Unlocking the Complexities of Tumor-Associated Regulatory T Cells Jaime L

Unlocking the Complexities of Tumor-Associated Regulatory T Cells Jaime L. Chao and Peter A. Savage This information is current as J Immunol 2018; 200:415-421; ; of September 25, 2021. doi: 10.4049/jimmunol.1701188 http://www.jimmunol.org/content/200/2/415 Downloaded from References This article cites 48 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/200/2/415.full#ref-list-1

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Unlocking the Complexities of Tumor-Associated Regulatory T Cells Jaime L. Chao and Peter A. Savage Regulatory T (Treg) cells are found at elevated densities prognostic significance (2, 3), suggesting that Treg cells may in many human cancers and are thought to be a major have a functional impact on tumor development and pro- barrier to the generation of robust antitumor T cell re- gression. Interestingly, in some cancers such as hepatocellular sponses. In this review, we discuss recent advances in the carcinoma, a high Treg cell density is predictive of poor understanding of tumor-associated Treg cell diversity clinical outcome, consistent with the paradigm that Treg cells and function. Emerging evidence indicates that the promote tumor progression by suppressing tumor-specific transcriptional program of Treg cells infiltrating human T cell responses. In contrast, a high Treg cell density is pre- cancers may represent a composite program blending a dictive of improved clinical outcome in other cancers such as Downloaded from tissue-associated expression signature with an addi- colorectal carcinoma. Although the precise mechanisms driving tional tumor-specific signature common to Treg cells this association are undefined, it has been proposed that the from multiple cancer types. Studies in mouse models favorable effect of Treg cells in colorectal carcinoma may reflect a role for Treg cells in suppressing tumor-promoting inflam- have defined unique molecular pathways required for mation in response to gut microbes (4). These disparate find- Treg cell function in the tumor context that can be ma- ings suggest that the role of Treg cells in shaping tumorigenesis http://www.jimmunol.org/ nipulated to selectively dampen intratumoral Treg cell may be highly context-dependent, varying considerably at activity. Finally, an expanding body of work has different organ sites. revealed diverse functions for Treg cells in nonlymphoid Given the pivotal role of Treg cells in immune suppression tissues that are unrelated to immune suppression, sug- and the prevalence of these cells in many human cancers, it is gesting a need to explore functions of intratumoral Treg thought that Treg cells constitute a major barrier to therapeutic cells beyond the regulation of antitumor immunity. efforts to mobilize the immune system to induce tumor re- The Journal of Immunology, 2018, 200: 415–421. gression. This idea has spurred concerted efforts to develop

modalities to enhance cancer immunotherapies by inducing by guest on September 25, 2021 he development and progression of cancer can be the selective depletion or modulation of intratumoral Treg cells profoundly impacted by tumor cell–extrinsic factors while simultaneously leaving Treg cells elsewhere in the body T such as cells of the immune system, which are unaffected. In this review, we highlight recent studies that thought to either promote or restrict tumor progression in advance our understanding of tumor-associated Treg cell bi- different contexts (1). Many human tumors contain immune ology and reveal potential paths for the selective manipulation cells localized diffusely or clustered within distinct regions, of these cells. First, we discuss evidence suggesting that ther- indicative of ongoing inflammatory reactions or antitumor apeutic Abs specific for T cell–expressed receptors such as immune responses. Regulatory T (Treg) cells expressing the CTLA-4 may function in part by inducing the specific de- transcription factor Foxp3 are common protagonists in these pletion of intratumoral Treg cells. We then review recent reactions, and they are often found at elevated densities in surveys of Treg cells isolated from human tumors, which tumor lesions relative to lymphoid and nonlymphoid sites. suggest that intratumoral Treg cells are broadly imprinted by Treg cells throughout the body are essential for the prevention the tissue microenvironment, but they also express a con- of autoimmunity and the maintenance of immune homeo- served tumor-specific signature that may be common to stasis, and they function by suppressing the activation and intratumoral Treg cells from multiple cancer types. Next, we differentiation of CD4+ Th cells and CD8+ cytotoxic T cells discuss work indicating that intratumoral Treg cells require reactive to autologous, environmental, or tumor-expressed unique molecular programs to function and thrive within Ags. Numerous correlative studies have revealed that for tumor lesions, and that these programs can be selectively some cancers, the density of tumor-infiltrating Treg cells has perturbed to modulate intratumoral Treg cell activity in

