Common gamma chain cytokines and CD8 T cells in cancer

Seminars in Immunology 42 (2019) 101307

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Review

Common gamma chain cytokines and CD8 T cells in cancer Mitra Shourian

a,b,1

c,d,1

, Jean-Christophe Beltra

a

, Benoîte Bourdin , Hélène Decaluwe

a,b,e,⁎

T

a

Cytokines and Adaptive Immunity Laboratory, CHU Sainte-Justine Research Center, Montreal, Quebec, Canada Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, USA d Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA e Immunology and Rheumatology Division, Department of Pediatrics, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada b c

ARTICLE INFO

ABSTRACT

Keywords: Cytokines Exhaustion Immune Checkpoint Adoptive Cell Therapy Immunotherapy Signaling JAK STAT PI3K

Overcoming exhaustion-associated dysfunctions and generating antigen-specific CD8 T cells with the ability to persist in the host and mediate effective long-term anti-tumor immunity is the final aim of cancer immunotherapy. To achieve this goal, immuno-modulatory properties of the common gamma-chain (γc) family of cytokines, that includes IL-2, IL-7, IL-15 and IL-21, have been used to fine-tune and/or complement current immunotherapeutic protocols. These agents potentiate CD8 T cell expansion and functions particularly in the context of immune checkpoint (IC) blockade, shape their differentiation, improve their persistence in vivo and alternatively, influence distinct aspects of the T cell exhaustion program. Despite these properties, the intrinsic impact of cytokines on CD8 T cell exhaustion has remained largely unexplored impeding optimal therapeutic use of these agents. In this review, we will discuss current knowledge regarding the influence of relevant γc cytokines on CD8 T cell differentiation and function based on clinical data and preclinical studies in murine models of cancer and chronic viral infection. We will restate the place of these agents in current immunotherapeutic regimens such as IC checkpoint blockade and adoptive cell therapy. Finally, we will discuss how γc cytokine signaling pathways regulate T cell immunity during cancer and whether targeting these pathways may sustain an effective and durable T cell response in patients.

1. Introduction For many years, first line treatments for cancers were based on surgery, radiation or chemotherapy. Efforts are now turning into immunotherapeutic strategies with the aim of re-arming our own immune system for efficient elimination of malignant cells. Targeting immune checkpoint (IC) molecules (i.e. PD-1, CTLA-4) efficiently revitalizes anti-tumor adaptive immunity leading to an unprecedent increase in objective response rates in a growing diversity of human cancers [1]. Adoptive cell therapies (ACT) consisting in infusion of autologous tumor reactive T cells expanded ex vivo or harboring a Chimeric Antigen Receptor directed against tumor antigens (CAR T cells) also represent promising avenues to treat solid and hematologic malignancies

[2,3]. Despite outstanding clinical results, the frequency of patients relapsing after immunotherapy remains elevated while a substantial fraction of patients appear to be refractory to immuno-modulatory agents. Limiting factors such as exhaustion of tumor-reactive CD8 T cells, lack of persistence of infused material in vivo and limited antitumor cytotoxicity hinder the efficacy of ACT and IC blockade and needs to be overcomed [4,5]. Fine-tuning combinatorial immunotherapeutic approaches and improving in vitro expansion protocols to achieve long-term protection in patients has become the new challenge. To this regard, cytokine properties notably that of the common gamma-chain (γc) family including interleukin-2 (IL-2), IL‐7, IL-15 and IL‐21 have been exploited to bypass limitations and improve the efficacy of cancer

Abbreviations: ACT, adoptive cell therapy; AICD, activation-induced cell death; AKT, protein kinase B; CAR, chimeric antigen receptor; FOXO1, forkhead box O1; GSK3, glycogen synthase kinase 3; IC, immune checkpoint; IFN, interferon; IL, interleukin; JAK, janus kinase; LCMV, lymphocytic choriomeningitis virus; MAPK, mitogen-activated protein kinase; MPEC, memory precursor effector cells; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositide-3 kinase; SLEC, shortlived effector cells; STAT, signal transducer and activator of transcription; TCM, central memory T cell; TEM, effector memory T cell; TEX, exhausted T cell; TSCM, stem cell memory T cells; TCR, T cell receptor; TF, transcription factor; TIL, tumor infiltrating lymphocyte; TME, tumor microenvironment; TNF, tumor necrosis factor; Tyk2, tyrosine kinase 2; γc, gamma chain ⁎ Corresponding author at: CHU Sainte-Justine, 3175 Cote-Sainte-Catherine, Montreal, Quebec, H3T 1C5, Canada. E-mail address: [email protected] (H. Decaluwe). 1 The authors contributed equally to the work. https://doi.org/10.1016/j.smim.2019.101307 Received 19 April 2019 1044-5323/ © 2019 Elsevier Ltd. All rights reserved.

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immunotherapy [6,7]. These cytokines signal through the JAK–STAT and PI3K-AKT-mTOR pathways, are powerful growth and survival factors for T cells and promote theircytotoxic functions [8–11]. Signals provided by these agents also direct the differentiation and cell-fate decisions of activated T cells. This offers the unique opportunity to orient anti-tumor T cell differentiation during ex vivo expansion of cells for ACT to achieve better persistence and tumor control in vivo. For instance, combining IL-21 with IL-7 and IL-15 allows development and expansion of stem cell memory T cells (TSCM), a T-cell subtype with superior anti-tumor efficacy and self-renewal potential [16–18]. Yet, γc cytokines remain pleiotropic factors with context-dependent impact on T cell development [19]. Indeed, while IL-2 supports effector CD8 T cell differentiation and acquisition of effector functions in mouse models of acute infection, this cytokine in combination with IL-15 direct some aspects of T cell exhaustion including the expression of specific IC in chronic settings [20,21]. Recent studies also suggest divergent usage of intracellular pathways by γc cytokines following acute or persistent antigenic stimulation coupled with alterations of these pathways due to distinct availability of suppressor of cytokine signaling (SOCS) proteins [22,23]. Getting a better understanding of the impact of these crucial cytokines in contexts of persistent antigen stimulation is now needed to optimize the use of these agents for cancer immunotherapy. In this review, we will discuss current knowledge regarding the therapeutic potential and limitations for using γc cytokines in current anti-cancer immunotherapeutic protocols including IC blockade and ACT with a particular focus on the impact of these agents on CD8 T cells. We will also discuss how γc cytokine signaling pathways regulate T cell immunity during cancer and how targeting these pathways may sustain effective T cell responses and memory development in patients with cancer.

