ImmunoTherapy of Cancer 5(87):P332.

310. Anonymous NKTR-255 in Relapsed/Refractory Multiple Myeloma & Non-

Hodgkin Lymphoma. https://ClinicalTrials.gov/show/NCT04136756.

311. Han KP, Zhu X, Liu B, Jeng E, Kong L, Yovandich JL, Vyas VV, Marcus WD,

Chavaillaz PA, Romero CA, Rhode PR, & Wong HC (2011) IL-15:IL-15 receptor

alpha superagonist complex: high-level co-expression in recombinant

mammalian cells, purification and characterization. Cytokine 56(3):804-810.

312. Zhu X, Marcus WD, Xu W, Lee HI, Han K, Egan JO, Yovandich JL, Rhode PR, &

Wong HC (2009) Novel human interleukin-15 agonists. J Immunol 183(6):3598-

3607.

313. Xu W, Jones M, Liu B, Zhu X, Johnson CB, Edwards AC, Kong L, Jeng EK, Han

K, Marcus WD, Rubinstein MP, Rhode PR, & Wong HC (2013) Efficacy and

mechanism-of-action of a novel superagonist interleukin-15: interleukin-15

receptor αSu/Fc fusion complex in syngeneic murine models of multiple

myeloma. Cancer Res 73(10):3075-3086.

185

314. Gomes-Giacoia E, Miyake M, Goodison S, Sriharan A, Zhang G, You L, Egan

JO, Rhode PR, Parker AS, Chai KX, Wong HC, & Rosser CJ (2014) Intravesical

ALT-803 and BCG treatment reduces tumor burden in a carcinogen induced

bladder cancer rat model; a role for cytokine production and NK cell expansion.

PLoS One 9(6):e96705.

315. Kim PS, Kwilas AR, Xu W, Alter S, Jeng EK, Wong HC, Schlom J, & Hodge JW

(2016) IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-

15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and

memory CD8+ T cells, and mediates potent anti-tumor activity against murine

breast and colon carcinomas. Oncotarget 7(13):16130-16145.

316. Mathios D, Park CK, Marcus WD, Alter S, Rhode PR, Jeng EK, Wong HC,

Pardoll DM, & Lim M (2016) Therapeutic administration of IL-15 superagonist

complex ALT-803 leads to long-term survival and durable antitumor immune

response in a murine glioblastoma model. Int J Cancer 138(1):187-194.

317. Rhode PR, Egan JO, Xu W, Hong H, Webb GM, Chen X, Liu B, Zhu X, Wen J,

You L, Kong L, Edwards AC, Han K, Shi S, Alter S, Sacha JB, Jeng EK, Cai W,

& Wong HC (2016) Comparison of the Superagonist Complex, ALT-803, to IL15

as Cancer Immunotherapeutics in Animal Models. Cancer Immunol Res 4(1):49-

60.

318. Rosario M, Liu B, Kong L, Collins LI, Schneider SE, Chen X, Han K, Jeng EK,

Rhode PR, Leong JW, Schappe T, Jewell BA, Keppel CR, Shah K, Hess B,

Romee R, Piwnica-Worms DR, Cashen AF, Bartlett NL, Wong HC, & Fehniger

TA (2016) The IL-15-Based ALT-803 Complex Enhances FcγRIIIa-Triggered NK

186

Cell Responses and In Vivo Clearance of B Cell Lymphomas. Clin Cancer Res

22(3):596-608.

319. Felices M, Chu S, Kodal B, Bendzick L, Ryan C, Lenvik AJ, Boylan KLM, Wong

HC, Skubitz APN, Miller JS, & Geller MA (2017) IL-15 super-agonist (ALT-803)

enhances natural killer (NK) cell function against ovarian cancer. Gynecol Oncol

145(3):453-461.

320. Robinson TO & Schluns KS (2017) The potential and promise of IL-15 in

immuno-oncogenic therapies. Immunol Lett 190:159-168.

321. Institute NC Clinical Trials Using Superagonist Interleukin-15:Interleukin-15

Receptor alphaSu/Fc Fusion Complex ALT-803. https://www.cancer.gov/about-

cancer/treatment/clinical-trials/intervention/C107503 Date Accessed: July 24,

2020.

322. Anonymous A Study of Intravesical BCG in Combination With ALT-803 in

Patients With Non-Muscle Invasive Bladder Cancer.

https://ClinicalTrials.gov/show/NCT02138734.

323. Anonymous QUILT-3.032: A Multicenter Clinical Trial of Intravesical Bacillus

Calmette-Guerin (BCG) in Combination With ALT-803 in Patients With BCG

Unresponsive High Grade Non-Muscle Invasive Bladder Cancer.

https://ClinicalTrials.gov/show/NCT03022825.

324. ImmunityBio (2019) ImmunityBio Granted FDA Breakthrough Therapy

Designation for N-803 IL-15 Superagonist in Non-Muscle Invasive Bladder

Cancer. https://immunitybio.com/immunitybio-granted-fda-breakthrough-therapy-

designation-for-n-803-il-15-superagonist-in-non-muscle-invasive-bladder-cancer/

Date Accessed: July 24, 2020.

187

325. Starnes CO (1992) Coley's toxins in perspective. Nature 357(6373):11-12.

326. Weninger W, Crowley MA, Manjunath N, & von Andrian UH (2001) Migratory

properties of naive, effector, and memory CD8(+) T cells. J Exp Med 194(7):953-

966.

327. Klebanoff CA, Gattinoni L, Torabi-Parizi P, Kerstann K, Cardones AR,

Finkelstein SE, Palmer DC, Antony PA, Hwang ST, Rosenberg SA, Waldmann

TA, & Restifo NP (2005) Central memory self/tumor-reactive CD8+ T cells

confer superior antitumor immunity compared with effector memory T cells. Proc

Natl Acad Sci U S A 102(27):9571-9576.

328. King JW, Thomas S, Corsi F, Gao L, Dina R, Gillmore R, Pigott K, Kaisary A,

Stauss HJ, & Waxman J (2009) IL15 can reverse the unresponsiveness of

Wilms' tumor antigen-specific CTL in patients with prostate cancer. Clin Cancer

Res 15(4):1145-1154.

