The phenotypic and functional consequences of ... - Wiley Online Library

IMMUNOLOGY

REVIEW ARTICLE

The phenotypic and functional consequences of tumour necrosis factor receptor type 2 expression on CD4+ FoxP3+ regulatory T cells

Xin Chen1 and Joost J. Oppenheim2 1

Basic Science Program, SAIC-Frederick, Inc., Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center for Cancer Research, NCI-Frederick, Frederick, MD, and 2Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center for Cancer Research, NCI-Frederick, Frederick, MD, USA

doi:10.1111/j.1365-2567.2011.03460.x Received 30 November 2010, revised 5 May 2011, accepted 6 May 2011. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. Correspondence: Dr X. Chen, BSP, SAICFrederick, Inc., Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center for Cancer Research, NCIFrederick, P.O. Box B, Bldg 560, Rm 31-19, Frederick, MD 21702-1201, USA. Email: [email protected] Senior author: Joost J. Oppenheim, email: [email protected]

Summary Cytokine receptors expressed by CD4+ FoxP3+ regulatory T cells (Treg cells) not only serve as a phenotypic marker for the identification of this important population of immunosuppressive cells, they also promote the function of Treg cells. CD25, the a-chain of interleukin-2 receptor, is a prototype of such a receptor, which enables Treg cells to be activated by interleukin-2. We and others have found that tumour necrosis factor receptor type 2 (TNFR2) is another important cytokine receptor preferentially expressed by Treg cells with important phenotypic and functional roles. TNFR2 is preferentially expressed by highly functional human and mouse Treg cells, and mediates the activating effect of TNF on Treg cells. We review here the studies of the regulation of expression of TNFR2 on functional Treg cells as well as on CD4+ FoxP3) effector T cells (Teff cells). We document the critical role of this receptor in the activation, proliferative expansion and survival of Treg cells. The contribution of TNFR2 expression on Treg and Teff cells to the beneficial and detrimental effects of anti-TNF treatment in autoimmune disorders will also be discussed. Keywords: CD4+ FoxP3+ regulatory T cells; CD4+ FoxP3) effector T cells; immunological phenotype; immunosuppression; tumour necrosis factor receptor 2

Introduction A sub-population of lymphocytes with immunological suppressive activity was detected over 30 years ago.1 However, lack of reliable surface markers impeded the study of these suppressive cells. In 1995, the existence of suppressive T cells was resurrected by the seminal discovery by Sakaguchi et al.2 that co-expression of CD25, the a-chain of the interleukin-2 (IL-2) receptor, on CD4 cells identified regulatory T (Treg) cells. Since then, extensive studies have provided compelling evidence that these thymus-derived suppressor cells, comprising 426

approximately 10% of peripheral CD4+ T cells and expressing the X chromosome-encoded forkhead transcription factor, FoxP3, play an important role in the maintenance of immune homeostasis and prevention of autoimmunity. Unfortunately, Treg cells also attenuate immune responses against tumour antigens.3,4 Besides FoxP3+ Treg cells, there are other types of Treg cells that can be induced from naive CD4 cells in the periphery during immune responses, such as IL-10-producing Tr1 cells5 and transforming growth factor-b-producing T helper type 3 (Th3) cells.6 In this review, our discussion will focus on the subset of naturally occurring, thymical-

Ó 2011 The Authors. Immunology Ó 2011 Blackwell Publishing Ltd, Immunology, 133, 426–433

TNFR2+ Treg cells ly derived and inducible contact-dependent suppressive FoxP3+ Treg cells. Tumour necrosis factor receptor type 1 (TNFR1; or p55) and TNFR2 (or p75) are two structurally related, but functionally distinct receptors that mediate the biological function of TNF.7 In contrast to the ubiquitous expression of TNFR1, TNFR2 is restricted to lymphocytes and is more efficiently activated by transmembrane TNF than by soluble TNF.8 With its death domain, TNFR1 is the primary signalling receptor on most cell types and accounts for the majority of the pro-inflammatory, cytotoxic and apoptotic effects classically attributed to TNF.8,9 In contrast, TNFR2 lacks an intracellular death domain and predominantly mediates signals promoting lymphocyte activation and proliferation.8,10 In T cells, TNFR2 has co-stimulatory function and enhances their responses to T-cell receptor (TCR) -mediated signalling.11–13 We and others have reported that TNFR2 is predominantly, but not exclusively, expressed by human and mouse Treg cells.14–16 In the era when anti-TNF reagents have been routinely used in the treatment of autoimmunity, it is particularly important to advance our understanding of the immunological relevance of TNFR2 expression and the interaction of TNF–TNFR2 in the function of Treg cells. For the first time, we have found that TNF activates Treg cells through TNFR2.17 Albeit counterintuitive and contradictory to a previous report,18 our observation is supported by the emerging evidence that TNF–TNFR2 interaction plays a critical role in the generation, expansion and function of human and mouse Treg cells.19–23 Inhibitors of TNF have clear beneficial effects in some autoimmune patients, despite the capacity of TNF to enhance the number and function of Treg cells. This puzzling issue will also be discussed in this review.