Divergent expression of these TFs within TEX cells notably TCF1, Eomes and T-bet distinguishes TEX subsets with inherent properties. TCF1 identifies TEX progenitors cells (TCF1+; CXCR5+Tim-3−), a specific population of TEX that resides in lymphoid tissues, retain some aspects associated with memory and Tfh cells and selectively respond to PD-L1 blockade [39,40]. This progenitor population converts to TCF1-negative terminally exhausted cells (TCF1−; CXCR5−Tim-3+) and this irreversible conversion is accompanied by increased Eomes levels at the expense of T-bet and increased susceptibility to apoptosis [39,43,287]. Continuous dynamic conversion of TEX progenitors to terminally exhausted cells is critical to maintain CD8 T cell responses during chronic viral infections and abrogation of either subset severely compromises responses [43]. Several factors intrinsically coordinate the establishment of exhaustion in CD8 T cells including persistent antigen stimulation, lack of co-stimulatory signals, the presence of regulatory cell types and the microenvironment including cytokines [49]. Cytokine signals govern CD8 T cell-fate decisions during acute viral infections. Recent work also enlighten a critical role for these soluble agents in controlling key aspects of T cell exhaustion [19,20]. The implication of cytokines and cytokine-dependent signaling pathways on T cell exhaustion development and persistence is an area of intense investigation. 2.2. CD8 T cell exhaustion: lessons from pre-clinical cancer models and human cancers Hallmarks of CD8 T cell exhaustion defined using the LCMV system have been reported on TILs from pre-clinical mouse tumor models and various human cancers [50–53]. TILs from numerous tumors express PD-1 but also several other IC molecules including CTLA-4, TIM-3, LAG3 or TIGIT [51,54–56]. High expression of PD-1 but also the ectoenzyme CD39 (ENTPD1) that selectively marks terminally exhausted cells in human and mouse chronic viral infections also identify tumor-reactive cells in the tumor microenvironment (TME) [51,57,58]. Similar to exhausted CD8 T cells in the LCMV system, the capacity of TILs to secrete cytokines (IFNγ, TNFα, IL-2) is severely compromised and the intensity and diversity of IRs expression correlates with the level of dysfunction in human and mouse tumors [51,53,54]. Integrative transcriptomic and epigenetic analyses comparing murine naïve, effector, memory and exhausted CD8 T cells allowed establishment of an exhaustion-specific gene signature that was largely enriched in CD8 T cells from HIV progressors and TILs from patients with melanoma [55]. Consistent with the idea of a common exhaustion developmental program during chronic viral infections and cancers, tumor-specific TCF1+ progenitors and TCF1− terminally exhausted cells were also evidenced in mouse and human tumors [53,59]. Tumor reactive TCF1+ CD8 T cells were more polyfunctional, conserved proliferative potential, were less apoptotic and converted to TCF1− terminally exhausted cells reminiscent of their biology in the LCMV system [39,40,53]. TCF1+ and TCF1− TILs also selectively shared epigenetic modules with their counterparts in the LCMV model [53]. This further highlights similarities in the developmental program of TEX subsets across pathologies. Albeit, transcriptional and epigenetic differences between TEX subsets subsist across chronic diseases demonstrating that factors inherent to a specific pathology (notably the highly immunosuppressive TME in the context of cancers) impacts TEX cell biology [53,55,60]. Of outmost importance, targeting ICs (i.e. PD-1, CTLA-4) in mouse and several human cancers revigorates TILs and improves patient outcome [50,53,61]. This demonstrates that parts of the biology of TEX cells notably the negative impact of ICs on T cell functions is transposable across pathologies and organisms. Hence, while IC blockade represents a key strategy to overthrow some exhaustion-associated dysfunctions, durable immune protection is not achieve in a majority of patients [50]. This underlines the need for combinatorial approaches capable of amplifying and sustaining the tremendous response elicited by checkpoint blockade therapy.

2. Exhaustion: A limiting factor for CD8 T cell function and persistence 2.1. CD8 T cell exhaustion: lessons from chronic lymphocytic choriomeningitis virus (LCMV) infection Exhaustion of CD8 T cells often occurs during chronic viral infections (HIV, HCV, HBV) and cancers [24,25]. First described as a gradual loss of functionnality by CD8 T cells facing chronic antigenic stimulation [26,31], it is now well-established that exhaustion represents a unique developmental branch of CD8 T cells differentiation, distinct from the canonical pathway toward effector and memory cells development. Indeed, exhausted CD8 T cells (TEX) acquire inherent transcriptional and epigenetic signatures [26–28,288,289,290] and once fully commited, TEX cannot retro-convert into prototypical effector or memory cells [291]. Recent studies identified the High-Mobility Group (HMG)-box protein TOX as the lineage-defining transcription factor (TF) essential for the establishment of exhaustion [292–296]. Features inherent to TEX development involve a gradual restriction of effector functions as compared to effector or memory cells. This includes an early loss of IL-2 and tumor necrosis factor α (TNFα) production, a reduced cytotoxicity, and a stepwise reduction of interferon-γ (IFNγ) secretion, proliferative potential and survival [26,31,32]. This limited pattern of effector functions largely imparts to the up-regulation of several inhibitory receptors (IRs) also referred as checkpoint molecules (PD-1, TIM-3, 2B4, LAG-3, Tigit, CD160, CTLA-4) [26,33,34]. Indeed, TEX loss of functions increases with the level of expression and the diversity of IRs present at the cell surface and blockade of these receptors notably the canonical PD-1/PD-L1 pathway temporarily revigorates TEX cells [27,32,33]. Despite restricted effector functions, TEX are critical for controling viral spread or tumor growth and establishing a balance between disease control and immunopathology [35,36]. Comparative analysis of TEX with conventional effector or memory cells evidenced TFs with key functions during exhaustion development including TCF1, Blimp-1, T-bet, Eomes, NFAT, Nr4a, IRF4, c-MAF, and BATF [39–48]. 2

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2.3. CD8 T cell exhaustion in adoptive cell therapies

[11,82]. IL-2 was identified decades ago as a powerful growth factor, promoting survival and expansion of T cells in vitro. Shortly after, the observation was made that cultivating tumor resections from murine or human tumors in the presence of IL-2 generated nearly pure cultures of TILs with heightened cytotoxicity against autologous tumor antigens [83–86]. Since, a variety of clinical trials were attempted using IL-2 as a monotherapy or as a tool to expand autologous tumor reactive T cells or T cells redirected against various tumor antigens for subsequent infusion to patients [87]. More recently, IL-2 has also been evaluated as a combinatorial agent to checkpoint blockade therapy [15,88–90]. Despite significant rates of objective and even complete responses, therapeutic use of IL-2 remains limited by the severe toxicity associated with infusion of this cytokine in patients, the pleiotropic stimulatory and suppressive effects of this cytokine on T cell responses and its impact on T cell terminal differentiation and exhaustion. The identification of IL-2 as the main factor promoting expansion and development of autologous immune cells with heightened antitumor cytotoxicity in vitro led to rapid translation of this cytokine to cancer therapy. Monotherapy by infusion of IL-2 was first attempted in preclinical cancer models. In rodents, infusion of IL-2 reduced established lung metastasis derived from various sarcomas or B16 melanoma cells [91]. IL-2 administration also reduced the growth of established subcutaneous tumors even leading to complete regression in some animals [91]. From this study, it is noteworthy that the beneficial impact of IL-2 monotherapy was only achieved at high dose regimens [91]. These encouraging results were rapidly translated to human through multiple clinical trials involving patients with various metastatic cancers [92–100]. These trials confirmed that IL-2 had to be administered repeatedly due to very short lifespan in vivo (7 min) and at high dose regimens to reach significant beneficial effects in patients [92,97]. Complete responses were often durable as > 80% of cured patients remained disease free up to 148 months post onset of therapy [101]. However, grade 3 and 4 toxicity that included hypotension, cardiac ischemia, vascular leak syndrome and sepsis were a clear limitation of IL-2-based therapy [97]. Despite improvement in the management of these unwanted side effects, the toxicity associated with IL-2 therapy remains a serious hindrance for therapeutic use of this cytokine. IL-2 is also extensively used for ex-vivo expansion of TILs or CAR T cells for ACT and is administered to patients to improve the proliferation and maintenance of adoptively transferred T cells [3]. TILs massively expand in IL-2 and conserve cytotoxicity directed against autologous tumor antigens. This was first described in mouse models of cancer and re-infusion of TILs expanded in IL-2 mediated rejection of liver and pulmonary metastasis more efficiently than IL-2 alone [83]. Of importance, non-myeloablative pre-conditioning regimen before TILs infusion and continuous administration of IL-2 in vivo post-transfer were essential for the persistence of transferred cells [84]. This protocol was rapidly translated to patients with various metastatic cancers through several clinical trials and achieved impressive rates of objective responses (up to 72%) [102–106]. Limitations to this therapeutic success included IL-2-mediated terminal differentiation of TILs expanded in vitro. This was reminiscent of the impact of IL-2-derived signals in promoting terminal differentiation of activated CD8 T cells at the expense of memory development [21,107]. Indeed, TILs from younger cultures and/or T cells exhibiting central memory and stem cell memory features were correlated with better persistence in vivo and improved tumor control [12,103,108,109]. IL-2 also causes antigen-induced cell death (AICD), promotes terminal exhaustion, suppresses memory development and controls the expression of specific inhibitory receptors (i.e. 2B4, Tim-3) that together also likely limit the persistence and function of transferred cells in vivo (Fig. 1) [20,21,110]. Optimizing in vitro protocols for TILs expansion to substantiate for IL-2 and accommodating for the generation of sufficient number of cells while conserving stem cell properties is an area of intense investigation. For instance, ex-vivo expansion of TILs or CAR T cells using cytokine cocktails or combinations of IL-15/