329. Teague RM, Sather BD, Sacks JA, Huang MZ, Dossett ML, Morimoto J, Tan X,

Sutton SE, Cooke MP, Ohlén C, & Greenberg PD (2006) Interleukin-15 rescues

tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors.

Nat Med 12(3):335-341.

330. Oelert T, Papatriantafyllou M, Pougialis G, Hämmerling GJ, Arnold B, & Schüler

T (2010) Irradiation and IL-15 promote loss of CD8 T-cell tolerance in response

to lymphopenia. Blood 115(11):2196-2202.

331. Hoyos V, Savoldo B, Quintarelli C, Mahendravada A, Zhang M, Vera J, Heslop

HE, Rooney CM, Brenner MK, & Dotti G (2010) Engineering CD19-specific T

lymphocytes with interleukin-15 and a suicide gene to enhance their anti-

lymphoma/leukemia effects and safety. Leukemia 24(6):1160-1170.

188

332. Hurton LV, Singh H, Najjar AM, Switzer KC, Mi T, Maiti S, Olivares S,

Rabinovich B, Huls H, Forget MA, Datar V, Kebriaei P, Lee DA, Champlin RE, &

Cooper LJ (2016) Tethered IL-15 augments antitumor activity and promotes a

stem-cell memory subset in tumor-specific T cells. Proc Natl Acad Sci U S A

113(48):E7788-e7797.

333. Krenciute G, Prinzing BL, Yi Z, Wu MF, Liu H, Dotti G, Balyasnikova IV, &

Gottschalk S (2017) Transgenic Expression of IL15 Improves Antiglioma Activity

of IL13Rα2-CAR T Cells but Results in Antigen Loss Variants. Cancer Immunol

Res 5(7):571-581.

334. Hodgins JJ, Khan ST, Park MM, Auer RC, & Ardolino M (2019) Killers 2.0: NK

cell therapies at the forefront of cancer control. J Clin Invest 129(9):3499-3510.

335. Liu E, Tong Y, Dotti G, Shaim H, Savoldo B, Mukherjee M, Orange J, Wan X, Lu

X, Reynolds A, Gagea M, Banerjee P, Cai R, Bdaiwi MH, Basar R, Muftuoglu M,

Li L, Marin D, Wierda W, Keating M, Champlin R, Shpall E, & Rezvani K (2018)

Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR

show long-term persistence and potent antitumor activity. Leukemia 32(2):520-

531.

336. Cooley S, He F, Bachanova V, Vercellotti GM, DeFor TE, Curtsinger JM,

Robertson P, Grzywacz B, Conlon KC, Waldmann TA, McKenna DH, Blazar BR,

Weisdorf DJ, & Miller JS (2019) First-in-human trial of rhIL-15 and haploidentical

natural killer cell therapy for advanced acute myeloid leukemia. Blood Adv

3(13):1970-1980.

337. Liu E, Marin D, Banerjee P, Macapinlac HA, Thompson P, Basar R, Nassif

Kerbauy L, Overman B, Thall P, Kaplan M, Nandivada V, Kaur I, Nunez Cortes

189

A, Cao K, Daher M, Hosing C, Cohen EN, Kebriaei P, Mehta R, Neelapu S,

Nieto Y, Wang M, Wierda W, Keating M, Champlin R, Shpall EJ, & Rezvani K

(2020) Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid

Tumors. N Engl J Med 382(6):545-553.

338. Mujib S, Jones RB, Lo C, Aidarus N, Clayton K, Sakhdari A, Benko E, Kovacs C,

& Ostrowski MA (2012) Antigen-independent induction of Tim-3 expression on

human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is

associated with proliferation and is dependent on the phosphoinositide 3-kinase

pathway. J Immunol 188(8):3745-3756.

339. Chew GM, Fujita T, Webb GM, Burwitz BJ, Wu HL, Reed JS, Hammond KB,

Clayton KL, Ishii N, Abdel-Mohsen M, Liegler T, Mitchell BI, Hecht FM,

Ostrowski M, Shikuma CM, Hansen SG, Maurer M, Korman AJ, Deeks SG,

Sacha JB, & Ndhlovu LC (2016) TIGIT Marks Exhausted T Cells, Correlates with

Disease Progression, and Serves as a Target for Immune Restoration in HIV

and SIV Infection. PLoS Pathog 12(1):e1005349.

340. Kinter AL, Godbout EJ, McNally JP, Sereti I, Roby GA, O'Shea MA, & Fauci AS

(2008) The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce

the expression of programmed death-1 and its ligands. J Immunol 181(10):6738-

6746.

341. Yu P, Steel JC, Zhang M, Morris JC, Waitz R, Fasso M, Allison JP, & Waldmann

TA (2012) Simultaneous inhibition of two regulatory T-cell subsets enhanced

Interleukin-15 efficacy in a prostate tumor model. Proc Natl Acad Sci U S A

109(16):6187-6192.

190

342. Yu P, Steel JC, Zhang M, Morris JC, & Waldmann TA (2010) Simultaneous

blockade of multiple immune system inhibitory checkpoints enhances antitumor

activity mediated by interleukin-15 in a murine metastatic colon carcinoma

model. Clin Cancer Res 16(24):6019-6028.

343. Zhao M, Luo M, Xie Y, Jiang H, Cagliero C, Li N, Ye H, Wu M, Hao S, Sun T,

Yang H, Zhang M, Lin T, Lu H, & Zhu J (2019) Development of a recombinant

human IL-15·sIL-15Rα/Fc superagonist with improved half-life and its antitumor

activity alone or in combination with PD-1 blockade in mouse model. Biomed

Pharmacother 112:108677.

344. Knudson KM, Hicks KC, Alter S, Schlom J, & Gameiro SR (2019) Mechanisms

involved in IL-15 superagonist enhancement of anti-PD-L1 therapy. J

Immunother Cancer 7(1):82.

