TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy

TNF has both proinflammatory and antiinflammatory effects. It binds to two structurally related but functionally distinct receptors TNFR1 and TNFR2. Unlike TNFR1 that is ubiquitously expressed, TNFR2 expression is more limited to myeloid and lymphoid cell lineages including a fraction of regulatory T cells (Treg). In general, TNFR1 is responsible for TNF-mediated cell apoptosis and death, and mostly induces proinflammatory reactions. However, TNFR2 mainly leads to functions related to cell survival and immune suppression. Treg play an indispensable role in maintaining immunological self-tolerance and restraining excessive immune reactions deleterious to the host. Impaired Treg-mediated immune regulation has been observed in various autoimmune diseases as well as in cancers. Therefore, Treg might provide an ideal therapeutic target for diseases where the immune balance is impaired and could benefit from the regulation of Treg properties. TNFR2 is highly expressed on Treg in mice and in humans, and TNFR2+ Treg reveal the most potent suppressive capacity. TNF-TNFR2 ligation benefits Treg proliferation, although the effect on Treg suppressive function remains controversial. Here, we will describe in detail the TNF-mediated regulation of Treg and the potential clinical applications in cancer immunotherapy as well as in autoimmune diseases, with the focus on human Treg subsets.


CD4+FOXP3+ regulatory T cells (Treg) have an indispensable role in maintain-
ing immune homeostasis and immune tolerance. They control unwanted immune responses that are involved in the regulation of immune tolerance to self as well as to foreign antigens. Loss-of-function mutation in FOXP3 locus, a gene encoding Treg lineage transcription factor FOXP3, leads to multiorgan associated autoimmunity. Abnormal numbers of Treg and/or impaired suppressive function of Treg are often found in various autoimmune diseases like type 1 diabetes (T1D) [1], multiple sclerosis (MS) [2], rheumatoid arthritis (RA) [3], psoriasis [4][5][6], and systemic lupus erythematosus (SLE) [7][8][9]. On the other hand, tumor-infiltrating Treg generally show potent suppressive functions, indicating that they regulate tumorspecific immune responses and might help tumor immune escape [10]. It seems logical to use Treg as a therapeutic target for diseases where the immune balance is impaired and could benefit from the regulation of Treg properties. Nevertheless, due to the intrinsic properties of Treg, i.e. heterogeneity and plasticity, several key questions need to be clarified before making Treg an ideal candidate for clinical applications.
Tumor necrosis factor (TNF) is initially expressed on cell surface as a membrane bound cytokine (mTNF), which can be cleaved by a metalloprotease TNF converting enzyme (TACE) to generate soluble form of TNF (sTNF) [11]. TNF binds to receptors, TNF receptor 1 (TNFR1) and 2 (TNFR2). In contrast to TNFR1, TNFR2 expression is restricted in certain cell types including lymphocytes [12]. TNF-TNFR1 interaction mostly induces proinflammatory reactions, whereas TNFR2 generally leads to the suppressive function of TNF [13]. It is known that TNFR2 is constitutively expressed on both murine and human Treg, and TNFR2+ Treg are the most suppressive Treg subpopulation [14][15][16][17]. The effect of TNF on Treg suppressor function remains controversial. In this chapter, we will describe in detail the TNFmediated signal transduction pathways, its effect on Treg cells, and the potential clinical applications in various immunopathologies.