Expression of TNFR2 identifies the potent suppressive mouse and humans Treg cells

ment,25 highlighting the critical role of TNFR2+ Treg cells in accounting for the tumour immunosuppressive environment.

TNFR2 is superior to CD25 as a surface marker of functional mouse Treg cells CD4+ CD25+ or CD4+ FoxP3+ cells that also express TNFR2 exhibited the most potent suppressive activity, whereas TNFR2) Treg cells, even if they are CD25+ and FoxP3+ cells from normal C57BL/6 mice, had only minimal or no suppressive ability.15,24 It is noteworthy that although not considered to be Treg cells, TNFR2-expressing CD4+ CD25) cells from normal mice have considerable immunosuppressive activity. Expression of TNFR2 can therefore identify highly suppressive FoxP3+ Treg cells present in the CD25) subset. Consequently, flow-sorted CD4+ TNFR2+ cells are up to approximately fourfold more suppressive than flow-sorted unfractionated CD4+ CD25+ cells,15 indicating that TNFR2 is superior to CD25 as a surface marker of functional Treg cells in mice.

Combination of TNFR2 and FoxP3 or CD25 defines the maximally suppressive mouse Treg cells Studies based on FoxP3/gfp knock-in mice revealed that FoxP3-expressing cells are solely responsible for the suppressive activity of CD4+ TNFR2+ cells, whereas CD4+ TNFR2+ FoxP3) cells have no suppressive function at all.24 Expression of TNFR2 per se is therefore not sufficient to endow CD4 cells with immunosuppressive capacity. In fact, FoxP3, CD25 and TNFR2 by themselves cannot be used to identify the functional Treg cells in mice. Combination of surface expression of TNFR2 and intracellular expression of FoxP3 (or FoxP3/gfp), or the combination of surface expression of both TNFR2 and CD25 as a surrogate, allows identification of highly suppressive cells. TNFR2 is also a marker of the maximal suppressive subset of Treg cells in other strains of mice, such as BALB/c.24

Expression of TNFR2 on mouse Treg cells In normal mice, the majority of thymic CD4+ FoxP3+ Treg cells (approximately 80%) express TNFR2. The expression of this receptor on Treg cells is decreased in the periphery, with the lowest expression on peripheral blood Treg cells (approximately 10%). In the peripheral lymphoid tissues, e.g. lymph nodes and spleen of normal mice, TNFR2 is preferentially expressed by 30–40% of Treg cells, whereas fewer than 10% of CD4+ FoxP3) effector T (Teff) cells express TNFR2 and at lower levels on a per cell basis.15,24 The majority of tumour-infiltrating Treg cells are highly expressing and suppressive TNFR2+ cells.15,24 Depletion of TNFR2+ Treg cells results in tumour eradication after cyclophosphamide treat-

TNFR2 is a better mouse functional Treg-cell marker than CD103 It has been reported that CD103 expression can define the most potent suppressive subset of Treg cells and expression of CD103 alone, regardless of CD25 expression, on CD4 cells is able to define highly suppressive cells.26 However, although TNFR2 identifies more mouse peripheral Treg cells (30–40%) than CD103 (10–30%), CD4+ TNFR2+ cells and CD4+ CD25+ TNFR2+ cells are markedly more suppressive than CD4+ CD103+ cells and CD4+ CD25+ CD103+ cells, respectively.15 Hence, TNFR2 is superior to CD103 as an indicator of more of the maximally suppressive Treg cells.


427

X. Chen and J. J. Oppenheim Expression of TNFR2 on human Treg cells TNFR2 is also constitutively expressed on human thymic Treg cells, but not on thymic Teff cells.27 Human circulating FoxP3+ cells present in CD25high, CD25low and even CD25) subsets of CD4+ cells expressed markedly higher levels of TNFR2 (approximately 70%), compared with CD4+ FoxP3) Teff cells (approximately 20%).14,16,28 Among subsets of human peripheral blood CD4 cells, FoxP3+ Treg cells expressed the highest levels of TNFR2 on a per cell basis.14,16 The TNFR2 is also expressed on antigen-specific CD4 Treg cells induced by tolerogenic dendritic cells (DCs)19 and CD8+ Treg cells generated by anti-CD3 treatment.20 Only a minority of resting mouse peripheral Treg cells (30–40%) express TNFR2,15 whereas the majority of normal human peripheral blood Treg cells (approximately 70%) express this molecule.16 Nevertheless, in an ongoing inflammatory condition, up-regulation of TNFR2 expression on human Treg cells can also serve as an indicator of more suppressive Treg cells, as shown in malaria patients.29 TNFR2 expression is also up-regulated on human Treg cells present in the synovial fluid of patients with rheumatoid arthritis,28 presumably reflecting their enhanced suppressive capacity.30 Human solid tumour tissue harbours high levels of FoxP3-expressing functional Treg cells.31 Elevated levels of TNFR2 are expressed by tumour-infiltrating lymphocytes in human solid tumour tissue.32–34 Consequently, most likely, similar to their mouse counterpart, human tumour-infiltrating Treg cells also up-regulate their TNFR2 expression.