345. Wrangle JM, Velcheti V, Patel MR, Garrett-Mayer E, Hill EG, Ravenel JG, Miller

JS, Farhad M, Anderton K, Lindsey K, Taffaro-Neskey M, Sherman C, Suriano

S, Swiderska-Syn M, Sion A, Harris J, Edwards AR, Rytlewski JA, Sanders CM,

Yusko EC, Robinson MD, Krieg C, Redmond WL, Egan JO, Rhode PR, Jeng

EK, Rock AD, Wong HC, & Rubinstein MP (2018) ALT-803, an IL-15

superagonist, in combination with nivolumab in patients with metastatic non-

small cell lung cancer: a non-randomised, open-label, phase 1b trial. Lancet

Oncol 19(5):694-704.

346. Mlecnik B, Bindea G, Angell HK, Sasso MS, Obenauf AC, Fredriksen T,

Lafontaine L, Bilocq AM, Kirilovsky A, Tosolini M, Waldner M, Berger A, Fridman

WH, Rafii A, Valge-Archer V, Pagès F, Speicher MR, & Galon J (2014)

Functional network pipeline reveals genetic determinants associated with in situ

191

lymphocyte proliferation and survival of cancer patients. Sci Transl Med

6(228):228ra237.

347. Bahri R, Pateras IS, D'Orlando O, Goyeneche-Patino DA, Campbell M, Polansky

JK, Sandig H, Papaioannou M, Evangelou K, Foukas PG, Gorgoulis VG, &

Bulfone-Paus S (2015) IL-15 suppresses colitis-associated colon carcinogenesis

by inducing antitumor immunity. Oncoimmunology 4(9):e1002721.

348. Tasaki K, Yoshida Y, Miyauchi M, Maeda T, Takenaga K, Kouzu T, Asano T,

Ochiai T, Sakiyamna S, & Tagawa M (2000) Transduction of murine colon

carcinoma cells with interleukin-15 gene induces antitumor effects in

immunocompetent and immunocompromised hosts. Cancer Gene Ther

7(2):255-261.

349. Hazama S, Noma T, Wang F, Iizuka N, Ogura Y, Yoshimura K, Inoguchi E,

Hakozaki M, Hirose K, Suzuki T, & Oka M (1999) Tumour cells engineered to

secrete interleukin-15 augment anti-tumour immune responses in vivo. Br J

Cancer 80(9):1420-1426.

350. Yajima T, Nishimura H, Wajjwalku W, Harada M, Kuwano H, & Yoshikai Y

(2002) Overexpression of interleukin-15 in vivo enhances antitumor activity

against MHC class I-negative and -positive malignant melanoma through

augmented NK activity and cytotoxic T-cell response. Int J Cancer 99(4):573-

578.

351. Meazza R, Lollini PL, Nanni P, De Giovanni C, Gaggero A, Comes A, Cilli M, Di

Carlo E, Ferrini S, & Musiani P (2000) Gene transfer of a secretable form of IL-

15 in murine adenocarcinoma cells: effects on tumorigenicity, metastatic

potential and immune response. Int J Cancer 87(4):574-581.

192

352. Kobayashi H, Dubois S, Sato N, Sabzevari H, Sakai Y, Waldmann TA, & Tagaya

Y (2005) Role of trans-cellular IL-15 presentation in the activation of NK cell-

mediated killing, which leads to enhanced tumor immunosurveillance. Blood

105(2):721-727.

353. Liu RB, Engels B, Arina A, Schreiber K, Hyjek E, Schietinger A, Binder DC, Butz

E, Krausz T, Rowley DA, Jabri B, & Schreiber H (2012) Densely granulated

murine NK cells eradicate large solid tumors. Cancer Res 72(8):1964-1974.

354. Liu RB, Engels B, Schreiber K, Ciszewski C, Schietinger A, Schreiber H, & Jabri

B (2013) IL-15 in tumor microenvironment causes rejection of large established

tumors by T cells in a noncognate T cell receptor-dependent manner. Proc Natl

Acad Sci U S A 110(20):8158-8163.

355. Rafiq S, Hackett CS, & Brentjens RJ (2020) Engineering strategies to overcome

the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol 17(3):147-167.

356. Vaddepally RK, Kharel P, Pandey R, Garje R, & Chandra AB (2020) Review of

Indications of FDA-Approved Immune Checkpoint Inhibitors per NCCN

Guidelines with the Level of Evidence. Cancers (Basel) 12(3).

357. Leick KM, Pinczewski J, Mauldin IS, Young SJ, Deacon DH, Woods AN,

Bosenberg MW, Engelhard VH, & Slingluff CL, Jr. (2019) Patterns of immune-

cell infiltration in murine models of melanoma: roles of antigen and tissue site in

creating inflamed tumors. Cancer Immunol Immunother 68(7):1121-1132.

358. Tang H, Wang Y, Chlewicki LK, Zhang Y, Guo J, Liang W, Wang J, Wang X, &

Fu YX (2016) Facilitating T Cell Infiltration in Tumor Microenvironment

Overcomes Resistance to PD-L1 Blockade. Cancer Cell 29(3):285-296.

193

359. Taylor MA, Hughes AM, Walton J, Coenen-Stass AML, Magiera L, Mooney L,

Bell S, Staniszewska AD, Sandin LC, Barry ST, Watkins A, Carnevalli LS, &

Hardaker EL (2019) Longitudinal immune characterization of syngeneic tumor

models to enable model selection for immune oncology drug discovery. J

Immunother Cancer 7(1):328.

360. Gajewski TF, Corrales L, Williams J, Horton B, Sivan A, & Spranger S (2017)

Cancer Immunotherapy Targets Based on Understanding the T Cell-Inflamed

Versus Non-T Cell-Inflamed Tumor Microenvironment. Adv Exp Med Biol

1036:19-31.

361. Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, McKee M, &

Gajewski TF (2009) Chemokine expression in melanoma metastases associated

with CD8+ T-cell recruitment. Cancer Res 69(7):3077-3085.

362. Kraman M, Bambrough PJ, Arnold JN, Roberts EW, Magiera L, Jones JO,

Gopinathan A, Tuveson DA, & Fearon DT (2010) Suppression of antitumor

immunity by stromal cells expressing fibroblast activation protein-alpha. Science

330(6005):827-830.