Defective sustainability and exhaustion of CD8 T cells is also an important limitation of ACT [4,62]. ACT relies on the isolation and in vitro expansion of TILs derived from tumor lesions or the ex vivo generation of genetically-engineered cells bearing specific T-cell receptors (TCRs) or CAR constructs prior to their re-infusion into patients [2,3]. For effective expansion, cells must be driven into cell division by an appropriate cytokine cocktail. Standard protocols include priming and rapid expansion phases, in which antigen-specific or anti-CD3 priming is complemented by medium to high doses of IL-2 to sustain T cell expansion. Further, exogenous administration of cytokines to patients is often used to complement adoptive cell transfer. Unfortunately, sustained TCR stimulation and cytokine-driven proliferation are known drivers of terminal T cell differentiation and exhaustion in chronic viral infection [19,63]. In consequence, one of the main hindrances in ACT is the failure to limit T-cell dysfunction and exhaustion following standard ex vivo expansion protocols. A cytokine such as IL-2 does indeed support T cell proliferation and expansion but to the detriment of increased IC expression at the surface of tumor-specific T cells. Further, cytokine stimulation supports the development of terminally differentiated cells with poor survival and decreased sustainability. In vivo, it may also act on other cell types, to induce the secretion of immunosuppressive cytokines (IL-10, TGFβ) and the proliferation and expansion of immunosuppressive cells (Treg, MDSC), further limiting efficient antitumor T cell responses. These factors collectively contribute to the reduced efficacy of ACT in vivo, especially in the setting of solid tumors [60,64]. Several studies have shown a linear correlation between the differentiation status of infused cells and the magnitude of tumor regression. TSCM are memory cells that share the CD44low CD62Lhigh phenotype with naïve T cells but are distinguishable by the co-expression of the memory markers CD95 and higher levels of the β chain of the IL-2 and IL-15 receptor (IL2Rβ) [65,66]. They have been shown to induce superior tumor regression compared to central memory cells (TCM) and effector memory cells (TEM), and are associated with improved therapeutic responses including targeting solid cancers [67]. Notably, less differentiated memory cells, being either TSCM or TCM, have the capacity to persist in vivo, sustaining long-term control of the tumor and limiting potential relapses [68–71]. Interestingly, it has been reported that γc cytokines, in particular IL-7 and IL-21, expand and sustain less-differentiated TSCM [16,72–74]. Thus, the capacity of γc-cytokines to improve T cell expansion, survival and orient their differentiation in vitro toward suitable cell-type for ACT renders these agents extremely attractive for cancer immunotherapy. Yet, recent studies including by our group showed that sustained cytokine signaling may limit effective T cell functions as it may impact on the T cell exhaustion program of the cells [19,20,75]. A better understanding of their role during ACT is critical to efficiently use these molecules for cancer immunotherapy. 3. Common gamma-chain cytokines: pros and cons in cancer immunotherapy 3.1. IL-2 IL-2 is predominately produced by activated CD4 T cells and to a lesser extent CD8 T cells, NK cells and NKT cells [76–78]. IL-2 binds with low affinity to its cognate IL-2 receptor alpha-chain (IL2Rα; CD25) which triggers recruitment of the IL-2Rβ (CD122) and IL-2Rγ (CD132) chains and formation of the trimeric high affinity IL-2 receptor. IL-2 also binds with intermediate affinity to the IL2-Rβγ complex in the absence of the IL2-Rα-chain. Engagement of the IL-2 receptor activates JAK1 and JAK3 with subsequent phosphorylation of STAT proteins predominantly STAT5A and STAT5B but also STAT1 and STAT3 (Fig. 1) [11,79–81]. IL-2 signaling also elicits the P13K-AKT and RAS-MAPK (mitogen-activated protein kinase) signaling pathways in T cells (Fig. 2) 3

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Fig. 1. Impact of the γc family of cytokines IL-2, IL-7, IL-15 and IL-21 on CD8 T cells during cancer. A. Structural diagram of the specific cytokine receptors and their JAK/STAT signaling molecules in response to IL2, IL-15, IL-21, and IL-7. Each cytokine binds its specific heteromeric receptor composed of two or three chains: a specific α chain, the common γ chain and the β chain for IL-2 and IL-15. Cytokine ligation leads to the recruitment and phosphorylation of JAK1 and JAK3, and the subsequent dimerization and phosphorylation of specific STATs in the cytosol. IL-2, IL-15 and IL-7 predominantly activate STAT5 while IL-21 activates STAT3. The STAT dimers translocate to the nucleus to activate or repress gene expression. B. Graphical representation (heatmap) of the physiologic impact of IL-2, IL-7, IL-15 and IL-21 on the survival, homeostasis and antigen-specific proliferation, differentiation, and expression of effector molecules and inhibitory receptors of CD8 T cells. The color key indicates that each cytokine limits/reduces (blue) or sustains (pink) the different parameters mentioned. Note that IL-21, and more modestly IL-7, limit the expression of inhibitory receptors and the terminal differentiation of CD8 T cells during cancer. Star indicates memory CD8 T cells, and empty circle indicates naïve CD8 T cells. TEM: effector memory T cell, TCM: central memory T cell TEX: exhausted T cell, MPEC: memory precursor effector cells, SLEC: short-lived effector cells.

IL-21 or IL-15/IL-7 are superior to IL-2 in driving T cells with less-differentiated phenotype, greater cytotoxic activity and more stem-like phenotype (see below) [111,112]. These cells displayed enhanced persistence and improved antitumor activity when transferred into humanized mouse models [4,113,114]. IL-2 also provides essential signals for the development and suppressive functions of Foxp3+ regulatory T cells (Tregs) [115,116]. Ex vivo expansion of TILs in IL-2 and in vivo infusion of the cytokine promotes Treg cells development, a parameter often negatively correlated with clinical responses [117]. Anti-cancer therapeutic strategies using IL-2 have demonstrated the curative potential of this cytokine. Effort are now turning into strategies to redirect IL-2 signals on effector T cells, preventing off target effects notably on Treg cells. Development of IL-2/IL-2Ab complexes that preferentially binds the intermediate affinity IL-2Rβγ complex allows IL-2 signals to be directed to T cells while limiting its impact on Treg cells on which constitutive expression of the IL-2Rα-chain provides higher sensitivity to native form of IL-2 [118–121]. A recent study also developed synthetic IL-2/IL-2Rβ pairs selectively interacting with each other while being insensitive to their native counterparts [122]. When expressed on T cells, the synthetic IL-2Rβ-chain (orthoIL-2Rβ) selectively responds to infusion of synthetic IL-2 (orthoIL-2) preventing off target effect in vivo on Treg cells. This strategy demonstrated efficiency at controlling tumors in a preclinical mouse model. Developing T cells

genetically modified to secrete IL-2 was also attempted in mice and humans to bypass the need for IL-2 infusion after adoptive transfer of expanded TILs [123–125]. These cells demonstrated higher antitumor potential in mice and could expand in vitro without exogenous IL-2. However, in vivo persistence of these IL-2 secreting T cells in patients was limited along with clinical effectiveness. Lastly, a new generation of CAR T cells was engineered to express a truncated version of the IL2Rβ intracytoplasmic domain capable of activating STAT5 and STAT3 under TCR signaling [126]. These CARs demonstrated higher persistence in vivo and superior effectiveness in eradicating not only liquid but also solid tumors in a preclinical model. Combining IL-2 therapy with checkpoint blockade was also tested. Neutralization of CTLA-4 elicits strong antitumor responses in preclinical mouse models and blocking this receptor in humans with specific antibodies (ipilimumab) promotes tumor regression and extends survival of patients with established metastatic melanoma [127–129]. CTLA-4 restrains T-cell responses by competing with CD28 for binding to B7.1/2 and blockade of this receptor also restrains Treg cell number and function [24,130–132]. De facto, the hypothesis was made that blocking CTLA-4 may complement IL-2 therapy by counteracting IL-2mediated accumulation of Treg cells. Despite synergistic effects of rIL-2 + CTLA-4 blockade in a preclinical cancer model, no synergistic effect was reported in patients [88,89]. Alternatively, IL-2 therapy showed 4