Department of Pathology, University of Chicago, Chicago, IL 60637 Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; Areg, amphiregulin; CRC, colorectal carcinoma; cTreg, central Treg; eTreg, effector Treg; ORCID: 0000-0003-1872-734X (J.L.C.). HFSC, hair follicle stem cell; Nrp1, neuropilin-1; NSCLC, non–small cell lung cancer; Received for publication August 17, 2017. Accepted for publication October 2, 2017. Sema4a, semaphorin-4a; SLO, secondary lymphoid organ; Treg, regulatory T; VAT, visceral adipose tissue; VEGF, vascular endothelial growth factor. This work was supported by National Institutes of Health Grants R01-AI126756 (to P.A.S.) and R01-AI110507 (to P.A.S.). J.L.C. was supported by National Institutes of Health Ó Training Grant T32 AI007090. Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 Address correspondence and reprint requests to Dr. Peter A. Savage, Department of Pathology, University of Chicago, 900 E. 57th Street, KCBD 6134, Chicago, IL 60637. E-mail address: [email protected]

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701188 416 BRIEF REVIEWS: TUMOR-ASSOCIATED REGULATORY T CELLS preclinical animal models. Finally, we discuss mounting evi- Despite clear mechanistic evidence in mice that Abs can dence that Treg cells resident in nonlymphoid organs can promote tumor rejection by inducing intratumoral Treg cell function to regulate diverse processes such as tissue homeo- depletion, the relevance of these concepts to the efficacy of Ab- stasis, repair, and metabolism, and we speculate about the based immunotherapies in human cancer patients remains potential implications of these findings on our understanding undefined. The clearest available evidence in support of this of tumor-associated Treg cells. We conclude by highlighting idea comes from the studies of Romano et al. (14), which critical gaps in knowledge in the field and outlining future compared samples from human melanoma patients who did inquiries needed to gain a more complete understanding of or did not respond to therapy using the anti–CTLA-4 Ab intratumoral Treg cells at different organ sites. ipilimumab. In ex vivo assays, it was found that nonclassical monocytes expressing the activating Fc receptor CD16 can Do “checkpoint blockade” Abs function by depleting intratumoral engage ipilimumab and induce ADCC-mediated lysis of Treg Treg cells? cells in vitro. Importantly, clinical responses to ipilimumab In the past decade, Abs specific for the T cell coinhibitory were associated with Treg cell depletion in tumor lesions, as receptors CTLA-4 and PD-1 have shown striking success in well as elevated densities of nonclassical monocytes in the inducing durable clinical benefit in a fraction of cancer patients peripheral blood at baseline compared with nonresponders spanning a variety of cancer types (5). Early in their devel- (14), suggestive of ADCC-dependent intratumoral Treg cell opment, these Abs were dubbed “checkpoint blockade” Abs ablation. Taken together, the studies above support the idea based on the idea that they were thought to function by that Abs specific for T cell–expressed receptors may function in Downloaded from blocking the binding of CTLA-4 or PD-1 to their ligands, part by inducing the selective elimination of tumor-infiltrating thereby releasing tumor-specific T cells from checkpoints, Treg cells in circumstances in which the Ab isotype can avidly limiting their activation and effector function (6). However, engage activating Fc receptors, and Fc receptor–expressing recent work has challenged this idea, suggesting that some of myeloid cells are present at sufficient densities. these Abs may function instead by inducing the depletion of The idea that intratumoral Treg cell depletion may underlie CTLA-4–expressing cells in the tumor environment by binding the efficacy of therapeutic Abs in some settings highlights the http://www.jimmunol.org/ to target cells and inducing Ab-dependent cellular cytotoxicity functional importance of Treg cells in the tumor context. (ADCC) (7, 8). Given that Treg cells generally express high Moreover, the notion that Abs that had previously been thought amounts of cell-surface CTLA-4, PD-1, and other immuno- of as “blocking” reagents may function in some contexts by modulatory receptors such as OX40, 4-1BB, and ICOS (dis- inducing ADCC has revitalized enthusiasm for older strategies cussed below), this suggests that therapeutic Abs targeting these for therapeutic Treg cell ablation. For example, an attractive proteins may function in part by driving the selective depletion marker for Treg cell targeting is the IL-2 receptor a-chain + of intratumoral Treg cells. CD25; relative to conventional T cells, Foxp3 Treg cells ex-