363. Demoulin S, Herfs M, Delvenne P, & Hubert P (2013) Tumor microenvironment

converts plasmacytoid dendritic cells into immunosuppressive/tolerogenic cells:

insight into the molecular mechanisms. J Leukoc Biol 93(3):343-352.

364. Buckanovich RJ, Facciabene A, Kim S, Benencia F, Sasaroli D, Balint K,

Katsaros D, O'Brien-Jenkins A, Gimotty PA, & Coukos G (2008) Endothelin B

receptor mediates the endothelial barrier to T cell homing to tumors and disables

immune therapy. Nat Med 14(1):28-36.

194

365. Spranger S, Luke JJ, Bao R, Zha Y, Hernandez KM, Li Y, Gajewski AP, Andrade

J, & Gajewski TF (2016) Density of immunogenic antigens does not explain the

presence or absence of the T-cell-inflamed tumor microenvironment in

melanoma. Proc Natl Acad Sci U S A 113(48):E7759-e7768.

366. Caton ML, Smith-Raska MR, & Reizis B (2007) Notch-RBP-J signaling controls

the homeostasis of CD8- dendritic cells in the spleen. J Exp Med 204(7):1653-

1664.

367. Clausen BE, Burkhardt C, Reith W, Renkawitz R, & Förster I (1999) Conditional

gene targeting in macrophages and granulocytes using LysMcre mice.

Transgenic Res 8(4):265-277.

368. Sauer JD, Sotelo-Troha K, von Moltke J, Monroe KM, Rae CS, Brubaker SW,

Hyodo M, Hayakawa Y, Woodward JJ, Portnoy DA, & Vance RE (2011) The N-

ethyl-N-nitrosourea-induced Goldenticket mouse mutant reveals an essential

function of Sting in the in vivo interferon response to Listeria monocytogenes

and cyclic dinucleotides. Infect Immun 79(2):688-694.

369. Hohl TM, Rivera A, Lipuma L, Gallegos A, Shi C, Mack M, & Pamer EG (2009)

Inflammatory monocytes facilitate adaptive CD4 T cell responses during

respiratory fungal infection. Cell Host Microbe 6(5):470-481.

370. Müller U, Steinhoff U, Reis LF, Hemmi S, Pavlovic J, Zinkernagel RM, & Aguet

M (1994) Functional role of type I and type II interferons in antiviral defense.

Science 264(5167):1918-1921.

371. Saederup N, Cardona AE, Croft K, Mizutani M, Cotleur AC, Tsou CL, Ransohoff

RM, & Charo IF (2010) Selective chemokine receptor usage by central nervous

195

system myeloid cells in CCR2-red fluorescent protein knock-in mice. PLoS One

5(10):e13693.

372. Lebrec H, Horner MJ, Gorski KS, Tsuji W, Xia D, Pan WJ, Means G, Pietz G, Li

N, Retter M, Shaffer K, Patel N, Narayanan PK, & Butz EA (2013) Homeostasis

of human NK cells is not IL-15 dependent. J Immunol 191(11):5551-5558.

373. Yu P, Bamford RN, & Waldmann TA (2014) IL-15-dependent CD8+ CD122+ T

cells ameliorate experimental autoimmune encephalomyelitis by modulating IL-

17 production by CD4+ T cells. Eur J Immunol 44(11):3330-3341.

374. Curran MA, Geiger TL, Montalvo W, Kim M, Reiner SL, Al-Shamkhani A, Sun

JC, & Allison JP (2013) Systemic 4-1BB activation induces a novel T cell

phenotype driven by high expression of Eomesodermin. J Exp Med 210(4):743-

755.

375. Burkholder B, Huang RY, Burgess R, Luo S, Jones VS, Zhang W, Lv ZQ, Gao

CY, Wang BL, Zhang YM, & Huang RP (2014) Tumor-induced perturbations of

cytokines and immune cell networks. Biochim Biophys Acta 1845(2):182-201.

376. Colpitts SL, Stonier SW, Stoklasek TA, Root SH, Aguila HL, Schluns KS, &

Lefrancois L (2013) Transcriptional regulation of IL-15 expression during

hematopoiesis. J Immunol 191(6):3017-3024.

377. Soudja SM, Ruiz AL, Marie JC, & Lauvau G (2012) Inflammatory monocytes

activate memory CD8(+) T and innate NK lymphocytes independent of cognate

antigen during microbial pathogen invasion. Immunity 37(3):549-562.

378. Yajima T, Yoshihara K, Nakazato K, Kumabe S, Koyasu S, Sad S, Shen H,

Kuwano H, & Yoshikai Y (2006) IL-15 regulates CD8+ T cell contraction during

primary infection. J Immunol 176(1):507-515.

196

379. Tan JT, Dudl E, LeRoy E, Murray R, Sprent J, Weinberg KI, & Surh CD (2001)

IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc Natl

Acad Sci U S A 98(15):8732-8737.

380. Yajima T, Nishimura H, Ishimitsu R, Watase T, Busch DH, Pamer EG, Kuwano

H, & Yoshikai Y (2002) Overexpression of IL-15 in vivo increases antigen-driven

memory CD8+ T cells following a microbe exposure. J Immunol 168(3):1198-

1203.

381. Sandau MM, Kohlmeier JE, Woodland DL, & Jameson SC (2010) IL-15

regulates both quantitative and qualitative features of the memory CD8 T cell

pool. J Immunol 184(1):35-44.

382. Huntington ND, Puthalakath H, Gunn P, Naik E, Michalak EM, Smyth MJ,

Tabarias H, Degli-Esposti MA, Dewson G, Willis SN, Motoyama N, Huang DC,

Nutt SL, Tarlinton DM, & Strasser A (2007) Interleukin 15-mediated survival of

natural killer cells is determined by interactions among Bim, Noxa and Mcl-1. Nat

Immunol 8(8):856-863.

383. Buck MD, Sowell RT, Kaech SM, & Pearce EL (2017) Metabolic Instruction of

Immunity. Cell 169(4):570-586.

