Benoit L Salomon
email: benoit.salomon@inserm.fr.

Insights into the biology and therapeutic implications of TNF and regulatory T cells

Treatments that block tumour necrosis factor (TNF) have major beneficial effects in several autoimmune and rheumatic diseases, including rheumatoid arthritis. However, some patients do not respond to TNF inhibitor treatment and some rare occurrences of paradoxical disease exacerbation have been reported. These limitations on the clinical efficacy of TNF inhibitors can be explained by the differences between TNF receptor 1 (TNFR1) and TNFR2 signalling and by the diverse effects of TNF on multiple immune cells, including FOXP3 + regulatory T (Treg) cells.

This basic knowledge sheds light on the consequences of TNF inhibitor therapies on Treg cells in treated patients and on the limitations of such treatment in the control of diseases with an autoimmune component. Accordingly, the next generation of drugs targeting TNF is likely to be based on agents that selectively block the binding of TNF to TNFR1 and on TNFR2 agonists. These approaches could improve the treatment of rheumatic diseases in the future.

[H1] Introduction

Tumour necrosis factor (TNF) is an inflammatory cytokine that is detected in the blood within minutes after an injury and has a major protective role in infectious diseases. In the late 1980s, TNF was detected in the joints of patients with rheumatoid arthritis (RA) [START_REF] Feldmann | Translating molecular insights in autoimmunity into effective therapy[END_REF][START_REF] Monaco | Anti-TNF therapy: past, present and future[END_REF] . A few years later, overexpression of TNF in transgenic mice was shown to induce autoimmune arthritis [START_REF] Keffer | Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis[END_REF] . Agents that block this cytokine, termed TNF inhibitors, include monoclonal antibodies (mAbs) and soluble TNF receptors. Anti-TNF therapy was first tested in patients with sepsis without clear success and then repurposed for the treatment of RA in the early 1990s [START_REF] Feldmann | Translating molecular insights in autoimmunity into effective therapy[END_REF][START_REF] Monaco | Anti-TNF therapy: past, present and future[END_REF] . TNF inhibitors are now widely used and have greatly improved the medical care of patients with RA, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis and ankylosing spondylitis. Five original TNF inhibitors and numerous biosimilars have been approved, mostly for the treatment of arthritis, psoriasis or ankylosing spondylitis (Table 1). However, not all patients respond to TNF inhibitor treatment. One-third of patients with RA have to stop taking these drugs within the first year because of insufficient efficacy or adverse events [START_REF] Silva-Fernandez | Rheumatoid arthritis: When TNF inhibitors fail in RA--weighing up the options[END_REF] . About 20% of patients with psoriasis do not respond to treatment with a TNF inhibitor and around one-third of initial responders lose response over time [START_REF] Roda | Loss of Response to Anti-TNFs: Definition, Epidemiology, and Management[END_REF] . Similar efficacy profiles are observed for patients with inflammatory bowel disease (IBD) [START_REF] Esposito | Survival rate of antitumour necrosis factor-alpha treatments for psoriasis in routine dermatological practice: a multicentre observational study[END_REF] . Although this Review mainly focuses on the effects of TNF inhibitors in rheumatic diseases, particularly RA, I also discuss their effects and use in the treatment of other autoimmune and inflammatory diseases to illustrate the role and mechanisms of these agents in general.

Treatment with TNF inhibitors is also associated with adverse effects, such as infections, which are explained by the intrinsic anti-inflammatory effects of these agents. More intriguing (and counterintuitive) is the paradoxical exacerbation of pre-existing autoimmune disease or the development of new-onset autoimmune disease following TNF inhibitor therapy. Rarely, treated patients can develop lupus-like syndrome, vasculitis, antiphospholipid syndrome or sarcoidosis.

For example, the reported prevalence of systemic lupus erythematosus (SLE) among recipients of TNF inhibitor therapy is 0.1-0.2% [START_REF] Ramos-Casals | Autoimmune diseases induced by TNF-targeted therapies[END_REF][START_REF] Ramos-Casals | Autoimmune diseases induced by biological agents: a doubleedged sword?[END_REF][START_REF] Brenner | Regulation of tumour necrosis factor signalling: live or let die[END_REF] . A few patients develop organ-specific autoimmune conditions, such as interstitial lung disease, optical neuritis, demyelinating neuropathy, multiple sclerosis (MS), psoriasis or autoimmune hepatitis, with the highest prevalence (2.00-5.00%) reported for psoriasis and the lowest (0.05-0.20%) for demyelinating disease. Several reviews have discussed in depth the spectrum of autoimmune diseases occurring in TNF inhibitor-treated patients [START_REF] Ramos-Casals | Autoimmune diseases induced by TNF-targeted therapies[END_REF][START_REF] Ramos-Casals | Autoimmune diseases induced by biological agents: a doubleedged sword?[END_REF][START_REF] Brenner | Regulation of tumour necrosis factor signalling: live or let die[END_REF][START_REF] Conrad | TNF blockade induces a dysregulated type 1 interferon response without autoimmunity in paradoxical psoriasis[END_REF] , among which MS is of particular interest. In the late 1990s, before the increased risk of demyelinating neuropathy associated with TNF inhibitor treatment was known, two clinical trials investigated the efficacy of TNF inhibitors in MS. However, these drugs induced unexpected disease exacerbations that led to the worldwide contraindication of these drugs in these patients [START_REF] Van Oosten | Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2[END_REF][START_REF] Study-Group | TNF neutralization in MS: results of a randomized, placebo-controlled multicenter study. The Lenercept Multiple Sclerosis Study Group and The University of British Columbia MS/MRI Analysis Group[END_REF] . These observations sparked intense interest in elucidating why not all patients respond to TNF inhibitor therapy, developing biomarkers to predict response, and understanding why some treated patients develop paradoxical autoimmunity. This Review focuses on the effects of TNF on inflammation and immunity. I describe the pro-inflammatory and regulatory roles of TNF, both of which are now well-established, and address the effects of this cytokine on diverse aspects of regulatory T (Treg) cell biology, including their expansion, differentiation and suppressive function. Finally, I describe the effect of TNF inhibitors on Treg cells and explore potential candidates for the next generation of drugs that target TNF or its receptors. Although TNF also has important roles in organogenesis and the development of lymphoid organs, protection of tissues in the nervous system, heart and joints [START_REF] Aggarwal | Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey[END_REF][START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF][START_REF] Atretkhany | Distinct modes of TNF signaling through its two receptors in health and disease[END_REF] and inhibition of tumorigenesis [START_REF] Salomon | Tumor Necrosis Factor alpha and Regulatory T Cells in Oncoimmunology[END_REF] , these topics are outside the scope of the present Review and will not be considered.

[H1] The two Janus faces of TNF TNF has complex regulatory and pro-inflammatory effects in diseases with an autoimmune component [START_REF] Aggarwal | Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey[END_REF][START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF][START_REF] Atretkhany | Distinct modes of TNF signaling through its two receptors in health and disease[END_REF] , such as RA [START_REF] Davignon | Modulation of T-cell responses by anti-tumor necrosis factor treatments in rheumatoid arthritis: a review[END_REF] . This cytokine is produced under various inflammatory conditions by multiple cell types and exists in two forms: a soluble form that acts as a ligand, and a membrane-bound form that can act as either a ligand or a receptor [START_REF] Horiuchi | Transmembrane TNF-a: structure, function and interaction with anti-TNF agents[END_REF][START_REF] Lee | Reverse Signaling of Tumor Necrosis Factor Superfamily Proteins in Macrophages and Microglia: Superfamily Portrait in the Neuroimmune Interface[END_REF][START_REF] Qu | Forward and Reverse Signaling Mediated by Transmembrane Tumor Necrosis Factor-Alpha and TNF Receptor 2: Potential Roles in an immunosuppressive Tumor Microenvironment[END_REF] . Furthermore, TNF can induce multiple downstream signalling pathways [START_REF] Brenner | Regulation of tumour necrosis factor signalling: live or let die[END_REF] as a result of binding to two different receptors, TNF receptor (TNFR) 1 and TNFR2, which are structurally related but have divergent biological properties. TNFR1 is broadly expressed whereas TNFR2 is expressed mostly by T cells, certain myeloid and endothelial cells and some cells of the central nervous system [START_REF] Aggarwal | Signalling pathways of the TNF superfamily: a double-edged sword[END_REF][START_REF] Chen | Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells[END_REF] .

The next sections describe the distinct functions of TNFR1 and TNFR2 and discuss the proinflammatory and anti-inflammatory effects of TNF on innate immune cells and lymphocytes, and present information on the cellular source of TNF.

[H2] TNFR1 and TNFR2

TNF is one of the most potent pro-inflammatory cytokines [START_REF] Ware | Network communications: lymphotoxins, LIGHT, and TNF[END_REF] , which explains the success of TNF inhibitor therapy in diseases with an inflammatory component. However, the paradoxical development or exacerbation of autoimmune disease in some patients treated with these drugs reveals the anti-inflammatory aspect of this cytokine, which is partly explained by effects downstream of TNFR2. Polymorphisms in TNFRSF1B, which encodes TNFR2, are frequently observed in patients with rheumatic diseases (RA, SLE, ankylosing spondylitis and systemic sclerosis) or IBD [START_REF] Yang | Role of TNF-TNF Receptor 2 Signal in Regulatory T Cells and Its Therapeutic Implications[END_REF] . These mutations seem to alter the binding kinetics between TNF and TNFR2 and lead to inhibition of downstream NF-KB signalling, which suggests that TNFR2 signalling has a protective role in these diseases [START_REF] Faustman | TNF receptor 2 pathway: drug target for autoimmune diseases[END_REF] . A single nucleotide polymorphism in TNFRSF1A, which encodes TNFR1, is specifically associated with an increased risk of MS. This allele results in the expression of a novel soluble form of TNFR1 that binds to and blocks TNF, and therefore mimics the MS-exacerbating effect of TNF inhibitor therapy [START_REF] Gregory | TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis[END_REF] . Other mutations in TNFRSF1A that cause TNFR1 misfolding and endothelium reticulum stress are found in patients with periodic fevers [START_REF] Park | Lighting the fires within: the cell biology of autoinflammatory diseases[END_REF] .

The differential functions of TNFR1 and TNFR2 in rheumatic and autoimmune diseases have been defined in mouse models. Generally, TNFR1-knockout mice have reduced disease severity whereas TNFR2-knockout mice develop exacerbated disease (Table 2). In addition, treatment with either TNFR1 antagonists or TNFR2 agonists suppresses disease symptoms in mouse models of arthritis and in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS, further supporting a pathogenic role of TNFR1 and a protective role of TNFR2 (Table 2). A pathogenic role of TNFR1 and a protective role of TNFR2 have also been observed in mouse models of IBD, at least during the chronic phase of the disease [START_REF] Atretkhany | Distinct modes of TNF signaling through its two receptors in health and disease[END_REF][START_REF] Yang | Differential roles of TNFalpha-TNFR1 and TNFalpha-TNFR2 in the differentiation and function of CD4(+)Foxp3(+) induced Treg cells in vitro and in vivo periphery in autoimmune diseases[END_REF] . Thus, TNFR1 and TNFR2 seem to be pathogenic and protective, respectively, in some autoimmune and chronic inflammatory diseases.