A combination of TNFR2 and CD25 identifies functional human Treg cells Preferential expression of TNFR2 on human Treg cells suggests that, in combination with other markers, expression of this receptor may be exploited to identify human

Treg cells even in the CD25low or CD25) sub-population. We have shown that TNFR2-expressing CD4+ CD25+ T cells include all of the FoxP3+ cells present in the CD4+ CD25high subset as well as a substantial proportion of the FoxP3+ cells present in the CD4+ CD25low subset. Hence, there are fourfold more FoxP3+ cells in the CD4+ CD25+ TNFR2+ population than in the CD4+ CD25high population in peripheral blood. Flow-sorted CD4+ CD25+ TNFR2+ Treg cells express high levels of FoxP3 and have potent suppressive activity.16 Interestingly, a recent study reported that the combination of CD25 and TNFR2 also identifies highly suppressive human CD8+ Treg cells.20 The identification of FoxP3+ Treg cells may be further improved by the combination of TNFR2 with other markers, such as CD127)/low or CD62L+. Even in the CD25) fraction, there are some TNFR2+ CD127)/low cells that comprise some FoxP3+ cells (Xin Chen, unpublished data). Additional surface marker(s) need to be identified to isolate the FoxP3+ Treg cells present in the TNFR2+ CD25) CD127)/low population. Naive FoxP3+ Treg cells, as shown by being CD45RA+ or CD45RO), also expressed higher levels of TNFR2, as compared with naive Teff cells (unpublished data). Hence, TNFR2 is not only helpful in the identification of the activated/memory subset of Treg cells,15 it may also be used to identify naive suppressive Treg cells.

Both human and mouse TNFR2-expressing Treg cells express high levels of cytotoxic T-lymphocyte antigen-4 TNFR2-expressing Treg cells in mouse and human generally demonstrate a memory/effector phenotype. Strikingly, they also express the highest levels of cytotoxic T-lymphocyte antigen-4,15,16 a molecule reported to play a critical role in Treg-cell activity.35 The phenotypic and functional properties of mouse and human TNFR2+ Treg cells are summarized in Tables 1 and 2.

Table 1. Phenotypic and functional property of mouse CD4 subsets

Subsets CD4+ CD4+ CD4+ CD4+

CD25+ CD25+ CD25) CD25)

TNFR2+ TNFR2) TNFR2+ TNFR2)

CD45RB (MFI)

CD62L (MFI)

CD44 (MFI)

CD69 (%)

GITR (MFI)

CTLA4 (MFI)

FoxP3 (%)

Responses to TCR stimulation

Suppressive activity

++ ++ +++ +++++

++ +++++ ++ +++++

+++++ ++ +++++ +

+++++ )/+ +++ +

+++++ +++ + )/+

+++++ +++ +++++ )

+++++ +++++ +++ )

) ) ) +++++

+++++ + +++ )

Splenocytes from normal C57BL/6 mice were stained with anti-mouse CD4, CD25, TNFR2 antibodies and antibody for phenotypic marker. Expression of phenotypic marker was analysed with FACS by gating on indicated CD4 subsets. The expression level is calculated based on the maximal expression (%, or MFI) on a subset (+++++). To examine the responses to T-cell receptor (TCR) stimulation, flow-sorted CD4 subsets were stimulated with antigen-presneting cells and anti-CD3 antibody for 3 days, the proliferation and interferon-c in the supernatant was determined. For regulatory T (Treg) cell function assay, flow-sorted CD4+ CD25) TNFR2) cells were labelled with carboxyfluorosuccinimidyl ester (CFSE) and cultured alone or co-cultured with the desired number of flow-sorted CD4 subsets from spleen and lymph nodes of normal C57BL/6 mice. The cells were stimulated with antigen-presenting cells/anti-CD3 antibody. After a 48-hr incubation, the proliferation was measured by CFSE dilution on effector T cells with FACS. MFI, mean fluorescence index; TNFR2, tumour necrosis factor receptor 2.

428


TNFR2+ Treg cells Table 2. Phenotypic and functional property of human CD4 subsets

Subsets CD4+ CD4+ CD4+ CD4+

CD25+ CD25+ CD25) CD25)

TNFR2+ TNFR2) TNFR2+ TNFR2)

CD45RO

CD45RA

CCR4

CCR7

CD127

HLA DR

CTLA-4

FoxP3

Responses to TCR stimulation

Suppressive activity

+++++ ++++ +++++ +/)

)/+ +++ + +++++

+++++ ++ ++ )/+

++ ++++ ++++ +++++

++ +++++ ++++ +++++

+++++ )/+ ++ )/+

+++++ ++ +++ )/+

+++++ ++ + )

) + ++ +++++

+++++ +/) +/) )