In support of this idea, Simpson et al. (9) demonstrated in press high amounts of CD25 (15), and CD25 expression is by guest on September 25, 2021 mice that administration of distinct anti–CTLA-4 Ab clones required for optimal Treg cell survival and fitness (16, 17). induced the depletion of intratumoral Treg cells without However, previous studies in mice and humans have failed to impacting intratumoral conventional T cells or Treg cells out- demonstrate consistent activity of anti-CD25 Abs in the tumor side of the tumor, resulting in slowed outgrowth of trans- context (15). To re-examine this phenomenon, Arce Vargas plantable melanomas. Interestingly, the efficacy of these Abs was et al. (15) demonstrated in mice that administration of a dependent on the presence of tumor-infiltrating myeloid cells common anti-CD25 Ab clone reduced Treg cell frequencies in expressing activating Fc receptors, implying a role for Fc the blood and secondary lymphoid organs (SLOs) but failed to receptor–dependent, ADCC-mediated Treg cell depletion. induce intratumoral Treg cell depletion. In contrast, use of a Additionally, the authors suggested that the selectivity of anti– variant of this clone engineered to preferentially engage acti- CTLA-4 Ab in driving the preferential depletion of Treg vating Fc receptors (at the expense of binding to inhibitory Fc cells was largely due to the elevated cell-surface expression of receptors) induced robust intratumoral Treg cell depletion and CTLA-4 by intratumoral Treg cells relative to conventional enhanced antitumor immunity (15). These findings provide T cells within the tumor, a concept that may be broadly relevant additional evidence that the design of Ab Fc regions is a key for therapeutic Abs targeting other T cell–expressed receptors. determinant of biological activity and in vivo efficacy of Ab Working in parallel, Selby et al. (10) performed similar studies therapeutics, and they suggest a need to re-evaluate Abs that in mice using an anti–CTLA-4 Ab clone of fixed Ag-binding had previously been deemed to be ineffective in the tumor specificity in which the C region of the Ab was changed to setting. Moreover, the collective findings discussed above sug- different isotypes known to engage activating Fc receptors with gest that Abs targeting different T cell–expressed cell-surface varying affinities. It was found that the therapeutic efficacy of receptors may function by a common mechanism, that is, anti–CTLA-4 Ab treatment correlated with the ability of the Fc binding avidly to Treg cells and marking these cells for region to bind activating Fc receptors, again suggesting a role for elimination by ADCC (7). ADCC in determining in vivo Ab activity. These two studies highlight the importance of both the Fab and Fc regions of an Analysis of tumor-infiltrating Treg cells in human cancers Ab in determining the therapeutic efficacy of an Ab of interest. Beyond CTLA-4, CD25, and other well-defined immuno- Consistent with these findings, additional studies in mice have modulatory receptors, an improved understanding of the demonstrated similar requirements for therapeutic Abs targeting unique features of tumor-infiltrating Treg cells may provide other T cell–expressed coreceptors such as GITR (11, 12) and key insight into the function of these cells in the tumor context OX40 (13), for which specific Abs are currently under and reveal novel targets for the selective modulation of Treg clinical development for the treatment of human cancer (7). cells for therapeutic benefit. In this light, three recent papers The Journal of Immunology 417 reported high-resolution surveys of Treg cells infiltrating transferrin receptor), FCRL3 (encoding an Fc receptor–like human cancers. In one study, Plitas et al. (18) performed protein), and CCR8. It remains unknown whether these pro- transcriptional and phenotypic analysis of Treg cells isolated teins are important for Treg cell function in the tumor envi- from breast cancer lesions, and they compared these to Treg ronment, or reflect adaptation required to survive and thrive cells isolated from normal breast parenchyma or peripheral within neoplastic lesions. blood. The authors found that breast tumors contained ele- Beyond the characterization of transcriptional profiles, the vated percentages of Treg cells relative to normal breast tissue, single-cell RNA sequencing studies of Zheng et al. (20) and that intratumoral Treg cells were highly proliferative and provided a unique glimpse into Treg cell clonality within expressed high amounts of CD25, CTLA-4, and PD-1 pro- human tumors. The investigators were able to determine teins. RNA sequencing of purified cell populations revealed abTCR sequences for the majority of single Treg cells, which that the transcriptional profiles of intratumoral Treg cells and revealed that a substantial fraction of intratumoral Treg cells Treg cells from normal breast tissue were very similar, sug- expressed abTCRs that were found recurrently in a given gesting that much of the transcriptional program of breast tumor. In contrast, Treg cells isolated from peripheral blood cancer–infiltrating Treg cells may be associated with