384. Kaech SM & Cui W (2012) Transcriptional control of effector and memory CD8+

T cell differentiation. Nat Rev Immunol 12(11):749-761.

385. Waickman AT & Powell JD (2012) mTOR, metabolism, and the regulation of T-

cell differentiation and function. Immunol Rev 249(1):43-58.

386. Laplante M & Sabatini DM (2009) mTOR signaling at a glance. J Cell Sci 122(Pt

20):3589-3594.

197

387. Laplante M & Sabatini DM (2012) mTOR signaling in growth control and

disease. Cell 149(2):274-293.

388. van der Windt GJ, Everts B, Chang CH, Curtis JD, Freitas TC, Amiel E, Pearce

EJ, & Pearce EL (2012) Mitochondrial respiratory capacity is a critical regulator

of CD8+ T cell memory development. Immunity 36(1):68-78.

389. Richer MJ, Pewe LL, Hancox LS, Hartwig SM, Varga SM, & Harty JT (2015)

Inflammatory IL-15 is required for optimal memory T cell responses. J Clin Invest

125(9):3477-3490.

390. Kluger HM, Zito CR, Barr ML, Baine MK, Chiang VL, Sznol M, Rimm DL, Chen

L, & Jilaveanu LB (2015) Characterization of PD-L1 Expression and Associated

T-cell Infiltrates in Metastatic Melanoma Samples from Variable Anatomic Sites.

Clin Cancer Res 21(13):3052-3060.

391. Knudson KM, Hodge JW, Schlom J, & Gameiro SR (2020) Rationale for IL-15

superagonists in cancer immunotherapy. Expert Opin Biol Ther 20(7):705-709.

392. Marabelle A, Tselikas L, de Baere T, & Houot R (2017) Intratumoral

immunotherapy: using the tumor as the remedy. Ann Oncol 28(suppl_12):xii33-

xii43.

393. Park BH, Hwang T, Liu TC, Sze DY, Kim JS, Kwon HC, Oh SY, Han SY, Yoon

JH, Hong SH, Moon A, Speth K, Park C, Ahn YJ, Daneshmand M, Rhee BG,

Pinedo HM, Bell JC, & Kirn DH (2008) Use of a targeted oncolytic poxvirus, JX-

594, in patients with refractory primary or metastatic liver cancer: a phase I trial.

Lancet Oncol 9(6):533-542.

394. Andtbacka RH, Kaufman HL, Collichio F, Amatruda T, Senzer N, Chesney J,

Delman KA, Spitler LE, Puzanov I, Agarwala SS, Milhem M, Cranmer L, Curti B,

198

Lewis K, Ross M, Guthrie T, Linette GP, Daniels GA, Harrington K, Middleton

MR, Miller WH, Jr., Zager JS, Ye Y, Yao B, Li A, Doleman S, VanderWalde A,

Gansert J, & Coffin RS (2015) Talimogene Laherparepvec Improves Durable

Response Rate in Patients With Advanced Melanoma. J Clin Oncol

33(25):2780-2788.

395. Andtbacka RH, Ross M, Puzanov I, Milhem M, Collichio F, Delman KA,

Amatruda T, Zager JS, Cranmer L, Hsueh E, Chen L, Shilkrut M, & Kaufman HL

(2016) Patterns of Clinical Response with Talimogene Laherparepvec (T-VEC)

in Patients with Melanoma Treated in the OPTiM Phase III Clinical Trial. Ann

Surg Oncol 23(13):4169-4177.

396. Heo J, Reid T, Ruo L, Breitbach CJ, Rose S, Bloomston M, Cho M, Lim HY,

Chung HC, Kim CW, Burke J, Lencioni R, Hickman T, Moon A, Lee YS, Kim MK,

Daneshmand M, Dubois K, Longpre L, Ngo M, Rooney C, Bell JC, Rhee BG,

Patt R, Hwang TH, & Kirn DH (2013) Randomized dose-finding clinical trial of

oncolytic immunotherapeutic vaccinia JX-594 in liver cancer. Nat Med 19(3):329-

336.

397. Diab A, Marcondes M, Kotzin B, Tagliaferri MA, Hoch U, Li Y, Cattaruzza F,

Zalevsky J, Brohl AS, Brugarolas J, Bilen MA, Gupta S, Musibay ED, Conley AP,

Borazanci EH, D'Angelo SP, & Curti BD (2019) Phase Ib: Preliminary clinical

activity and immune activation for NKTR-262 [TLR 7/8 agonist] plus NKTR-214

[CD122-biased agonist] in patients (pts) with locally advanced or metastatic solid

tumors (REVEAL Phase Ib/II Trial). Journal of Clinical Oncology 37(8_suppl):26-

26.

199

398. Anonymous A Study of NKTR-262 in Combination With Bempegaldesleukin

(NKTR-214) and With Bempegaldesleukin Plus Nivolumab in Patients With

Locally Advanced or Metastatic Solid Tumor Malignancies.

https://ClinicalTrials.gov/show/NCT03435640.

399. Plote D, Choi W, Mokkapati S, Sundi D, Ferguson JE, 3rd, Duplisea J, Parker

NR, Yla-Herttuala S, Committee SCB, McConkey D, Schluns KS, & Dinney CP

(2019) Inhibition of urothelial carcinoma through targeted type I interferon-

mediated immune activation. Oncoimmunology 8(5):e1577125.

400. Gadkaree SK, Fu J, Sen R, Korrer MJ, Allen C, & Kim YJ (2017) Induction of

tumor regression by intratumoral STING agonists combined with anti-

programmed death-L1 blocking antibody in a preclinical squamous cell

carcinoma model. Head Neck 39(6):1086-1094.

401. Hong WX, Haebe S, Lee AS, Westphalen CB, Norton JA, Jiang W, & Levy R

(2020) Intratumoral Immunotherapy for Early-stage Solid Tumors. Clin Cancer

Res 26(13):3091-3099.

402. Anonymous CMP-001 in Combo With Nivolumab in Stage IIIB/C/D Melanoma

Patients With Clinically Apparent Lymph Node Disease.

https://ClinicalTrials.gov/show/NCT03618641.