[H2] Effects of TNF on innate immunity

[H3] Pro-inflammatory effects.

The pro-inflammatory effects of TNF on innate immunity involve several distinct mechanisms (Figure 1). TNF is one of the main drivers of acute inflammation because it activates endothelial cells, induces chemokine release and promotes intense and early (within hours) recruitment of neutrophils and monocytes, via both TNFR1 and TNFR2 [START_REF] Rampart | Inflammatory properties of recombinant tumor necrosis factor in rabbit skin in vivo[END_REF][START_REF] Venkatesh | Endothelial TNF receptor 2 induces IRF1 transcription factordependent interferon-beta autocrine signaling to promote monocyte recruitment[END_REF] . Acute inflammation is also attributed to the TNF-mediated activation of canonical NF-KB signalling, which leads to the early induction of inflammatory cytokines, including TNF itself, IL-6, IL-8 and IL-1β [START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF] . TNF also sustains inflammation through the activation of receptor-interacting protein kinase (RIPK) 1 and RIPK3, which promote necroptosis and the release of inflammatory compounds termed damage-associated molecular patterns (DAMPs) [START_REF] Duprez | RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome[END_REF] . In addition, via TNFR1 signalling, TNF promotes innate immunity by favouring the maturation of dendritic cells [START_REF] Ding | TNF receptor 1 mediates dendritic cell maturation and CD8 T cell response through two distinct mechanisms[END_REF][START_REF] Menges | Repetitive injections of dendritic cells matured with tumor necrosis factor alpha induce antigen-specific protection of mice from autoimmunity[END_REF] .

[H3] Regulatory effects.

The immunoregulatory functions of TNF are likely to involve multiple mechanisms (Figure 1).

TNF might promote the extra-adrenal production of immunoregulatory glucocorticoids [START_REF] Noti | TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis[END_REF] and inhibit haematopoiesis [START_REF] Wang | Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell-mediated TNFalpha signaling[END_REF] . TNF also stimulates innate immunosuppressive cells (via TNFR2) and activates mesenchymal stem cells, which produce increased levels of immunosuppressive prostaglandin E2 (PGE2), as has been shown in synovial fluid from patients with RA [START_REF] Ghannam | Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype[END_REF][START_REF] Sayegh | Rheumatoid Synovial Fluids Regulate the Immunomodulatory Potential of Adipose-Derived Mesenchymal Stem Cells Through a TNF/NF-kappaB-Dependent Mechanism[END_REF] . TNF also promotes immunosuppression by favouring either the differentiation or the suppressive function of myeloid-derived suppressor cells via increasing their production of reactive oxygen species, arginase 1 and inducible nitric oxide synthase [START_REF] Raveney | TNFR1-dependent regulation of myeloid cell function in experimental autoimmune uveoretinitis[END_REF][START_REF] Zhao | TNF signaling drives myeloid-derived suppressor cell accumulation[END_REF][START_REF] Chavez-Galan | Transmembrane Tumor Necrosis Factor Controls Myeloid-Derived Suppressor Cell Activity via TNF Receptor 2 and Protects from Excessive Inflammation during BCG-Induced Pleurisy[END_REF][START_REF] Hu | Transmembrane TNF-alpha promotes suppressive activities of myeloidderived suppressor cells via TNFR2[END_REF][START_REF] Sade-Feldman | Tumor necrosis factor-alpha blocks differentiation and enhances suppressive activity of immature myeloid cells during chronic inflammation[END_REF] .

[H3] Effects on dendritic cells, monocytes and macrophages.

Although TNF seems to favour the production of T-bet and IL-12 by dendritic cells [START_REF] Bachus | Impaired Tumor-Necrosis-Factor-alpha-driven Dendritic Cell Activation Limits Lipopolysaccharide-Induced Protection from Allergic Inflammation in Infants[END_REF] , other studies suggest that the presence of TNF inhibits the production of p40 (the common chain of IL-12 and IL-23) by dendritic cells, macrophages and monocytes [START_REF] Alzabin | Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway[END_REF][START_REF] Mayordomo | IL-12/23p40 overproduction by dendritic cells leads to an increased Th1 and Th17 polarization in a model of Yersinia enterocolitica-induced reactive arthritis in TNFRp55-/-mice[END_REF][START_REF] Notley | Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells[END_REF] . These divergent findings could be explained by differential actions of TNF depending on the maturation stage and type of both antigen presenting cells and dendritic cell subsets.

In addition, TNF can either promote or inhibit macrophage activation, effects that are both probably mediated by TNFR1 (reviewed elsewhere [START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF] ). The early response of macrophages to incubation with TNF, observed after a few hours, is both NF-KB-dependent and MAPKdependent and involves the expression of genes encoding various inflammatory molecules and cytokines. This initial response is followed (after 24 hours) by a state of desensitization, also called cross-tolerance or endotoxin tolerance. Desensitized (also termed tolerized) macrophages are unable to produce inflammatory factors when stimulated by potent activators such as Tolllike receptor ligands. The mechanism of desensitization involves NF-KB inhibition following activation of glycogen synthase kinase 3 (GSK3) and TNF-induced protein 3 (TNFAIP3) [START_REF] Park | Tumor necrosis factor induces GSK3 kinase-mediated cross-tolerance to endotoxin in macrophages[END_REF] .

Tolerized macrophages have a transiently reduced capacity to produce IL-12 and IL-23, which are pro-inflammatory [START_REF] Notley | Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells[END_REF][START_REF] Zakharova | Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells[END_REF] . The physiological role of cross-tolerance is probably the prevention of life-threatening inflammation in the context of overwhelming macrophage activation by pathogens and Toll-like receptor ligands. Ultimately, after prolonged incubation with TNF, specifically in the presence of type 1 interferons, macrophages overcome this desensitized state and recover their inflammatory function by modifying their metabolism and epigenetic status [START_REF] Kusnadi | The Cytokine TNF Promotes Transcription Factor SREBP Activity and Binding to Inflammatory Genes to Activate Macrophages and Limit Tissue Repair[END_REF][START_REF] Park | Type I interferons and the cytokine TNF cooperatively reprogram the macrophage epigenome to promote inflammatory activation[END_REF] .

[H2] Effects of TNF on lymphocytes

[H3] Pro-inflammatory effects.

TNF can either promote or suppress immunity through its differential effects on lymphocytes (Figure 1). The pro-inflammatory effects of TNF result from co-stimulation of T cells, mainly via TNFR2. TNF activates NF-KB and AKT signalling pathways that lead to increased T cell proliferation and survival, which are associated with increased levels of BCL2, BCLXL, IL-2 and survivin [START_REF] Tartaglia | Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor receptor[END_REF][START_REF] Kim | Critical role of TNF receptor type-2 (p75) as a costimulator for IL-2 induction and T cell survival: a functional link to CD28[END_REF][START_REF] Kim | TNF receptor type 2 (p75) functions as a costimulator for antigen-driven T cell responses in vivo[END_REF][START_REF] Calzascia | TNF-alpha is critical for antitumor but not antiviral T cell immunity in mice[END_REF][START_REF] Chen | TNFR2 expression by CD4 effector T cells is required to induce fullfledged experimental colitis[END_REF][START_REF] Soloviova | In vivo maturation of allo-specific CD8 CTL and prevention of lupus-like graft-versus-host disease is critically dependent on T cell signaling through the TNF p75 receptor but not the TNF p55 receptor[END_REF] . However, the co-stimulatory effect of TNF binding to TNFR2 on conventional T cells seems to be of marginal importance compared with its strong effect on Treg cells [START_REF] De Kivit | Stable human regulatory T cells switch to glycolysis following TNF receptor 2 costimulation[END_REF] , which is extensively discussed below.

[H3] Regulatory effects.

Although one report suggests that TNF promotes the expression of IL-10 by B regulatory (Breg) cells [START_REF] Schioppa | B regulatory cells and the tumor-promoting actions of TNF-alpha during squamous carcinogenesis[END_REF] , much more is known about the inhibitory effects of TNF on T cells. Prolonged exposure to TNF attenuates T cell receptor signalling by impairing store-operated calcium influx [START_REF] Cope | Chronic tumor necrosis factor alters T cell responses by attenuating T cell receptor signaling[END_REF][START_REF] Aspalter | Chronic TNF-alpha exposure impairs TCRsignaling via TNF-RII but not TNF-RI[END_REF] and also favours T cell exhaustion; in one report, TNF blockade during chronic infection with lymphocytic choriomeningitis virus abrogated the inhibitory gene expression signature [START_REF] Beyer | Tumor-necrosis factor impairs CD4(+) T cell-mediated immunological control in chronic viral infection[END_REF] . TNF is able to induce activation-induced cell death via TNFR1 engagement [START_REF] Qin | In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-gamma/TNF-alpha[END_REF] . Interestingly, TNFR2 signalling also seems to increase T-cell apoptosis by interfering with signalling pathways downstream of TNFR1 [START_REF] Naude | Tumor necrosis factor receptor cross-talk[END_REF] . However, TNFR2-dependent cell death might specifically occur in autoreactive T cells, which have altered TNFR2 signalling [START_REF] Faustman | TNF receptor 2 pathway: drug target for autoimmune diseases[END_REF][START_REF] Qin | In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-gamma/TNF-alpha[END_REF][START_REF] Lin | TNF receptor-2-triggered apoptosis is associated with the down-regulation of Bcl-xL on activated T cells and can be prevented by CD28 costimulation[END_REF][START_REF] Ban | Selective death of autoreactive T cells in human diabetes by TNF or TNF receptor 2 agonism[END_REF][START_REF] Bhattacharyya | Tumor-induced oxidative stress perturbs nuclear factor-kappaB activity-augmenting tumor necrosis factor-alpha-mediated T-cell death: protection by curcumin[END_REF][START_REF] Kim | TNFR2-deficient memory CD8 T cells provide superior protection against tumor cell growth[END_REF][START_REF] Luckey | T cell killing by tolerogenic dendritic cells protects mice from allergy[END_REF] . Cross-talk between TNFR1 and TNFR2 signalling is discussed in more detail in subsequent sections.