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great synergistic effects with PD-L1 blockade in a mouse model of chronic viral infection. This combination promoted tremendous accumulation of Ag-specific CD8 T cells and decreased viral loads more efficiently than either therapy alone (IL-2 or PD-L1 alone) [15]. Albeit IL-2 still increased the number of activated Treg cells even in the context of PD-L1 blockade, potential suppressive impact of these cells was overridden by the strong CD8 T cell response elicited by the combinatorial therapy [15]. Concomitantly, rIL-2 + anti-PD-1 mediated regression of large established tumors in a preclinical mouse model in combination with antibodies targeting tumor antigens and vaccination protocol [90]. Together, IL-2 demonstrated great therapeutic potential in various settings. However, its capacity to promote T cell terminal differentiation and exhaustion is a critical limiting factor and must be carefully considered. A better appreciation of the cellular response elicited by IL-2 notably in a context of chronic antigen stimulation will help to optimize the therapeutic use of this agent.

It was originally noticed that the better persistence of TILs expanded ex vivo achieved after lymphodepletion was dependent on increased circulating levels of IL-7 and IL-15 [148]. This observation coupled with the ability of IL-15 to promote proliferation and survival of memory CD8 T cells, NK cells and mount cytotoxic functions without causing suppressive effects often associated with the use of IL-2 prompted rapid evaluation of the anticancer efficacy of this cytokine [149–152]. Administration of exogenous IL-15 delayed tumor growth in a variety of preclinical tumor models including melanoma (pmel-1), colon adenocarcinoma (CT26) or lung adenocarcinoma (LA795) and in the later, IL15 even outcompeted IL-2 (at similar dosage) at controlling tumors and increasing survival rates [153–156]. In one study, depletion of CD8 T cells largely reduced the beneficial impact of IL-15 demonstrating the implication of this cell type together with NK cells in IL-15-mediated therapy [154]. The administration of recombinant IL-15 was subsequently evaluated in patients. A phase I clinical trial in patients with metastatic melanoma and renal cell carcinoma determined that IL-15 could be delivered without unwanted adverse effects [157]. Administration of IL-15 caused a significant increase in the frequency of circulating NK, γδ and CD8 memory T cells as expected from initial preclinical studies. However, albeit some patients were able to clear lung lesions, few objective responses were reported across multiple clinical trials [157]. Several parameters likely accounted for these disappointing results. First, reminiscent of issues discussed above with IL2, IL-15 had a short lifespan in vivo (1–2.5 hours). Second, IL-15 was more effective at higher doses in promoting expansion of memory CD8 T cells and NK cells but providing high dose regimens to patients was associated with toxicity that included hypotension, thrombocytopenia, persistent elevated levels of alanine aminotransferase and aspartate aminotransferase and increased levels of several pro-inflammatory cytokines including IL-6, IFNγ, TNFα, IL-1β. Lastly, the efficacy of IL-15 administration might also be limited by the expression of IL-15Rα in vivo as trans-presentation through this receptor chain in required for IL15 to mediate its biological effects. De facto, efforts are now turning into designing IL-15/IL-15Rα fusion proteins to overcome this issue and increase the in vivo stability of the cytokine. Several fusion proteins with superior therapeutic potential compared to native IL-15 were developed and are reviewed in detail elsewhere (reference [158]). It became rapidly evident that expansion of TILs in high concentration of IL-2 for extended periods (up to two weeks) promoted terminal differentiation [159]. This IL-2-driven differentiation was associated with loss of essential functions for successful engraftment and antitumor efficacy of ACT including self-renewal potential, expansion capacity and expression of homing molecules (CD62 L) [159]. Terminal effectors were also more apoptotic as assessed by elevated levels of BID, BAD and CD95 L compared to early effectors [71,160]. These parameters were reminiscent of the known impact of IL-2 in driving terminal differentiation of CD8 T cell effectors and promoting AICD [81,110]. Concomitantly, the differentiation state of expanded TILs was anti-correlated with persistence in vivo and anti-tumor activity [159–161]. In regards of the aforementioned limitations of using IL-2 for expanding TILs, IL-15 appeared as a relevant alternative. Indeed, IL15 was shown to expand TILs as efficiently as IL-2 without promoting terminal differentiation, and activated CD8 T cells cultured in IL-15 preferentially conserved a central memory phenotype (Tcm) as opposed to IL-2-based cultures [153,160,162,163]. Consistent with their less differentiated state, TILs expanded in IL-15 exhibited higher antitumor efficacy coupled with better persistence compared with TILs expanded in IL-2 in several preclinical models [71,153,160]. These data demonstrated that IL-15 might be superior to IL-2 for expansion of TILs in the context of ACT. IL-15 also plays a central role in ACT of CAR T cells. As opposed to ACT of TILs, CAR T cells are usually derived from peripheral blood of patients that contains naïve T cells [2]. Expansion of human naïve T cells in cultures complemented with IL-15 and IL-7 allows development of cells with a Tscm phenotype and IL-15 was essential for the development of murine Tscm [72,164]. Tscm cells are considered

3.2. IL-15 IL-15 is produced by numerous cell-types including hematopoietic and non-hematopoietic cells. DCs, macrophages and monocytes are the main in vivo source of IL-15, but this cytokine is also produced by mastocytes, B and T cells (low levels) as well as epithelial and stromal cells [133]. Similar to IL-2, IL-15 binds to and signals through the IL2Rβγ complex on T cells and triggers recruitment and activation of JAK1/STAT3 through the shared β chain and JAK3/STAT5 through the common γ-chain (Fig. 1) [134]. IL-15-derived signals also initiate AKT/ P13 K and RAS/MAPK signaling pathways activation in T cells (Fig. 2) [134]. While IL-2 and IL-15 share IL2Rβ and γc, the cytokines possess inherent α-chains (CD25 [IL2Rα] and IL15Rα [CD215] respectively) and signal on T cells through distinct mechanisms. Autocrine or paracrine soluble IL-2 is captured by the IL2Rα-chain on T cells which triggers formation of the high affinity IL-2R (described above). In contrast, IL-15 is often complexed with its α-chain and directly transpresented to the IL2Rβγ heterodimer on T cells via IL-15/IL-15Rα expressing cells [135]. Albeit the IL-15/IL-15Rα complex can be cleaved from cell surface and detected in the serum, trans-presentation is thought to be the main mechanism by which IL-15 mediates its biologic activity [136–139]. Because they share similar signaling pathways, IL-2 and IL-15 mediate redundant and/or complementary functions during T cell responses (Fig. 1). Both cytokines promote the expansion of activated CD8 T cells, sustain acquisition of effector functions (granzyme B, perforin) and direct the differentiation of short-lived effector cells (SLECs) in acute infection settings [21,140–142]. In vitro, IL-2 and IL-15 similarly trigger the expression of specific inhibitory receptors (i.e. 2B4 and Tim-3) on CD8 T cells and targeted disruption of the IL-2Rβ-chain common to both cytokines abrogates the induction of these two receptors in a mouse model of chronic viral infection [20]. Albeit it is admitted that IL-2 might dominate over IL-15 in directing effector CD8 T cell differentiation during acute infections, the rapid loss of IL-2 secretion capacity by CD4 and CD8 T cells in chronic infection settings suggests a prominent role for IL-15 in this context [31]. Besides, IL-15 also exert inherent functions including the homeostatic maintenance of memory CD8 T cells as well as the development, homeostasis and function of NK, NKT, γδ , and resident memory T cells (Trm) [137,143–145]. From a therapeutic perspective, IL-15 is of particular interest as this cytokine expands TILs as efficiently as IL-2 ex vivo without the suppressive effects associated with the later and TILs grown in IL-15 mediate tumor regression more efficiently than TILs grown in IL-2 [117]. As opposed to IL-2, IL-15 does not promote Treg cell expansion and this cytokine supports T cell survival through induction of BCL2 while IL-2 causes termination of T cell responses and promotes AICD [146,147]. De facto, the therapeutic potential of IL-15 was evaluated as a monotherapy or in combination with checkpoint blockade. This cytokine is also central for the generation of Tscm in ACT expansion protocols. 5

