Roles of IL‐2 in bridging adaptive and innate immunity, and as a tool for cellular immunotherapy

Abstract IL‐2 was initially characterized as a T cell growth factor in the 1970s, and has been studied intensively ever since. Decades of research have revealed multiple and diverse roles for this potent cytokine, indicating a unique linking role between adaptive and innate arms of the immune system. Here, we review the literature showing that IL‐2 is expressed in a plethora of cell types across the immune system, where it has indispensable functions in orchestrating cellular interactions and shaping the nature and magnitude of immune responses. Emerging from the basic research that has revealed the molecular mechanisms and the complexity of the biologic actions of IL‐2, several immunotherapeutic approaches have now focused on manipulating the levels of this cytokine in patients. These strategies range from inhibition of IL‐2 to achieve immunosuppression, to the application of IL‐2 as a vaccine adjuvant and in cancer therapies. This review will systematically summarize the major findings in the field and identify key areas requiring further research in order to realize the potential of IL‐2 in the treatment of human diseases.


INTRODUCTION
IL-2 was originally identified in the 1970's as the first T cell growth factor. 1,2 As a result of this key property, it has been intensively researched ever since: thousands of papers now describe the details of IL-2's molecular and cellular biology (reviewed in Refs. 3,4 and others). What has emerged from these studies is a complex picture that extends well beyond the limited scope of a prototypical T cell growth factor; in fact, IL-2 occupies a central position in all immune responses and during homeostasis, being produced by, and acting upon, a plethora of cell types, with its effects determined by source, target, dose, and context. Our understanding of IL-2's roles is underpinned by early studies that elucidated its molecular mechanism of expression in T cells, which led to the pivotal discovery of the NFAT family of transcrip-Abbreviations: AML, acute myeloid leukemia; CAR, chimeric antigen receptor; CN, calcineurin; CsA, cyclosporine A; DC, dendritic cell; GVHD, graft-versus-host disease; TIL, tumor-infiltrating lymphocytes; Treg, T regulatory cells. transcription factors including NF-kB, 12 13 or T-bet 12 to promote IL-2 expression in different cell types. In contrast, in T regulatory cells (Treg), the binding of NFAT together with the FoxP3 transcription factor represses IL-2 expression, 14,15 explaining the dependence of Treg on IL-2 from other cellular sources. Altogether these molecular interactions aim to assure balanced expression of IL-2 in innate and adaptive immune cells, which is essential for orchestrating an optimal immune response.
One reason that the discovery of NFAT as a molecular regulator of IL-2 signaling was so important is that it led to a means to target IL-2 expression and achieve clinical immunosuppression. It was first shown that inhibition of the phosphatase activity of CN using the fungal isolate cyclosporine A (CsA) or the synthetic inhibitor FK506 resulted in impaired NFAT signaling, 8,16 leading to reduced expression of IL-2, and an efficient immunosuppressive effect. 16 These immunosuppressive therapeutic approaches have since revolutionized the field of organ transplantation, reducing the rejection of solid grafts, 17 and limiting graft-versus-host disease (GVHD) in hematopoietic stem cell transplant recipients. 18,19 The success of CN inhibitors in the transplantation setting led to trials and eventual widespread use of CsA and FK506 in other pathologies, including psoriasis, eczema, rheumatoid arthritis, and Crohn's disease. 20 However, simultaneously, the recognition of the importance of IL-2 for maintaining immune-suppressive Treg has led to the somewhat counterintuitive strategy of treating some autoimmune conditions with low doses of exogenous IL-2 summarized in Table 1.
Together, these opposing strategies demonstrate the complexity of IL-2's roles across the immune system, and the ongoing challenges of understanding this enigmatic cytokine.
Alongside progress in understanding NFAT-mediated signaling leading to IL-2 production, a further layer of complexity has more recently become apparent from studies of IL-2's downstream signaling in target cells. Complex molecular mechanisms control cellular sensitivity to IL-2. One mechanism is through variable subunit composition of the heterotrimeric IL-2 receptor (IL-2R), whose affinity for the cytokine is determined by the combinations of the IL-2R (CD25), IL-2R (CD122), and the common gamma or c (CD132) chains. 3,54 The "low affinity" IL-2R is formed only by IL-2R , whereas the "intermediate affinity IL-2R" consists of IL-2R and c , and the "high affinity IL-2R" is comprised of 1 of each of the 3 chains. 55 The expression of different versions of the IL-2R on responder cells (also shown in Therefore, although the first role of IL-2 in T cells was described 50 years ago, advances in our understanding of the regulation of its production and signaling, and how it might be used effectively and precisely to modulate patients' immune systems, are still very much a topic of ongoing research. This review will examine recent findings in these areas, with particular emphasis on IL-2's emerging roles in the crosstalk between innate and adaptive immune cells, and its current and future uses in clinical immunotherapy.