TNF also inhibits the differentiation of TH17 cells by increasing IL-2 production [START_REF] Miller | Transmembrane TNF-TNFR2 Impairs Th17 Differentiation by Promoting Il2 Expression[END_REF] , and decreases IL-17 production by conventional T cells and effector Treg cells via activation of TNFAIP3 [START_REF] Urbano | TNF-alpha-induced protein 3 (TNFAIP3)/A20 acts as a master switch in TNF-alpha blockade-driven IL-17A expression[END_REF][START_REF] Urbano | TNFalpha-Signaling Modulates the Kinase Activity of Human Effector Treg and Regulates IL-17A Expression[END_REF] . This mechanism might explain the increase in numbers of TH17 cells described in TNFR1-knockout mice or after treatment with TNF inhibitors in mouse models of RA and psoriasis [START_REF] Alzabin | Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway[END_REF][START_REF] Mayordomo | IL-12/23p40 overproduction by dendritic cells leads to an increased Th1 and Th17 polarization in a model of Yersinia enterocolitica-induced reactive arthritis in TNFRp55-/-mice[END_REF][START_REF] Notley | Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells[END_REF][START_REF] Elicabe | Lack of TNFR p55 results in heightened expression of IFN-gamma and IL-17 during the development of reactive arthritis[END_REF][START_REF] Ma | Tumor necrosis factor alpha blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells[END_REF] . A similar increase in TH17 cells has been reported specifically in nonresponding patients with RA treated with TNF inhibitors [START_REF] Alzabin | Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway[END_REF][START_REF] Hull | Anti-tumour necrosis factor treatment increases circulating T helper type 17 cells similarly in different types of inflammatory arthritis[END_REF] . Interestingly, these non-responding patients showed a TH1-mediated and TH17-mediated immune response against the TNF inhibitor, which might have precipitated their lack of clinical response [START_REF] Talotta | Paradoxical Expansion of Th1 and Th17 Lymphocytes in Rheumatoid Arthritis Following Infliximab Treatment: a Possible Explanation for a Lack of Clinical Response[END_REF] . Finally, in the late 2000s, regulatory properties of TNF were proposed to result from its effects on Treg cells. These mechanisms are extensively discussed below.

[H2] Cellular sources of TNF Multiple cell types are able to produce TNF, but the immune cells that produce this cytokine in the highest amounts are myeloid cells and activated T cells [START_REF] Kruglov | Pathogenic and protective functions of TNF in neuroinflammation are defined by its expression in T lymphocytes and myeloid cells[END_REF] . The role of TNF produced by these two cell types in rheumatic and autoimmune diseases has been investigated using genetically modified mice with conditional knock-out of TNF only in myeloid cells or only in T cells. In mice with collagen-induced arthritis, conditional knockout of TNF in myeloid cells leads to reduced disease severity, showing that the TNF produced by these cells contributes to the pathology. By contrast, mice with TNF-deficient T cells have exacerbated arthritis, suggesting a protective role of the TNF produced by T cells [START_REF] Kruglov | Contrasting contributions of TNF from distinct cellular sources in arthritis[END_REF] . Similarly, mice with EAE and TNF-deficient myeloid cells have attenuated disease, which is either delayed in onset or reduced in severity depending on the model [START_REF] Kruglov | Pathogenic and protective functions of TNF in neuroinflammation are defined by its expression in T lymphocytes and myeloid cells[END_REF][START_REF] Wolf | Autonomous TNF is critical for in vivo monocyte survival in steady state and inflammation[END_REF] . Finally, the role of TNF produced by B cells has been analyzed in mice with TNF-deficient B cells. These mice have reduced arthritis and reduced levels of autoantibodies [START_REF] Kruglov | Contrasting contributions of TNF from distinct cellular sources in arthritis[END_REF] .

[H2] TNF structure and signalling

[H3] Soluble and transmembrane TNF.

Crystallographic studies show that trimers of TNF interact with trimers of either TNFR1 or TNFR2 [START_REF] Mukai | Solution of the structure of the TNF-TNFR2 complex[END_REF][START_REF] Chan | The pre-ligand binding assembly domain: a potential target of inhibition of tumour necrosis factor receptor function[END_REF] . This trimeric association of the cytokine with its receptor is characteristic of the TNF superfamily and is critical for downstream signalling [START_REF] Naude | Tumor necrosis factor receptor cross-talk[END_REF][START_REF] Croft | Beyond TNF: TNF superfamily cytokines as targets for the treatment of rheumatic diseases[END_REF] .

TNF is initially produced as a transmembrane molecule that can be processed by disintegrin and metalloproteinase domain-containing protein 17 (also known as TNF converting enzyme (TACE)) encoded by the ADAM17 gene [START_REF] Van Hauwermeiren | Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1[END_REF] . Thus, activated myeloid and T cells produce transmembrane TNF and secreted soluble TNF, which are both biologically active [START_REF] Kruglov | Contrasting contributions of TNF from distinct cellular sources in arthritis[END_REF][START_REF] Alexopoulou | Transmembrane TNF protects mutant mice against intracellular bacterial infections, chronic inflammation and autoimmunity[END_REF][START_REF] Nguyen | Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis[END_REF] . The role of soluble TNF in the pathophysiology of rheumatic and autoimmune diseases has been assessed in mice engineered to express a TNF protein that cannot be cleaved by TACE. Thus, these mice produce normal levels of transmembrane TNF but no soluble TNF [START_REF] Alexopoulou | Transmembrane TNF protects mutant mice against intracellular bacterial infections, chronic inflammation and autoimmunity[END_REF] . Importantly, such mice do not develop EAE or arthritis, showing that soluble TNF but not transmembrane TNF contributes to these diseases [START_REF] Kruglov | Contrasting contributions of TNF from distinct cellular sources in arthritis[END_REF][START_REF] Alexopoulou | Transmembrane TNF protects mutant mice against intracellular bacterial infections, chronic inflammation and autoimmunity[END_REF][START_REF] Nguyen | Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis[END_REF] . By contrast, mice with global deletion of TNF (full knock-out) still develop EAE, which suggests that transmembrane TNF has protective effects in the disease [START_REF] Alexopoulou | Transmembrane TNF protects mutant mice against intracellular bacterial infections, chronic inflammation and autoimmunity[END_REF] .

Despite a similarly high binding affinity for its two receptors, trimeric soluble TNF triggers TNFR1 signalling much more efficiently than it does TNFR2 signalling [START_REF] Grell | The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor[END_REF] . Although this observation requires further confirmation, TNFR1 signalling is usually considered to be triggered by both soluble and transmembrane TNF whereas TNFR2 signalling is preferentially triggered by transmembrane TNF [START_REF] Medler | Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target[END_REF] . These observations suggest that soluble TNF (notably that produced by myeloid cells at the onset of a rheumatic or autoimmune disease) binds to TNFR1 to promote inflammation and precipitate the disease, whereas transmembrane TNF (probably that expressed by both myeloid and T cells) has regulatory effects mostly derived from triggering TNFR2. These observations might have implications for the use of TNF inhibitors. For example, etanercept (a TNFR2-Fc fusion protein) can efficiently block soluble TNF (as well as α3 and α2β1 lymphotoxins) but not transmembrane TNF, whereas anti-TNF monoclonal antibodies block both soluble and transmembrane TNF [START_REF] Van Hauwermeiren | Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1[END_REF] . This concept also has implications for the design of next-generation TNF inhibitors, as discussed below.

[H3] TNFR1 and TNFR2 signalling pathways.

TNFR1 and TNFR2 signalling pathways are complex and have been extensively reviewed elsewhere [START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF][START_REF] Atretkhany | Distinct modes of TNF signaling through its two receptors in health and disease[END_REF][START_REF] Naude | Tumor necrosis factor receptor cross-talk[END_REF][START_REF] Medler | Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target[END_REF] . Accordingly, only the pathways most relevant to this review are outlined here. Most of the available knowledge has been obtained in cell lines and non-immune cells and deserves further investigation to confirm its relevance in immune cells.

Upon binding of trimeric TNF to TNFR1, the cytoplasmic tail of the receptor recruits the adaptor protein TNFR1-associated death domain (TRADD) via its death domain. TRADD can then interact with other adaptor proteins, such as TNF receptor associated factor 2 (TRAF2), and kinases, such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1) or cellular inhibitor of apoptosis (cIAP) 1 and cIAP2. The resulting molecular complex, named complex 1, is able to phosphorylate and ubiquitinate several other molecules, ultimately leading to potent activation of canonical NF-KB and MAPK pathways. Members of these pathways, such as c-Jun N-terminal kinase (JNK) and p38, in turn, activate AP1 complex [START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF][START_REF] Atretkhany | Distinct modes of TNF signaling through its two receptors in health and disease[END_REF][START_REF] Naude | Tumor necrosis factor receptor cross-talk[END_REF][START_REF] Medler | Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target[END_REF] . This complex-1dependent signalling pathway favours cell proliferation and survival. Alternatively, TNFR1 and TRADD interact with the Fas associated death domain (FADD) adaptors RIPK1 and RIPK3, forming the complex 2 interactome, which is able to induce cell death: either apoptosis (via caspase 8 activation) or necroptosis (via mixed lineage kinase domain-like (MLKL) protein activation) [START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF][START_REF] Atretkhany | Distinct modes of TNF signaling through its two receptors in health and disease[END_REF][START_REF] Naude | Tumor necrosis factor receptor cross-talk[END_REF][START_REF] Medler | Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target[END_REF] .

Complex 1 and complex 2 are downstream effectors of TNFR1 signalling. Complex 1 is probably involved in most of the effects of TNF on dendritic cells and macrophages, including activation of inflammatory target genes and production of inflammatory cytokines. Complex 2 is involved in TNF-dependent, activation-induced cell death and the formation of inflammationdependent DAMPs.

The signal transduction pathway downstream of TNFR2 lacks a death domain and involves different adaptors. Binding of transmembrane TNF to TNFR2 recruits TRAF1 or TRAF2 adaptors to this receptor, leading to activation of cIAP1 or cIAP2 kinases and activation of canonical and non-canonical NF-KB, JNK and AKT pathways that promote cell proliferation and survival [START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF][START_REF] Atretkhany | Distinct modes of TNF signaling through its two receptors in health and disease[END_REF][START_REF] Naude | Tumor necrosis factor receptor cross-talk[END_REF][START_REF] Medler | Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target[END_REF][START_REF] So | Regulation of PI-3-Kinase and Akt Signaling in T Lymphocytes and Other Cells by TNFR Family Molecules[END_REF][START_REF] Wajant | Targeting Regulatory T Cells by Addressing Tumor Necrosis Factor and Its Receptors in Allogeneic Hematopoietic Cell Transplantation and Cancer[END_REF] . These pathways are likely to be involved in the TNF-dependent activation of mesenchymal stem cells and myeloid-derived stem cells as well as T cell costimulation. TRAF2 recruitment to TNFR2 also decreases the amount of cytoplasmic TRAF2, which interferes with TNFR1 signalling by favouring the formation of (cell death-promoting) complex 2 to the detriment of (survival-promoting) complex 1 [START_REF] Naude | Tumor necrosis factor receptor cross-talk[END_REF] . This cross-talk between TNFR1 and TNFR2 signalling pathways seems to be responsible for TNFR2-dependent T cell death [START_REF] Twu | TNFR1 delivers pro-survival signals that are required for limiting TNFR2-dependent activation-induced cell death (AICD) in CD8+ T cells[END_REF] .

[H3] TNF reverse signalling.

Reverse (extracellular to intracellular) signalling induced by transmembrane TNF has been described but remains poorly documented. This phenomenon is only outlined here as it has been reviewed elsewhere [START_REF] Horiuchi | Transmembrane TNF-a: structure, function and interaction with anti-TNF agents[END_REF][START_REF] Lee | Reverse Signaling of Tumor Necrosis Factor Superfamily Proteins in Macrophages and Microglia: Superfamily Portrait in the Neuroimmune Interface[END_REF][START_REF] Qu | Forward and Reverse Signaling Mediated by Transmembrane Tumor Necrosis Factor-Alpha and TNF Receptor 2: Potential Roles in an immunosuppressive Tumor Microenvironment[END_REF] . In this context, TNFR1 or TNFR2 can act as ligands for transmembrane TNF, which can function as a cell receptor transducing a signal in several different situations.