Normal human peripheral blood mononuclear cells were stained with anti-human CD4, CD25, tumour necrosis factor receptor 2 (TNFR2) antibodies and antibody for phenotypic marker. The expression of phenotypic marker was analysed by FACS, gating on indicated CD4 subsets. The expression level is calculated from the maximal percentage of positive cells (%) on a subset (+++++). To examine the responses to T-cell receptor (TCR) stimulation, flow-sorted CD4 subsets were stimulated with antigen-presenting cells and anti-CD3 antibody for 3 days, the proliferation and interferon-c in the supernatant was determined. For regulatory T (Treg) cell function assay, flow-sorted CD4+ CD25) TNFR2) cells were labelled with carboxyfluorosuccinimidyl ester (CFSE) and cultured alone or co-cultured with the desired number of flow-sorted CD4 subsets. The cells were stimulated with antigen-presenting cells/anti-CD3 antibody. After 48-hr incubation, the proliferation was measured by CFSE dilution on effector T cells with FACS.

Implications of TNFR2 expression in the function of Treg cells TNFR2 mediates the Treg-activating effect of TNF Our previous studies show that TNF exerted a negative feedback effect on inflammatory responses by activating and expanding mouse Treg cells through TNFR217,36 (reviewed in ref. 17). Intriguingly, Treg cells do not produce TNF, whereas activated Teff cells are major producers of TNF. As a consequence, TNF produced by activated autoreactive Teff cells paradoxically stimulated the activation and expansion of Treg cells, which eventually gained sufficient potency to attenuate the autoimmune inflammatory response.23,37

TNFR2 induces suppressive function of human Treg cells Vitamin D3-treated human dendritic cells (VD3-DCs) were able to induce functional human CD4+ FoxP3+ Treg cells capable of suppressing the proliferation of responder T cells in vitro.38 The VD3-DCs expressed markedly higher levels of membrane-bound TNF, a form of TNF that preferentially interacts with TNFR2.19 Importantly, inhibition of TNF or blockade of TNFR2 abrogated the TNF-dependent induction of functional Treg cells by VD3-DCs.19 Furthermore, induction of human CD8+ Treg cells by anti-CD3 monoclonal antibody was also found to be TNF-dependent.20

TNFR2 promotes survival of human Treg cells in an inflammatory environment Human Treg cells express and secrete higher levels of thioredoxin-1 (Trx-1), a major antioxidative molecule, and therefore have a greater capacity to resist oxidative stress in the tumour or inflammatory environment. Although

inflammatory stimuli up-regulated Trx-1 expression on Treg cells, only TNF increased the release of Trx-1. In conjunction with increased expression and secretion of Trx-1 on Treg cells, but not on Teff cells, TNF also enhanced surface thiol expression and consequently increased their resistance to H2O2.22 Hence, TNF promotes Treg-cell survival and activity within inflammatory milieus.

Shed soluble TNFR2 inhibits inflammatory response The contributions of soluble TNFR2 to Treg-cell activity are also becoming evident. For example, both activated human and mouse Treg cells produced high levels of functional soluble TNFR2 with the capacity to capture TNF and block its activating effects on Teff and Treg cells. The Treg cells deficient in TNFR2 were not able to control inflammatory responses in vivo,14 underscoring the likelihood that shed TNFR2 is an effector molecule that contributes to the immunosuppressive effects of Treg cells.

TNF enhances TNFR superfamily co-stimulatory signal to Treg cells Our recent study found that TNF preferentially up-regulated TNFR2 on Treg cells, resulting in a self-amplification loop in the activation of Treg cells. Furthermore, other co-stimulatory TNFR superfamily members OX40 and 4-1BB were also preferentially up-regulated on Treg cells by TNF stimulation. As agonistic antibodies to these receptors activated Treg cells, this further augmented the Treg-activating effect of TNF–TNFR2 interaction.39 The TNFR2+ Treg cells are highly proliferative in vivo,24 which presumably is a result of these multiple activation pathways initiated by TNF–TNFR2 signalling.


429

X. Chen and J. J. Oppenheim TNF synergizes with IL-2 in activation of Treg cells As shown by our previous observation, TNF in concert with IL-2 up-regulated expression of CD25 on Treg cells and consequently increased the phospho-Stat5 levels.36 Therefore, TNF is able to enhance the Treg-cell-activating activity of IL-2. Furthermore, both IL-2 and TNF each and together also up-regulated expression of TNFR2 on Treg cells,39 so these two cytokines form a reciprocal amplification network and synergize in the activation of Treg cells. This raises the question concerning the sequence of in vivo events at inflammatory sites. CD25+ FoxP3+ TNFR2) cells presumably can be stimulated by Teff-cell-derived IL-2 to become TNFR2+ and functional. Conversely, CD25) FoxP3+ TNFR2+ cells can be induced by TNF to become CD25+ and responsive to IL-2. Expression of both CD25 and TNFR2 can be induced and up-regulated by TCR stimulation.24,36,40 Hence, TCR stimulation by antigen, CD25 stimulation by IL-2, and TNFR2 stimulation by TNF can either independently or cooperatively promote the function of Treg cells.