Regulatory T cells and its plasticity
Treg exert their function in primary and secondary lymphoid organs and nonlymphoid tissues. FOXP3, as the lineage transcription factor of Treg, facilitates Treg thymic development by stabilizing its own expression and inhibiting transcription factors needed for the development of other helper T-cell (Th) lineages like T-bet for Th1, GATA3 for Th2, and RORγt for Th17 cells [18]. Next to FOXP3, Treg constitutively express a high level of the IL-2 receptor α chain (CD25) and a low level of the IL-7 receptor α chain (CD127) compared to human activated non-Treg. The combination of CD4+, CD25high, and CD127low has been used to isolate Treg for functional studies and for adoptive immunotherapy [19]. However, no unique Treg marker has been identified so far, although many molecules are proposed. These Treg-related cell markers include CD27 [20], CD62L [21], CTLA4 (cytotoxic T-lymphocyte-associated protein) [22], CD39 and CD73 ectoenzymes [23], Helios [24], Neuropilin-1 [25], HLA-DR [26], and the most recently identified combination of TIGIT and FcRL3, which results in the identification of human Helios+ memory Treg [27].
Compelling evidence indicates that both mouse and human Treg consist of various subpopulations and have a more or less plastic phenotype depending on the microenvironment they are in [28]. Based on the site of Treg generation, two major Treg subsets are classified, namely, thymus-derived Treg (tTreg) that develop in the thymus from CD4 single positive thymocytes which in general display high-affinity self-reactive T-cell receptors (TCRs), and peripherally induced Treg (pTreg) which emerge in the periphery from conventional CD4+ T lymphocytes (Tconv) in response to environmental antigens and tolerogenic stimuli. Studies in mice have shown that pTreg and tTreg are both required for full protection against colitis and lymphoproliferative disease [29,30], indicating that these two Treg subsets play distinct roles in protecting against immunopathology. However, the relative contribution of tTreg and pTreg in human immune tolerance remains a major unresolved issue, partially due to the lack of specific markers to definitively distinguish them. In fact, the transcription factor Helios was the first marker proposed to distinguish both mice and human tTreg from pTreg [31]. However, this has been disputed by studies showing that Helios can also be expressed by activated Tconv [32] and by pTreg upon in vitro and in vivo stimulation [33], precluding its TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.85632 use as tTreg-specific marker. Another cell surface marker that has been proposed to harbor the specificity necessary to distinguish between murine tTreg and pTreg is the coreceptor Neuropilin-1 [25]. Unfortunately, human Treg do not uniquely express Neuropilin-1 [34].

TNF/TNFR signaling pathways
TNF is firstly discovered as an inflammatory cytokine that is induced by the endotoxin [35]. Various immune cells produce TNF including macrophages, monocytes, dendritic cells, B cells, activated natural killer cells, and activated T cells. TNF is initially expressed on the cell surface as a trimeric type II transmembrane protein mTNF, which is then cleaved by the metalloproteinase TACE (also known as ADAM17) and released as soluble extracellular sTNF [36]. Both forms of TNF are present as bioactive homotrimers. There exist two structurally related but functionally distinct receptors, TNFR1 (p55) and TNFR2 (p75). TNFR1 is ubiquitously expressed on most mammalian cell types, and it binds to mTNF as well as sTNF, whereas TNFR2 expression is restricted to immune cells, neurons, and endothelial cells. TNFR2 binds with higher affinity to mTNF than sTNF compared to TNFR1. TNFR1 and TNFR2 share the similar extracellular TNF-binding motifs but differ in their intracellular domains. Both receptors lack intrinsic enzyme activity; thus, upon the ligand binding, they need to recruit the cytosolic proteins to initiate the intracellular signal transduction. Specifically, TNFR1 contains a homologous intracellular region called "death domain", which preferentially interacts with the adaptor protein named TNFR1-associated death-domain (TRADD) protein [37]. TRADD further recruits another two adaptor proteins, receptor interacting protein kinase 1 (RIPK1) and TNFR-associated factor (TRAF) 2, thus forming an enzymatic complex signalosome, which is also known as signaling complex 1. One of the main targets of the complex 1 is the enzyme complex called IkB kinase (IKK). Phosphorylation of IKK in turn leads to the canonical activation of the transcription factor NFkB as well as members of the family of MAPKs such as c-jun kinase (JNK) and p38 MAPK. The TRADD containing signaling complex 1 may further be converted to a death-inducing signaling complex, so-called complex 2, by adaptor protein Fas-associated protein with death domain (FADD). The complex 2 is able to further initiate downstream caspase cascades, thus inducing cell apoptosis and cell death [37].
The pathways induced by TNFR2 are slightly different from TNFR1. Due to the lack of death domain, TNFR2 is unable to recruit TRADD protein, but it can directly interact with TRAF2 [38]. In contrast to TNFR1 that drives apoptosis and cell death, TNFR2 induces the noncanonical activation of NFκB via the activation of the NFκB-inducing kinase (NIK), which further leads to the phosphorylation of IKKα and the processing of p100, a crucial step in the nuclear translocation of p52/RelB [38,39]. Interestingly, TRAF2 binding to TNFR2 is considerably weaker than its binding to TRADD protein. Upon binding to TRAF2, TNFR2 could also recruit cIAP1/2 proteins [39] that are involved in the TNFR1-mediated NFκB activation, indicating that there exists a crosstalk between TNFR1 and TNFR2 pathways. Another interesting adaptor protein called endothelial/epithelial protein tyrosine kinase (Etk) interacts with the C-terminal domain of TNFR2 in a ligandindependent manner [40]. TNFR2-mediated Etk phosphorylation is able to partially activate the growth factor receptor VEGFR2, which in turn results in the activation of PI3K/Akt pathway and cell survival.
A number of proteins are essential for the negative regulation of the TNF-TNFR pathways. A20, also named as TNF alpha-induced protein 3, is one of the most studied negative regulatory proteins. A20 is an ubiquitin editing enzyme. It limits NFκB signaling after activation by TNF [41]. Consistent with this, A20-deficient mice are hypersensitive to TNF exposure and die perinatally because of severe inflammation and multiorgan failure [42]. Intriguingly, A20 is recently shown to regulate the de novo generation of tTreg in a cell-intrinsic manner, while the suppressor function of A20-deficient Treg is unchanged in vitro [43].