For example, TNFR2-expressing T cells promote the increased expression of TNF in monocytes and/or macrophages via transmembrane TNF, a phenomenon that has been observed in the joints of patients with RA. Also, TNFR1-expressing endothelial cells induce cross-tolerance in monocytes and/or macrophages via transmembrane TNF. Finally, TNF inhibitors are also able to bind to transmembrane TNF and thereby induce the apoptosis of transmembrane TNF-expressing cells; this phenomenon has been observed for instance in T cells and synovial macrophages from patients with RA [START_REF] Catrina | Evidence that anti-tumor necrosis factor therapy with both etanercept and infliximab induces apoptosis in macrophages, but not lymphocytes, in rheumatoid arthritis joints: extended report[END_REF][START_REF] Mitoma | Mechanisms for cytotocxic effects of anti-tumor necrosis factors agents on transmembrane tumor necrosis factor a-expressing cells[END_REF] . The mechanism of TNF reverse signalling involves increased intracellular levels of calcium and TGF-β and activation of the MAPK-ERK pathway. However, the in vivo relevance of reverse signalling is difficult to assess because this phenomenon has been poorly described. I consider that reverse signalling might contribute to the spectrum of effects of TNF and might have an important role in inducing cross-tolerance of macrophages and in the death of transmembrane TNF-expressing cells induced by administration of TNF inhibitors.

[H2] Integrative view of TNF functions

Here, I present a simplified and integrated view of what I believe is the major role of TNF in immunity (Figure 1). TNF is one of the most potent inflammatory cytokines owing to its capacity to activate endothelial cells, neutrophils, macrophages and dendritic cells, leading to leukocyte recruitment and massive release of inflammatory cytokines at sites of inflammation.

Most of these pro-inflammatory phenomena are mediated by TNFR1 signalling. Besides its proinflammatory functions, TNF also has anti-inflammatory (regulatory) functions, although their role and mechanisms in immunity are yet to be clarified. The regulatory functions involve TNFdependent activation of suppressive cells, such as mesenchymal stem cells, myeloid-derived stem cells and of course Treg cells (which are extensively discussed below). TNF might also promote death or exhaustion of T cells and inhibit pathogenic TH17 cells. Most of these regulatory phenomena are mediated by TNFR2 signalling.

The end result of TNF blockade depends on the type of autoimmune disease present and the timing of treatment. Blocking the interaction between TNF and TNFR1 led to increased numbers of pathogenic TH1 and TH17 cells in mouse models of arthritis and psoriasis [START_REF] Alzabin | Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway[END_REF][START_REF] Mayordomo | IL-12/23p40 overproduction by dendritic cells leads to an increased Th1 and Th17 polarization in a model of Yersinia enterocolitica-induced reactive arthritis in TNFRp55-/-mice[END_REF][START_REF] Notley | Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells[END_REF][START_REF] Elicabe | Lack of TNFR p55 results in heightened expression of IFN-gamma and IL-17 during the development of reactive arthritis[END_REF][START_REF] Ma | Tumor necrosis factor alpha blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells[END_REF] .

This increase was associated with exacerbation of psoriasis (as might logically be expected) but surprisingly with attenuation of arthritis because this treatment also blocked the migration of pathogenic T cells to the joints [START_REF] Notley | Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells[END_REF] . Similarly, patients with RA treated with TNF inhibitors have increased levels of circulating TH1 and TH17 cells [START_REF] Alzabin | Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway[END_REF][START_REF] Hull | Anti-tumour necrosis factor treatment increases circulating T helper type 17 cells similarly in different types of inflammatory arthritis[END_REF][START_REF] Talotta | Paradoxical Expansion of Th1 and Th17 Lymphocytes in Rheumatoid Arthritis Following Infliximab Treatment: a Possible Explanation for a Lack of Clinical Response[END_REF] , which could explain some of the paradoxical inflammation observed in a subset of these patients. The effects of TNF blockade could also depend on the timing of treatment in relation to the course of disease. To investigate the role of TNF signalling via TNFR2 in a model of collagen-induced arthritis, TNFR1-knockout mice were treated with TNF on either days 2-20 or days 22-40 after disease induction [START_REF] Tada | Collagen-induced arthritis in TNF receptor-1-deficient mice: TNF receptor-2 can modulate arthritis in the absence of TNF receptor-1[END_REF] .

Interestingly, early TNF treatment led to disease exacerbation whereas late TNF treatment led to attenuation of arthritis. An opposite effect of TNF that is similarly dependent on the stage of disease progression has been documented in non-obese diabetic mice; TNF seems to exacerbate diabetes in young mice by activating dendritic cells and to attenuate it in adult mice by inhibiting conventional T cells and promoting Treg cell activation [START_REF] Cope | Chronic tumor necrosis factor alters T cell responses by attenuating T cell receptor signaling[END_REF][START_REF] Lee | The role of TNF-alpha in the pathogenesis of type 1 diabetes in the nonobese diabetic mouse: analysis of dendritic cell maturation[END_REF][START_REF] Mcdevitt | Multiple roles for tumor necrosis factor-alpha and lymphotoxin alpha/beta in immunity and autoimmunity[END_REF][START_REF] Green | The temporal importance of TNFalpha expression in the development of diabetes[END_REF] .

[H1] Effects of TNF on Treg cells

Treg cells are master regulators of autoimmune diseases. Mice and humans that are genetically deficient in Treg cells die soon after birth from a massive and systemic autoimmune syndrome, which reveals the critical role of these cells in the suppression of autoimmunity [START_REF] Sakaguchi | Regulatory T cells and immune tolerance[END_REF] . Functional or quantitative defects of Treg cells have been reported in many human autoimmune diseases [START_REF] Buckner | Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+) regulatory T cells in human autoimmune diseases[END_REF] .

Other indirect evidence supports the concept that Treg cells contribute to human autoimmune diseases. For instance, some biomarkers of disease activity, such as C reactive protein levels, are inversely correlated with Treg cell proportion in patients with RA [START_REF] Ehrenstein | Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy[END_REF] or IBD [START_REF] Dige | Adalimumab treatment in Crohn's disease does not induce early changes in regulatory T cells[END_REF]100 . Moreover, Treg cell transfer seems to have beneficial effects in patients with various autoimmune diseases 101 .

Transcriptomic analyses that compared Treg with conventional T cells in lymphoid tissues

showed that several members of the TNFR superfamily, including TNFR2, TNFR superfamily member 4 (OX40), TNFR superfamily member 9 (41BB) and TNFR superfamily member 18 (GITR) are included in the Treg cell signature 102 . These molecules are further upregulated upon stimulation of either the T cell receptor (TCR) or T cell-specific surface glycoprotein CD28 and are therefore preferentially expressed by effector Treg cells rather than resting Treg cells 103,104 . At steady state, 30% of Treg cells express TNFR2 and most of this subset are effector Treg cells that have a stronger suppressive function in vitro than do TNFR2 -resting Treg cells 105,106 . Thus, TNFR2 belongs to the Treg cell signature and is a marker of highly suppressive Treg cells.

[H2] Effects on Treg cell expansion

Expansion is defined as an increase in cell numbers, and results from a combination of increased proliferation, prolonged survival and phenotypic stability. TNFR2 signalling seems to expand Treg cells by increasing all three of these factors.

Initially, TNF and/or TNFR2 co-stimulation were shown to increase Treg cell proliferation in mice [START_REF] Chen | Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells[END_REF]107 . Our group and others showed that effector T cells, in particular TH17 cells, are a major source of the TNF that induces this increase in the Treg cell population in vivo [108][109][110] . Similar findings were obtained for human Treg cells [START_REF] Nguyen | Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis[END_REF]111,112 . TNF can also substantially prolong Treg cell survival 103 . Indirect evidence indicates that TNFR2 signalling also maintains FOXP3 expression, which increases Treg cell phenotypic stability and therefore their long-term expansion [112][113][114][115] .

In many of these in vitro studies, soluble TNF was capable of boosting Treg cell expansion. Although transmembrane TNF has a stronger effect than soluble TNF on induction of TNFR2 signalling [START_REF] Grell | The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor[END_REF] , strong evidence indicates that soluble TNF can indeed stimulate the expansion of Treg cells by binding to TNFR2. Furthermore, TNFR1 expression has not been detected on Treg cells (unlike TNFR2 expression) [START_REF] Chen | Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells[END_REF] . The expansion-promoting effect of soluble TNF on Treg cells was lost in TNFR2-deficient Treg cells and when TNFR2, but not TNFR1, was blocked 113 . Finally, treatment with TNF or TNFR2 agonists induced similar co-stimulation of Treg cells 111 . The capacity of soluble TNF to efficiently induce TNFR2 signalling could be explained by the use of high concentrations of this cytokine or the presence of TNF aggregates with crosslinking properties in the preparations. TNFR2 agonists, which are either multimers of mutated TNF or mAbs that bind only to TNFR2 (discussed in more detail below), strongly costimulate Treg cells in both mice and humans [START_REF] De Kivit | Stable human regulatory T cells switch to glycolysis following TNF receptor 2 costimulation[END_REF]103,111,[116][117][118] . In a study of pre-activated T cells, TNFR2 co-stimulation strongly increased the proliferation of Treg but had no effect on conventional T cells [START_REF] De Kivit | Stable human regulatory T cells switch to glycolysis following TNF receptor 2 costimulation[END_REF] . The capacity of TNFR2 co-stimulation to promote Treg cell expansion was confirmed in vivo in animals treated with TNFR2 agonists [START_REF] Medler | Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target[END_REF]117,[119][120][121] .

Although very little is known about TNFR2 signal transduction in Treg cells, transcriptomic analyses showed that binding of TNF to TNFR2 on purified mouse or human Treg cells induced a gene expression signature indicative of NF-κB pathway activation 103,122 . More precisely, TNFR2 signalling induced nuclear translocation and binding of RelA to its DNA target sequence, which suggests that the canonical NF-κB pathway is activated by TNFR2 signalling in Treg cells. Importantly, the increased proliferation and prolonged survival of Treg cells induced by TNFR2 triggering was severely attenuated in RelA-deficient Treg cells 103,104 . Some evidence also suggests that the non-canonical NF-κB pathway is also activated by TNFR2 signalling in Treg cells but this observation has to be treated with caution because these assays were conducted on a cell population with low Treg cell purity 123 . Other data suggest that TNFR2 signalling induces activation of the MAPK pathway, notably via p38 124,125 . TNFR2-mediated costimulation of Treg cells also induced a glycolytic switch associated with activation of mammalian target of rapamycin complex 1 (mTORC1) signalling via phosphoinositide-3 kinase (PI3K), although the signalling pathway connecting TNFR2 to PI3K was not identified [START_REF] De Kivit | Stable human regulatory T cells switch to glycolysis following TNF receptor 2 costimulation[END_REF] .

Overall, strong evidence indicates that the boost in Treg cell numbers induced by TNFR2 signalling involves activation of the canonical NF-κB pathway. The role of the other signalling pathways mentioned here requires further documentation.