Treg cells out-compete Teff cells for co-stimulation by TNF–TNFR2 Expression of TNFR2 can be up-regulated on Teff cells with enhanced Treg-resistant capacity Although preferentially expressed by Treg cells, TNFR2 is also expressed by a smaller fraction of resting mouse Teff cells (< 10%) and relatively more human peripheral blood Teff cells (approximately 20%).15,16,24,36 On TCR stimulation, expression of TNFR2 on both human and mouse Teff cells was enhanced by up to 30%.24,28,36 TNFR2-expressing Teff cells were more proliferative than TNFR2) Teff cells in vivo in normal mice in the presence of Treg cells,24 suggesting that TNFR2+ Teff cells are more resistant to inhibition. Indeed, Teff cells with constitutive or TCR-stimulation induced TNFR2 expression, such as tumour-infiltrating lymphocytes, were much more resistant to Treg-mediated inhibition.24

Activated Treg cells exhibited higher levels of TNFR2 and dominant suppressive capacity In both mouse and humans, resting Treg cells express markedly higher levels of TNFR2 than resting Teff cells. This relationship is also true for activated T cells: TNFR2 expression on activated Teff cells is consistently lower than that on Treg cells activated in the same way, which acquire a TNFR2high phenotype.24,28,36 In the pro-inflammatory tumour environment, Teff cells exhibited elevated TNFR2 expression and were resistant to inhibition by lymph node-derived Treg cells. However, these TNFR2430

expressing Teff cells were still markedly inhibited by tumour-derived Treg cells with a TNFR2high phenotype.24 Hence, Treg cells with persistently higher levels of TNFR2 expression appear to be more competitive than Teff cells in the use of TNF–TNFR2 co-stimulation. TNFR2 is preferentially expressed on the surface of both human and mouse Treg cells, in either the steady state or the activated state. This may enable Treg cells to outcompete Teff cells in binding with TNF. Further, once shed by Treg cells, soluble TNFR2 further reduces the availability of TNF to Teff cells, by binding and neutralizing TNF. Hence, by depriving Teff cells of TNF, Treg cells acts as ‘living’ enbrel-like decoys. Presumably, the preferential expression of TNFR2 on Treg cells is critical for retaining their capacity to negative feedback control of inflammatory responses, by efficiently competing with Teff cells for the TNF–TNFR2 co-stimulation.

Effect of ant-TNF on Treg-cell activity Anti-TNF has contrasting anti-inflammatory and pro-inflammatory effects in autoimmune disorders It is generally considered that TNF has major pathogenic effects in autoimmune inflammatory responses, which provided the rationale to develop anti-TNF therapy.41 Although anti-TNF therapy clearly has beneficial effects in most rheumatoid arthritis and some other autoimmune diseases, this treatment does not always result in a positive response, and can at times induce the onset of autoimmune inflammation (reviewed in ref. 42 and others). For example, anti-TNF treatment almost uniformly resulted in immune activation and exacerbation of disease in patients with multiple sclerosis 43 Furthermore, a minority of patients with rheumatoid arthritis and inflammatory bowel disease develop lupus erythematosus and neuroinflammatory diseases after anti-TNF therapy.43 The puzzling effect of anti-TNF therapy is likely to be attributable to the pleiotropic action of TNF in immune responses. Although generally considered to be a master pro-inflammatory cytokine, TNF nevertheless also has unexpected anti-inflammatory and immunosuppressive effects, especially after prolonged exposure (reviewed in ref. 44–46). The mechanism underlying the contrasting effects of anti-TNF as well as of TNF by itself is not fully understood.

Effects of anti-TNF therapy on Treg activity Assuming the primary biological function of Treg cells is to maintain immune homeostasis and prevent autoimmunity, a convenient and logical explanation for the beneficial effect of anti-TNF could be to promote Treg-cell activity, and based on this hypothesis, down-regulation of Treg-cell activity by TNF could be further extrapolated.


TNFR2+ Treg cells As a matter of fact, this notion is favoured by a number of studies.18,47–50 Nevertheless, recent studies point to the opposite direction. For example, induction of suppressive function of antigen-specific human CD4+ Treg cells by vitamin D3-treated tolerogenic DCs19 or human CD8+ Treg cells treated with anti-CD3 monoclonal antibody were blocked by anti-TNF or anti-TNFR2 antibodies.20 The stimulatory effect of pathogenic Teff cells on Treg cells could also be attenuated by neutralization of TNF.51 Furthermore, in the mouse psoriasis model, anti-TNF therapy suppressed FoxP3 expression in the skin and the number of FoxP3+ Treg cells was reduced in the draining lymph nodes, accompanied by the exacerbation of inflammation.21 It is therefore possible that greater inhibition of Teff cells than Treg cells may account for the beneficial effects of anti-TNF in some autoimmune patients. Based on careful analysis, we expressed reservations regarding the interpretation of data presented in these studies assuming that anti-TNF promotes Treg activity (as previously reviewed17). For example, in a recent study aiming to examine the interplay between TNF and Treg cells, it was reported that anti-TNF reagents could increase the number and function of Treg cells in a mouse arthritis model that was driven by over-expression of human TNF.52 However, it is well established that human TNF can only bind to mouse TNFR1, but not TNFR2,53 so this model fails to reveal the interaction between TNF and Treg cells through TNFR2. Furthermore, the suppressive cells reportedly found in patients with rheumatoid arthritis after anti-TNF therapy were actually Tr1 and/or Th3 suppressor cells, not FoxP3+ Treg cells which exert their suppressive function in a direct cell-to-cell contact manner.49,50 Therefore, the current studies do not provide convincing evidence that anti-TNF increases the function and number of CD4+ FoxP3+ Treg cells.

