Unlocking the complexities of tumor-associated regulatory T cells

Do “checkpoint blockade” antibodies function by depleting intratumoral Treg cells?

In the past decade, antibodies specific for the T cell co-inhibitory receptors CTLA-4 and PD-1 have shown striking success in inducing durable clinical benefit in a fraction of cancer patients spanning a variety of cancer types (5). Early in their development, these antibodies were dubbed “checkpoint blockade” antibodies based on the idea that they were thought to function by blocking the binding of CTLA-4 or PD-1 to their ligands, thereby releasing tumor-specific T cells from checkpoints limiting their activation and effector function (6). However, recent work has challenged this idea, suggesting that some of these antibodies may function instead by inducing the depletion of CTLA-4-expressing cells in the tumor environment by binding to target cells and inducing antibody-dependent cellular cytotoxicity (ADCC) (7, 8). Given that Treg cells generally express high amounts of cell-surface CTLA-4, PD-1, and other immunomodulatory receptors such as OX40, 4-1BB, and ICOS (discussed below), this suggests that therapeutic antibodies targeting these proteins may function in part by driving the selective depletion of intratumoral Treg cells.

In support of this idea, Simpson and colleagues demonstrated in mice that administration of distinct anti-CTLA-4 antibody clones induced the depletion of intratumoral Treg cells without impacting intratumoral conventional T cells or Treg cells outside of the tumor, resulting in slowed outgrowth of transplantable melanomas (9). Interestingly, the efficacy of these antibodies was dependent on the presence of tumor-infiltrating myeloid cells expressing activating Fc receptors, implying a role for Fc receptor-dependent, ADCC-mediated Treg cell depletion. In addition, the authors suggested that the selectivity of anti-CTLA-4 antibody in driving the preferential depletion of Treg cells was largely due to the elevated cell-surface expression of CTLA-4 by intratumoral Treg cells relative to conventional T cells within the tumor, a concept that may be broadly relevant for therapeutic antibodies targeting other T cell-expressed receptors. Working in parallel, Selby and colleagues performed similar studies in mice using an anti-CTLA-4 antibody clone of fixed antigen-binding specificity in which the constant region of the antibody was changed to different isotypes known to engage activating Fc receptors with varying affinities (10). It was found that the therapeutic efficacy of anti-CTLA-4 antibody treatment correlated with the ability of the Fc region to bind activating Fc receptors, again suggesting a role for ADCC in determining in vivo antibody activity. These two studies highlight the importance of both the Fab and Fc regions of an antibody in determining the therapeutic efficacy of an antibody of interest. Consistent with these findings, additional studies in mice have demonstrated similar requirements for therapeutic antibodies targeting other T cell-expressed co-receptors such as GITR (11, 12) and OX40 (13), for which specific antibodies are currently under clinical development for the treatment of human cancer (7).

Despite clear mechanistic evidence in mice that antibodies can promote tumor rejection by inducing intratumoral Treg cell depletion, the relevance of these concepts to the efficacy of antibody-based immunotherapies in human cancer patients remains undefined. The clearest available evidence in support of this idea comes from the studies of Romano et al., which compared samples from human melanoma patients who did or did not respond to therapy using the anti-CTLA-4 antibody ipilimumab (14). In ex vivo assays, it was found that non-classical monocytes expressing the activating Fc receptor CD16 can engage ipilimumab and induce ADCC-mediated lysis of Treg cells in vitro. Importantly, clinical responses to ipilimumab were associated with Treg cell depletion in tumor lesions, as well as elevated densities of non-classical monocytes in the peripheral blood at baseline compared to non-responders (14), suggestive of ADCC-dependent intratumoral Treg cell ablation. Taken together, the studies above support the idea that antibodies specific for T cell-expressed receptors may function in part by inducing the selective elimination of tumor-infiltrating Treg cells in circumstances in which the antibody isotype can avidly engage activating Fc receptors, and Fc receptor-expressing myeloid cells are present at sufficient densities.