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the most undifferentiated cell-type after naïve cells and demonstrated superior persistence and antitumor efficacy compared to Tem and even Tcm [65]. In addition to directing the development of Tscm, IL-15 is also essential for their self-renewal [65]. Consistently, the durability of CAR T cells expanded in IL-15/IL-7 was superior to that generated from IL-2 cultures in a humanized mouse model [114]. Together, these data demonstrate that IL-15 should be further considered for anticancer adoptive cell therapy protocols. Similar to IL-2, the combinatorial potential of IL-15 with immune checkpoint inhibition was evaluated. Two subsequent reports investigated the potential synergistic impact of blocking PD-L1, CTLA-4 or both receptors in addition to IL-15 administration in preclinical cancer models of prostate cancer (TRAMP-C2) and colon adenocarcinoma (CT-26) [154,165]. Albeit the survival rate of CT-26 tumor bearing mice increased with either combination therapy (IL-15+αPDL1 or IL-15+αCTLA-4), it was not the case for TRAMP-C2 tumor carrying animals [154,165]. Further, no synergistic impact of combination therapy versus IL-15 alone was observed regarding tumor growth. These results revealed the limits of IL-15 in combination to checkpoint blockade therapy. However, further investigations are needed to determine whether newly designed IL-15/IL-15Ra complexes that present higher stability and biological activity will be more efficient than native IL-15 in this context. Globally, IL-15 regulates multiple biological processes and particularly dictate the expansion and differentiation of CD8 T cells, with potent anti-tumor cytotoxic functions and preserved memory-like features. Although these characteristics puts this cytokine at the forefront of ACT and cancer immunotherapy, its toxicity and limited clinical improvement in vivo and its potential to sustain T cell exhaustion in chronic viral infection, urges us to use this cytokine cautiously in human cancer patients.

of CARs ex vivo was somewhat limited [74]. The beneficial effect of IL21 on the expansion of TILs ex vivo was confirmed by other studies, further sustaining the demonstration that IL-21 favors the expansion of a less differentiated pool of effectors [111,171]. This was validated in human patients suffering from high-risk leukemia and treated with WT1-specific CD8 T cells expanded in the presence of IL-21. The authors could demonstrate long-lasting persistence of the adoptively transferred T cells for more than a year, and the absence of leukemic relapse in these high-risk patients, suggesting effective cytotoxic functions [172]. A recent study demonstrates that IL-21 reprograms the metabolism of human expanded T cells from aerobic glycolysis towards fatty acid oxidation (FAO) and mitochondrial metabolism, promoting the development of CD62L+ CD127+ CD28+ memory-like cells [173]. This is in accordance with the demonstration that the metabolic switch towards FAO and mitochondrial oxidative phosphorylation is the favored metabolic pathway used to sustain CD8 memory T cell differentiation and survival [174,175]. Interestingly, expansion of human T cells in the presence of IL-21 also reduced PD1 and CD57, markers of exhaustion and senescence respectively, in continuity with the protective role of IL-21 in preventing T cell exhaustion (see below) [173]. In vivo, IL-21 has a minimal impact on CD8 T cells during acute viral infection in help-independent infection models [176,177]. However, STAT3-dependent signals, through IL-21 and IL-10, promote the differentiation of memory precursor effector cells (MPECs) and the persistence of a functional memory T cell pool, in a Bcl6 and Eomes dependent manner [170]. In contrast, IL-21 plays a pivotal role in the context of chronic antigenic stimulation such as chronic LCMV infection. The absence of IL-21 signaling prevents the downregulation of IL-2 secretion by CD4 T cells, and results in the development of CD8 T cell exhaustion and viral persistence. Indeed, IL-21−/− or IL-21R−/− mice present rapid functional exhaustion upon infection with LCMV clone 13 and are unable to clear the virus [178,179]. Likewise, IL-21 treatment of chronically infected CD4-/- mice rescues the functionality of the CD8 T cell pool and accelerates viral clearance [180]. In the context of chronic viral infection, CD4 T cell help is driven by IL-21 signals, CD4 T cells switching from being IL-2-producers to becoming IL-21 producers [178]. Mechanistically, IL-21 maintains functional CD8 T cells during chronic LCMV infection by sustaining the expression of the transcription factor BATF via STAT3 [181]. IL-21-induced BATF likely cooperates with IRF4 to induce the expression of the transcriptional regulator Blimp-1, which promotes effector functions [181,182]. This is in contrast to the demonstrate role of BATF, Blimp-1 and IRF4 in sustaining exhaustion and the expression of inhibitory receptors [41,46,48]. Numerous preclinical mouse models of cancer demonstrate that IL21 enhances the proliferation and effector function of CD8 T cells, which resulted in the inhibition of tumor growth and increased mice survival [183,184]. Moreover, IL-21 injections led to long-term persistence of functional memory cells able to control secondary tumor challenge [183]. Interestingly, IL-21 synergizes with other γc cytokines to sustain potent anti-tumor functions. Administration of IL-21 in combination with IL-15 enhanced cytotoxic CD8 T and NK cell numbers and functions which correlated with marked regression of established tumors and improved mice survival [18,185]. Collectively, these reports confirm that IL-21 enhance CD8 T cell effector functions in the context of cancer, as it does during chronic viral infections. Of particular interest, IL-21 appears to synergize with IC blockade in multiple pre-clinical cancer models. Tumor regression was associated with increased intratumoral infiltration and effector memory differentiation of CD8 TILs when IL-21 was used in combination with either anti-PD1 or anti-CTLA4 blocking antibodies [14]. However, IL-21 has been suggested to be pro-tumorigenic in various cancer types -breast [186], T cell leukemia [187], multiple myeloma [188], and Hodgkin lymphoma [189]-, which renders its administration in vivo delicate in these contexts. Thus, the benefit of IL-21 in cancer is likely through its capacity to direct Tscm differentiation ex vivo while furthering effector functions,

3.3. IL-21 IL-21, the most recently discovered member of the γc cytokine family, is mainly produced by NKT cells and CD4 T cell subsets including Tfh cells and Th17 cells [166]. Its receptor, composed of the IL21Rα and the γc chain, is expressed constitutively on hematopoietic cells, including CD8 T cells, and upregulated in response to TCR or IL-21 itself. IL-21 signals via the JAK/ STAT, the MAPK and PI3K-AKT signalling pathways (Fig. 2). In contrast to other members of the γc family of cytokines, IL-21 predominantly activates STAT3, while it may also activate STAT1, STAT5A, and STAT5B (Fig. 1) [167]. In addition to STATs, IL-21 regulates the expression of several TF involved in the differentiation and cell fate decisions of CD4 and CD8 T cells such as Tbet, Eomes, Bcl-6, Blimp-1, BATF and IRF4 [166,167]. As such, IL-21 has been shown to play an important role in the differentiation of CD4 T cell subsets, promoting Tfh cell development and Th17 differentiation, and inhibiting Treg differentiation [10,166,167]. The role of IL-21 on CD8 T cells depends on the stimulation context (Fig. 1). In vitro, IL-21 sustains the antigen-specific proliferation of CD8 T cells in concert with other γc cytokines [18,168]. Granzyme A and B being known IL-21 target genes, IL-21 further sustains the cytotoxic functions of CD8 T cells, while it increases their cytokine secretion capacities [169]. More importantly, IL-21 preserves CD8 T cells in a less differentiated, memory-like state, thanks to its capacity to promote Bcl6 and TCF1 expression while inhibiting IL-2-induced Eomes transcription, in a STAT3 dependent manner [12,170]. This protective function of IL21 puts this cytokine at the forefront of ACT. Multiple preclinical models of cancer have demonstrated that IL-21-primed CD8 T cells present potent anti-tumoral effector functions when adoptively transferred in tumor-bearing mice [12]. This was confirmed in a preclinical model of lymphoma using human T cells transfected with retroviral constructs containing a CD19 CAR and each γc cytokine individually. In this model, CD19 CAR T cells overexpressing IL-21 demonstrated increased efficacy and prolonged persistence in vivo, although expansion 6

