The importance of T_regs was first described in studies of autoimmunity in animal model systems dating back to the mid-1960s. These early studies showed that removal of the thymus from neonatal mice led to severe autoimmunity in many organs, including hematological disorders, endocrinopathies, gastritis, and oophoritis/orchitis [3]. These studies demonstrated an important role for T cells derived from the thymus in suppressing immune responses in a variety of tissues. Importantly, these early studies also demonstrated that a T cell subpopulation was required to prevent autoimmunity as the transfer of CD4⁺CD8⁻, but not CD4⁻CD8⁺, thymocytes was sufficient to abrogate autoimmunity (Fig. 6.2a).

Subsequent studies sought to define cell-intrinsic markers of this suppressive cell population. Studies to identify markers of T_regs first focused on the observation that T_regs appeared to be an activated T cell population. A cell surface marker that is associated with T cell activation and function is the high-affinity IL-2 receptor α-chain (IL2Rα or CD25). The importance of CD25 in T_reg biology was discovered by comparing reconstitution of athymic nude mice with CD4⁺CD25⁺ versus CD4⁺CD25⁻ splenocytes. Reconstitution of nude mice with the CD4⁺CD25⁺ subpopulation led to suppression of autoimmunity, while the CD4⁺CD25⁻ T cell-reconstituted mice succumbed to autoimmunity (Fig. 6.2b) [4]. This study was the first to highlight the importance of CD25 as a marker of T_regs.

Advancing these observations demonstrated that both CD25 and IL-2 are essential for T_reg development and survival [5]. Although IL-2 contributes to survival and function of all T cells via CD25, it is critically important for T_regs, so much so that CD4⁺CD25⁺ T cells are highly enriched for T_regs. Importantly, T_regs do not secrete IL-2 themselves and therefore require paracrine production of IL-2 by other cell types to exert their effector function. Following the identification of CD4⁺CD25⁺ T cells as a suppressive cell population, additional studies sought to further define markers of T_regs.

The discovery of a genetic mutation that led to a severe autoimmune disease in mice (known as scurfy), and a similar disease in humans (known as immunodysregulation polyendocrinopathy enteropathy X-linked syndrome or IPEX), led investigators to consider the role of this gene in T_regs. This single X-linked gene (now known as Foxp3) was found to encode a key transcription factor (FOXP3) that directs the formation and function of T_regs [6]. FOXP3 has been shown to be critical in driving the development and suppressive function of T_regs by controlling the transcription of key genes required for the maintenance and function of T_regs. FOXP3 drives or enhances the transcription of genes associated with suppression, such as CD25 and CTLA-4, while simultaneously suppressing the transcription of inflammatory genes, such as IFN-γ and IL-2. Expression of FOXP3 itself is driven by the epigenetic hypomethylation of the Foxp3 promoter, which is considered a hallmark of T_regs [7]. While FOXP3 is a specific marker for T_regs, it should be noted that FOXP3 can also be transiently expressed in activated human CD4⁺ T cells [8]. This transient expression of FOXP3 following activation can cause some effector T cells to appear to be FOXP3⁺ T_regs, and therefore phenotypic identification of human T_regs should rely on a combination of markers (i.e., coexpression of CD25 and FOXP3 or the absence of CD127, as described below). Additionally, FOXP3 cannot be used to sort T_regs from unmanipulated samples from mouse or human donors, as it is a transcription factor that is expressed in the nucleus. Consequently, murine studies in which FOXP3⁺ cells are purified routinely rely on the use of genetic reporters.

More recently, the absence of the interleukin-7 (IL-7) receptor (CD127) on CD4⁺CD25⁺ T cells has been described as a population that is enriched for FOXP3⁺ T_regs. Conventional memory T cells require signals from IL-7 for their maintenance and as such express high levels of CD127 [9]. Conversely, T_regs do not express CD127 because FOXP3 suppresses transcription of the Il7r gene, leading to the absence of CD127 on cells that express FOXP3 [10]. In addition to being enriched for expression of FOXP3, cells that are CD4⁺CD25⁺CD127⁻ are highly suppressive in vitro, demonstrating that this population is functionally T_regs. Collectively, these markers have facilitated the purification and analysis of T_regs. However, identification of additional markers would be beneficial.