residency and intratumoral conventional T cells exhibited diverse in mammary tissue. The analysis also revealed that CCR8, a abTCR sequences with few recurrent clones. In accordance receptor for chemokines such as CCL1 and CCL18, was se- with this, Plitas et al. (18) used bTCR sequencing of lectively expressed by both intratumoral Treg cells and Treg bulk T cell populations to demonstrate that breast cancer– cells in normal breast parenchyma, suggesting that CCR8- infiltrating Treg cells exhibited reduced clonal diversity relative Downloaded from dependent signals may be important for Treg cell recruit- to naive-phenotype peripheral Treg cells. These findings provide ment, positioning, or function at these sites. Notably, the direct evidence of expansion or enrichment of distinct Treg cell authors demonstrated that CCR8 protein was also expressed clones in human tumors, a finding that is consistent with pre- by intratumoral Treg cells from human colorectal carcinoma vious work in mice suggesting that developing tumors drive the (CRC), melanoma, and lung adenocarcinoma samples, sug- enrichment of oligoclonal Treg cell populations (22). gesting that CCR8 may be relevant in multiple cancer types. Taken together, these studies demonstrate that in the human http://www.jimmunol.org/ In parallel studies, De Simone et al. (19) analyzed bulk Treg cancers analyzed thus far, the transcriptional program of cells and CD4+ Th cell subsets isolated from CRCs and non– tumor-infiltrating Treg cells may represent a composite sig- small cell lung cancers (NSCLC),enablingadirectcomparison nature blending a tissue-specific signature associated with the of the transcriptomes of Treg cells from cancers originating at organ of cancer origin with an additional tumor-specific sig- different organ sites. Interestingly, this revealed a set of tran- nature that may be common to Treg cells from many cancer scripts that were preferentially upregulated by Treg cells in both types (Fig. 1). These findings pave the way for future mech- CRC and NSCLC compared with Treg cells and conventional anistic studies probing the functional role of these pathways in

T cells from normal nonlymphoid tissues and blood. Single-cell coordinating intratumoral Treg cell activity and determining by guest on September 25, 2021 analysis of intratumoral Treg cells using quantitative PCR the feasibility of targeting these pathways using Abs or small- confirmed that many of these signature genes were also upreg- molecule agents. ulated in Treg cells isolated from other cancer types, including breast cancer, gastric cancer, and metastases of NSCLC and Intratumoral Treg cells exhibit unique requirements that may enable CRC. More recently, Zheng et al. (20) performed single-cell selective targeting or modulation RNA sequencing of T cells isolated from the blood, tumor, and In recent years, there has been a growing appreciation that Treg normal tissue of patients with hepatocellular carcinoma. Tran- cells exhibit substantial phenotypic and functional diversity scriptional analysis of single FOXP3-expressing Treg cells [reviewed extensively by others (23–25)]. In the SLOs, Treg identified ∼400 genes that were preferentially upregulated by cells can be broadly categorized as quiescent, naive-phenotype intratumoral Treg cells relative to Treg cells isolated from central Treg (cTreg) cells or highly proliferative effector Treg normal adjacent tissue. Importantly, Zheng et al. (20) per- (eTreg) cells exhibiting an activated phenotype (26, 27) formed a meta-analysis and noted that 31 genes in the intra- (Fig. 1). Conditional deletion of the TCR on Foxp3- tumoral Treg cell signature were identified in the three human expressing cells blocks the differentiation of cTreg cells into studies highlighted in this review, as well as a fourth study of eTreg cells and leads to the development of autoimmunity, cells isolated from human melanomas (21), indicating that indicating that eTreg cell differentiation is required for Treg intratumoral Treg cells isolated from different cancer types may cell–mediated immune suppression (28, 29). Additionally, express a conserved “tumor-specific” Treg cell signature. This eTreg cells in the SLOs exhibit specialized transcriptional signature includes many genes encoding well-characterized programs associated with common Th cell differentiation immunomodulatory cell-surface receptors, including CTLA-4, pathways such as Th1, Th2, Th17, and T follicular helper cell GITR, 4-1BB, TIGIT, OX40, ICOS, and CD27. Given programs, and, in some cases, adoption of these programs by that these markers are also expressed by a fraction of Treg cells Treg cells is essential for the regulation of inflammatory re- throughout the body, these proteins may represent the “usual sponses. Beyond Treg cell diversity in the SLOs, as discussed suspects” that appear in the conserved tumor-specific Treg cell below, it is now evident that additional differentiation steps signature based on upregulation in the tumor environment. are