Roles of IL-2 in orchestrating adaptive immunity
IL-2 is a major modulator of the development, homeostasis and functions of various T cell subsets, and therefore has key role in orchestrating the balance of adaptive immune responsiveness. It has long been known that in the thymus IL-2 fuels the initial proliferation of naïve T cells 59 and is essential for maturation of Treg. 60 At the same time, IL-2 is also responsible for the expansion and cytotoxicity of effector T cells. 56 What remains debated is which cell types in the thymus are the key cellular sources of IL-2 for the different lymphocyte subtypes.
For example, in the case of murine Treg, while 1 study showed that DCderived IL-2 was important for their development in an ex vivo thymic slice model, 61 another group using IL-15 −/− mice with il-2 also deleted in T cells, B cells and DC, reported that only T cell-derived IL-2, and not IL-2 from B cells or DC, was essential for Treg development in the thymus in vivo. 62 In the periphery, IL-2 is a master regulator of T cell biology. Effector T cells are the main producers of IL-2 that they use for autocrine stimulation of their own proliferation, cytotoxicity, and the downstream development of memory T cells. 63 T cell homeostasis also relies on paracrine IL-2 signaling. 64 Interestingly, studies on human DC have revealed their ability to capture and present either DC-or T-cell produced IL-2 at the immunologic synapse in order to stimulate antigen-specific T cell proliferation. 65 These findings highlight a novel mechanism by which even extremely small amounts of IL-2 can be critical for the initiation of immune responses by acting, quite literally, as a molecular bridge/connection between the effector cells of the innate and adaptive arms of immunity.
Although the roles of IL-2 in stimulating immune responses are well known, early studies in mice lacking IL-2 or its or receptor chains also uncovered the role of IL-2 in preventing autoimmunity, 66 Selective Tregs responses to low IL-2 through IL-2-dependent transcriptional amplification mechanism.
Alopecia areata 31 Low IL-2 led to increased Treg count. No adverse event was reported.
Rheumatoid arthritis 25 Low IL-2 induced Treg expansion and activation without effector T cell activation.
Crohn's disease, ulcerative colitis 25 Low IL-2 induced Treg expansion and activation without effector T cell activation.
Transplantations Graft-versus-host disease [32][33][34][35] Low IL-2 administration was associated with preferential, sustained expansion of functional Tregs (while maintaining the immune response to infections) resulting in reduced chronic GVHD. IL-2 restores homeostasis of CD4 + T cell subsets through selective increase of Stat5 phosphorylation in Tregs and a decrease of phosphorylated Stat5 in conventional CD4 + T cells.

Inflammatory condition
Chronic kidney disease (CKD) 36 Low Treg count is lower in CKD patients. IL-2 selectively expanded CD4 + High IL-2 therapy displayed durable response and antitumor activity in some patient with metastatic melanoma. Selective inhibition of IL-2-mediated enhancement of T may be beneficial for IL-2 therapy.
Renal cancer 45,46 High IL-2 therapy displayed durable response in subset of patients. Selective inhibition of IL-2-mediated enhancement of T may be beneficial for IL-2 therapy.

Myeloid cells and IL-2
Although IL-2 was long-considered purely a T cell cytokine, there is clear evidence that functional calcium-NFAT signaling also occurs within some myeloid cell subsets, as reviewed. 20,76,77 Activation of the CN-NFAT pathway was first described in DC in response to whole bacteria or LPS, 78,79 and since, also in response to stimulation with the fungal components zymosan, 80 or curdlan. 81 These findings led others to investigate the activation of the CN-NFAT pathway in macrophages, which was found to be stimulated upon phagocytosis of fungal conidia, 82,83 and in human macrophages by exposure to Aspergillus fumigatus. 84 Intensive research followed these initial findings, aiming to establish the molecular mechanisms of NFAT activation in myeloid cells.
Together, they revealed multiple pathways leading to CN-NFAT signaling: in murine macrophages and DC, Dectin-1 ligation by yeast or zymosan particles resulted in NFAT activation 85 ; whereas murine DC exposed to LPS or whole bacteria showed CN-NFAT activation followed by IL-2 expression that relied on TLR4 ligation. 78,79 Later studies refined this work by showing that CD14 was capable of mediating bacterial ligand-induced CN-NFAT activation alone in these cells. 86 Furthermore, the interaction of TLR-

NK cells and IL-2
Immune surveillance provided by NK cells is a key mechanism to eliminate infected or cancerous cells. NK cell expansion, maturation, activity, and cytotoxicity are strongly dependent on levels of IL-2. 54,93,94 Although NK cells are able to express their own IL-2 upon activation of the CN-NFAT pathway, 95 other reports have described their dependence on IL-2 produced from T cells, 96 and, more recently, on DCderived IL-2 for activation of IFN-production. 97,98 IL-2 also sits at the crossroads of innate NK cell regulation and the regulation of Treg from the adaptive arm of the immune system.
In NK cells, the absence of various chains of the IL-2 receptor, or the lack of IL-2, results in impaired NK cell homeostasis. 56 In particular, CD127 + immature NK cells expand in an IL-2-dependent manner, which is strongly inhibited by presence of Treg due to competition for IL-2; accordingly, depletion of Treg results in expansion of NK cell numbers. 99 Mechanistically, CD127 + NK cells are thought to compete for IL-2 binding with Treg via expression of the high affinity IL-2R , whereas the IL-2R c chain, which recognizes IL-2, IL-15, and IL-21, mediates the multiple facets of NK cell activation, maturation, and proliferation that are modulated by these closely-related cytokines. 100,101