The role of T_regs in preventing autoimmunity was described using thymus-derived T_regs (tT_regs). However, populations of suppressive CD4⁺FOXP3⁺ T cells can also be generated outside of the thymus [11]. T_regs that develop in vivo outside of the thymus are known as peripheral T_regs (pT_regs). pT_regs differentiate from naïve CD4⁺ T cells in the periphery following activation of naïve CD4+ T cells with suboptimal doses of antigen in the presence of transforming growth factor β (TGF-β). Activation of naïve CD4+ T cells under these conditions leads to the induction of FOXP3, the inability to secrete the effector cytokines IFN-γ and IL-2, and the ability to suppress proliferation of effector T cells in vitro. While tT_regs clearly limit autoimmunity in vivo, the role that pT_regs play is less clear [12]. One proposed function of pT_regs is to suppress immune responses to potentially damaging antigens, such as the gut microbiota, that are not recognized by the self-directed T cell receptor repertoire of tT_regs. Alternatively, pT_regs may be important for controlling immune responses in specific situations, such as in response to mucosal inflammation [13] or in controlling fetal-maternal tolerance [14]. It is clear that pT_regs can be induced in specific situations or to specific antigens in vivo, but their contribution relative to tT_regs in controlling autoimmunity and ultimately their role in cancer immunology requires further investigation. Taken together, tT_regs are indispensable for limiting autoimmunity in vivo, while pT_regs most likely play a role in controlling immune activation in specific scenarios where exogenous antigen-specific T_regs are required.

The identification of a CD4⁺ T cell population that is essential to the prevention of autoimmunity has led to an entire branch of immunology dedicated to their study. Progress over several decades has led to substantial insight into the role of T_regs in suppressing autoimmunity, the origin and development of T_regs, and the appreciation for the essential role that FOXP3 plays in driving T_reg development and function. Identification of cell surface markers for T_regs has also accelerated their analysis in vitro and in vivo.

Early studies suggested that T_regs required direct contact with effector T cells or antigen-presenting cells to mediate suppression [16, 17]. Through physical interaction with either conventional CD4+ CD25- T cells or antigen-presenting cells, T_regs limited the production of IL-2 from effector T cells and prevented co-stimulation of effector T cells by antigen-presenting cells. T_reg-mediated suppression was lost following the addition of IL-2 or anti-CD28, underscoring that suppression by T_regs relied on deprivation of IL-2 and co-stimulation. These initial studies laid the framework for more in-depth analysis of the contact-dependent mechanisms used by T_regs to suppress immune responses.

One surface molecule expressed by T_regs that mediates contact-dependent suppression is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). CTLA-4 is required to prevent systemic autoimmunity, and knockout of this gene in mice leads to fatal autoimmune-mediated destruction of multiple tissues [18]. CTLA-4 competes with CD28 for binding to the dendritic cell-expressed co-stimulatory molecules CD80 and CD86. Compared with CD28, CTLA-4 binds to CD80/CD86 with a higher affinity, effectively depriving conventional T cells of co-stimulation. Importantly, CTLA-4 is constituteively expressed by T_regs and mediates one form of contact-dependent suppression. T_regs can also further deprive effector T cells of co-stimulation through trans-endocytosis and subsequent degradation of CD80/CD86 from antigen-presenting cells [19]. In head and neck cancer patients, treatment with targeted chemotherapy led to an increase in intratumoral CTLA-4⁺ T_regs, which was associated with poor clinical outcome [20]. Contact-dependent immunosuppression by CTLA-4⁺ T_regs is necessary to maintain immune homeostasis, and CTLA-4⁺ T_regs are likely to play a role in suppressing antitumor immunity.

Another molecule expressed on T_regs that contributes to contact-dependent suppression is lymphocyte-activation gene 3 (LAG-3) [21]. In addition to expression on T_regs, LAG-3 is upregulated on the surface of conventional T cells following activation. LAG-3 is associated with the T cell receptor (TCR) on the surface of T cells and binds major histocompatibility complex class II (MHC-II) molecules. This interaction between LAG-3 and MHC-II leads to inhibition of TCR/CD3-mediated T cell activation. LAG-3 can also directly modulate dendritic cell function by interacting with MHC-II and preventing dendritic cell maturation by depriving them of activating signals from conventional CD4⁺ T cells. Consistent with a role for LAG-3 in contact-dependent suppression by T_regs, in vitro or in vivo blockade of LAG-3 reduces suppression by T_regs [22]. Further, genetic deletion of Lag3 in mice also led to reduced suppressive activity [23]. In melanoma and colorectal cancer patients, LAG-3⁺ T_regs are expanded in peripheral blood compared with healthy donors and are present at a higher frequency within lymph nodes containing tumor metastases compared with normal lymph nodes [24]. Furthermore, FOXP3⁺LAG-3⁺ cells were found to secrete IL-10 and TGF-β and to potently suppress proliferation in a contact-dependent manner in vitro.