required for the trafficking, function, and fitness of Treg Importantly, the conserved 31-gene signature also includes cells infiltrating nonlymphoid organs and inflammatory sites. genes encoding proteins not previously implicated in tumor- Building from these concepts, the demonstration that Treg infiltrating Treg cell biology, such as MAGEH1 (encoding a cells infiltrating human tumors express unique transcriptional type II MAGE family protein of unknown function), IL1R2 signatures suggests that Treg cells may have distinct require- (encoding a decoy receptor for IL-1), TFRC (encoding a ments to function and thrive in the unique inflammatory 418 BRIEF REVIEWS: TUMOR-ASSOCIATED REGULATORY T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 1. Hallmarks of tumor-infiltrating Treg cells. A conceptual model highlighting the phenotypic and functional diversity of Treg cells in lymphoid organs, nonlymphoid tissue sites, and tumor lesions is shown. cTreg cells and eTreg cells in the lymph nodes and spleen are thought to mediate the suppression of autoimmune reactions and the maintenance of immune homeostasis. cTreg cells are characterized by a quiescent “naive” phenotype and can differentiate into activated-phenotype eTreg cells in an Ag-dependent process. Tissue-associated Treg cells present in different nonlymphoid sites exhibit tissue-specific transcriptional programs unique to the tissue of residence, and they can mediate diverse context-dependent functions that are independent of immune suppression. The mechanisms driving Treg cell enrichment and expansion within tumors are unknown but may involve responsiveness to Ag, chemokines, and inflammatory mediators. Intratumoral Treg cells are characterized by the following hallmarks: 1) evidence of oligoclonal expansion; 2) a composite transcriptional signature that blends a tissue-specific Treg cell signature associated with the tissue of cancer origin with a conserved tumor-specific signature that is common to Treg cells from different cancer types; 3) high expression of common Treg markers known to function as immunomodulatory receptors, including CTLA-4, PD-1, TIGIT, by guest on September 25, 2021 OX40, and GITR; and 4) expression of transcripts encoding proteins with undefined roles in intratumoral Treg cell biology, including CCR8, MAGEH1, IL1R2, TFRC, and FCRL3. Beyond suppression of antitumor T cell responses, additional functions of intratumoral Treg cells have yet to be clearly defined. The arrow suggests a precursor–product relationship, which has been defined for the cTreg-to-eTreg cell transition, but remains hypothetical for tissue-associated and intratumoral Treg cell populations. milieus associated with cancer, including tissue remodeling, connection between VEGF production and Treg cell–specific angiogenesis, hypoxia, and altered metabolic states (1). This Nrp1 function has yet to be demonstrated in vivo, these raises the possibility that unique requirements of intratumoral findings raise the intriguing possibility that Nrp1 may play a Treg cells, if identified, could be targeted to specifically key role in coordinating Treg cell responsiveness to VEGF- modulate tumor-infiltrating Treg cells without impacting dependent angiogenic signals in the tumor environment. In Treg cells elsewhere in the body. As discussed below, recent parallel studies, work from Vignali and colleagues (33, 34) advances have identified such pathways and provided func- demonstrated that Nrp1 functions as a receptor for Sema4a tional evidence in support of this idea. on Treg cells, and they revealed a key role for Nrp1 ligand Some of the first work demonstrating this concept came engagement in maintaining Treg cell functionality within from studies examining the role of neuropilin-1 (Nrp1) ex- tumors. Using mutant mice in which Nrp1 is conditionally pression by Treg cells. Nrp1 is a type I transmembrane protein deleted on Treg cells, they showed that Nrp1 expression was that is highly expressed by Treg cells and has been suggested to dispensable for Treg cell–mediated maintenance of systemic play a key role in Treg cell function (30, 31). Nrp1 functions tolerance and immune homeostasis (33). However, challenge as a receptor for both vascular endothelial growth factor of Nrp1 mutant mice with transplantable B16 melanomas (VEGF), a critical regulator of blood vessel formation, and revealed slower tumor outgrowth and enhanced antitumor semaphorin-4a (Sema4a), a cell-surface protein that has well- T cell responses, consistent with the findings of Hansen et al. defined roles in axonal guidance but is also expressed by Blockade of the Sema4a–Nrp1 axis using mAbs significantly dendritic cells, B cells, and T cells. Work from Hansen et al. slowed tumor growth, indicating that transient Nrp1 block- (32) demonstrated that Nrp1-expressing Treg cells migrate in ade can have measurable effects. Mechanistic analysis showed response to tumor-derived VEGF in vitro, and they showed that tumor-infiltrating Treg cells in Nrp1 mutant mice that Treg cell–specific deletion of Nrp1 slowed the outgrowth maintained normal levels of Foxp3 expression but exhibited a of transplantable and autochthonous tumors without induc- dysregulated transcriptional program and aberrant production ing systemic autoimmunity, suggesting a role for Nrp1 ex- of cytokines such as IFN-g (34). Interestingly, this effect was pression on tumor-associated Treg cells. Although a direct dominant, in that conditional deletion of Nrp1 on 50% of The Journal of Immunology 419