Effect of TNFR2 on Treg
Although TNFR1 expression is not different between Treg and non-Treg cells, human Treg constitutively express high levels of TNFR2 compared to CD25-Tconv. Moreover, TNFR2+ Treg reveal the most potent suppressive capacity [14,44]. The effect of TNF on Treg suppressor function remains controversial. Several groups including ours demonstrated that sTNF preserved or even increased FOXP3 expression as well as Treg suppressive capacity in both mice and humans [15,[45][46][47]. The TNF-TNFR2 is crucial for sustaining FOXP3 expression and maintaining the stability of murine Treg in an inflammatory environment [44]. A similar phenomenon is also observed for human Treg in vitro [48]. There is also evidence for the negative effects of TNF on Treg function. Studies show that TNF impairs Treg function by reducing FOXP3 expression or enhancing its dephosphorylation [47,49]. In clinical practices, RA patients responding to anti-TNF antibody adalimumab showed an increased percentage of FOXP3 + cells as well as the restored regulatory function [50]. It should be noted that the nature of the TNFR2 antibodies used in these studies was likely different (agonistic versus antagonistic) [46]. Recent studies highlight that TNFR2 agonisms and antagonisms might regulate the phenotype and the suppressor function of Treg in a complete different way [46]. TNF priming induces the proliferation and activation of Treg in vitro [15,51] as well as in vivo via TNFR2 in an acute mouse GvHD model [52]. Our group have found that stimulation of human Treg with a TNFR2-agonist antibody preserved a stable Treg phenotype and function after ex vivo expansion [48]. Using TNFR2 agonist only was enough to prevent the loss of FOXP3 expression, whereas the sustained hypomethylation of TSDR (Treg-specific demethylated region) of FOXP3 gene locus required both rapamycin and TNFR2 agonist, suggesting that stabilization of FOXP3 expression requires both mTOR and NFκB signal pathways. In vitro restimulation of TNFR2 agonist plus rapamycin-expanded Treg led neither to the loss of FOXP3 protein nor the enhancement of IL-17A production, especially under proinflammatory conditions, indicating a well-preserved Treg stability. TNFR2 knockout CD4+ T cells have increased expression of RORγt and IL-17 production, which is dependent on the impairment of TNFR2-mediated activation of NFκB [53]. We speculate that a similar process of regulation may exist in human Treg where TNFR2/NFκB signaling might act as a double-edged sword to enhance FOXP3 but also to inhibit RORγt expression, thus contributing to Treg stability. Another possible explanation is that TNFR2 engagement results in an autocrine TNF-TNFR2 loop, which further regulates the expression of histone methyltransferase EZH2 [51], a subunit of the polycomb repressor complex 2 (PRC2). EZH2 is known to bind to FOXP3 thus helping FOXP3 to regulate the gene transcriptional repression [54].