The idea that intratumoral Treg cell depletion may underlie the efficacy of therapeutic antibodies in some settings highlights the functional importance of Treg cells in the tumor context. Moreover, the notion that antibodies that had previously been thought of as “blocking” reagents may function in some contexts by inducing ADCC has revitalized enthusiasm for older strategies for therapeutic Treg cell ablation. For example, an attractive marker for Treg cell targeting is the IL-2 receptor alpha chain CD25; relative to conventional T cells, Foxp3⁺ Treg cells express high amounts of CD25 (15), and CD25 expression is required for optimal Treg cell survival and fitness (16, 17). However, previous studies in mice and humans have failed to demonstrate consistent activity of anti-CD25 antibodies in the tumor context (15). To re-examine this phenomenon, Arce Vargas et al. demonstrated in mice that administration of a common anti-CD25 antibody clone reduced Treg cell frequencies in the blood and secondary lymphoid organs (SLOs) but failed to induce intratumoral Treg cell depletion (15). In contrast, use of a variant of this clone engineered to preferentially engage activating Fc receptors (at the expense of binding to inhibitory Fc receptors) induced robust intratumoral Treg cell depletion and enhanced anti-tumor immunity (15). These findings provide additional evidence that the design of antibody Fc regions is a key determinant of biological activity and in vivo efficacy of antibody therapeutics, and suggests a need to re-evaluate antibodies that had previously been deemed to be ineffective in the tumor setting. Moreover, the collective findings discussed above suggest that antibodies targeting different T cell-expressed cell-surface receptors may function by a common mechanism - binding avidly to Treg cells and marking these cells for elimination by ADCC (7).

Analysis of tumor-infiltrating Treg cells in human cancers

Beyond CTLA-4, CD25, and other well-defined immunomodulatory receptors, an improved understanding of the unique features of tumor-infiltrating Treg cells may provide key insight into the function of these cells in the tumor context, and reveal novel targets for the selective modulation of Treg cells for therapeutic benefit. In this light, three recent papers report high-resolution surveys of Treg cells infiltrating human cancers. In one study, Plitas et al. performed transcriptional and phenotypic analysis of Treg cells isolated from breast cancer lesions, and compared these to Treg cells isolated from normal breast parenchyma or peripheral blood (18). The authors found that breast tumors contained elevated percentages of Treg cells relative to normal breast tissue, and that intratumoral Treg cells were highly proliferative and expressed high amounts of CD25, CTLA-4, and PD-1 proteins. RNA sequencing of purified cell populations revealed that the transcriptional profiles of intratumoral Treg cells and Treg cells from normal breast tissue were very similar, suggesting that much of the transcriptional program of breast cancer-infiltrating Treg cells may be associated with residency in mammary tissue. The analysis also revealed that CCR8, a receptor for chemokines such as CCL1 and CCL18, was selectively expressed by both intratumoral Treg cells and Treg cells in normal breast parenchyma, suggesting that CCR8-dependent signals may be important for Treg cell recruitment, positioning, or function at these sites. Notably, the authors demonstrated that CCR8 protein was also expressed by intratumoral Treg cells from human colorectal carcinoma (CRC), melanoma, and lung adenocarcinoma samples, suggesting that CCR8 may be relevant in multiple cancer types.