[H2] Effects on Treg suppressive function

The effects of TNF on the suppressive function of mouse and human Treg cells have been assessed in vitro (Table 3). The first of these studies showed no effect of low-dose (≤5 ng/ml) TNF in human cells [START_REF] Ehrenstein | Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy[END_REF] . Five subsequent reports showed that treatment with TNF, usually at a high dose (50 ng/ml), reduced the suppression of conventional T cell activation by human Treg cells 122,[126][127][128][129] . By contrast, in vitro studies performed in mouse cells showed that the presence of high amounts of TNF either had no effect or even increased Treg cell-mediated suppression of conventional T cell activation [START_REF] Chen | Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells[END_REF]107 . Moreover, other evidence also suggests that TNF does not inhibit Treg cell-mediated suppression of conventional T cells, and might even increase it. For instance, administration of a TNFR2 agonist to mice with graft versus host disease (GvHD) or collagen-induced arthritis promoted Treg cell expansion and had a therapeutic effect 117,119,121 . Also, treatment of cultured Treg cells with TNF increased their capacity to suppress colitis or GvHD after transfer 103,130 , whereas TNFR2-deficient Treg cells had a reduced capacity to suppress colitis or GvHD 114,131 . However, these observations provide only indirect evidence that TNF either had no effect on or increased Treg cell suppressive function in mice. Indeed, this cytokine might influence other parameters of Treg cell biology (such as proliferation, survival, functional stability or migration). Interestingly, EAE was exacerbated in genetically modified mice in which TNFR2 was ablated only in Treg cells. Ablation of TNFR2 in Treg cells seems to decrease their suppressive function specifically in the inflamed central nervous system 132 . In this context, the expression of TNFR2 by Treg cells might be essential for their suppressive function and their capacity to control EAE.

Our group also performed an analysis of the suppressive capacity of Treg cells from numerous different human donors under three different T cell activation conditions. We consistently found that TNF (added either before or during the suppression assay) either had no effect on or even slightly increased the suppressive activity of human Treg cells 133 . The preservation of Treg cell suppressive activity after TNFR2 co-stimulation (achieved using a TNFR2 agonist) in humans has also been confirmed [START_REF] De Kivit | Stable human regulatory T cells switch to glycolysis following TNF receptor 2 costimulation[END_REF] .

Several factors might account for the contrasting findings in mouse and human cells.

First, as none of the available markers can exclusively characterize the population of human Treg cells, the purified Treg cell populations used in some of these studies might still have some level of contamination by activated conventional T cells, especially when only CD4 and CD25 expression was used to sort the cells 134 . Second, given the high inter-individual variability in Treg cell phenotypes, responses to TNF and suppressive activity, it is important to collect data from a sufficiently large sample of individuals. Finally, a Treg cell functional defect identified in a suppression assay could be due either to intrinsic Treg cell dysfunction or to the presence of contaminating conventional T cells that are resistant to Treg cell suppression. This last point is critical with regard to the effects of TNF. Indeed, in addition to its proliferation-promoting effect on Treg cells, TNF not only increases the proliferation of conventional T cells [START_REF] Tartaglia | Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor receptor[END_REF][START_REF] Kim | TNF receptor type 2 (p75) functions as a costimulator for antigen-driven T cell responses in vivo[END_REF]133 but also increases their resistance to Treg cell-mediated suppression 135 . In several studies performed in human cells, TNF was present during the suppression assays and might act on any contaminating conventional T cells, which would impair the evaluation of Treg cell suppressive function (Table 3). Accordingly, pre-incubation of the Treg cells with TNF is appropriate before testing their capacity to suppress conventional T cells. For this reason, to accurately determine whether TNF alters the suppressive function of Treg cells, we recommend that TNF is added only during the pre-incubation phase (that is, before the suppressive assay), and that activation of only the conventional T cells is measured by FACS.

The absence of these two precautionary measures in some of the reports claiming that TNF inhibits Treg cell suppressive activity in humans undermines their conclusions (Table 3).

To conclude, weak evidence indicates that TNF is able to either inhibit or increase the suppressive activity of Treg cells. After careful analyses of the data from in vitro assays, I would say that TNF has no or only a minor effect on Treg cell suppressive function in this context. TNF also synergizes with IL-2 to increase the expression of CD25 (the IL-2 receptor α-chain) and FOXP3 [START_REF] Chen | Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells[END_REF]133 . Moreover, TNF increases the IL-2-induced phosphorylation of STAT5 [START_REF] Chen | Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells[END_REF] However, whereas TNF alone has no effect on thymic Treg cell differentiation, experiments in mice show that TNF inhibits the differentiation of induced Treg cells, whereas treatment with TNF inhibitors increased the differentiation of induced Treg cells 143,144 . This inhibitory effect of TNF was also observed on peripheral Treg cells in vivo. In mice with EAE, injection of anti-TNF or anti-TNFR2 mAbs at the time of disease induction led to reduced disease severity, which was associated with an increased proportion of Treg cells and evidence of increased peripheral Treg cell differentiation 144 . Two other papers do not support this observation and even suggest that the TNF-TNFR2 axis promotes the differentiation of induced Treg and peripheral Treg cells [START_REF] Yang | Differential roles of TNFalpha-TNFR1 and TNFalpha-TNFR2 in the differentiation and function of CD4(+)Foxp3(+) induced Treg cells in vitro and in vivo periphery in autoimmune diseases[END_REF][START_REF] Ma | Tumor necrosis factor alpha blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells[END_REF] . However, the design of these two studies meant that contaminating natural Treg cells were present in the starting inoculum, and thus treatment with TNF might boost the expansion of these contaminating cells rather than increase the differentiation of induced Treg cells [START_REF] Yang | Differential roles of TNFalpha-TNFR1 and TNFalpha-TNFR2 in the differentiation and function of CD4(+)Foxp3(+) induced Treg cells in vitro and in vivo periphery in autoimmune diseases[END_REF][START_REF] Ma | Tumor necrosis factor alpha blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells[END_REF] . TNF does not seem to affect thymic Treg cell differentiation at steady state, because mice lacking TNFR2 have normal thymic Treg cell numbers. However, ablation or neutralization of TNFR2 combined with ablation or neutralization of two other members of the TNFR superfamily, namely OX40 and GITR, led to reduced differentiation of thymic Treg cells 145 .

Overall, whereas the effect of TNF on Treg cell differentiation is still open to discussion, an excess of TNF seems to impair the differentiation of induced Treg cells and peripheral Treg cells in mice.

In humans, the inhibition of Treg cell differentiation by TNF was first observed in patients with RA. TNF inhibitor treatment increased the in vitro differentiation of induced Treg cells derived from patients with RA but not those from healthy controls 146 . This observation explained why blood samples from patients with RA treated with infliximab had an increased proportion of Treg cells, which might result from increased differentiation of peripheral Treg cells [START_REF] Ehrenstein | Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy[END_REF]146 . Other members of the TNF family, such as 41BB, OX40 or TNFR superfamily member 25 (also known as death receptor 3 (DR3)), can also inhibit the differentiation of induced Treg cells [147][148][149] .

These observations suggest that a shared mechanism is involved, perhaps implicating NF-KB, PI3K or MAPK pathways. IFNγ produced by T cells following TNFR co-stimulation has also been proposed to inhibit the differentiation of induced Treg cells. Alternatively, the increased activation of the PI3K-AKT pathway resulting from TNFR signalling could lead to reduced activation of phosphorylated SMAD3, which transactivates Foxp3 expression in mouse induced Treg cells 144 .

[H2] Overall effects of TNF on Treg cells

In summary, TNF has multiple negative and positive effects on Treg cell biology, most probably resulting from TNFR2 rather than TNFR1 signalling (Figure 2). The best-characterized of the positive effects of TNF are increased Treg cell proliferation and expansion. TNF also seems to promote Treg cell survival in vitro, although the relevance of this effect in vivo is difficult to evaluate. The TNF-dependent increases in Treg cell proliferation and survival are at least partially dependent on RelA and activation of the canonical NF-KB pathway. Involvement of p38 and PI3K-AKT pathway activation has also been suggested but requires further investigation.

Finally, weak evidence indicates that TNF increases the stability and suppressive function of Treg cells, a phenomenon that might be partially due to TNF signalling synergizing with IL-2 signalling and with phosphorylation of STAT5. Other reports suggest a negative effect of TNF on Treg cell biology in vitro. Whether this cytokine truly has a negative effect on Treg cell function is questionable. By contrast, TNF seems to increase Treg cell suppressive function in vivo, at least in some inflammatory contexts. However, the evidence of an inhibitory effect of TNF on differentiation of induced Treg cells is fairly solid and might involve the PI3K-AKT pathway (Figure 2).

[H1] Treg cells in RA

As Treg cells have an important role in the suppression of autoimmunity, numerous studies have attempted to identify whether these cells have a quantitative or functional defect in patients with autoimmune diseases. Major drawbacks of these studies include the use of sample sizes too small to account for interindividual variability and the absence of a specific marker for human Treg cells, which has led to the utilization of different combinations of markers. As a result, the literature is full of conflicting data.

I present here the main findings on Treg cell proportion and function in patients with RA. This disease is particularly interesting as Treg cells can be obtained from both the blood and joints (the target tissue of the disease), which are easily accessible for analysis. Data obtained in other autoimmune diseases are also included where relevant.

[H2] Treg cell proportion

Contrasting findings have been reported in studies of the proportion of Treg cells in the blood of patients with RA receiving conventional immunosuppressive treatment (the effects of TNF inhibitors are discussed below). Among studies that compared patients with RA with healthy control individuals, four described a decreased Treg cell proportion [START_REF] Talotta | Paradoxical Expansion of Th1 and Th17 Lymphocytes in Rheumatoid Arthritis Following Infliximab Treatment: a Possible Explanation for a Lack of Clinical Response[END_REF][150][151][152] , five found no difference [START_REF] Ehrenstein | Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy[END_REF]126,146,153,154 and one found an increased Treg cell proportion in the patients with RA 155 .

Most studies that have analyzed both blood and synovial fluid of patients with RA concluded that the proportion of Treg cells was higher in synovial fluid than in blood, and remained stable over time in individual patients 152,[155][156][157] . The Treg cells isolated from synovial fluid seem to be bona fide Treg cells because they exhibit FOXP3 promoter demethylation. Also, the phenotype of these Treg cells indicates that they have an activated status 157 . The synovial fluid of patients with RA contains high amounts of IL-6, TNF and IFNγ, low levels of IL-17A, IL-10 or IL-13 and does not contain IL-1 126,157 . Which of these factors is responsible for the increased proportion and activation of synovial Treg cells remains unclear. However, IL-6 is not likely to be involved, because this cytokine (which is produced by joint fibroblasts) induces transdifferentiation of Treg cells into highly pathogenic TH17 cells in a mouse model of autoimmune arthritis, a phenomenon that might also take place in patients with RA 158 . IL-6 also induced proteasomal degradation of FOXP3 and loss of the suppressive activity of Treg cells 159,160 . We do not know much about the effect of IFNγ on Treg cells. Therefore, the activation and/or expansion of Treg cells in the synovial fluid of patients with RA is likely to be caused by high local levels of TNF.