A separate contact-dependent mechanism by which T_regs can exert effector function is through the release of cytolytic granules containing granzyme and perforin. Although this is a feature that is normally restricted to CD8⁺ T cells, T_regs can express granzyme and perforin and can eliminate autologous cells through a perforin-dependent pathway [25]. T_regs expressing granzyme and perforin are therefore able to suppress immune responses through the direct elimination of effector T cells. Finally, one study demonstrated the importance of granzyme and perforin in the suppression of antitumor immunity in a murine cancer model, underscoring the importance of this contact-dependent mechanism in promoting tumor growth [26].

The second general mechanism by which T_regs can exert their suppressive function is either by secretion, uptake, or generation of soluble molecules. As discussed earlier, T_regs are characterized by constitutive expression of the IL-2 receptor CD25. This high expression of CD25 causes T_regs to preferentially bind IL-2, depriving conventional T cells of this important stimulatory cytokine. Effector T cell deprivation of IL-2 at inflammatory sites subsequently leads to loss of their effector function and apoptosis [27].

A second soluble mechanism used by T_regs to suppress immune responses is the secretion of cytokines, such as interleukin-10 (IL-10) [28]. In particular, IL-10 production by T_regs plays a role in controlling inflammation at mucosal sites, and mice that lack the Il10 gene in T_regs develop spontaneous colitis and inflammation of the skin and lungs [29]. IL-10 can directly inhibit effector T cells through interaction with the hetero-tetrameric IL-10 receptor complex, leading to activation of STAT3 and transcription of anti-inflammatory genes [30]. In addition to direct suppression of effector T cells, IL-10 can also suppress immune responses by limiting the ability of macrophages to produce inflammatory cytokines [31]. Similarly, IL-10 also prevents the maturation of dendritic cells and inhibits their expression of co-stimulatory molecules [32]. While T_regs are noted for their ability to secrete IL-10, other cell types, such as macrophages under certain conditions, also secrete IL-10 [28]. Although IL-10 is a highly pleiotropic cytokine, it has a clear role in suppression of immune responses by T_regs.

Another important cytokine produced by T_regs that has been shown to play a broad and important role in the immune system is TGF-β [33]. Unlike other cytokines, TGF-β is initially translated as an inactive protein that requires proteolysis for activation. Inactive TGF-β is non-covalently bound to latency-associated peptide (LAP) through an association with GARP on the surface of T_regs [34]. This membrane-bound form of TGF-β is then activated through several possible proteolytic pathways, allowing the activated form of TGF-β to perform its immunosuppressive function [35]. One of the first descriptions of a connection between TGF-β and T_regs was in a model of experimental autoimmune encephalitis (EAE) in mice, where oral tolerance was induced by feeding mice myelin basic protein [36]. Analysis of the CD4⁺ T cells that infiltrated the nervous system to facilitate tolerance revealed that these cells produced TGF-β and prevented EAE. As with other T_reg molecules, knockout of TGF-β from murine T_regs leads to induction of autoimmune disease, underscoring the importance of TGF-β in immune homeostasis [37]. TGF-β suppresses effector T cell responses in several ways, including inhibiting IL-2 production and IFN-γ and perforin production in CD8+ T cells [38]. In head and neck cancer patients, an important role of TGF-β secreting T_regs has been described [39]. Taken together, secretion of TGF-β by T_regs plays an important role in maintaining immune homeostasis and can inhibit antitumor immunity.

Another important cytokine produced by T_regs to facilitate immunosuppression in murine models is interleukin-35 (IL-35) [40]. IL-35 is a member of the IL-12 family of heterodimeric cytokines and consists of one IL-12α subunit and one IL-27β/Ebi3 (Epstein-Barr virus-induced gene 3) subunit [41]. These cytokine genes are constitutively expressed in a subpopulation of murine T_regs, but not conventional T cells, and are upregulated following T_reg activation. IL-35 confers suppressive activity on naïve CD4+ T cells and directly suppresses division of conventional cells. Like other inhibitory cytokines, IL-35 can also drive the development of an induced T_reg population, called iTr35, that can suppress effector T cells via IL-35 [42]. IL-35 mediates signaling via a unique IL12rβ2:gp130 receptor heterodimer and a STAT1:STAT4 heterodimer [43]. In murine cancer models, IL-35 has recently been shown to play an important role in promoting tumor growth by contributing to T cell exhaustion in the tumor microenvironment [44]. Consistent with increased IL-35 production from highly activated T_regs, an IL-35 reporter mouse revealed enrichment of IL-35⁺ T_regs in the tumor microenvironment, and neutralization of IL-35 or T_reg-specific genetic deletion of Ebi3 led to enhanced antitumor immunity, which was mediated via enhanced cell proliferation and effector function and improved memory cell generation of effector T cells. T_reg-restricted deletion of Ebi3 also led to reduced expression of the inhibitory receptors PD-1, LAG-3, and TIM-3, suggesting that IL-35 may promote exhaustion through upregulation of multiple inhibitory receptors [44].