In parallel studies, De Simone and colleagues analyzed bulk Treg cells and CD4⁺ T helper cell subsets isolated from CRCs and non-small-cell lung cancers (NSCLC), enabling a direct comparison of the transcriptomes of Treg cells from cancers originating at different organ sites (19). Interestingly, this revealed a set of transcripts that were preferentially upregulated by Treg cells in both CRC and NSCLC compared to Treg cells and conventional T cells from normal non-lymphoid tissues and blood. Single-cell analysis of intratumoral Treg cells using quantitative PCR confirmed that many of these signature genes were also upregulated in Treg cells isolated from other cancer types, including breast cancer, gastric cancer, and metastases of NSCLC and CRC. More recently, Zheng and colleagues performed single-cell RNA sequencing of T cells isolated from the blood, tumor, and normal tissue of patients with hepatocellular carcinoma (20). Transcriptional analysis of single FOXP3-expressing Treg cells identified ~400 genes that were preferentially upregulated by intratumoral Treg cells relative to Treg cells isolated from normal adjacent tissue. Importantly, Zheng et al. performed a meta-analysis and noted that 31 genes in the intratumoral Treg cell signature were identified in the three human studies highlighted here, as well as a fourth study of cells isolated from human melanomas (21), indicating that intratumoral Treg cells isolated from different cancer types may express a conserved “tumor specific” Treg cell signature. This signature includes many genes encoding well characterized immunomodulatory cell-surface receptors, including CTLA-4, GITR, 4-1BB, TIGIT, OX40, ICOS, and CD27. Given that these markers are also expressed by a fraction of Treg cells throughout the body, these proteins may represent the “usual suspects” that appear in the conserved tumor-specific Treg cell signature based on upregulation in the tumor environment. Importantly, the conserved 31-gene signature also includes genes encoding proteins not previously implicated in tumor-infiltrating Treg cell biology, such as MAGEH1 (encoding a type II MAGE family protein of unknown function), IL1R2 (encoding a decoy receptor for IL-1), TFRC (encoding a transferrin receptor), FCRL3 (encoding an Fc receptor-like protein), and CCR8. It remains unknown whether these proteins are important for Treg cell function in the tumor environment, or reflect adaptation required to survive and thrive within neoplastic lesions.

Beyond the characterization of transcriptional profiles, the single-cell RNA sequencing studies of Zheng et al. provided a unique glimpse into Treg cell clonality within human tumors. The investigators were able to determine αβTCR sequences for the majority of single Treg cells, which revealed that a substantial fraction of intratumoral Treg cells expressed αβTCRs that were found recurrently in a given tumor. In contrast, Treg cells isolated from peripheral blood and intratumoral conventional T cells exhibited diverse αβTCR sequences with few recurrent clones. In accordance with this, Plitas et al. utilized βTCR sequencing of bulk T cell populations to demonstrate that breast cancer-infiltrating Treg cells exhibited reduced clonal diversity relative to naive-phenotype peripheral Treg cells (18). These findings provide direct evidence of expansion or enrichment of distinct Treg cell clones in human tumors, a finding that is consistent with previous work in mice suggesting that developing tumors drive the enrichment of oligoclonal Treg cell populations (22).

Taken together, these studies demonstrate that in the human cancers analyzed thus far, the transcriptional program of tumor-infiltrating Treg cells may represent a composite signature blending a tissue-specific signature associated with the organ of cancer origin with an additional tumor-specific signature that may be common to Treg cells from many cancer types (Figure 1). Theses findings pave the way for future mechanistic studies probing the functional role of these pathways in coordinating intratumoral Treg cell activity, and determining the feasibility of targeting these pathways using antibodies or small-molecule agents.

Intratumoral Treg cells exhibit unique requirements that may enable selective targeting or modulation

In recent years, there has been a growing appreciation that Treg cells exhibit substantial phenotypic and functional diversity (Reviewed extensively by others (23–25)). In the SLOs, Treg cells can be broadly categorized as quiescent, naïve-phenotype “central” Tregs (cTregs), or highly proliferative “effector” Tregs (eTregs) exhibiting an activated phenotype (26, 27) (Figure 1). Conditional deletion of the TCR on Foxp3-expressing cells blocks the differentiation of cTregs into eTreg cells and leads to the development of autoimmunity, indicating that eTreg differentiation is required for Treg-mediated immune suppression (28, 29). In addition, eTreg cells in the SLOs exhibit specialized transcriptional programs associated with common T helper cell differentiation pathways such as Th1, Th2, Th17, and Tfh programs, and in some cases, adoption of these programs by Treg cells is essential for the regulation of inflammatory responses. Beyond Treg cell diversity in the SLOs, as discussed below, it is now evident that additional differentiation steps are required for the trafficking, function, and fitness of Treg cells infiltrating non-lymphoid organs and inflammatory sites. Building from these concepts, the demonstration that Treg cells infiltrating human tumors express unique transcriptional signatures suggests that Treg cells may have distinct requirements to function and thrive in the unique inflammatory milieus associated with cancer, including tissue remodeling, angiogenesis, hypoxia, and altered metabolic states (1). This raises the possibility that unique requirements of intratumoral Treg cells, if identified, could be targeted to specifically modulate tumor-infiltrating Treg cells without impacting Treg cells elsewhere in the body. As discussed below, recent advances have identified such pathways and provided functional evidence in support of this idea.