Treg cells induced T cell–intrinsic defects but also led to the suppressing adaptive immunity and maintaining immune dysregulation of the remaining Nrp1-expressing intratumoral homeostasis. Although the relevance of these functions to Treg cells (34). Taken together, these studies in mice dem- tumor-associated Treg cells is highly speculative at this time, onstrate that disruption of Nrp1 expression by Treg cells these findings expand our view of Treg cell functional di- specifically affects Treg cell activity in the tumor environment versity, suggest that the functions of intratumoral Treg cells without impacting Treg cell function elsewhere in the body, may be manifold and context-dependent, and force a revis- revealing the existence of a distinct pathway that can be itation of the paradigm that the primary function of tumor- specifically targeted to modulate intratumoral Treg cell ac- infiltrating Treg cells is to suppress antitumor immunity. tivity. Further work will be needed to determine whether In seminal work, Mathis, Benoist, and colleagues (36–38) Nrp1 signaling plays a key role in Treg cell function in hu- demonstrated that Treg cells play a key role in regulating man cancers, and to assess the relative contributions of VEGF obesity-related inflammation of the visceral adipose tissue and Sema4a signals in coordinating the activity of Nrp1- (VAT). The investigators showed that the densities of Treg expressing Treg cells in the tumor environment. cells in the VAT are greatly diminished in insulin-resistant In other areas of inquiry, Luo et al. (35) demonstrated that mouse models of obesity, and that sustained expansion of the transcription factor Foxo1 antagonizes the eTreg cell Treg cells induced normalization of blood glucose levels in transcriptional program, and must be downregulated for mice fed a high-fat diet, indicating that Treg cells can affect proper eTreg cell differentiation and tumor infiltration. Using adipose-associated inflammation and metabolic function. The mutant mice in which Foxo1 expression in Treg cells is re- investigators used transcriptional profiling to reveal that VAT Downloaded from sistant to downregulation, the authors observed that the ho- Treg cells preferentially express an array of transcripts not mozygous mutant mice exhibited defects in eTreg cell typically expressed by Treg cells at other sites, and they differentiation associated with decreased Treg cell trafficking showed that at least two of these factors are critical for the to nonlymphoid organs, resulting in autoimmune reactions optimal accumulation of Treg cells in VAT. First, it was mediated by CD8+ T cells. Moreover, the outgrowth of shown that PPAR-g, a receptor that is important for adipo- transplantable and genetically driven tumors was slowed in cyte differentiation, is highly expressed by VAT Treg cells and http://www.jimmunol.org/ homozygous mutant mice, indicative of impaired Treg cell is required for Treg cell enrichment in adipose tissue. Second, activity and enhanced antitumor immunity. Interestingly, the it was shown that most VAT Treg cells express the IL-33 investigators also identified a “sweet spot” in which hetero- receptor ST2, and that Treg cell density in the VAT was zygosity of the constitutively activated Foxo1 mutant allele led diminished in ST2-deficient mice. Notably, IL-33 has been to enhanced antitumor immunity without inducing wide- shown to function as an “alarmin” that triggers inflammatory spread autoimmunity. Thus, this study identified Foxo1 ac- responses when released from stressed or dying cells (39), tivity as a key determinant of the differentiation of eTreg cells suggesting a role for the IL-33–ST2 axis in driving the

and demonstrated that this axis can be modulated genetically homing and/or expansion of Treg cells at sites of tissue by guest on September 25, 2021 to restrict the trafficking and fitness of intratumoral Treg cells damage. Taken together, these studies laid the groundwork without altering Treg cell–mediated immune regulation in the for a paradigm suggesting that Treg cells at nonlymphoid SLOs. Given that Foxo1 is an intracellular transcription factor sites mediate functions that are independent of immune that is widely expressed by many cell types and functions suppression, express unique gene expression profiles that downstream of the PI3K–Akt pathway, it is unclear whether are required for optimal function and fitness in the local the “tuning” of Foxo1 activity in genetic mouse models can be