TCR-independent CD137 (4-1BB) signaling promotes CD8+-exhausted T cell proliferation and terminal differentiation.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

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 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 cells. This basic knowledge sheds light on the consequences of TNF inhibitor therapies on regulatory T 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.

Circulating Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Contribute to Sepsis-Induced Immunosuppression in Patients During Septic Shock.

BACKGROUND: Septic shock remains a major cause of death that can be complicated by long-term impairment in immune function. Among regulatory T (Treg) cells, the tumor necrosis factor receptor 2 positive (TNFR2pos) Treg-cell subset endorses significant immunosuppressive functions in human tumors and a sepsis mouse model but has not been investigated during septic shock in humans. METHODS: We prospectively enrolled patients with septic shock hospitalized in intensive care units (ICU). We performed immunophenotyping and functional tests of CD4+ T cells, Treg cells, and TNFR2pos Treg cells on blood samples collected 1, 4, and 7 days after admission to ICU. RESULTS: We investigated 10 patients with septic shock compared to 10 healthy controls. Although the proportions of circulating Treg cells and TNFR2pos Treg-cell subsets were not increased, their CTLA4 expression and suppressive functions in vitro were increased at 4 days of septic shock. Peripheral blood mononuclear cells from healthy donors cultured with serum from septic shock patients had increased CTLA4 expression in TNFR2pos Treg cells compared to TNFR2neg Treg cells. CONCLUSIONS: In patients with septic shock, CTLA4 expression and suppressive function were increased in circulating TNFR2pos Treg cells. We identify TNFR2pos Treg cells as a potential attractive target for therapeutic intervention.

Tissue-restricted control of established central nervous system autoimmunity by TNF receptor 2-expressing Treg cells.

CD4+Foxp3+ regulatory T (Treg) cells are central modulators of autoimmune diseases. However, the timing and location of Treg cell-mediated suppression of tissue-specific autoimmunity remain undefined. Here, we addressed these questions by investigating the role of tumor necrosis factor (TNF) receptor 2 (TNFR2) signaling in Treg cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We found that TNFR2-expressing Treg cells were critical to suppress EAE at peak disease in the central nervous system but had no impact on T cell priming in lymphoid tissues at disease onset. Mechanistically, TNFR2 signaling maintained functional Treg cells with sustained expression of CTLA-4 and Blimp-1, allowing active suppression of pathogenic T cells in the inflamed central nervous system. This late effect of Treg cells was further confirmed by treating mice with TNF and TNFR2 agonists and antagonists. Our findings show that endogenous Treg cells specifically suppress an autoimmune disease by acting in the target tissue during overt inflammation. Moreover, they bring a mechanistic insight to some of the adverse effects of anti-TNF therapy in patients.

Deletion of intestinal epithelial AMP-activated protein kinase alters distal colon permeability but not glucose homeostasis.

OBJECTIVE: The intestinal epithelial barrier (IEB) restricts the passage of microbes and potentially harmful substances from the lumen through the paracellular space, and rupture of its integrity is associated with a variety of gastrointestinal disorders and extra-digestive diseases. Increased IEB permeability has been linked to disruption of metabolic homeostasis leading to obesity and type 2 diabetes. Interestingly, recent studies have uncovered compelling evidence that the AMP-activated protein kinase (AMPK) signaling pathway plays an important role in maintaining epithelial cell barrier function. However, our understanding of the function of intestinal AMPK in regulating IEB and glucose homeostasis remains sparse. METHODS: We generated mice lacking the two α1 and α2 AMPK catalytic subunits specifically in intestinal epithelial cells (IEC AMPK KO) and determined the physiological consequences of intestinal-specific deletion of AMPK in response to high-fat diet (HFD)-induced obesity. We combined histological, functional, and integrative analyses to ascertain the effects of gut AMPK loss on intestinal permeability in vivo and ex vivo and on the development of obesity and metabolic dysfunction. We also determined the impact of intestinal AMPK deletion in an inducible mouse model (i-IEC AMPK KO) by measuring IEB function, glucose homeostasis, and the composition of gut microbiota via fecal 16S rRNA sequencing. RESULTS: While there were no differences in in vivo intestinal permeability in WT and IEC AMPK KO mice, ex vivo transcellular and paracellular permeability measured in Ussing chambers was significantly increased in the distal colon of IEC AMPK KO mice. This was associated with a reduction in pSer425 GIV phosphorylation, a marker of leaky gut barrier. However, the expression of tight junction proteins in intestinal epithelial cells and pro-inflammatory cytokines in the lamina propria were not different between genotypes. Although the HFD-fed AMPK KO mice displayed suppression of the stress polarity signaling pathway and a concomitant increase in colon permeability, loss of intestinal AMPK did not exacerbate body weight gain or adiposity. Deletion of AMPK was also not sufficient to alter glucose homeostasis or the acute glucose-lowering action of metformin in control diet (CD)- or HFD-fed mice. CD-fed i-IEC AMPK KO mice also presented higher permeability in the distal colon under homeostatic conditions but, surprisingly, this was not detected upon HFD feeding. Alteration in epithelial barrier function in the i-IEC AMPK KO mice was associated with a shift in the gut microbiota composition with higher levels of Clostridiales and Desulfovibrionales. CONCLUSIONS: Altogether, our results revealed a significant role of intestinal AMPK in maintaining IEB integrity in the distal colon but not in regulating glucose homeostasis. Our data also highlight the complex interaction between gut microbiota and host AMPK.

Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Play a Major Role in CD4+ T-Cell Impairment During Sepsis.

Sepsis causes inflammation-induced immunosuppression with lymphopenia and alterations of CD4+ T-cell functions that renders the host prone to secondary infections. Whether and how regulatory T cells (Treg) are involved in this postseptic immunosuppression is unknown. We observed in vivo that early activation of Treg during Staphylococcus aureus sepsis induces CD4+ T-cell impairment and increases susceptibility to secondary pneumonia. The tumor necrosis factor receptor 2 positive (TNFR2pos) Treg subset endorsed the majority of effector immunosuppressive functions, and TNRF2 was particularly associated with activation of genes involved in cell cycle and replication in Treg, probably explaining their maintenance. Blocking or deleting TNFR2 during sepsis decreased the susceptibility to secondary infection. In humans, our data paralleled those in mice; the expression of CTLA-4 was dramatically increased in TNFR2pos Treg after culture in vitro with S. aureus. Our findings describe in vivo mechanisms underlying sepsis-induced immunosuppression and identify TNFR2pos Treg as targets for therapeutic intervention.

TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function.

BACKGROUND: In addition to their multilineage potential, mesenchymal stem cells (MSCs) have a broad range of functions from tissue regeneration to immunomodulation. MSCs have the ability to modulate the immune response and change the progression of different inflammatory and autoimmune disorders. However, there are still many challenges to overcome before their widespread clinical administration including the mechanisms behind their immunoregulatory function. MSCs inhibit effector T cells and other immune cells, while inducing regulatory T cells (T regs), thus, reducing directly and indirectly the production of pro-inflammatory cytokines. TNF/TNFR signaling plays a dual role: while the interaction of TNFα with TNFR1 mediates pro-inflammatory effects and cell death, its interaction with TNFR2 mediates anti-inflammatory effects and cell survival. Many immunosuppressive cells like T regs, regulatory B cells (B regs), endothelial progenitor cells (EPCs), and myeloid-derived suppressor cells (MDSCs) express TNFR2, and this is directly related to their immunosuppression efficiency. In this article, we investigated the role of the TNFα/TNFR2 immune checkpoint signaling pathway in the immunomodulatory capacities of MSCs. METHODS: Co-cultures of MSCs from wild-type (WT) and TNFR2 knocked-out (TNFR2 KO) mice with T cells (WT and TNFα KO) were performed under various experimental conditions. RESULTS: We demonstrate that TNFR2 is a key regulatory molecule which is strongly involved in the immunomodulatory properties of MSCs. This includes their ability to suppress T cell proliferation, activation, and pro-inflammatory cytokine production, in addition to their capacity to induce active T regs. CONCLUSIONS: Our results reveal for the first time the importance of the TNFα/TNFR2 axis as an active immune checkpoint regulating MSC immunological functions.

The TNF/TNFR2 signaling pathway is a key regulatory factor in endothelial progenitor cell immunosuppressive effect.

BACKGROUND: Endothelial progenitor cells (EPCs) are non-differentiated endothelial cells (ECs) present in blood circulation that are involved in neo-vascularization and correction of damaged endothelial sites. Since EPCs from patients with vascular disorders are impaired and inefficient, allogenic sources from adult or cord blood are considered as good alternatives. However, due to the reaction of immune system against allogenic cells which usually lead to their elimination, we focused on the exact role of EPCs on immune cells, particularly, T cells which are the most important cells applied in immune rejection. TNFα is one of the main activators of EPCs that recognizes two distinct receptors. TNFR1 is expressed ubiquitously and its interaction with TNFα leads to differentiation and apoptosis, whereas, TNFR2 is expressed predominantly on ECs, immune cells and neural cells and is involved in cell survival and proliferation. Interestingly, it has been shown that different immunosuppressive cells express TNFR2 and this is directly related to their immunosuppressive efficiency. However, little is known about immunological profile and function of TNFR2 in EPCs. METHODS: Using different in-vitro combinations, we performed co-cultures of ECs and T cells to investigate the immunological effect of EPCs on T cells. We interrupted in the TNFα/TNFR2 axis either by blocking the receptor using TNFR2 antagonist or blocking the ligand using T cells derived from TNFα KO mice. RESULTS: We demonstrated that EPCs are able to suppress T cell proliferation and modulate them towards less pro-inflammatory and active phenotypes. Moreover, we showed that TNFα/TNFR2 immune-checkpoint pathway is critical in EPC immunomodulatory effect. CONCLUSIONS: Our results reveal for the first time a mechanism that EPCs use to suppress immune cells, therefore, enabling them to form new immunosuppressive vessels. Furthermore, we have shown the importance of TNFα/TNFR2 axis in EPCs as an immune checkpoint pathway. We believe that targeting TNFR2 is especially crucial in cancer immune therapy since it controls two crucial aspects of tumor microenvironment: 1) Immunosuppression and 2) Angiogenesis. Video Abstract. (MP4 46355 kb).