Some of the first work demonstrating this concept came from studies examining the role of neuropilin-1 (Nrp1) expression by Treg cells. Nrp1 is a type I transmembrane protein that is highly expressed by Treg cells and has been suggested to play a key role in Treg cell function (30, 31). Nrp1 functions as a receptor for both vascular endothelial growth factor (VEGF), a critical regulator of blood vessel formation, and semaphorin-4a (Sema4a), a cell-surface protein that has well-defined roles in axonal guidance but is also expressed by dendritic cells, B cells, and T cells. Work from Hansen and colleagues demonstrated that Nrp1-expressing Treg cells migrate in response to tumor-derived VEGF in vitro, and showed that Treg-specific deletion of Nrp1 slowed the outgrowth of transplantable and autochthonous tumors without inducing systemic autoimmunity, suggesting a role for Nrp1 expression on tumor-associated Treg cells (32). Although a direct connection between VEGF production and Treg-specific Nrp1 function has yet to be demonstrated in vivo, these findings raise the intriguing possibility that Nrp1 may play a key role in coordinating Treg cell responsiveness to VEGF-dependent angiogenic signals in the tumor environment. In parallel studies, work from Vignali and colleagues demonstrated that Nrp1 functions as a receptor for Sema4a on Treg cells, and revealed a key role for Nrp1 ligand engagement in maintaining Treg cell functionality within tumors (33, 34). Using mutant mice in which Nrp1 is conditionally deleted on Treg cells, they showed that Nrp1 expression was dispensable for Treg-mediated maintenance of systemic tolerance and immune homeostasis (33). However, challenge of Nrp1 mutant mice with transplantable B16 melanomas revealed slower tumor outgrowth and enhanced anti-tumor T cell responses, consistent with the findings of Hansen et al. Blockade of the Sema4a-Nrp1 axis using monoclonal antibodies significantly slowed tumor growth, indicating that transient Nrp1 blockade can have measurable effects. Mechanistic analysis showed that tumor-infiltrating Treg cells in Nrp1 mutant mice maintained normal levels of Foxp3 expression, but exhibited a dysregulated transcriptional program and aberrant production of cytokines such as IFN-γ (34). Interestingly, this effect was dominant, in that conditional deletion of Nrp1 on 50% of Treg cells induced T cell-intrinsic defects but also led to the dysregulation of the remaining Nrp1-expressing intratumoral Treg cells (34). Together, these studies in mice demonstrate that disruption of Nrp1 expression by Treg cells specifically affects Treg cell activity in the tumor environment without impacting Treg cell function elsewhere in the body, revealing the existence of a distinct pathway that can be specifically targeted to modulate intratumoral Treg cell activity. Further work will be needed to determine whether Nrp1 signaling plays a key role in Treg cell function in human cancers, and to assess the relative contributions of VEGF and Sema4a-signals in coordinating the activity of Nrp1-expressing Treg cells in the tumor environment.