Finally, T_regs can also mediate immunosuppression via the generation of adenosine, a labile, highly suppressive molecule [45]. Extracellular adenosine accumulates at sites of ischemia and inflammation in vivo. In the extracellular space, adenosine is generated by T_regs via breakdown of ATP. The extracellular ectoenzymes CD39 and CD73 on T_regs, or cells in close proximity, in tandem catalyze the breakdown of ATP to adenosine. While CD73 is broadly expressed on activated T cells and other cell types, CD39 expression is largely restricted to T_regs. Increased levels of adenosine at sites of inflammation inhibit immune responses through interaction with either the adenosine A_2A receptor on effector T cells or the adenosine A_2B receptor on antigen-presenting cells. The interaction of extracellular adenosine with either receptor leads to increased intracellular levels of cAMP and limits the release of inflammatory cytokines from both effector T cells and antigen-presenting cells. T_regs can therefore limit the production of inflammatory cytokines locally by breaking down ATP into adenosine through extracellular ectoenzymes.

As described in this section, T_regs use multiple contact-dependent and contact-independent/contact-soluble mechanisms to suppress effector T cell responses and antigen-presenting cell development and function. Given the detrimental effects of autoimmunity and excessive immune responses and the diversity of cell populations and effector mechanisms they need to control, T_regs likely have evolved multiple immunosuppressive mechanisms to adequately control autoimmunity and inflammation in a variety of settings. An important question is whether certain mechanisms are more dominantly or preferentially utilized by T_regs in tumors and thus may be targeted therapeutically without substantially impacting the ability of T_regs to maintain immune homeostasis and peripheral tolerance.

T_regs function in diverse environments and suppress a variety of cell types. Consequently, their function and survival are likely modulated or potentiated by a variety of environmental cues, many of which are likely poorly understood or have yet to be defined. Early studies suggested that T_reg suppression was contact dependent [16, 17]. However, this notion was inconsistent with the growing appreciation of the importance of cytokines in mediating T_reg-dependent suppression. This conundrum was resolved when a more recent study showed that it was not suppression by T_regs per se that was exclusively contact dependent but rather the boosting/potentiation of their suppressive activity that was contact dependent [46]. This study found that co-culture of T_regs with fixed or live conventional CD4⁺ T cells or antigen-presenting cells was sufficient to boost the capacity of T_regs to suppress effector T cells across a permeable transwell membrane via IL-10 and IL-35.

The potentiation of T_reg function and survival was found to be mediated by neuropilin-1 (Nrp1) on the surface of T_regs via interaction with Sema4a [47]. Nrp1 is involved in normal neural and vascular development and also plays a role in tumor angiogenesis [48]. Signaling through the Nrp1/Sema4a interaction is necessary for T_reg suppression by soluble cytokines in vitro [47]. Nrp1 on T_regs limits Akt (protein kinase B [PKB]) activity via phosphatase and tensin homolog (PTEN), which in turn stabilizes the T_reg phenotype and enhances their survival and function. Consistent with a requirement for Nrp1 to mediate T_reg potentiation, genetic ablation of Nrp1 in murine T_regs led to a significant enhancement of antitumor immunity in vivo but did not lead to overt autoimmunity or peripheral inflammation [47, 49]. These observations highlighted the unique role of Nrp1 in stabilizing and potentiating the survival and suppressive function of T_regs in the tumor microenvironment. However, it remains to be determined if the Nrp1 pathway is only utilized in the tumor microenvironment and if so why and if there are other mechanisms that regulate T_reg fate and function.

The role that T_regs play in the suppression of antitumor immunity has been demonstrated in several mouse models. Mutation or elimination of the Foxp3 gene in mice and humans leads to fatal autoimmunity, so germline deletion of Foxp3 in mice cannot be used to study antitumor immunity. While T_regs can be limited or depleted in adult mice with antibodies targeting CD4 or CD25 or drugs such as cyclophosphamide, these treatments also impact activated effector T cells, confounding experimental interpretation [50]. Instead, inducible T_reg-targeting genetic systems have been used to transiently deplete T_regs in adult mice to study their role in suppressing antitumor immunity. One model that has been utilized in mice is insertion of the human diphtheria toxin receptor (DTR) under control of the Foxp3 locus (Foxp3 ^DTR) [51, 52]. Following the administration of diphtheria toxin, all cells expressing FOXP3 are depleted, allowing for a direct assessment of the role of T_regs in suppressing antitumor immunity.