[H2] Treg cell function

Compared with Treg cells obtained from the blood of healthy control individuals, Treg cells isolated from the blood of patients with RA were shown to have similar suppressive activity in one study 155 and decreased suppressive activity in another 128 . In a third study, the capacity of these cells to suppress conventional T cell proliferation was maintained but their cytokine production was reduced [START_REF] Ehrenstein | Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy[END_REF] . Contrasting findings have also been reported for the suppressive activity of Treg cells isolated from the synovial fluid of patients with RA. Several studies showed that synovial fluid Treg cells from patients with RA were as active, or were more active, than blood Treg cells from either patients with RA or healthy control individuals in terms of suppression of proliferation or IFNγ production 152,[155][156][157] . Another publication reported that synovial fluid Treg cells from patients with RA had decreased suppressive activity 126 .

Importantly, these studies noted considerable variation between patients, with Treg cells from some individuals but not others showing a high level of suppression 157 . This observation might explain the contrasting results and further emphasizes the importance of generating data from at least 7-10 different patients, which was not the case for most of these studies.

Firm conclusions are difficult to draw because the available evidence does not provide a clear picture of whether Treg cells in the blood of patients with RA have similar proportions and functions to those of healthy control individuals. The situation is a little bit clearer for synovial fluid Treg cells, which seem to be present at an increased proportion in patients with RA.

[H2] Effects of TNF inhibitors [H3] Treg cell proportion

The proportion of Treg cells in the blood has been analyzed in many studies of patients with RA 3-6 months (typically 3 months) after initiation of TNF inhibitor treatment. In studies of infliximab-treated patients with RA, the Treg cell proportion increased [START_REF] Talotta | Paradoxical Expansion of Th1 and Th17 Lymphocytes in Rheumatoid Arthritis Following Infliximab Treatment: a Possible Explanation for a Lack of Clinical Response[END_REF][START_REF] Ehrenstein | Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy[END_REF]115,146,151 after treatment (Table 4). In studies of patients with RA treated with either adalimumab or etanercept, the Treg cell proportion was either increased 150,154,161 or unchanged 153,154,162 (Table 4). This Treg cell increase was more often observed in responding than in non-responding patients.

Moreover, in studies of infliximab-treated patients with Crohn disease or IBD (Supplementary Table 1), the Treg cell proportion was also either unchanged [START_REF] Dige | Adalimumab treatment in Crohn's disease does not induce early changes in regulatory T cells[END_REF]163 or increased 100,115,[163][164][165][166][167][168] . Some of the studies in patients with IBD or Crohn disease also analyzed the kinetics of this treatment-related increase in Treg cell proportion. In a study of patients with Crohn disease, the increase was transient and only occurred after the first injection 165 . In two studies of patients with IBD, the increase occurred 2 weeks after the first injection and was maintained for ≥22 weeks 100,166 , whereas in another study in patients with Crohn disease no increase was detected after 1 week but an increase was detected at week 24 in patients who had low Treg cell proportions before treatment [START_REF] Dige | Adalimumab treatment in Crohn's disease does not induce early changes in regulatory T cells[END_REF] (Supplementary Table 1).

Two studies in patients with uveitis 169,170 and one in patients with ankylosing spondylitis 171 showed an increase in the Treg cell proportion after TNF inhibitor therapy.

However, one study in patients with juvenile idiopathic arthritis observed no difference 172 and one in patients with sarcoidosis observed a decrease in the Treg cell proportion 173 following TNF inhibitor therapy (Table 4).

Some general conclusions can be drawn from these data. Most publications described an increase in the proportion of Treg cells in blood after TNF inhibitor therapy. Discrepancies between some studies could be due to the following factors: first, infliximab seems to induce an increase in the Treg cell proportion more consistently than either adalimumab or etanercept.

Second, a Treg cell increase seems to be more consistent among patients who responded to TNF inhibitor treatment. The type of concomitant medications might also matter. For instance, although methotrexate monotherapy induces an increase in Treg cell proportion 150 , combination therapy with methotrexate and a TNF inhibitor provided an optimal increase in Treg cells in vitro 174 . Also, steroid treatment might increase Treg cell proportion and function 175,176 . Finally, as discussed above, technical factors related to the way that Treg cells were purified might influence the conclusions of these studies. Some activated conventional T cells (which also express CD25) are likely to contaminate the population identified as Treg cells. Thus, the findings of these studies have to be considered carefully because the level of conventional T cell contamination could differ between healthy control individuals and patients with rheumatic or autoimmune disease, or before and after TNF inhibitor treatment. Use of the CD45RA (naive T cell) or CD45RO (memory T cell) markers, in addition to CD25 or CD127, would help to limit the risk of such contamination 134 . Several mechanisms by which Treg cells might increase after TNF inhibitor treatment are supported by experimental evidence. First, Treg cells might increase because treatment with TNF blockers such as infliximab favour the differentiation of peripheral Treg cells 146 . Second, Treg cells might increase because treatment with anti-TNF mAbs such as adalimumab augments the expression of transmembrane TNF on monocytes, which then triggers Treg cell expansion via TNFR2 signalling. Thus, anti-TNF mAbs that are intended to inhibit TNF might paradoxically increase its activity [START_REF] Nguyen | Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis[END_REF] . The preferential expansion of activated Treg cells rather than resting Treg cells in patients receiving anti-TNF mAbs supports this hypothesis 100 . In patients with RA 151 or IBD 168 , Treg cells that are more sensitive to spontaneous apoptosis than are those of healthy control individuals, might be present at an increased proportion in patients treated with TNF inhibitors because they are protected from cell death by this therapy. In patients with IBD, TNF inhibitor therapy blocks Treg cell migration to inflamed tissues, which results in increased Treg cell levels in blood and decreased levels in the intestinal mucosa 166 . Finally, TNF inhibitor therapy leads to a decrease in inflammatory cytokine levels and pathogenic T cells while sparing Treg cells in patients with Crohn disease 161 or ankylosing spondylitis 165,171 . Therefore, this treatment might target conventional T cells in preference to Treg cells, thereby explaining the relative increase in the Treg cell proportion within the population of CD4 + T cells. As the increased proportion of blood Treg cells following TNF inhibitor treatment is an in vivo phenomenon that occurs over a long time period, determining which of the above-described mechanisms is most relevant remains a challenge.

[H3] Suppressive function.

Treatment with mAb TNF inhibitors affects not only the Treg cell proportion but also their suppressive function. Early work showed that Treg cells from patients with RA obtained before the initiation of TNF inhibitor therapy had a poor capacity to suppress cytokine production by conventional T cells, and that the suppressive activity of these Treg cells was restored following anti-TNF treatment [START_REF] Ehrenstein | Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNFalpha therapy[END_REF] . These functional Treg cells resulted from either the generation of new peripheral Treg cells following infliximab treatment 146 or from the expansion of differentiated Treg cells following adalimumab treatment [START_REF] Nguyen | Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis[END_REF] . These restored Treg cells were even able to suppress pathogenic TH17 cells, unlike the Treg cells of healthy control individuals 154 . Dysfunction of Treg cells obtained from the blood or synovial fluid of patients with RA and restoration of their suppressive function after TNF inhibitor treatment (infliximab) were also confirmed in two other studies 126,128 . Restoration of functional blood Treg cells after TNF inhibitor treatment has also been described in patients with IBD 164 . TNF inhibitors based on mAbs seem to act, at least in part, by restoring the functional Treg cell compartment. By contrast, etanercept is likely to act by suppressing conventional T cells and/or rendering them sensitive to the suppressive effects of Treg cells 172,177 . In another study, Treg cells obtained from patients with Crohn disease were shown to be functional even before initiation of infliximab treatment 178 . However, the Treg cell purification strategy used in this paper meant that activated conventional T cells might have contaminated the population of Treg cells, thereby resulting in an inaccurate measurement of the suppressive activity of genuine Treg cells. Therefore, the conclusions of this report have to be interpreted with caution.

In summary, the beneficial effects of TNF inhibitor therapies could be due to either restoration of fully functional Treg cells or to an increased susceptibility of conventional T cells to the suppressive effects of Treg cells.

[H3] Treg cell biomarkers of response.

The development of biomarkers to identify the 20-30% of patients with RA or IBD who will not respond to TNF inhibitor therapy is highly desirable, and some Treg cell-related biomarkers are potential candidates. In some studies, an increase in the Treg cell proportion after TNF inhibitor treatment was observed only in patients who responded to this therapy (Table 4). Thus, the Treg cell proportion before TNF inhibitor treatment has been proposed as a predictive biomarker to predict treatment response. However, patients with IBD who respond to TNF inhibitors could have Treg cell proportions before therapy that are either higher [START_REF] Dige | Adalimumab treatment in Crohn's disease does not induce early changes in regulatory T cells[END_REF]100 or lower 178 than those of nonresponding patients. Moreover, in patients with ankylosing spondylitis 171,174 or RA 171,174 , the Treg cell proportion before TNF inhibitor therapy was not predictive of treatment efficacy.

As discussed above, a possible mechanism for the observed increase of Treg cells upon mAb TNF inhibitor treatment is binding of the mAb to transmembrane TNF on myeloid cells, leading first to its increased expression and then to a boost in Treg cell numbers mediated by TNFR2 signalling [START_REF] Nguyen | Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF-TNF-RII binding in rheumatoid arthritis[END_REF] . As the expression of transmembrane TNF on monocytes can be readily assessed by flow cytometry, the capacity of adalimumab to provoke an increase in Treg cell numbers in a 3-day culture has been used to identify which patients with RA would respond to this treatment 174 .

In summary, pretreatment Treg cell proportion does not seem to be a reliable biomarker of response to anti-TNF therapies. The expression of transmembrane TNF on myeloid cells as a biomarker of treatment response deserves to be confirmed in other studies.

[H1] Next-generation drugs targeting TNF

The putative mechanisms underlying non-response and paradoxical autoimmunity to TNF inhibitor treatment could be explained by the regulatory aspect of TNF. Blocking TNF is associated with an increased risk of impairing the activity of some suppressor cells, including Treg cells, or increasing the activation of autoreactive T cells. The overall effect of these treatments is likely to depend on the specific autoimmune disease present, its stage and severity, and on genetic and environmental factors unique to each patient. At the time of treatment, if TNF has a dominant inflammatory and pathogenic role, TNF inhibitors will be beneficial. By contrast, if TNF has a dominant regulatory and protective role, TNF inhibitors will be detrimental.

Given that most of the pro-inflammatory properties of TNF are due to TNFR1 signalling induced by soluble TNF and most of the regulatory properties of are due to TNFR2 signalling induced by transmembrane TNF, the next generation of TNF inhibitors might preferentially target TNFR1 or TNFR2 [START_REF] Kalliolias | TNF biology, pathogenic mechanisms and emerging therapeutic strategies[END_REF][START_REF] Faustman | TNF receptor 2 pathway: drug target for autoimmune diseases[END_REF][START_REF] Van Hauwermeiren | Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1[END_REF][START_REF] Medler | Tumor necrosis factor receptor-2 (TNFR2): an overview of an emerging drug target[END_REF][START_REF] Wajant | Targeting Regulatory T Cells by Addressing Tumor Necrosis Factor and Its Receptors in Allogeneic Hematopoietic Cell Transplantation and Cancer[END_REF]179,180 . Two types of TNFR-specific agents have been proposed: mAbs and so-called TNF muteins, which are forms of this cytokine harbouring mutations in the receptor-interacting domains [181][182][183] .