In other areas of inquiry, Luo et al. demonstrated that the transcription factor Foxo1 antagonizes the eTreg transcriptional program, and must be downregulated for proper eTreg differentiation and tumor infiltration (35). Using mutant mice in which Foxo1 expression in Treg cells is resistant to downregulation, the authors observed that the homozygous mutant mice exhibited defects in eTreg differentiation associated with decreased Treg cell trafficking to non-lymphoid organs, resulting in autoimmune reactions mediated by CD8⁺ T cells. Moreover, the outgrowth of transplantable and genetically driven tumors was slowed in homozygous mutant mice, indicative of impaired Treg cell activity and enhanced anti-tumor immunity. Interestingly, the investigators also identified a “sweet spot” in which heterozygosity of the constitutively-activate Foxo1 mutant allele led to enhanced anti-tumor immunity without inducing widespread autoimmunity. Thus, this study identified Foxo1 activity as a key determinant of the differentiation of eTregs, and demonstrated that this axis can be modulated genetically to restrict the trafficking and fitness of intratumoral Treg cells without altering Treg-mediated immune regulation in the SLOs. Given that Foxo1 is an intracellular transcription factor that is widely expressed by many cell types and functions downstream of the PI3K-Akt pathway, it is unclear whether the “tuning” of Foxo1 activity in genetic mouse models can be recapitulated using small-molecule drugs targeting this pathway. Regardless, these data reveal that subtle perturbations in Treg cell signaling can induce major functional changes that dissociate Treg cell function in the tumor environment from Treg cell activity elsewhere in the body.

Collectively, these studies demonstrate that in mice, intratumoral Treg cells exhibit unique requirements for proper function and fitness in the tumor context, and show that these requirements are divergent from the functional requirements of Treg cells tasked with preventing autoimmunity and maintaining immune homeostasis in the SLOs. Moving forward, it is possible that the intratumoral Treg cell signatures identified in the studies discussed above may help illuminate molecular pathways required for Treg cell activity in human cancers, providing new opportunities to selectively target intratumoral Treg cells while maintaining systemic immune regulation.

Beyond immune suppression: newly defined functions of Treg cells in non-lymphoid tissues

In recent years, a growing number of studies have revealed unique functions of Foxp3⁺ Treg cells in non-lymphoid sites that appear to be independent of their well-defined roles in suppressing adaptive immunity and maintaining immune homeostasis. While the relevance of these functions to tumor-associated Treg cells is highly speculative at this time, these findings expand our view of Treg cell functional diversity, suggest that the functions of intratumoral Treg cells may be manifold and context-dependent, and force a re-visitation of the paradigm that the primary function of tumor-infiltrating Treg cells is to suppress anti-tumor immunity.

In seminal work, Mathis and Benoist demonstrated that Treg cells play a key role in regulating obesity-related inflammation of the visceral adipose tissue (VAT) (36–38). The investigators showed that the densities of Treg cells in the VAT are greatly diminished in insulin-resistant mouse models of obesity, and that sustained expansion of Treg cells induced normalization of blood glucose levels in mice fed a high fat diet, indicating that Treg cells can affect adipose-associated inflammation and metabolic function. The investigators used transcriptional profiling to reveal that VAT Treg cells preferentially express an array of transcripts not typically expressed by Treg cells at other sites, and showed that at least two of these factors are critical for the optimal accumulation of Treg cells in VAT. First, it was shown that PPAR-γ, a receptor that is important for adipocyte differentiation, is highly expressed by VAT Treg cells and is required for Treg cell enrichment in adipose tissue. Second, it was shown that the majority of VAT Treg cells express the IL-33 receptor ST2, and that Treg cell density in the VAT was diminished in ST2-deficient mice. Notably, IL-33 has been shown to function as an “alarmin” that triggers inflammatory responses when released from stressed or dying cells (39), suggesting a role for the IL-33/ST2 axis in driving the homing and/or expansion of Treg cells at sites of tissue damage. Together, these studies laid the groundwork for a paradigm suggesting that Treg cells at non-lymphoid sites mediate functions that are independent of immune suppression, express unique gene expression profiles that are required for optimal function and fitness in the local environment, and are responsive to antigen-independent inflammatory mediators (25).