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in synergy with IC blockade, without the risk of overt T cell exhaustion. This therapeutic potential has recently been demonstrated in patients treated for stage IV metastatic melanoma with IL-21-expanded TILs and CTLA-4 blockade, supporting further development for this adjuvant cytokine in cancer immunotherapies [190,191].

studies further demonstrate that IL-7 combined to IL-15 promotes the development of long-lasting less differentiated TSCM cells. Indeed, IL-7 and IL-15 were critical to promote the development of T cells with a CD62L+ CCR7+ CD45RA+ CD45RO+ CD95+ IL7Rα+ phenotype when naïve T cell precursors from healthy donors were stimulated through CD3/CD28 engagement [72]. These TSCM cells manipulated to express tumor-specific TCRs demonstrated augmented expansion and potent effector functions in response to the specific peptide, while conserving self-renewal abilities [72]. Similarly, CD19 CAR T cells expanded in the presence of IL-7 and IL-15 led to increased frequency of CD45RA+ CCR7+ T cells, preferential homing to secondary lymphoid organs through CCR7, enhanced in vivo persistence and improved antitumor immunity in xenograft models of cancer [114]. GD2 and EPHA2 CAR T cells engineered for constitutive IL7R-STAT5 signaling equally sustained potent T cell expansion and function, eradicating metastatic neuroblastoma and glioblastoma respectively [213]. These results support the concept that IL-7 drives the differentiation of TSCM cells during ACT, a prerequisite for potent anti-tumor efficacy after any ex vivo expanded T cell product. Altogether, these studies suggest that IL-7 therapy is a potential strategy to sustain immune reconstitution in lymphodepleted patients, augment T cell responses against cancer and chronic viral infections and direct the lineage fate and expansion of ex vivo expanded T cells for ACT. A direct impact of IL-7 in preventing or reversing T cell exhaustion remains to be investigated.

3.4. IL-7 IL-7 is primarily produced by non-hematopoietic stromal and epithelial cells in the bone marrow and thymus, and by fibroblastic reticular cells in the T cell zones of secondary lymphoid organs. The receptor for IL-7 is composed of the IL7Rα-chain (CD127) and the γc chain (CD132). IL7Rα, which is also a component of the receptor for thymic stromal lymphopoietin (TSLP), is mainly expressed by cells of lymphoid lineage. Ligation of IL-7 to its receptor triggers JAK1/JAK3 and subsequently STAT5 phosphorylation, while it can also activate the PI3K/AKT and MAPK pathways (Fig. 2) [192,193]. Hence, IL-7 has pleiotropic functions on T cell biology, regulating survival, cell cycle entry and metabolism (Fig. 1) [194]. In particular, IL-7 supports the survival of naive and memory CD8 T cells through the expression of Bcl2, Bcl-XL and Mcl-1, in a STAT5-dependent manner [163,195–198]. Further, the re-expression of IL7Rα at the surface of effector cells has been key to identify subsets of cells destined to become memory (memory precursor effector cells; MPEC) [199,200]. The unique capacity of IL-7 to promote both T cell survival and homeostasis and the development of memory precursors opened the door for this cytokine to be used as an immunotherapeutic agent. As expected, in tumor-bearing hosts, IL-7 administration as an adjuvant to vaccination approaches increased the pool of effector and memory CD8 T cells, prolonging mice survival through improved tumor control [201–203]. In patients with advanced cancers, recombinant IL-7 sustained the survival and proliferation of CD4 and CD8 T cells with robust proliferative abilities. Cell cycling as demonstrated by Ki-67 staining was sustained in 50% of CD8 T cells at the peak of the response and survival was mediated by persistent Bcl-2 expression. IL-7 administration also increased the number of recent thymic emigrants and the diversity of the TCR Vβ repertoire, rejuvenating the peripheral T cell pool [204,205]. IL-7 may also limit the development of T cell exhaustion. In the context of chronic antigenic stimulation, such as chronic LCMV infection or cancer, loss of IL7Rα expression correlated to the severity of CD8 T cell exhaustion [206,207]. Further, IL-7 therapy reduced the expression of PD-1 on antigen-specific CD8 T cells in both chronic viral infection and cancer models [203,208]. It remains to be demonstrated if this effect on PD-1 is direct or secondary to reduced tumor or viral burden. Finally, IL-7 may also be promising as an adjuvant to IC blockade. One recent pre-clinical model suggests that IL-7 may potentiate PD-1 blockade in vivo, although the mechanisms for this potential synergy remains to be identified [209]. Despite the above encouraging observations, the potential for IL-7 as an adjuvant in cancer immunotherapy needs to be evaluated cautiously. First, IL-7 is pro-tumorigenic in multiple cancer subtypes, through its capacity to sustain the expression of Bcl-2, cyclin D1, c-FOS and c-JUN [210]. Second, recent phase 1 clinical trials in humans, although well-tolerated and immunologically promising, have not been translated in improved clinical outcome yet [204,205,211]. These suggest that manipulating IL-7 pathways may be best ex vivo. Multiple studies support the notion that IL-7 may enhance the effectiveness of ACT, thanks to its capacity to promote the survival and proliferation of antigen-induced T cells, and to regulate fate decisions of these cells [192,211]. Initial studies with murine CTLs extracted from lymph nodes of tumor bearing mice and re-expanded in vitro in the presence of IL-7 demonstrated increased anti-tumor activity of these CTLs when adoptively transferred into syngenic tumor-bearing mice, compared to CTLs expanded in IL-2 or IL-4 containing media [73,212]. These pioneered studies formed the premises of current ACT expansion protocols and identified IL-7 as a potential adjuvant. More recent

4. Signaling pathways regulated by common gamma chain cytokines: therapeutic opportunities 4.1. JAK-STAT signaling cascade 4.1.1. The biology of JAKs and STATs Cytokines activating the JAK-STAT pathway induce a very conserved and simple signaling cascade (Fig. 2) [214,215]. Because the cytokinespecific dimeric or trimeric receptor does not possess intrinsic kinase activity in its cytoplasmic tail, they rely on extrinsic kinases to signal. These JAKs trans-phosphorylate each other and then phosphorylate downstream molecules, including STATs and tyrosine residues on the receptor tail itself [216]. STATs are activated through their Srchomology 2 (SH2) domain, dimerize and enter the nucleus to bind consensus DNA sequence called IFNγ-activated sites (GAS) motifs and induce the transcription of a whole array of genes [217–219]. STATs may also form more complex oligomers (tetramers) in the nucleus, to regulate the expression of target genes [220]. Although STATs generally induce the activation of transcription, they may repress transcription in particular cases [221–223]. The activation of specific JAKs or STATs will depend on: 1) the tissue distribution of these above proteins, 2) the presence of specific cytokines in the environment and, 3) the expression of the receptor chains at the surface of the cell, and will be influenced by members of other signaling pathways (PI3K, MAPK) [224]. There are four JAKs and seven STATs in mammalian cells [214]. In the context of common γc dependent cytokines specific to this review, JAK1 will bind the cytokine-specific receptor chain (IL2Rβ for IL-2 and IL-15, IL7Rα for IL-7, IL21Rα for IL-21) while JAK3 will bind the associated γc [225,226]. The γc chain is critical for transducing downstream signals and inducing JAK transphosphorylation while the cytokine-specific receptor chain provides docking of STAT SH2 domains to its phosphotyrosine binding sites [220]. These docking sites determine which STAT will be activated by a given cytokine. IL-2, IL-7 and IL-15 predominantly activate STAT5 (A and B), and to a lesser extent STAT1 and STAT3, while IL-21 predominantly activates STAT3, and to a lesser extent STAT1 and STAT5 (Fig. 1) [10]. Collectively, JAK-STATs regulate canonical immune responses, from survival, proliferation, differentiation, migration to apoptosis. Some of their cytokine-specific role have been mentioned above and have been reviewed in details by others [229–236]. 7