Anti-TNF may sensitize Teff cells to Treg-mediated inhibition We favour the idea that the beneficial effect of anti-TNF in autoimmunity is based on the inhibition of Teff cells and increasing the sensitivity of Teff cells to Treg-medi-

ated inhibition, rather than by promoting Treg-cell activity. As mentioned previously, TNFR2 expression on Teff cells enhances their resistance to Treg-mediated inhibition.24 TNFR2 expression on Teff cells is induced or upregulated by TCR stimulation24,28,36 in a TNF-dependent manner, because it could be blocked by neutralization of TNF (unpublished data). Our preliminary data showed that anti-TNF antibody and Treg cells had additive, if not synergistic, effects on the inhibition of Teff-cell activation. These data suggest that Teff cells are more sensitive to anti-TNF than Treg cells, presumably because Treg cells can still outcompete Teff cells in use of the remaining limited TNF for co-stimulation. Consequently, anti-TNF therapy by preferentially depriving Teff cells of activation by TNF would enhance their susceptibility to the suppressive effects of residual Treg cells.

Concluding remarks There is increasing evidence that TNFR2 expression is an indicator of highly potent suppressor Treg cells, and by mediating the effect of TNF, TNFR2 promotes the activation, expansion and survival of Treg cells, especially in the inflammatory environment. At the inflammatory site, multiple pathways are likely to contribute cooperatively to the optimal activation of Treg cells initiated by TNF– TNFR2 interaction, including: (i) TNF–TNFR2 self-amplification loop, (ii) TNF/IL-2 and TNFR2/CD25 reciprocal amplification loop, (iii) synergistic co-stimulation of TNFR2 and other TNFR superfamily members, and (iv) positive feedback regulation between TNFR2/CD25 and TCR signalling (Fig. 1). Both Teff and Treg cells are likely to use co-stimulation of TNF–TNFR2 for activation, expansion and maintenance of immune equilibrium. Based on the reviewed experimental evidence, we propose that, at the initial stage, TNF co-stimulates the activation of Teff cells and liberates them from Treg cells to mount an effective immune response against pathogens; whereas at a later stage in the inflammatory responses, higher levels of TNFR2 expression by Treg cells may enable them to outcompete Teff cells for TNF and to down-regulate

Amplified TNFR2 expression Amplified CD25 expression TNFR2 TNF

Treg

Amplified TNFRSF (OX40, 4-1BB) expression

Amplified TCR signaling

Biological effect on Treg cells Treg Treg Treg Treg Treg Treg Treg Treg Treg Treg Treg Treg

• Activation • Proliferative expansion • Up-regulation of FoxP3 • Increase of suppressive function • Survival

Figure 1. At inflammatory site, multiple pathways contribute to the optional regulatory T (Treg) cell activation initiated by tumour necrosis factor (TNF) –TNF receptor 2 (TNFR2) interaction. TNFRSF, TNFR superfamily.


431

X. Chen and J. J. Oppenheim inflammatory responses. The stimulatory effect of TNF on Treg cells therefore represents an important negative feedback mechanism that results in the attenuation and termination of prolonged or excessive immune responses, which otherwise may cause severe collateral damage (as reviewed in ref. 17). The clinical relevance of the TNF–TNFR2 pathway in the activation of Treg cells should also be addressed in future studies, which may improve the safety of the current anti-TNF treatment regimen. Further elucidation of signalling pathways and molecular basis of TNFR2 in the activation of Treg cells may yield novel strategy to upregulate or down-regulate Treg-cell activity for therapeutic purposes.

Acknowledgements This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. This research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

Disclosures The authors have no financial and commercial conflicts of interest.