403. Anonymous Combined Immunotherapy and Radiosurgery for Metastatic

Colorectal Cancer. https://ClinicalTrials.gov/show/NCT03507699.

404. Anonymous A Study of IMO-2125 in Combination With Ipilimumab Versus

Ipilimumab Alone in Subjects With Anti-PD-1 Refractory Melanoma

(ILLUMINATE-301). https://ClinicalTrials.gov/show/NCT03445533.

200

405. Anonymous Study of the Safety and Efficacy of MIW815 With PDR001 to

Patients With Advanced/Metastatic Solid Tumors or Lymphomas.

https://ClinicalTrials.gov/show/NCT03172936.

406. Anonymous Pembrolizumab +/- SD-101 in Hormone-Naïve Oligometastatic

Prostate Cancer With RT and iADT.

https://ClinicalTrials.gov/show/NCT03007732.

407. Anonymous Clinical Study of CMP-001 in Combination With Pembrolizumab or

as a Monotherapy. https://ClinicalTrials.gov/show/NCT02680184.

408. Anonymous A Study to Assess the Safety and Efficacy of Intratumoral IMO-

2125 in Combination With Ipilimumab or Pembrolizumab in Patients With

Metastatic Melanoma. https://ClinicalTrials.gov/show/NCT02644967.

409. Anonymous Pembrolizumab With or Without Talimogene Laherparepvec or

Talimogene Laherparepvec Placebo in Unresected Melanoma (KEYNOTE-034).

https://ClinicalTrials.gov/show/NCT02263508.

410. Hamid O, Ismail R, & Puzanov I (2020) Intratumoral Immunotherapy-Update

2019. Oncologist 25(3):e423-e438.

411. Aznar MA, Tinari N, Rullán AJ, Sánchez-Paulete AR, Rodriguez-Ruiz ME, &

Melero I (2017) Intratumoral Delivery of Immunotherapy-Act Locally, Think

Globally. J Immunol 198(1):31-39.

412. Sowell RT, Goldufsky JW, Rogozinska M, Quiles Z, Cao Y, Castillo EF,

Finnegan A, & Marzo AL (2017) IL-15 Complexes Induce Migration of Resting

Memory CD8 T Cells into Mucosal Tissues. J Immunol 199(7):2536-2546.

201

413. Brunet JF, Denizot F, Luciani MF, Roux-Dosseto M, Suzan M, Mattei MG, &

Golstein P (1987) A new member of the immunoglobulin superfamily--CTLA-4.

Nature 328(6127):267-270.

414. Ishida Y, Agata Y, Shibahara K, & Honjo T (1992) Induced expression of PD-1, a

novel member of the immunoglobulin gene superfamily, upon programmed cell

death. Embo j 11(11):3887-3895.

415. Krummel MF & Allison JP (1995) CD28 and CTLA-4 have opposing effects on

the response of T cells to stimulation. J Exp Med 182(2):459-465.

416. Leach DR, Krummel MF, & Allison JP (1996) Enhancement of antitumor

immunity by CTLA-4 blockade. Science 271(5256):1734-1736.

417. Yang YF, Zou JP, Mu J, Wijesuriya R, Ono S, Walunas T, Bluestone J, Fujiwara

H, & Hamaoka T (1997) Enhanced induction of antitumor T-cell responses by

cytotoxic T lymphocyte-associated molecule-4 blockade: the effect is manifested

only at the restricted tumor-bearing stages. Cancer Res 57(18):4036-4041.

418. Kwon ED, Hurwitz AA, Foster BA, Madias C, Feldhaus AL, Greenberg NM, Burg

MB, & Allison JP (1997) Manipulation of T cell costimulatory and inhibitory

signals for immunotherapy of prostate cancer. Proc Natl Acad Sci U S A

94(15):8099-8103.

419. Shrikant P, Khoruts A, & Mescher MF (1999) CTLA-4 blockade reverses CD8+ T

cell tolerance to tumor by a CD4+ T cell- and IL-2-dependent mechanism.

Immunity 11(4):483-493.

420. Hurwitz AA, Yu TF, Leach DR, & Allison JP (1998) CTLA-4 blockade synergizes

with tumor-derived granulocyte-macrophage colony-stimulating factor for

202

treatment of an experimental mammary carcinoma. Proc Natl Acad Sci U S A

95(17):10067-10071.

421. Hurwitz AA, Foster BA, Kwon ED, Truong T, Choi EM, Greenberg NM, Burg MB,

& Allison JP (2000) Combination immunotherapy of primary prostate cancer in a

transgenic mouse model using CTLA-4 blockade. Cancer Res 60(9):2444-2448.

422. van Elsas A, Hurwitz AA, & Allison JP (1999) Combination immunotherapy of

B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)

and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing

vaccines induces rejection of subcutaneous and metastatic tumors accompanied

by autoimmune depigmentation. J Exp Med 190(3):355-366.

423. Kwon ED, Foster BA, Hurwitz AA, Madias C, Allison JP, Greenberg NM, & Burg

MB (1999) Elimination of residual metastatic prostate cancer after surgery and

adjunctive cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade

immunotherapy. Proc Natl Acad Sci U S A 96(26):15074-15079.

424. Mokyr MB, Kalinichenko T, Gorelik L, & Bluestone JA (1998) Realization of the

therapeutic potential of CTLA-4 blockade in low-dose chemotherapy-treated

tumor-bearing mice. Cancer Res 58(23):5301-5304.

425. Keler T, Halk E, Vitale L, O'Neill T, Blanset D, Lee S, Srinivasan M, Graziano

RF, Davis T, Lonberg N, & Korman A (2003) Activity and safety of CTLA-4

blockade combined with vaccines in cynomolgus macaques. J Immunol

171(11):6251-6259.

426. Phan GQ, Yang JC, Sherry RM, Hwu P, Topalian SL, Schwartzentruber DJ,

Restifo NP, Haworth LR, Seipp CA, Freezer LJ, Morton KE, Mavroukakis SA,

Duray PH, Steinberg SM, Allison JP, Davis TA, & Rosenberg SA (2003) Cancer

203

regression and autoimmunity induced by cytotoxic T lymphocyte-associated

antigen 4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci U

S A 100(14):8372-8377.