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Fig. 2. Overview of the IL-15 signaling pathway in CD8 T cells. IL-15 is presented in trans by its IL-15Rα to the receptor dimer IL2Rβ and γc expressed at the surface of CD8 T cells. The interleukin-receptor association leads to the rapid activation of three classes of kinases including JAK, Shc and PI3K leading to activation of the JAK-STAT, MAPK/ ERK and PI3K/AKT/mTOR downstream pathways. Following a cascade of signaling events, the transcription factors STAT5, ERK, S6, and FOXO1 will promote or inhibit the expression of a multitude of genes essential for the survival, homeostatic proliferation, expansion, migration, metabolism and differentiation of CD8 T cells. JAK: Janus Kinase, STAT: Signal Transducer and Activator of Transcription, SH2: Src Homology 2 domain, Shc: Grb2: Growth factor receptor-bound protein 2, Gab2: GRB2 Associated Binding Protein 2, MAPK: MitogenActivated Protein Kinase, MEK: Mitogen-activated protein kinase kinase, ERK: Extracellular Signal–Regulated Kinase, AKT: Protein Kinase B, mTORC1: mammalian target of rapamycin complex 1, mTORC2: mammalian target of rapamycin complex 2, S6K: Ribosomal protein S6 Kinase, S6: ribosomal protein S6, FOXO1: Forkhead Box O1, GSK3: Glycogen synthase kinase 3, PDK1: 3-Phosphoinositide-Dependent Protein Kinase-1, PIP2: Phosphatidylinositol 4,5-bisphosphate, PIP3: Phosphatidylinositol (3,4,5)-trisphosphate, IC: intracellular, Bcl-2 B: B-cell lymphoma 2, Blimp-1 transcription factor B lymphocyte-induced maturation protein 1, Grz: granzyme, T-bet: T-box transcription factor, Eomes: eomesodermin, Bim: Bcl-2-like protein 11, Mcl1: myeloid cell leukemia sequence 1, NFkB: nuclear factor kappa-lightchain-enhancer of activated B cells, c-myc: cellular myelocytomatosis, CCR7: C-C chemokine receptor type 7, CD62L: L-selectin, IL7R: IL-7 receptor, KLF2: Kruppel Like Factor 2.

4.1.2. JAK-STAT in cancer Constitutive JAK-STAT signaling was revealed in multiple cancers and has evidenced the contribution of this signaling pathway to tumorigenesis. Functional activation of JAK2 drives the development of myeloproliferative neoplasms, and has fueled the development of novel therapies targeting the JAK pathway (see below). Since this initial description, multiple other gain-of-function mutations involving activating JAKs (JAK1, JAK2, JAK3) or their downstream STATs (STAT3, STAT5, STAT6) have been identified and appear to trigger the development of hematologic malignancies and solid tumors (reviewed in [233,237,238]). In these cases, activated STATs exert their pro-survival and proliferative effect directly in cancer cells, and even cancer stem cells, promoting cancer growth and spread. Further, cytokine-dependent STAT activation prompted by the inflammatory microenvironment sustain angiogenesis and the development of tumor cell niches, in stroma and at metastatic sites. Targeting STAT pathways with specific small molecule inhibitors appeared to be promising approaches to intrinsically limit tumor progression (reviewed in [234,239]). This led to the approval of ruxolitinib, a selective inhibitor of JAK1 and JAK2, for the treatment of myeloproliferative neoplasms. Multiple other JAK inhibitors are now in development, clinical trials or have been licensed to treat various malignancies or autoimmune diseases, each of them with distinct specificities. Despite the excitement for targeting this signaling pathway in cancer treatment in recent years, clinical responses to JAK inhibitors remain modest, and JAK2-expressing tumors eventually evade JAK inhibition and become drug resistant through the formation of new signaling heterodimers (with

JAK1 or TYK2) [240]. However, they could be useful drugs in combination with other targeted therapies [238,241,242]. 4.1.3. STAT-dependent T cell immune responses Detailed evaluation of the impact of JAK inhibitors on T cell functions in cancer models and in treated patients is lacking. Very few studies have evaluated specific T cell responses during treatment with JAK inhibitors. However, knowing the critical role of cytokines in regulating adaptive immune responses, as discussed above, it might be envisioned that JAK and STAT inhibition must be finely tuned to sustain effective anti-tumor T cell responses, while not inducing over immune suppression. Indeed, STAT5 is well described for its fundamental role in the survival of naïve and memory CD8 T cells, and for sustaining the proliferation of effector CD8 T cells. Constitutive activation of STAT5 increases the pool of effector and memory CD8 T cells in response to an acute viral infection, through increased Bcl-2 dependent survival and cell proliferation [243–245]. It sustains the persistence of KLRG1high CD127low short-lived effector cells and promotes the increased survival of CD127high KLRG1low memory precursors. Conversely, lack of STAT3 signaling in both mouse models of acute viral infection and patients with STAT3 deficiencies led to the development of dysfunctional memory T cells [170,246]. Further, absence of signaling downstream of STAT3 sustained the development of terminally differentiated CD8 T cell effectors, as did constitute activation of STAT5. This STAT3/STAT5 duality is not restricted to CD8 T cells, as reciprocal interactions have been demonstrated in CD4 T cell subsets, including T follicular helper 8

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cells (Tfh), and B cells [248–253]. The reciprocal impact of STAT3 and STAT5 signaling on CD8 T cells during infection is further supported by our demonstration that combined IL-2 and IL-15, that mainly signal through STAT5, drive the differentiation of terminally exhausted CD8 T cells and the expression of numerous immune checkpoints (PD1, LAG3, CD160, 2B4, and Tim3) during chronic viral infection [20], while previous studies reported that IL-21, that mainly signals through STAT3, has a protective role during chronic antigen persistence [178–180,247].

fundamental “signaling nodes” will be either activated or inhibited by AKT: FOXO1, mTORC1 and glycogen synthase kinase 3 (GSK3) [261]. Phosphorylated AKT will decrease FOXO1 activity through nuclear export and degradation [262]. It will also inhibit GSK3 function by blocking accessibility to its phosphate-binding motif in the catalytic subunit [263,264]. Finally, it will activate the mTORC1 pathway and its downstream ribosomal protein kinases (p70 S6K) by inhibiting the tuberous sclerosis complex, a potent inhibitor of mTORC1 [265,266]. In response to γc cytokines, only the FOXO1 and mTORC1 nodes will be altered. These modifications will have significant impact on the T cell immune responses.