[H2] Selective TNFR1 antagonists

The capacity of TNFR1 antagonists to block the pro-inflammatory interaction of TNF with TNFR1 has been investigated in mouse models of autoimmune diseases.

Both mAbs and TNF muteins have been developed that have potent TNFR1 antagonist activity and a strong therapeutic effect in mouse models of autoimmune diseases (Table 5). For example, the mAb DMS5540 was as effective as etanercept in the treatment of collagen-induced arthritis. In addition, the effects of DMS5540 on immune cells were superior to those of etanercept, as DMS5540 induced Treg cell activation and reduced the activation of conventional T cells, a phenomenon not observed with etanercept 184 . Several anti-TNFR1 mAbs (namely atrosab, trivalent nanobody TNFR1 silencer (TROS) and HM1097) were able to suppress EAE [185][186][187] . Finally, the muteins XPro1595 and R1antTNF had therapeutic effects in arthritis or EAE; when these agents were compared with etanercept, they sometimes demonstrated improved efficacy [188][189][190][191][192] .

[H2] TNFR2 agonists

As TNFR2 signalling stimulates the expansion of Treg cells, TNFR2 agonists such as the mAbs MR2-1 and another unnamed version are interesting candidates for improving Treg cell therapy in autoimmune diseases (Table 5). When added to human Treg cell cultures, these mAbs promote the expansion and improve the stability and purity of Treg cells over time 111,118 .

Two additional TNF muteins with human TNFR2 agonist activity (TNF07 and TNC-scTNFR2) have been generated 116,193 and TNF07 has been shown to promote Treg cell activation in vitro. In the future, mAbs or TNF muteins with TNFR2 agonist activity might be used to improve cell culture methods used to generate Treg cell preparations for use in cell therapy. This notion is supported by mouse studies showing that adding TNF or a TNFR2 agonist to Treg cell cultures increased the capacity of these cells to suppress colitis 103,130 or GvHD 103,130 after their reintroduction in vivo 103,130 .

The capacity of TNFR2 agonists to stimulate Treg cells in vivo has been tested in mouse models. Treatment with either of two TNF muteins with TNFR2 agonist activity (STAR2 and EHD2-sc-mTNFR2) 194,195 induced in vivo Treg cell activation and expansion 117,120 that was associated with prevention or amelioration of arthritis 119,121 , EAE 132 or GvHD 117 . These agents also protected the central nervous system of treated animals from inflammation and neuronal injury induced by chronic nerve constriction or drug treatment, respectively 195,196 .

Whereas classical immunosuppressive drugs aim to suppress autoimmunity by neutralizing pathogenic cells, an alternative approach is based on increasing the expansion or suppressive capacity of Treg cells. A prototype of this new class of drugs is IL-2 197 . Our group showed that administration of low-dose IL-2 boosts the proliferation of Treg cells and induces remission of type 1 diabetes mellitus in non-obese diabetic mice 198,199 . Low-dose IL-2 is now being investigated as a treatment for other autoimmune diseases in multiple clinical trials. One study has investigated this treatment in 14 different autoimmune diseases, including RA and ankylosing spondylitis (NCT01988506). TNFR2 agonists are another type of drug that are able to boost the number or function of Treg cells. No clinical trial has so far investigated the use of TNFR2 agonists to treat an autoimmune disease. However, bacillus Calmette-Guérin (BCG) vaccine can induce TNF release without secondary effects, thereby providing an indirect way to trigger TNFR2 signalling. BCG vaccine has been tested for efficacy in type 1 diabetes (NCT00607230 and NCT02081326).

[H1] Conclusions

TNF has a long and fascinating yet chaotic history. This cytokine was discovered in the mid-1970s and named for its effect as a tumour cell killer. Major milestones in its history include its cloning in the mid-1980s, the discovery that TNF binds to two receptors, that its signalling transduction is highly complex (and still remains to be fully explored), and that it has multiple effects at steady state.

TNF is now known to be one of the most important inflammatory cytokines. Although TNF is critical for beneficial immune responses, the realization that TNF is also harmful in many autoimmune diseases led to the great success of TNF inhibitors and ultimately to the flowering of research into other biological therapies. The regulatory role of this cytokine is also important to consider. Here again, the mechanisms underlying the immunosuppressive activity of TNF are complex. However, one of its main features seems to involve the expression of transmembrane TNF on myeloid or T cells, which interacts with TNFR2 on Treg cells to boost their proliferation and maybe also their stability and suppressive function.

The inflammatory and regulatory roles of TNF are both essential to take into account in the design of future generations of TNF inhibitors. Preclinical studies have shown that selective antagonists of TNFR1 inhibit the inflammatory action of TNF whereas selective agonists of TNFR2 boost Treg cell numbers and potentially also improve their function. Therefore, TNFR1 antagonists and TNFR2 agonists could be beneficial in future treatments of several diseases with an autoimmune component.. In the future, biotechnology and pharmaceutical companies are expected to work hand in hand with academic laboratories towards the successful translation of these fascinating observations into the clinic. 28,187,203-205,28,187,191,203,204, 206,207,208,132 DTHA, delayed-type hypersensitivity arthritis; EAE, experimental autoimmune encephalomyelitis; ND, not determined.

  Another critical point is the choice of parameter used to assess the activation of conventional T cells. As TNF strongly increases Treg cell proliferation (and possibly also cytokine production), measuring the activation of only the conventional T cells within the population is critical. This measurement can be done by analyzing fluorescent marker dilution or assessing intracellular cytokine production using flow cytometry techniques such as fluorescence-activated cell sorting (FACS). Researchers should not use thymidine incorporation or enzyme-linked immunosorbent assays (ELISA) to measure the proliferation or cytokine production of the whole cell population, which includes both conventional T cells and Treg cells.

  However, this cytokine seems to have an essential role in stimulation of Treg cell function in some conditions associated with inflammation. The data derived from in vitro studies of mechanisms underlying the suppressive activity of Treg cells reflect only the tip of the iceberg, as only two or three suppressive mechanisms have been analyzed in these studies to date. However, it is now well established that Treg cells in vivo are able to use a wide range of suppressive mechanisms depending on their tissue localization and the type of inflammation present 136,137 . The suppressive activity of Treg cells also involves many different effector molecules. Some have been thoroughly studied and shown to be essential for aspects of Treg cell suppression, such as cytotoxic T-lymphocyte protein 4 (CTLA4) and IL-10 138 . FOXP3 expression is also critical because its loss leads to loss of Treg cell function 138 , but no single marker has been shown to easily quantify the level of Treg cell suppression. Several mechanisms have been suggested to explain how TNF might increase the suppressive function of Treg cells in mice. TNF promotes full differentiation of effector Treg cells by stimulating NF-KB, which might increase some of these cells' suppressive functions 103,104 .

  and limits the loss of FOXP3 expression in cultured cells by preventing re-methylation of the Foxp3 promoter 113,115 . Thus, TNF might increase Treg cell suppression and stability by favouring both phosphorylation of STAT5 and FOXP3 expression, which are key determinants of these Treg cell features 139,140 . Finally, TNF limits IL-17 production by Treg cells by activating TNFAIP3 71 . Other mechanisms have been proposed to explain how TNF might decrease Treg cell function. TNF decreases FOXP3 expression by increasing the expression of deleted in breast cancer 1 (DBC1) and miR-34a, which respectively promote FOXP3 degradation and reduce FOXP3 transcription and translation 128,141,142 . Alternatively, TNF might increase the expression of serine/threonine-protein phosphatase PP1, which dephosphorylates FOXP3, thereby decreasing its effect on Treg cell suppressive function 126 . [H2] Effects on Treg cell differentiation The population of FOXP3 + Treg cells is composed of thymic Treg cells, which acquire their Treg cell state during their development in the thymus, and peripheral Treg cells, which acquire their Treg cell state during peripheral differentiation of mature naive conventional T cells. Finally, induced Treg cells can be differentiated in vitro from naive conventional T cells by TCR stimulation in the presence of IL-2 and TGF-β. Thus, induced Treg cells are the in vitro counterpart of peripheral Treg cells.

Figure 1 .

 1 Figure 1. The proinflammatory and anti-inflammatory activities of TNF are driven by effects on innate and adaptive immunity. Tumour necrosis factor (TNF) is a major proinflammatory cytokine (top panel) that activates both innate (left side) and adaptive (right side) immunity. TNF promotes recruitment of leukocytes, favours the production of other proinflammatory cytokines, activates neutrophils and participates in co-stimulation of conventional T cells. TNF also has regulatory activities (bottom panel) such as inhibition of haematopoiesis, increased glucocorticoid production, activation of suppressive cells (such as mesenchymal stem cells (MSC) and myeloid-derived suppressor cells (MDSC)) or altering the function of dendritic cells (DCs) and macrophages. TNF also regulates immunity by promoting IL-10-producing B cells, inducing T cell apoptosis, altering T cell receptor (TCR) signalling, inhibiting TH17 cell differentiation and boosting numbers and function of regulatory T (Treg) cells. APC, antigen-presenting cell; FLS, fibroblast-like synoviocyte; HSC, haematopoietic stem cell.

Figure 2 .

 2 Figure 2. The overall effects of TNF on regulatory T cells. Most of the effects of tumour necrosis factor (TNF) on regulatory T (Treg) cells are due to induction of TNF receptor 1 (TNFR2) signalling, which is probably preferentially mediated by transmembrane TNF rather than soluble TNF. Signal transduction downstream of TNFR2 that does not involve kinase activity involves TNF receptor-associated factor (TRAF) adaptor proteins. Multiple downstream signalling pathways lead to positive (left) and negative (right) effects on Treg cell biology. TNFR2 signalling strongly induces Treg cell proliferation and has a moderate survival-promoting effect on Treg cells; both of these effects depend on RelA and probably also on the activation of p38, AKT and mTORC1 by phosphorylation (p). Weak evidence indicates that TNF also promotes the stability and suppressive function of Treg cells, perhaps via TNF-induced protein 3 (TNFAIP3)and signal transducer and activator of transcription 5 (STAT5) signalling pathways.In addition to these positive effects of TNF, the negative effects of this cytokine are clear in relation to the inhibition of induced Treg cell differentiation (which involves phosphoinositide 3kinase (PI3K) and/or phosphorylated RACα serine/threonine-protein kinase (AKT) pathway activation). Weak evidence suggests that TNF induces Treg cell dysfunction, perhaps via a mechanism involving deleted in breast cancer 1 (DBC1), microRNA 34a (miR-34a) and serine/threonine-protein phosphatase PP1. Arrow thickness and box shading intensity is proportional to the importance of the effect or the likelihood that a given molecule is involved in the pathway.

Table 1 . Clinically approved TNF inhibitors in the USA and Europe

 1 Disease indications for biosimilars can differ from those of the original drug and depend on the countries where they are registered. AS, ankylosing spondylitis; JIA, juvenile idiopathic arthritis; mAb, monoclonal antibody; NA, not applicable; RA, rheumatoid arthritis.