427. Attia P, Phan GQ, Maker AV, Robinson MR, Quezado MM, Yang JC, Sherry

RM, Topalian SL, Kammula US, Royal RE, Restifo NP, Haworth LR, Levy C,

Mavroukakis SA, Nichol G, Yellin MJ, & Rosenberg SA (2005) Autoimmunity

correlates with tumor regression in patients with metastatic melanoma treated

with anti-cytotoxic T-lymphocyte antigen-4. J Clin Oncol 23(25):6043-6053.

428. Maker AV, Attia P, & Rosenberg SA (2005) Analysis of the cellular mechanism

of antitumor responses and autoimmunity in patients treated with CTLA-4

blockade. J Immunol 175(11):7746-7754.

429. Maker AV, Phan GQ, Attia P, Yang JC, Sherry RM, Topalian SL, Kammula US,

Royal RE, Haworth LR, Levy C, Kleiner D, Mavroukakis SA, Yellin M, &

Rosenberg SA (2005) Tumor regression and autoimmunity in patients treated

with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2: a

phase I/II study. Ann Surg Oncol 12(12):1005-1016.

430. Maker AV, Yang JC, Sherry RM, Topalian SL, Kammula US, Royal RE, Hughes

M, Yellin MJ, Haworth LR, Levy C, Allen T, Mavroukakis SA, Attia P, &

Rosenberg SA (2006) Intrapatient dose escalation of anti-CTLA-4 antibody in

patients with metastatic melanoma. J Immunother 29(4):455-463.

431. Ji RR, Chasalow SD, Wang L, Hamid O, Schmidt H, Cogswell J, Alaparthy S,

Berman D, Jure-Kunkel M, Siemers NO, Jackson JR, & Shahabi V (2012) An

immune-active tumor microenvironment favors clinical response to ipilimumab.

Cancer Immunol Immunother 61(7):1019-1031.

204

432. Ayers M, Lunceford J, Nebozhyn M, Murphy E, Loboda A, Kaufman DR, Albright

A, Cheng JD, Kang SP, Shankaran V, Piha-Paul SA, Yearley J, Seiwert TY,

Ribas A, & McClanahan TK (2017) IFN-γ-related mRNA profile predicts clinical

response to PD-1 blockade. J Clin Invest 127(8):2930-2940.

433. Curran MA & Allison JP (2009) Tumor vaccines expressing flt3 ligand synergize

with ctla-4 blockade to reject preimplanted tumors. Cancer Res 69(19):7747-

7755.

434. Curran MA, Montalvo W, Yagita H, & Allison JP (2010) PD-1 and CTLA-4

combination blockade expands infiltrating T cells and reduces regulatory T and

myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A

107(9):4275-4280.

435. Li B, VanRoey M, Wang C, Chen TH, Korman A, & Jooss K (2009) Anti-

programmed death-1 synergizes with granulocyte macrophage colony-

stimulating factor--secreting tumor cell immunotherapy providing therapeutic

benefit to mice with established tumors. Clin Cancer Res 15(5):1623-1634.

436. Pardoll DM (2012) The blockade of immune checkpoints in cancer

immunotherapy. Nat Rev Cancer 12(4):252-264.

437. Fridman WH, Pagès F, Sautès-Fridman C, & Galon J (2012) The immune

contexture in human tumours: impact on clinical outcome. Nat Rev Cancer

12(4):298-306.

438. Bald T, Landsberg J, Lopez-Ramos D, Renn M, Glodde N, Jansen P, Gaffal E,

Steitz J, Tolba R, Kalinke U, Limmer A, Jönsson G, Hölzel M, & Tüting T (2014)

Immune cell-poor melanomas benefit from PD-1 blockade after targeted type I

IFN activation. Cancer Discov 4(6):674-687.

205

439. Buck MD, O'Sullivan D, & Pearce EL (2015) T cell metabolism drives immunity.

J Exp Med 212(9):1345-1360.

440. O'Brien KL & Finlay DK (2019) Immunometabolism and natural killer cell

responses. Nat Rev Immunol 19(5):282-290.

441. O'Neill LA, Kishton RJ, & Rathmell J (2016) A guide to immunometabolism for

immunologists. Nat Rev Immunol 16(9):553-565.

442. Shrestha S, Yang K, Wei J, Karmaus PW, Neale G, & Chi H (2014) Tsc1

promotes the differentiation of memory CD8+ T cells via orchestrating the

transcriptional and metabolic programs. Proc Natl Acad Sci U S A

111(41):14858-14863.

443. Nandagopal N, Ali AK, Komal AK, & Lee SH (2014) The Critical Role of IL-15-

PI3K-mTOR Pathway in Natural Killer Cell Effector Functions. Front Immunol

5:187.

444. Mao Y, van Hoef V, Zhang X, Wennerberg E, Lorent J, Witt K, Masvidal L, Liang

S, Murray S, Larsson O, Kiessling R, & Lundqvist A (2016) IL-15 activates

mTOR and primes stress-activated gene expression leading to prolonged

antitumor capacity of NK cells. Blood 128(11):1475-1489.

445. Boissonnas A, Fetler L, Zeelenberg IS, Hugues S, & Amigorena S (2007) In vivo

imaging of cytotoxic T cell infiltration and elimination of a solid tumor. J Exp Med

204(2):345-356.

446. Anikeeva N, Somersalo K, Sims TN, Thomas VK, Dustin ML, & Sykulev Y

(2005) Distinct role of lymphocyte function-associated antigen-1 in mediating

effective cytolytic activity by cytotoxic T lymphocytes. Proc Natl Acad Sci U S A

102(18):6437-6442.

206

447. Jayaraman A, Jackson DJ, Message SD, Pearson RM, Aniscenko J, Caramori

G, Mallia P, Papi A, Shamji B, Edwards M, Westwick J, Hansel T, Stanciu LA,

Johnston SL, & Bartlett NW (2014) IL-15 complexes induce NK- and T-cell

responses independent of type I IFN signaling during rhinovirus infection.