Using the Foxp3 ^DTR mice, depletion of FOXP3⁺ T_regs has been performed in mice with a variety of implanted tumors. Following depletion of T_regs, mice had reduced tumor growth and prolonged survival compared with littermates that did not have their T_regs depleted [47, 53]. These mechanistic studies in mice have demonstrated that T_regs play an important role in suppressing antitumor immunity and that specific depletion of T_regs is sufficient to prevent tumor growth and prolong survival in mice. However, depletion of T_regs following administration of diphtheria toxin in the Foxp3 ^DTR mice is not specific for T_regs in the tumor microenvironment, and these mice quickly succumb to autoimmune disease despite their antitumor immune responses. This once again highlights the importance of T_regs in maintaining peripheral tolerance throughout life and suggests that systemic depletion of T_regs may not be a viable treatment option for cancer patients.

T_regs are present in many healthy tissues, and as tumors grow, T_regs can possibly expand locally through increased antigenic stimulation and subsequent proliferation (Fig. 6.4a). In the context of a tumor, tissue-resident T_regs specific for self-peptides presented on MHC receive additional antigenic stimulation as the tumor grows, leading to expansion of local T_regs. In support of this concept, studies have found that T_regs within tumors have a distinct T cell receptor repertoire from conventional CD4⁺ T cells [54] and that the T cell receptor repertoire of T_regs is largely skewed toward a few clonally expanded populations [55]. Also, T_regs are often the most proliferative immune cell type in tumors [47]. Expansion of T_regs specific for antigens present in normal tissue may partially explain the increased presence of T_regs within tumors, although this mechanism could exist in conjunction with enhanced trafficking of T_regs to tumors.

To perform their effector function, activated T_regs need to traffic to sites of inflammation within tissues. Trafficking of leukocytes is generally controlled via chemotactic cytokines known as chemokines. These chemokines interact with a specific array of cell surface transmembrane G protein-coupled receptors. For example, under normal physiologic conditions in the lymph node, expression of the chemokine receptor CCR7 on T_regs leads to their recruitment to T cell zones, where they have access to abundant IL-2 [56]. The recruitment of T_regs to T cell zones within lymph nodes highlights the function of chemokine receptors and ligands to target trafficking to specific anatomical locations. Similarly, secretion of specific chemokines by tumors or other immune cells within the tumor microenvironment can actively recruit T_regs through interaction with specific homing receptors on T_regs.

In cancer, specific chemokine/receptor interactions that recruit T_regs to the tumor are dependent on the tissue origin of the tumor and the cytokine milieu produced in the tumor microenvironment. The most commonly reported mechanism by which T_regs are recruited to the tumor microenvironment is through interaction of the chemokine CCL22 with CCR4 expressed on T_regs. First described in breast cancer, this pathway has been found to play an important role in recruiting activated T_regs to other tumor types including ovarian cancer, colorectal cancer, and head and neck cancer [57]. While tumor cells may actively secrete chemokines, tumor-associated macrophages (TAMs) are another major source of chemokines. TAMs were shown to be the major source of chemokines responsible for the recruitment of T_regs in ovarian cancer [58]. CCL22 secretion by tumor-infiltrating CD8⁺ T cells can also drive T_reg recruitment into the tumor [59].

In the normal setting of inflammation, T_regs are recruited to limit tissue damage from the ensuing immune response. This is analogous to recruitment of T_regs to tumors via local expansion or chemokine-mediated recruitment. This highlights the co-opting of a normal biological process to promote tumor-induced tolerance. Studies in a wide variety of murine tumor models have demonstrated that T_regs play a central role in preventing antitumor immunity.

Many studies have evaluated associations between the frequency of T_regs in the tumor microenvironment and clinical outcome, including in head and neck [60], ovarian [58, 62], breast [63], pancreatic [64, 65], gastric [66, 67], lung [68], renal [69, 70], and liver cancers [71] and melanoma [61]. Studies assessing the frequency of T_regs within tumors have generally used tissue sections and have looked for FOXP3+ cells in the presence or absence of additional markers. Other studies have looked at the frequency of T_regs within tumors as a ratio of CD8⁺ T cells to T_regs [72–75]. A recent meta-analysis of 17 different types of cancer across more than 15,500 cancer cases found that a higher frequency of T_regs in tumors was associated with poorer overall survival when considering all cancer types [76] (Fig. 6.5). Many of these studies used histology of tissue sections and have relied on identifying T_regs using staining for FOXP3 alone or with one or two other markers.