TNFR2 agonists and autoimmune diseases
Defect in the function of Treg as well as the low numbers are the main properties of various autoimmune diseases. Therefore, restoring the proper functional Treg TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.85632 thus favoring the immune tolerance induction has become a final goal of treatment for patients with autoimmune diseases. As discussed above, ample studies show that either TNF and/or TNFR2 agonism has capacity to enhance Treg proliferation and activation. Furthermore, TNF-TNFR2 is essential to maintain the Treg function and stability in the inflammatory environment [44,48]. Impaired TNF-TNFR signaling pathways occur in several human diseases including T1D, SLE, IBD, and MS. For instance, a single-nucleotide polymorphism (SNP) in the first intron is linked to a decreased level of TNFR2 in carriers of the SNP and a high risk of disease susceptibility [55]. T1D patients have higher TNFR2 + Treg compared to healthy controls. The rationale for using TNFR2 agonists as a therapeutic option for autoimmune diseases was first shown in T1D. Using blood from patients with T1D, a dose-response relationship between TNFR2 agonism and the destroying of pathogenic autoreactive CD8 T cells was observed [56], suggesting inducing of TNF-TNFR2 pathway is an effective approach of selectively killing autoreactive T cells.
Currently used biologics targeting TNF include the anti-TNF antibodies infliximab, adalimumab, certolizumab, and the decoy receptor etanercept that binds to sTNF. Although they have a good safety profile, with increasing use of these drugs, paradoxical adverse events involving the skin, joints, and lungs have been described [57]. Skin manifestations are the most common adverse event and occur in about 25% of patients receiving anti-TNFs. The underlying mechanism is recently attributed to the TNFR2/A20 signal axis which is specifically responsible for TNF-mediated IL-17A inhibition [58]. Termination of NFκB activation is critical to prevent aberrant inflammatory responses. In memory CD4 T cells, A20 is identified as one of the strongest TNF-responsive genes with a strong inverse correlation to IL-17A expression.

TNFR2 antagonists and cancer immunotherapy
Tumor microenvironment preferably recruits TNFR2+ Treg cells which possess a highly immunosuppressive capacity, thus facilitating tumor immune escape. That TNFR2 knockout mice show improved immune responses to tumors might be caused by the lack of TNFR2 expressing Treg or have failed to develop systemic autoimmunity [59] or the decreased numbers and the impaired function of MDSCs [60]. In humans, the high level of TNFR2+ Treg is found in the peripheral blood of lung cancer patients [10] and in the tumor-associated ascites in ovarian cancer patients [61]. Moreover, the increased TNFR2 gene expression on Treg cells has been shown to be associated with exhaustion of CD8 cytotoxic T lymphocytes in metastatic melanoma patients.
In addition to being an inducer of Treg expansion, TNFR2 also acts as an oncogene which has been identified on at least 25 tumor types. Enhanced expression of TNFR2 on tumor itself has been also reported but not limited in human renal cell carcinoma, multiple myeloma, colon cancer, ovarian cancer, and cutaneous T-cell lymphomas (CTCL) [62]. In general, the overexpression of TNFR2 exploits this cytokine receptor for increased tumor cell proliferation and tumor growth. Genetic mutation/genomic gains of TNFRSF1B, a gene encoding TNFR2 protein, occur in patients with Sézary syndrome (SS), a rare form of CTCL often refractory to treatment. SS is characterized with high expression of TNFR2 on the tumor cells and Treg. Such gain-of-function mutation in TNFR2 leads to the enhanced noncanonical NKκB activation [63], a pathway primarily involved in cell expansion and growth. It seems being desirable to apply one approach that could successfully inhibit potent suppressive Treg and also directly prevent tumor growth by using the antagonistic molecules against TNFR2. Such TNFR2-specific blocking molecules would ideally inhibit Treg and permit Tconv proliferation and function, thus enabling to restore the antitumor immune responses and to induce tumor regression.