IL-2 IN IMMUNOTHERAPY
The prominent role of IL-2 in T cell stimulation led to it being the first human cytokine employed therapeutically. Almost 4 decades ago, IL-2 was used with some success to treat cancer, 102 and today, researchers continue to dissect its importance in this disease, with a recent study showing that single nucleotide polymorphisms in the IL-2 gene are associated with colorectal cancer prognosis. 103 However, the range of conditions that IL-2 is used to help treat stretches beyond cancer to

Direct uses of IL-2 in vivo or in combination with adoptive cell transfer protocols
Intense research and numerous clinical trials culminated, in 1992, in the approval of IL-2 infusion as the first licensed immunotherapy for the treatment of renal cancer. 102 Since then many clinical trials testing IL-2's ability, either alone or in combination with other therapeutic approaches, to treat a range of conditions have been conducted or are ongoing (Fig. 2). Although high-dose IL-2 remains a good treatment option for a subgroup of metastatic renal cancer and melanoma patients, its more widespread use has been limited by its toxicity, relatively short half-life, and variability in patient responses. 104 Despite this, therapy related mortality remains very low, and IL-2 treatment can offer significant improvements to survival, particularly in patients with metastatic melanoma. 43 Although high-dose IL-2 therapy has mostly been used to target late-stage cancers, low-dose infusion therapies were developed to promote Treg expansion and thereby treat autoimmunity and disorders of inflammation. Following promising data from experimental models, the first clinical studies in 2011 confirmed that low-doses of IL-2 were effective in treating GVHD and hepatitis virus C-induced vasculitis via their proliferative effects on Treg. 32,26 This proven potential leads to ongoing research into ways to optimize IL-2 therapy, by manipulating, for example, dosage or route of administration, or via the administration of engineered IL-2 derivatives (see trails listed in Table 1 and strategies illustrated in Fig. 2). An important development has been the generation of protocols for the production of clinical grade recombinant fusion proteins. This allowed the first progress towards extending IL-2's half-life, by using an engineered IL-2 analog with improved binding and activator function. 105 The same technology also permitted the investigation of strategies targeting IL-2 to specific cells at the site of action. For example, a fusion molecule of a fragment of diphtheria toxin conjugated to IL-2 (Ontak R ) or its improved equivalent E7777 has been investigated for its effectiveness in Treg depletion in clinical trials. 106,107 In this case, when the fusion protein is bound and internalized by cells expressing IL2-R , the diphtheria toxin is released from acidic vesicles into the cytoplasm where it inhibits protein synthesis, leading to subsequent cell death. Nevertheless, the safety, efficacy as well as exact effect of this agent on immune cells remain to be elucidated.
Other novel approaches include administration of IL-2 in immune complexes. These have been used to activate the immune system during chronic viral infections, such as HIV or herpes, where the IL-2 therapy resulted in increased numbers of Th cells. [40][41][42] Recent reports also describe the successful combination of IL-2 treatment with cell cycle checkpoint inhibitors, which was able to overcome previous resistance to these drugs in patients with advanced melanoma, 108 paving the way for further studies on the adjuvant use of IL-2 to improve responses to existing chemotherapeutic agents.
In summary, IL-2 continues to be used and explored as part of the therapeutic protocol for a large number of diseases (Table 1); however, its success has until now been limited due to a short in vivo halflife, its toxicity, and the cytokine's ability to amplify Treg. More recent studies have focused on how to overcome these drawbacks, and have identified the use of engineered forms of IL-2, 109,110 and its combination with traditional chemotherapeutic drugs 108 as highly promising strategies. A pressing area in need of further work is how to understand which patients will benefit most from IL-2 immunotherapies, and the mechanisms underlying variable responses to these treatments.

Uses of IL-2 in cellular immunotherapy expansion protocols
Together with the success of IL-2 direct infusion therapies, this cytokine is also an indispensable tool for the in vitro expansion and activation of T cells including chimeric antigen receptor (CAR) T cells,  120 This approach is paving the way for the use of genetically-modified NK cells in the clinic.
As well as NK cells, T cells also have the potential to express strong antitumor activities. 121

CONCLUSIONS AND PERSPECTIVES
It is a testament to IL-2's importance and enduring intrigue that it has been so actively researched for the past 50 years, and we are still making new discoveries even now. Here, we have highlighted the roles of IL-2 in adaptive and innate immunity, and at their intersection, confirming IL-2 as a key factor in the maintenance of immune homeostasis across multiple cell types (Fig. 1). Furthermore, many of these findings have now been successfully translated into effective immunotherapies. The overview of currently running clinical studies assures that IL-2 use in immunotherapy will be further expanding in the future, most probably broadening amount of clinical protocols and well as targeted disorders.

AUTHORSHIP
KB wrote manuscript and prepared the figures, JF conceptualized and wrote manuscript.

DISCLOSURES
The authors declare no conflicts of interest.
authors would like to thank Dr Lucy Robinson of Insight Editing London for reviewing and editing the manuscript prior to submission.