environment, and are responsive to Ag-independent inflam- recapitulated using small-molecule drugs targeting this path- matory mediators (25). way. Regardless, these data reveal that subtle perturbations in In other areas, additional work has defined a unique role for Treg cell signaling can induce major functional changes that Treg cells in promoting tissue repair in nonlymphoid organs dissociate Treg cell function in the tumor environment from (40, 41) and identified amphiregulin (Areg) as a key factor Treg cell activity elsewhere in the body. produced by Treg cells at sites of tissue damage. In one study, Collectively, these studies demonstrate that in mice, intra- Burzyn et al. (40) demonstrated that clonally expanded Treg tumoral Treg cells exhibit unique requirements for proper cell populations accumulate in acutely injured muscle tissue function and fitness in the tumor context, and they show that and in the damaged muscle of Dmd mutant mice, a mouse these requirements are divergent from the functional re- model of muscular dystrophy. Transient depletion of Treg quirements of Treg cells tasked with preventing autoimmunity cells impaired muscle repair and was associated with increased and maintaining immune homeostasis in the SLOs. Moving cellular infiltrates, increased fibrosis, and a failure of myeloid forward, it is possible that the intratumoral Treg cell signatures cells to switch to a proregenerative phenotype. As with VAT- identified in the studies discussed above may help illuminate associated Treg cells, muscle-infiltrating Treg cells exhibited a molecular pathways required for Treg cell activity in human unique transcriptional program that was distinct from that of cancers, providing new opportunities to selectively target splenic Treg cells. One of the genes preferentially expressed by intratumoral Treg cells while maintaining systemic immune muscle-infiltrating Treg cells was the gene encoding Areg, an regulation. epidermal growth factor family ligand that signals through the epidermal growth factor receptor system. Treg cells were Beyond immune suppression: newly defined functions of Treg cells in found in close proximity to regenerating muscle myofibers in nonlymphoid tissues situ, and they promoted the differentiation of muscle satellite In recent years, a growing number of studies have revealed cells in vitro, suggesting that Treg cells may function to unique functions of Foxp3+ Treg cells in nonlymphoid sites promote muscle regeneration in part by direct communica- that appear to be independent of their well-defined roles in tion with muscle progenitor cells. In a separate study, Arpaia 420 BRIEF REVIEWS: TUMOR-ASSOCIATED REGULATORY T CELLS et al. (41) generated mice with Treg cell–specific deletion of cell activity in the tumor context. This suggests that the Areg and examined the impact of this deficiency on immune functions of intratumoral Treg cells may be diverse and highly regulation and tissue repair in the lung in the context of an context-dependent, and highlights a key need to examine ongoing viral infection. Notably, the authors found that Areg additional functions of tumor-infiltrating Treg cells beyond expression by Treg cells was dispensable for the maintenance their defined roles in the suppression of antitumor immunity. of immune homeostasis and the regulation of virus-specific T cell responses. In contrast, Areg mutant mice exhibited Conclusions increased tissue damage and a rapid decline in lung function Evidence that intratumoral Treg cell depletion may underlie following influenza virus infection, demonstrating that Treg the clinical efficacy of therapeutic Abs such as anti–CTLA-4 cells restrict tissue damage via Areg production in this setting. serves as a testament to the functional importance of Treg Additional mechanistic data revealed that Areg production by cells in the tumor context and provides the impetus for Treg cells in vitro was triggered by exposure to IL-18 and continued efforts to understand the fundamental biology of IL-33, but not by TCR stimulation, suggesting that TCR- Treg cells and the functional diversity of these cells in dif- independent cues in the microenvironment may be the ferent cancers. Given that many current and emerging ther- primary drivers of Areg production by Treg cells. Taken to- apeutic Abs target cell-surface receptors that are expressed at gether, these findings raise the intriguing possibility that a key high amounts by tumor-infiltrating Treg cells, it will be function of intratumoral Treg cells in lung cancers and other critical to determine the extent to which these Abs function by solid cancers may lie in regulating tissue remodeling and re- direct action on Treg cells, either through modulation of Treg Downloaded from pair through the production of Areg and other factors. No- cell activity or through ADCC-mediated Treg cell depletion. tably, the studies of Plitas et al. (18) revealed that Areg is In this regard, it will be interesting to compare the clinical significantly