In other areas, additional work has defined a unique role for Treg cells in promoting tissue repair in non-lymphoid organs (40, 41), and identified Amphiregulin (Areg) as a key factor produced by Treg cells at sites of tissue damage. In one study, Burzyn and colleagues demonstrated that clonally expanded Treg populations accumulate in acutely injured muscle tissue and in the damaged muscle of Dmd mutant mice, a mouse model of muscular dystrophy. Transient depletion of Treg cells impaired muscle repair, and was associated with increased cellular infiltrates, increased fibrosis, and a failure of myeloid cells to switch to a pro-regenerative phenotype. As with VAT-associated Treg cells, muscle-infiltrating Treg cells exhibited a unique transcriptional program that was distinct from that of splenic Treg cells. One of the genes preferentially expressed by muscle-infiltrating Treg cells was the gene encoding Areg, an epidermal growth factor (EGF) family ligand that signals through the EGF receptor system. Treg cells were found in close proximity to regenerating muscle myofibers in situ, and promoted the differentiation of muscle satellite cells in vitro, suggesting that Treg cells may function to promote muscle regeneration in part by direct communication with muscle progenitor cells. In a separate study, Arpaia and colleagues generated mice with Treg-specific deletion of Areg, and examined the impact of this deficiency on immune regulation and tissue repair in the lung in the context of an ongoing viral infection (41). Notably, the authors found that Areg expression by Treg cells was dispensable for the maintenance of immune homeostasis and the regulation of virus-specific T cell responses. In contrast, Areg mutant mice exhibited increased tissue damage and a rapid decline in lung function following influenza virus infection, demonstrating that Treg cells restrict tissue damage via Areg production in this setting. Additional mechanistic data revealed that Areg production by Treg cells in vitro was triggered by exposure to IL-18 and IL-33, but not by TCR stimulation, suggesting that TCR-independent cues in the microenvironment may be the primary drivers of Areg production by Treg cells. Taken together, these findings raise the intriguing possibility that a key function of intratumoral Treg cells in lung cancers and other solid cancers may lie in regulating tissue remodeling and repair through the production of Areg and other factors. Notably, the studies of Plitas et al. revealed that Areg is significantly upregulated by a subset of intratumoral Treg cells in breast cancer lesions (18), lending credence to this idea.

Finally, a recent study by Ali et al. further expanded the newly defined roles of tissue-associated Treg cells, demonstrating a pivotal role for skin-resident Treg cells in promoting the differentiation of hair follicle stem cells (HFSCs) (42). The authors demonstrated that Treg cells are localized in close proximity to the HFSC niche within hair follicles, and that inducible ablation of Treg cells suppressed hair growth. Interestingly, it was also shown that Treg cell expression of Jagged1 (Jag1), a Notch ligand that is highly expressed by Treg cells at this site, was required for optimal HFSC proliferation and induction of the active growth stage of hair follicle formation. Thus, these findings suggest a model in which skin-associated Treg cells function at steady state to promote hair follicle development via engagement of Notch receptors on HFSCs. Interestingly, Notch pathway activation or inhibition has been implicated as a driver of tumorigenesis in some human malignancies, including melanoma (43–45). These concepts raise the possibility that Treg cells may shape cancer development by direct cell-cell crosstalk with cancer cells or cancer stem cells via Jag1-Notch interactions.

Collectively, the studies discussed above highlight our expanding view of the functional diversity and specialization of Treg cells in distinct tissue environments throughout the body, and reveal broad principles regarding Treg cell biology in non-lymphoid tissues (Figure 1). Specifically, tissue-associated Treg cells can respond to antigen-independent inflammatory mediators, and express unique gene expression profiles that are required for optimal function and survival in the local environment. From a functional standpoint, tissue-associated Treg cells exhibit diverse functions that are independent of immune suppression; these functions may include conserved mechanisms such as the augmentation of tissue repair via Areg production, or specialized tissue-specific functions such as metabolic regulation or stimulation of stem cell differentiation. The finding that the transcriptional programs of intratumoral Treg cells from various human cancer types are similar to the programs of their Treg cell counterparts in normal adjacent tissue suggests that the principles defined for tissue-associated Treg cells may be directly relevant for Treg cell activity in the tumor context. This suggests that the functions of intratumoral Treg cells may be diverse and highly context-dependent, and highlights a key need to examine additional functions of tumor-infiltrating Treg cells beyond their defined roles in the suppression of anti-tumor immunity.