References 1 Gershon RK, Kondo K. Cell interactions in the induction of tolerance: the role of thymic lymphocytes. Immunology 1970; 18:723–37. 2 Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995; 155:1151–64. 3 Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 2005; 6:345–52. 4 Shevach EM. Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity 2009; 30:636–45. 5 Roncarolo MG, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings MK. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev 2006; 212:28–50. 6 Weiner HL. Induction and mechanism of action of transforming growth factor-betasecreting Th3 regulatory cells. Immunol Rev 2001; 182:207–14. 7 Vandenabeele P, Declercq W, Beyaert R, Fiers W. Two tumour necrosis factor receptors: structure and function. Trends Cell Biol 1995; 5:392–9. 8 Grell M, Douni E, Wajant H et al. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 1995; 83:793–802. 9 Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science 2002; 296:1634–5. 10 Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting JP. TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination. Nat Neurosci 2001; 4:1116–22. 11 Kim EY, Priatel JJ, Teh SJ, Teh HS. TNF receptor type 2 (p75) functions as a costimulator for antigen-driven T cell responses in vivo. J Immunol 2006; 176:1026–35. 12 Kim EY, Teh HS. TNF type 2 receptor (p75) lowers the threshold of T cell activation. J Immunol 2001; 167:6812–20. 13 Kim EY, Teh HS. Critical role of TNF receptor type-2 (p75) as a costimulator for IL2 induction and T cell survival: a functional link to CD28. J Immunol 2004; 173:4500–9.

432

14 van Mierlo GJ, Scherer HU, Hameetman M, Morgan ME, Flierman R, Huizinga TW, Toes RE. Cutting edge: TNFR-shedding by CD4+CD25+ regulatory T cells inhibits the induction of inflammatory mediators. J Immunol 2008; 180:2747–51. 15 Chen X, Subleski JJ, Kopf H, Howard OM, Mannel DN, Oppenheim JJ. Cutting edge: expression of TNFR2 defines a maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory cells: applicability to tumor-infiltrating T regulatory cells. J Immunol 2008; 180:6467–71. 16 Chen X, Subleski JJ, Hamano R, Howard OM, Wiltrout RH, Oppenheim JJ. Co-expression of TNFR2 and CD25 identifies more of the functional CD4+FoxP3+ regulatory T cells in human peripheral blood. Eur J Immunol 2010; 40:1099–106. 17 Chen X, Oppenheim JJ. TNF-alpha: an activator of CD4+FoxP3+TNFR2+ regulatory T cells. Curr Dir Autoimmun 2010; 11:119–34. 18 Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF down-modulates the function of human CD4+CD25hi T regulatory cells. Blood 2006; 108:253–61. 19 Kleijwegt FS, Laban S, Duinkerken G, Joosten AM, Zaldumbide A, Nikolic T, Roep BO. Critical role for TNF in the induction of human antigen-specific regulatory T cells by tolerogenic dendritic cells. J Immunol 2010; 185:1412–8. 20 Ablamunits V, Bisikirska B, Herold KC. Acquisition of regulatory function by human CD8+ T cells treated with anti-CD3 antibody requires TNF. Eur J Immunol 2010; 40:2891–901. 21 Ma HL, Napierata L, Stedman N, Benoit S, Collins M, Nickerson-Nutter C, Young DA. Tumor necrosis factor alpha blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells. Arthritis Rheum 2010; 62:430–40. 22 Mougiakakos D, Johansson CC, Jitschin R, Bottcher M, Kiessling R. Increased thioredoxin-1 production in human naturally occurring regulatory T cells confers enhanced tolerance to oxidative stress. Blood 2011; 117:854–61. 23 Grinberg-Bleyer Y, Saadoun D, Baeyens A et al. Pathogenic T cells have a paradoxical protective effect in murine autoimmune diabetes by boosting Tregs. J Clin Invest 2010; 120:4558–68. 24 Chen X, Hamano R, Subleski JJ, Hurwitz AA, Howard OM, Oppenheim JJ. Expression of costimulatory TNFR2 induces resistance of CD4+FoxP3) conventional T cells to suppression by CD4+FoxP3+ regulatory T cells. J Immunol 2010; 185:174–82. 25 van der Most RG, Currie AJ, Mahendran S, Prosser A, Darabi A, Robinson BW, Nowak AK, Lake RA. Tumor eradication after cyclophosphamide depends on concurrent depletion of regulatory T cells: a role for cycling TNFR2-expressing effector-suppressor T cells in limiting effective chemotherapy. Cancer Immunol Immunother 2009; 58:1219–28. 26 Lehmann J, Huehn J, de la Rosa M et al. Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25) regulatory T cells. Proc Natl Acad Sci USA 2002; 99:13031–6. 27 Annunziato F, Cosmi L, Liotta F et al. Phenotype, localization, and mechanism of suppression of CD4+CD25+ human thymocytes. J Exp Med 2002; 196:379–87. 28 Nagar M, Jacob-Hirsch J, Vernitsky H et al. TNF activates a NF-kappaB-regulated cellular program in human CD45RA– regulatory T cells that modulates their suppressive function. J Immunol 2010; 184:3570–81. 29 Minigo G, Woodberry T, Piera KA et al. Parasite-dependent expansion of TNF receptor II-positive regulatory T cells with enhanced suppressive activity in adults with severe malaria. PLoS Pathog 2009; 5:e1000402. 30 van Amelsfort JM, Jacobs KM, Bijlsma JW, Lafeber FP, Taams LS. CD4+CD25+ regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid. Arthritis Rheum 2004; 50:2775–85. 31 Kryczek I, Liu R, Wang G et al. FOXP3 defines regulatory T cells in human tumor and autoimmune disease. Cancer Res 2009; 69:3995–4000. 32 Miles DW, Happerfield LC, Naylor MS, Bobrow LG, Rubens RD, Balkwill FR. Expression of tumour necrosis factor (TNF alpha) and its receptors in benign and malignant breast tissue. Int J Cancer 1994; 56:777–82. 33 Pusztai L, Clover LM, Cooper K, Starkey PM, Lewis CE, McGee JO. Expression of tumour necrosis factor alpha and its receptors in carcinoma of the breast. Br J Cancer 1994; 70:289–92. 34 Trentin L, Zambello R, Bulian P et al. Tumour-infiltrating lymphocytes bear the 75 kDa tumour necrosis factor receptor. Br J Cancer 1995; 71:240–5. 35 Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T, Sakaguchi S. CTLA-4 control over Foxp3+ regulatory T cell function. Science 2008; 322:271–5. 36 Chen X, Baumel M, Mannel DN, Howard OM, Oppenheim JJ. Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. J Immunol 2007; 179:154–61. 37 Bilate AM, Lafaille JJ. Can TNF-alpha boost regulatory T cells? J Clin Invest 2010; 120:4190–2. 38 Penna G, Roncari A, Amuchastegui S, Daniel KC, Berti E, Colonna M, Adorini L. Expression of the inhibitory receptor ILT3 on dendritic cells is dispensable for