Strategies for blocking of TNF/TNFR2 signaling
A number of agonistic or antagonistic biological agents targeting to TNF and/or TNFR2 have been developed. Two potent dominant TNFR2 antagonist antibodies are developed by Faustman et al. group [64]. They report that these TNFR2 antagonists lock the TNFR2 receptor in the form of antiparallel dimmers, which further prevents the TNF binding as well as the intracellular scaffolding. Consequently, these dominant TNFR2 antagonists, even in the presence of TNF, could kill Treg isolated from ovarian cancer ascites more potently than it kills Treg from healthy donors. Interestingly, TNFR2 antagonistic mAbs are also able to directly kill TNFR2-expression ovarian cancer cell lines in vitro [64]. Similar effect is observed in another in vitro study where the cancer cells and lymphocytes were isolated from the end-stage SS patients [65]. In mouse model of colon and breast cancers, combining a blocking TNFR2 antibody with a kind of immune stimulant markedly enhances the antitumor efficacy of immunotherapy through reducing the number of tumor-infiltrating TNFR2+ Treg and increasing the number of IFNγ-producing CD8 cells [66].
Some pharmacological agents are found to regulate TNF and/or its receptors expression. Thalidomide and its analogues prevent the surface expression of TNFR2 on activated T cells, which is associated with the inhibition of TNFR2 protein trafficking to the cell membrane [67]. Treating acute myeloid leukemia patients with azacitidine and lenalidomide, a thalidomide derivative can reduce TNFR2 expression on T cells as well as TNFR2+ Treg in vivo, leading to enhanced effector immune function [68]. Cyclophosphamide is a DNA alkylating agent. It is commonly used as a cytotoxic chemotherapy in cancer treatment. In a mouse model, it is shown that cyclophosphamide treatment depletes TNFR2+ Treg via inducing the death of replicating Treg that co-express TNFR2 and KI-67 [69]. A re-expansion of Treg from lymphodepletion suppresses the effective antitumor immunity developed after cyclophosphamide treatment. Intriguingly, blockade of TNF signaling using etanercept inhibits TNFR2+ Treg cell expansion during recovery from cyclophosphamide-induced lymphodepletion and markedly inhibits the growth of established CT26 tumors in mice [70]. Altogether, it suggests that a TNFR2-targeted approach to inactive host Treg, especially in only tumor microenvironment, may offer optimal options for antitumor immune reactions.

Conclusions
Many surface receptors of Treg are also expressed on other immune cells, with TNFR2 being a prominent exception with highest density in the tumor microenvironment. TNFR2 is a functional receptor on Treg. Cell surface expression of TNFR2 not only identifies the potent Treg subsets but also is the property of tumor-infiltrating Treg. TNFR2 expression on some cancer-infiltrating Treg is about 100 times higher than on circulating Treg in control subjects. In other types of cancer, the abundance of TNFR2+ Treg in peripheral blood is higher than healthy ones. Targeting TNFR2 using small molecule agonists or antagonists is a promising but also a challenging task. Considering the suppressive property of Treg and its impaired functions in various immunopathologies, there is no doubt that novel (tumor-specific) antagonists against TNFR2 are promising for cancer immunotherapy. From the clinical utilities point of view, combination of TNFR2 inhibition with immune checkpoint inhibitors seems to be an attractive approach in reshaping modern cancer immunotherapy.