upregulated by a subset of intratumoral Treg cells efficacy of Abs bearing isotypes that vary with respect to in breast cancer lesions, lending credence to this idea. ADCC triggering (7). Such mechanistic understanding may Finally, a recent study by Ali et al. (42) further expanded the be crucial for identifying patients who are likely to benefit newly defined roles of tissue-associated Treg cells, demon- from treatment, versus those who are not. Additionally, for http://www.jimmunol.org/ strating a pivotal role for skin-resident Treg cells in promoting cancers such as CRC in which Treg cells may play a key role the differentiation of hair follicle stem cells (HFSCs). The in restricting cancer progression, if a given Ab is shown to authors demonstrated that Treg cells are localized in close directly dampen intratumoral Treg cell activity, it may be proximity to the HFSC niche within hair follicles, and that unwise to use such Abs in these clinical contexts. inducible ablation of Treg cells suppressed hair growth. In- Moving forward, mechanistic experiments in tractable terestingly, it was also shown that Treg cell expression of mouse models of autochthonous cancer and human cell culture Jagged1, a Notch ligand that is highly expressed by Treg cells systems are needed to elucidate the role of key molecular

at this site, was required for optimal HFSC proliferation and pathways implicated in intratumoral Treg cell biology. For by guest on September 25, 2021 induction of the active growth stage of hair follicle formation. example, the human studies discussed above demonstrated that Thus, these findings suggest a model in which skin-associated CCR8 is upregulated by tumor-infiltrating Treg cells in Treg cells function at steady-state to promote hair follicle multiple cancer types, suggesting that CCR8-dependent sig- development via engagement of Notch receptors on HFSCs. nals may be important for Treg cell homing, positioning, or Interestingly, Notch pathway activation or inhibition has been function within tumor lesions. However, given that there are implicated as a driver of tumorigenesis in some human ma- additional chemokine receptors expressed by tumor-associated lignancies, including melanoma (43–45). These concepts raise Treg cells (18), and considerable redundancy in many che- the possibility that Treg cells may shape cancer development mokine–receptor axes, further investigation will be needed to by direct cell–cell crosstalk with cancer cells or cancer stem determine the requirement for CCR8 in intratumoral Treg cells via Jagged1–Notch interactions. cell activity. Furthermore, CCR8 expression is not unique to Collectively, the studies discussed above highlight our tumor-infiltrating Treg cells, but it is also expressed by tumor- expanding view of the functional diversity and specialization of infiltrating NKT cells and a fraction of Treg cells resident in Treg cells in distinct tissue environments throughout the body, some healthy tissues (18). Future studies in preclinical cancer and they reveal broad principles regarding Treg cell biology in models can be used to gauge the feasibility of targeting CCR8 nonlymphoid tissues (Fig. 1). Specifically, tissue-associated to either dampen intratumoral Treg cell function or induce Treg cells can respond to Ag-independent inflammatory the selective depletion of these cells. mediators and express unique gene expression profiles that are Finally, an undefined facet of intratumoral Treg cell biology required for optimal function and survival in the local envi- lies in understanding the cellular partners that Treg cells ronment. From a functional standpoint, tissue-associated interface with in the tumor environment. Do Treg cells directly Treg cells exhibit diverse functions that are independent of interact with tumor cells or cancer progenitors cells, or is immune suppression; these functions may include conserved their activity limited to interactions with APC populations mechanisms such as the augmentation of tissue repair via Areg such as dendritic cells or macrophages? Several imaging-based production, or specialized tissue-specific functions such as studies have demonstrated direct Treg cell interactions with metabolic regulation or stimulation of stem cell differentia- Ag-bearing dendritic cells within transplantable and geneti- tion. The finding that the transcriptional programs of intra- cally engineered murine tumors (46–48). However, given tumoral Treg cells from various human cancer types are emerging data indicating that Treg cells may be responsive to similar to the programs of their Treg cell counterparts in Ag-independent environmental cues within nonlymphoid normal adjacent tissue suggests that the principles defined for tissues, it will be important to determine whether additional tissue-associated Treg cells may be directly relevant for Treg Ag-independent liaisons take place in distinct regions of a The Journal of Immunology 421 tumor. 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