4.1.4. Modulation of STAT signaling in adoptive cell therapy for cancer A novel adoptively transferred CAR T cell construct has been designed based on the particularly specific effect of STAT signals to increase the efficacy and longevity of adoptively transferred CAR T cells [126]. This construct associates a truncated IL2Rβ cytoplasmic domain that drives STAT5 signaling with a STAT3 binding motif on a second generation CD28 CAR T cell construct reinforcing TCR and co-stimulatory signals by providing combined STAT3 and STAT5 signals. It drove the expression of IL-21 dependent target genes and increased levels of cytotoxic molecules (probably STAT5 mediated). Functionally, these motifs increase the proliferation and polyfunctionality of CAR T cell in response to tumor antigens, which led to increased cytolytic activity and more rapid leukemia control in vivo. Interestingly, the STAT3 motif, but not the STAT5 motif, was demonstrated to sustain a less differentiated TSCM state, with increased expression of CD27, CD28 and CD95. It allowed for the adoptively transferred CARs to better persist in vivo. Unfortunately, these CAR T cells eventually developed functional exhaustion. After repeated antigenic stimulation, they expressed some inhibitory receptors (PD1, LAG-3, Tim-3) and showed reduced proliferation and cytokine secretion capacities, although less strongly than second generation CARs. Collectively, these data suggest that manipulating STAT signals are fundamental tools to control T cell responses during cancer. Fine tuning of the timing of this manipulation, with specific inhibitors or targeted constructs, will be necessary to preserve the differentiation of TSCM ex vivo, to limit the development of T cell exhaustion in vivo, and to sustain their longevity after adoptive transfer in patients with cancer.

4.2.2. PI3K in cancer Because of its central role in the entry in cell cycle, survival, growth and migration, it is understandable that aberrant activation of PI3K had been shown to drive carcinogenesis and metastasis. Indeed, somatic mutations in the PI3K/AKT pathway are identified in the majority of human cancers, in both solid and hematopoietic malignancies (particularly breast cancer and B cell lymphoproliferative tumors). They are either gain-of-function mutations in one of the subunits of PI3K or downstream kinases; or are loss-of-function mutations of phosphatases regulating termination of AKT signaling (in PTEN for example) (reviewed in [267]). This dependency on PI3K activation for tumor growth led to the development of a whole array of specific PI3K inhibitors [267]. Some of these inhibitors will target all p100 isoforms (pan-PI3K inhibitors), while others will be more specific (p110α inhibitor, p110β inhibitor, p110δ inhibitor). Collectively, when used alone, these inhibitors have shown limited efficacy, except maybe for specific inhibitors of the mTOR pathway (rapamycin (sirolimus) and rapalogs (everolimus, temsirolimus, deforolimus)) in specific contexts, with high rates of adaptation by the tumor to preserve signaling activity [254]. Thus, combination therapies with other targeted therapies, either downstream of AKT (e.g. dual PI3K/mTOR inhibitors), or against a distinct signaling pathway (e.g. JAK/STAT inhibitors, inhibitors targeting the c-Myc pathway etc…) are currently envisioned [268,269]. 4.2.3. PI3K-dependent T cell immune responses The importance of PI3K in T cell responses stems from its fundamental role in the regulation of cell survival, proliferation and migration. Critical to the metabolism of the cell, it sustains glucose and nutrient uptake in the thymus and in the periphery [270]. At steady state, unphosphorylated FOXO1 proteins will induce the transcription of IL7Ra, Klf2 and its downstream target genes, Sell (CD62 L), Edg1 (S1P1) and CCR7, thus sustaining the survival, homeostatic proliferation and homing of naïve CD8 T cells to secondary lymphoid organs (Fig. 2) [271–273]. Upon TCR or cytokine stimulation, PI3K will promote the differentiation of short-lived effector cells (SLECs) and effector memory T (TEM) cells, the expression of cytolytic effector molecules [274], and the migration of effector cells to inflamed tissues through the regulation of chemokines and adhesion receptors [266]. In contrast, mTORC1 inhibition will favor MPECs and central memory T (TCM) cells [275] through the FOXO1dependent repression of Tbx21 and the enhancement of Eomes and Kruppel-like factor 2 (Klf2) transcription [273,276–278]. To regulate T cell differentiation, PI3K and its downstream signaling molecules, in particular mTORC1, serve as metabolic switches in CD8 T cells, sustaining glucose uptake by effector CD8 T cells through Glut1 expression and favoring glycolysis and lipid synthesis by controlling the expression of rate-limiting glycolytic enzymes [279]. In contrast, inhibition of mTORC1 will favor fatty acid oxidation and mitochondrial spare respiratory capacity, to sustain long-term survival of CD8 memory T cells [266]. Unphosphorylated FOXO1 presents in the nucleus will further sustain CD8 memory T generation and homeostasis through the expression of IL7Ra, Eomes, Klf2 and other memory-favoring genes [280].

4.2. PI3K signaling cascade Kinases that phosphorylate phosphatidyl-inositol lipids play a fundamental role in T cell biology [254]. This pathway can be activated by TCRs, co-stimulatory receptors and cytokine receptors, including the common γc receptor. 4.2.1. The biology of PI3K PI3K catalyzes the phosphorylation of phosphatidylinositol (4,5)-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3) (Fig. 2) [255]. This fundamental kinase is heterodimeric and is composed of one of four catalytic subunit p110 (p110α, β, δ, γ) which associates with one of five regulatory subunit p85 (p85α, p55α, p50α, p85β, and p55γ). p110α and p100β are ubiquitously expressed, while p110δ and p110γ are exclusively expressed in leucocytes. In the absence of stimulation, p85 interacts with p110δ through its SH2 domain and inhibits kinase activity [256]. In response to cytokines such as IL-7 and IL21, p85 will bind tyrosine residues on the IL7Rα and IL21Rα chains, releasing the p110δ subunit [257–259]. Similarly, p85 has been shown to bind IL2Rβ through distinct motifs and/or adaptor proteins [259,260]. The released p110δ is recruited to the plasma membrane where it phosphorylates PIP2 to generate PIP3. This activated phospholipid will act as a second messenger to recruit multiple proteins at the plasma membrane, forming signaling complexes. One of these complexes includes the serine/threonine kinase AKT (also called protein kinase B) and its activating kinase phosphoinositide-dependent kinase-1 (PDK-1). Upon formation of the complex, AKT will be phosphorylated by PDK-1 and mTORC2 and will undergo strong conformational changes to regulate multiple cellular networks controlling cell growth, cell cycle, cell survival and cell migration. In T cells, three

4.2.4. Targeting PI3K to increase T cell fitness during cancer treatment Targeting AKT, mTORC1 and other PI3K signaling molecules to sustain anti-tumoral T cell responses during cancer is appealing, 9

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knowing how PI3K regulates both innate and adaptive immunity. While recent murine studies have demonstrated that PI3Kγ inhibition sustains T cell recruitment to tumor sites, tempering tumor growth, it appears that the impact on increased cytolytic and cytokinic functions of T cells against the tumor may in part be indirect, through the reduction of immunosuppressive cytokines in the TME [281]. Similarly, the efficacy of DC-based vaccination approaches to promote effective anti-tumor T cell responses were enhanced when DCs favored pro-inflammatory cytokines over immunosuppressive cytokines in response to PI3K inhibition [282]. Thus, modification of innate immune responses upon PI3K inhibition enhance effective T cell responses in cancer. Other reports have demonstrated that pharmacologic inhibition of mTORC1, AKT or PI3Kδ in ex vivo expanded T cells have also a direct effect on T cell functions, favoring the development of “memory-like” cells with enhanced tumor control [276,283]. AKT inhibition did not hamper CAR or TCR retroviral transduction of T cells for ACT, while it sustained potent anti-leukemic responses through FOXO1-dependent gene transcription [284]. Similarly, gene-targeted inhibition of mTOR in vivo sustained memory CD8 T cell differentiation with enhanced proliferative and cytotoxic functions once challenged with a tumor [285]. These findings are in accordance with the concept that less differentiated T cells are more potent effectors for anti-tumor responses and thus for ACT of cancer. Finally, it is interesting to note that targeting PI3K, through its impact on macrophage reprogramming, shows synergy with immune checkpoint blockade in mice bearing HNSCC tumors [281]. Not only mice treated with combined PI3Kγ inhibition and PD1 blockade demonstrated increased tumor clearance, they also developed functional memory, remaining cancer free after re-challenge with tumor cells. It remains to be demonstrated if the development of long-lasting memory cells in this context were directly regulated by FOXO1 and/or mTORC1 inhibition in CD8 T cells.

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