TNFR2+ Treg cells induction of CD4+Foxp3+ regulatory T cells by 1,25-dihydroxyvitamin D3. Blood 2005; 106:3490–7.

47 Ehrenstein MR, Evans JG, Singh A, Moore S, Warnes G, Isenberg DA, Mauri C. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-

39 Hamano R, Huang J, Yoshimura T, Oppenheim JJ, Chen X. TNF by inducing co-stimulatory TNF receptor superfamily members TNFR2, 4-1BB and OX40 augments the number and function of mouse CD4+FoxP3+ regulatory T cells. Eur J Immunol 2011; In press. doi: 10.1002/eji.201041205. 40 Kim HP, Leonard WJ. The basis for TCR-mediated regulation of the IL-2 receptor alpha chain gene: role of widely separated regulatory elements. EMBO J 2002; 21:3051–9. 41 Feldmann M. Development of anti-TNF therapy for rheumatoid arthritis. Nat Rev

TNFalpha therapy. J Exp Med 2004; 200:277–85. 48 Toubi E, Kessel A, Mahmudov Z, Hallas K, Rozenbaum M, Rosner I. Increased spontaneous apoptosis of CD4+CD25+ T cells in patients with active rheumatoid arthritis is reduced by infliximab. Ann N Y Acad Sci 2005; 1051:506–14. 49 Nadkarni S, Mauri C, Ehrenstein MR. Anti-TNF-alpha therapy induces a distinct regulatory T cell population in patients with rheumatoid arthritis via TGF-beta. J Exp Med 2007; 204:33–9.

Immunol 2002; 2:364–71. 42 Ramos-Casals M, Brito-Zeron P, Munoz S, Soria N, Galiana D, Bertolaccini L, Cuadrado MJ, Khamashta MA. Autoimmune diseases induced by TNF-targeted therapies: analysis of 233 cases. Medicine (Baltimore) 2007; 86:242–51. 43 Kollias G, Kontoyiannis D, Douni E, Kassiotis G. The role of TNF/TNFR in organ-specific and systemic autoimmunity: implications for the design of optimized ‘anti-TNF’

50 Deepe GS Jr, Gibbons RS. TNF-alpha antagonism generates a population of antigenspecific CD4+CD25+ T cells that inhibit protective immunity in murine histoplasmosis. J Immunol 2008; 180:1088–97. 51 Thornton AM, Piccirillo CA, Shevach EM. Activation requirements for the induction of CD4+CD25+ T cell suppressor function. Eur J Immunol 2004; 34:366–76. 52 Biton J, Semerano L, Delavallee L, Lemeiter D, Laborie M, Grouard-Vogel G, Boissier

therapies. Curr Dir Autoimmun 2002; 5:30–50. 44 Cope AP. Regulation of autoimmunity by proinflammatory cytokines. Curr Opin Immunol 1998; 10:669–76. 45 Clark J, Vagenas P, Panesar M, Cope AP. What does tumour necrosis factor excess do to the immune system long term? Ann Rheum Dis 2005; 64(Suppl. 4):iv70–6. 46 Kollias G, Kontoyiannis D. Role of TNF/TNFR in autoimmunity: specific TNF receptor blockade may be advantageous to anti-TNF treatments. Cytokine Growth Factor Rev

MC, Bessis N. Interplay between TNF and regulatory T cells in a TNF-driven murine model of arthritis. J Immunol 2011; 186:3899–910. 53 Lewis M, Tartaglia LA, Lee A, Bennett GL, Rice GC, Wong GH, Chen EY, Goeddel DV. Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc Natl Acad Sci USA 1991; 88:2830–4.

2002; 13:315–21.


433