					Commenté [JM1]: Caroline: Check against table 1 in the
					published paper below for overlap:
	Drug	Molecule	Biosimilars	Approved rheumatic
				disease indications a	https://www.mdpi.com/2073-4468/4/1/48/html?
	Etanercept	Human TNFR2--IgG1-Fc	Benpali, Erelzi, Nepexto	RA, JIA, psoriatic arthritis,
		fusion protein		plaque psoriasis, AS	This table might need to be adjusted to avoid overlap (or else
	Infliximab	Humanized chimeric anti-	Remsima, Inflectra, Flixabi, Ixifi,	RA, psoriatic arthritis,	might need permissions).
		TNF IgG1/κ mAb	Zessly, Avsola	plaque psoriasis, AS
	Adalimumab	Fully human anti-TNF	Exemptia, Adfrar, Amjevita, Cyltezo,	RA, JIA, psoriatic arthritis,	(see handover sheet for one idea of alt layout)
		IgG1/κ mAb	Amgevita, Solymbic, Imraldi, Cyltezo,	plaque psoriasis, AS,
			Halimatoz, Hefiya, Hyrimoz, Hulio,	hidradenitis suppurativa,
			Idacio, Kromeya, Hadlima, Abrilada,	non-infectious uveitis
			Amsparity
	Certolizumab pegol PEGylated human Fab	NA	RA (Europe only),
		fragment of anti-TNF mAb		psoriatic arthritis
	Golimumab	Fully human anti-TNF	NA	RA, psoriatic arthritis, AS
		IgG1/κ mAb

a

Table 2 .

 2 Pathogenic and protective roles of TNFR1 and TNFR2 in models of rheumatic and autoimmune diseases

	Mouse model	TNFR1	TNFR2 knockout TNFR1	TNFR2 agonist Refs
		knockout		antagonist
	Collagen-induced	Attenuated	Exacerbated	Attenuated	Attenuated	200,201,184,119,121
	arthritis
	Antigen-induced	ND	Exacerbated	ND	ND	201
	arthritis
	DTHA	ND	Exacerbated	ND	ND	115
	Arthritis in TNF-	Attenuated	Exacerbated	ND	ND	202
	transgenic mice
	EAE	Attenuated	Exacerbated	Attenuated	Attenuated

Table 3 .

 3 Effect of TNF on Treg cell function in vitro.

	Treg cell population	Culture condition(s)	n	TNF added	Assay type	Effect of TNF suppression on Treg cell	Refs
	Human CD4 + CD25 hi	Soluble anti-CD3 and anti-CD28 mAbs	6	Before or during	Cytokine FACS	No change
	Human CD4 + CD25 hi	Coated anti-CD3 mAbs	6	Before	Proliferation 3 H	Decreased
	Human CD4 + CD25 +	HBV e-antigen	7	None	Proliferation 3 H	Decreased
					Proliferation
	Human CD4 + CD25 +	Coated anti-CD3 mAbs	NR	During	FACS, cytokine	Decreased
					ELISA
	Human CD4 + CD25 hi	Coated anti-CD3 mAbs	3	Before or during	Proliferation FACS	Decreased
	Human CD4 + CD25 hi CD127 low	Coated anti-CD3 and anti-CD28 mAbs	5?	Before	Proliferation FACS	Decreased
		Coated anti-CD3 and
	Human CD4 + CD25 hi CD127 low CD45RA -	anti-CD28 mAbs; APC and soluble anti-CD3 mAbs; APC and coated	28	Before or during	Proliferation FACS	No change or increased
		anti-CD3 mAbs
	Mouse CD4 + CD25 +	APC and soluble anti-CD3 mAbs	6	Before or during	Proliferation FACS	No change or increased
	Mouse CD4 + CD25 +	APC and soluble anti-CD3 mAbs	3	Before	Proliferation FACS	Increased

APC, antigen presenting cells; ELISA, enzyme-linked immunosorbent assay; FACS, 812 fluorescence-activated cell sorting; n, number of healthy individuals; NR, not reported; TNF,

Table 4 .

 4 Treg cell proportions in blood before and after TNF inhibitor therapy Baseline (before initiation of TNF inhibitor treatment). b In patients versus controls. c Absolute number. d Three patients also received golimumab, adalimumab or certolizumab. e Versus patients treated only with ciclosporine or colchicine. AS, ankylosing spondylitis; JIA, juvenile idiopathic arthritis; ND, not determined; NR, not reported; NSAID, nonsteroidal anti-inflammatory drug.

					Treg cells
	Study population	TNF inhibitor (concomitant medications)	Sampling time points a	Cell population	Pre-treatment (proportion) b	Post-treatment (proportion)	Refs
	27 patients with RA; 8 healthy controls	Infliximab (NSAIDs, methotrexate)	Baseline, 1.5 and 3.0 months	CD4 + CD25 hi	Same	Increased from baseline; increased in responders vs nonresponders
	17 patients with RA; 15 healthy controls	Infliximab (NSAIDs, methotrexate)	Baseline and 3.0 months	CD4 + CD25 hi	Decreased c	Increased c from baseline
	31 patients with RA; 20 healthy controls	Infliximab (NSAIDs, methotrexate)	Baseline and months 4.0-6.0	CD4 + FOXP3 +	Same	Increased from baseline
		Infliximab	NR
	40 patients with RA; 10 healthy controls	(methotrexate, salazopyrin, hydroxychloroquine,		CD4 + CD25 + FOX P3 +	Decreased	Increased from baseline and in responders vs nonresponders
		steroids)
	10 patients with RA; 10 healthy controls	Adalimumab steroids) (NSAIDs methotrexate,	Baseline and 3.0 months	CD4 + CD25 hi	Same	No change from baseline
						Increased from
						baseline; increased in
	50 patients with RA; 15 healthy controls	Adalimumab or etanercept (NR)	NR	CD4 + FOXP3 +	Same	responders vs nonresponders to adalimumab; no change
						from baseline with
						etanercept
	48 patients with RA	Adalimumab or etanercept (methotrexate, leflunomide)	Baseline, 1.5 and 3.0 months	CD4 + FOXP3 + , CD25 hi 127 low	ND	No change from baseline, no difference between responders and nonresponders
	20 patients with RA; 10 healthy controls	Etanercept (methotrexate)	Baseline and 3.0 months	CD4 + CD25 hi FOX P3 +	Decreased	Increased from baseline
	33 patients with RA	Etanercept (methotrexate)	Baseline, 3.0 and 6.0 months	CD4 + CD25 + FOX P3 +	ND	Increased from baseline
	16 patients with RA	Infliximab or etanercept d (NR)	Baseline and 3.0 months	CD4 + CD25 + 127 lo w FOXP3 +	ND	Increased from baseline
	7 patients with JIA	Etanercept (NSAIDs, methotrexate)	Baseline and 1.0-5.0 months	CD4 + FOXP3 +	ND	No change from baseline
	222 patients with AS; 68 healthy controls	Infliximab or etanercept (NSAIDs)	Baseline and 6.0 months	CD4 + CD25 hi FOX P3 +	Decreased	Increased from baseline; increased in responders versus nonresponders
	46 patients with sarcoidosis; 26 healthy controls	Infliximab (NR)	Baseline, 3.5 and 6.0 months	CD4 + CD25 hi	Increased	Decreased from baseline
	12 patients with uveitis	Adalimumab (NR)	Baseline, 1.0 and 6.0 months	CD4 + CD25 hi 127 lo w FOXP3 +	ND	Increased from baseline
	16 patients with uveitis; 15 healthy controls	Infliximab (NR)	Baseline, 4.0-27.0 months	CD4 + FOXP3 +	Same	Increased from baseline e

a

Table 5 . Therapeutic effects of drugs targeting TNFRs in autoimmune disease models

 5

	Agent	Structure	Therapeutic efficacy	Refs
		Antagonists of TNFR1
	DMS5540	Bispecific anti-TNFR1 and anti-albumin mAb	Arthritis (CIA)	184
	ASTROSAB	Humanized anti-TNFR1 IgG1 mAb, mutated in	EAE	185
		the Fc fragment to abrogate complement and
		immune complex activation
	TROS	Trivalent nanobody comprising two mAb	EAE	186
		domains binding to TNFR1 and one mAb domain
		binding to albumin
	HM1097	Hamster IgG	EAE	187
	XPro1595	Dominant-negative PEGylated TNF muteins that	Arthritis (CIA) and EAE	188,190,192
		interact with soluble TNF to form inactive
		heterotrimers, which have low binding and
		signalling activity
	R1antTNF	PEGylated TNF mutein that binds specifically to	Arthritis (CIA) and EAE	189,191
		TNFR1 without signalling activity
		Agonists of TNFR2
	MR2-1	Mouse mAb against human TNFR2	Increased expansion and stability of Treg	57, 118
			cells; not tested in vivo
	Unnamed	Mouse mAb against human TNFR2	Increased expansion and stability of Treg	111
			cells; not tested in vivo
	TNF07	Human TNF mutein trimer	Increased expansion of Treg cells; not	116
			tested in vivo
	TNC-	Human TNF mutein trimer	Not tested in vitro or vivo	193
	scTNFR2
	STAR2	Mouse TNF mutein nanomer	Increased expansion, survival and	121,103,117,12
			function of Treg cells; effective in CIA,	0,132
			EAE and GvHD
	EHD2-sc-	Mouse TNF mutein hexamer	Increased expansion of Treg cells;	119,208
	mTNFR2		effective in EAE and CIA

CIA, collagen-induced arthritis; EAE, experimental autoimmune encephalomyelitis; GvHD, graft versus host disease; mAb, monoclonal antibody; TNF, tumour necrosis factor; TNFR, TNF receptor.

Acknowledgements

The author thanks current and past members of his laboratory for their hard work and fruitful and passionate exchanges on the effects of TNF on Treg cells. His research work is supported by Agence Nationale de la Recherche (grants ANR-15-CE15-0015-03 and ANR-17-CE15-0030-01) and Fondation pour la Recherche Médicale (équipe FRM).

Competing interests

B.S. declares that he received consultancy fees from HiFiBio Therapeutics regarding the applications of TNFR2 agonists and antagonists in cancer and autoimmunity.

Peer review information

Nature Reviews Rheumatology thanks [Referee#1 name], [Referee#2 name] and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Supplementary information

Supplementary information is available for this paper at https://doi.org/10.1038/s415XX-XXX-XXXX-X

Key points

• Tumour necrosis factor (TNF) is a major inflammatory cytokine that has deleterious effects in several rheumatic and autoimmune diseases, as attested by the success of TNF inhibitor therapy.

• Some patients do not respond to TNF inhibitors and others develop paradoxical autoimmune exacerbations that can be explained by the immunoregulatory role of TNF.

• The pro-inflammatory and anti-inflammatory properties of TNF are largely segregated by the capacity of this cytokine to bind to TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), respectively.

• The anti-inflammatory effects of TNF are explained by its capacity to increase the proliferation, stability and suppressive function of FOXP3 + regulatory T cells via TNFR2 signalling.

• Antagonists of TNFR1 and agonists of TNFR2 constitute a new generation of drugs that might be more effective and have fewer adverse effects than classical TNF inhibitors. tumour necrosis factor.