Mucosal Immunol 7(5):1151-1164.

448. Mikucki ME, Fisher DT, Matsuzaki J, Skitzki JJ, Gaulin NB, Muhitch JB, Ku AW,

Frelinger JG, Odunsi K, Gajewski TF, Luster AD, & Evans SS (2015) Non-

redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking

across tumour vascular checkpoints. Nat Commun 6:7458.

449. Padovan E, Spagnoli GC, Ferrantini M, & Heberer M (2002) IFN-alpha2a

induces IP-10/CXCL10 and MIG/CXCL9 production in monocyte-derived

dendritic cells and enhances their capacity to attract and stimulate CD8+ effector

T cells. J Leukoc Biol 71(4):669-676.

450. Guirnalda P, Wood L, Goenka R, Crespo J, & Paterson Y (2013) Interferon γ-

induced intratumoral expression of CXCL9 alters the local distribution of T cells

following immunotherapy with Listeria monocytogenes. Oncoimmunology

2(8):e25752.

451. Mulligan AM, Raitman I, Feeley L, Pinnaduwage D, Nguyen LT, O'Malley FP,

Ohashi PS, & Andrulis IL (2013) Tumoral lymphocytic infiltration and expression

of the chemokine CXCL10 in breast cancers from the Ontario Familial Breast

Cancer Registry. Clin Cancer Res 19(2):336-346.

452. Bergamaschi C, Pandit H, Nagy BA, Stellas D, Jensen SM, Bear J, Cam M,

Valentin A, Fox BA, Felber BK, & Pavlakis GN (2020) Heterodimeric IL-15

delays tumor growth and promotes intratumoral CTL and dendritic cell

207

accumulation by a cytokine network involving XCL1, IFN-γ, CXCL9 and

CXCL10. J Immunother Cancer 8(1).

453. Okada S, Han S, Patel ES, Yang LJ, & Chang LJ (2015) STAT3 signaling

contributes to the high effector activities of interleukin-15-derived dendritic cells.

Immunol Cell Biol 93(5):461-471.

454. Van Acker HH, Beretta O, Anguille S, De Caluwé L, Papagna A, Van den Bergh

JM, Willemen Y, Goossens H, Berneman ZN, Van Tendeloo VF, Smits EL, Foti

M, & Lion E (2017) Desirable cytolytic immune effector cell recruitment by

interleukin-15 dendritic cells. Oncotarget 8(8):13652-13665.

455. Norii M, Yamamura M, Iwahashi M, Ueno A, Yamana J, & Makino H (2006)

Selective recruitment of CXCR3+ and CCR5+ CCR4+ T cells into synovial tissue

in patients with rheumatoid arthritis. Acta Med Okayama 60(3):149-157.

456. Milling L, Zhang Y, & Irvine DJ (2017) Delivering safer immunotherapies for

cancer. Adv Drug Deliv Rev 114:79-101.

457. Marabelle A, Andtbacka R, Harrington K, Melero I, Leidner R, de Baere T,

Robert C, Ascierto PA, Baurain JF, Imperiale M, Rahimian S, Tersago D,

Klumper E, Hendriks M, Kumar R, Stern M, Öhrling K, Massacesi C, Tchakov I,

Tse A, Douillard JY, Tabernero J, Haanen J, & Brody J (2018) Starting the fight

in the tumor: expert recommendations for the development of human

intratumoral immunotherapy (HIT-IT). Ann Oncol 29(11):2163-2174.

458. Hanes J, Sills A, Zhao Z, Suh KW, Tyler B, DiMeco F, Brat DJ, Choti MA, Leong

KW, Pardoll DM, & Brem H (2001) Controlled local delivery of interleukin-2 by

biodegradable polymers protects animals from experimental brain tumors and

liver tumors. Pharm Res 18(7):899-906.

208

459. Kwong B, Liu H, & Irvine DJ (2011) Induction of potent anti-tumor responses

while eliminating systemic side effects via liposome-anchored combinatorial

immunotherapy. Biomaterials 32(22):5134-5147.

460. Fransen MF, van der Sluis TC, Ossendorp F, Arens R, & Melief CJ (2013)

Controlled local delivery of CTLA-4 blocking antibody induces CD8+ T-cell-

dependent tumor eradication and decreases risk of toxic side effects. Clin

Cancer Res 19(19):5381-5389.

461. Hori Y, Stern PJ, Hynes RO, & Irvine DJ (2009) Engulfing tumors with synthetic

extracellular matrices for cancer immunotherapy. Biomaterials 30(35):6757-

6767.

462. Wang C, Ye Y, Hochu GM, Sadeghifar H, & Gu Z (2016) Enhanced Cancer

Immunotherapy by Microneedle Patch-Assisted Delivery of Anti-PD1 Antibody.

Nano Lett 16(4):2334-2340.

463. Zitvogel L, Pitt JM, Daillère R, Smyth MJ, & Kroemer G (2016) Mouse models in

oncoimmunology. Nat Rev Cancer 16(12):759-773.

464. Olson B, Li Y, Lin Y, Liu ET, & Patnaik A (2018) Mouse Models for Cancer

Immunotherapy Research. Cancer Discov 8(11):1358-1365.

209

VITA

Rosa Maria Santana Carrero was born in Mayaguez, Puerto Rico in 1989, the daughter of Jose L. Santana and Rosalia Carrero. After completing her work at Ines Maria

Mendoza, Cabo Rojo, Puerto Rico in 2007, she entered The University of

Puerto Rico at Mayaguez (UPRM), Mayaguez, Puerto Rico. She received the degree of Bachelor of Science with a major in industrial biotechnology from

UPRM in June 2012. During her undergraduate studies Rosa performed research in the laboratory of Dr. Carlos Rios Velazquez studying radiation- resistant bacteria and she was part of the Minority Access to Research

Careers (MARC) program. In August of 2015 she joined The University of

Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical

Sciences.

Permanent Address:

4000 Willowrun Ln

Arlington, TX 76013

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