Tumor necrosis factor receptor family costimulation increases regulatory T-cell activation and function via NF-κB.

Several drugs targeting members of the TNF superfamily or TNF receptor superfamily (TNFRSF) are widely used in medicine or are currently being tested in therapeutic trials. However, their mechanism of action remains poorly understood. Here, we explored the effects of TNFRSF co-stimulation on murine Foxp3+ regulatory T cell (Treg) biology, as they are pivotal modulators of immune responses. We show that engagement of TNFR2, 4-1BB, GITR, and DR3, but not OX40, increases Treg proliferation and survival. Triggering these TNFRSF in Tregs induces similar changes in gene expression patterns, suggesting that they engage common signal transduction pathways. Among them, we identified a major role of canonical NF-κB. Importantly, TNFRSF co-stimulation improves the ability of Tregs to suppress colitis. Our data demonstrate that stimulation of discrete TNFRSF members enhances Treg activation and function through a shared mechanism. Consequently, therapeutic effects of drugs targeting TNFRSF or their ligands may be mediated by their effect on Tregs.

The NF-κB RelA Transcription Factor Is Critical for Regulatory T Cell Activation and Stability.

Regulatory T cells (Tregs) play a major role in immune homeostasis and in the prevention of autoimmune diseases. It has been shown that c-Rel is critical in Treg thymic differentiation, but little is known on the role of NF-κB on mature Treg biology. We thus generated mice with a specific knockout of RelA, a key member of NF-κB, in Tregs. These mice developed a severe autoimmune syndrome with multi-organ immune infiltration and high activation of lymphoid and myeloid cells. Phenotypic and transcriptomic analyses showed that RelA is critical in the acquisition of the effector Treg state independently of surrounding inflammatory environment. Unexpectedly, RelA-deficient Tregs also displayed reduced stability and cells that had lost Foxp3 produced inflammatory cytokines. Overall, we show that RelA is critical for Treg biology as it promotes both the generation of their effector phenotype and the maintenance of their identity.

CCR2-Dependent Recruitment of Tregs and Monocytes Following Radiotherapy Is Associated with TNFα-Mediated Resistance.

Radiotherapy (RT) represents one of the main anticancer approaches for the treatment of solid tumors. Beyond the expected direct effects of RT on tumor cells, evidence supporting the importance of an immune response to RT is growing. The balance between RT-mediated immunogenic and tolerogenic activity is ill-defined and deserves more attention. Herein, a murine model of head and neck squamous cell carcinoma was used to demonstrate that RT upregulated CCL2 chemokine production in tumor cells, leading to a CCR2-dependent accumulation of tumor necrosis factor alpha (TNFα)-producing monocytes and CCR2+ regulatory T cells (Treg). This corecruitment was associated with a TNFα-dependent activation of Tregs, dampening the efficacy of RT. Our results highlight an unexpected cross-talk between innate and adaptive immune system components and indicate CCL2/CCR2 and TNFα as potential clinical candidates to counterbalance the radioprotective action of monocyte-derived cells and Tregs, paving the way for potent combined radioimmunotherapies.

TNFR2/BIRC3-TRAF1 signaling pathway as a novel NK cell immune checkpoint in cancer.

Natural Killer (NK) cells control metastatic dissemination of murine tumors and are an important prognostic factor in several human malignancies. However, tumor cells hijack many of the NK cell functional features compromising their tumoricidal activity. Here, we show a deleterious role of the TNFα/TNFR2/BIRC3/TRAF1 signaling cascade in NK cells from the tumor microenvironment (TME). TNFα induces BIRC3/cIAP2 transcripts and reduces NKp46/NCR1 transcription and surface expression on NK cells, promoting metastases dissemination in mice and poor prognosis in GIST patients. NKp30 engagement, by promoting the release of TNFα, also contributes to BIRC3 upregulation, and more so in patients expressing predominantly NKp30C isoforms. These findings reveal that in the absence of IL-12 or a Th1-geared TME, TNFα can be considered as a negative regulatory cytokine for innate effectors.

Tumor Necrosis Factor α and Regulatory T Cells in Oncoimmunology.

Tumor necrosis factor α (TNF) is a potent pro-inflammatory cytokine that has deleterious effect in some autoimmune diseases, which led to the use of anti-TNF drugs in some of these diseases. However, some rare patients treated with these drugs paradoxically develop an aggravation of their disease or new onset autoimmunity, revealing an immunosuppressive facet of TNF. A possible mechanism of this observation is the direct and positive effect of TNF on regulatory T cells (Tregs) through its binding to the TNF receptor type 2 (TNFR2). Indeed, TNF is able to increase expansion, stability, and possibly function of Tregs via TNFR2. In this review, we discuss the role of TNF in graft-versus-host disease as an example of the ambivalence of this cytokine in the pathophysiology of an immunopathology, highlighting the therapeutic potential of triggering TNFR2 to boost Treg expansion. We also describe new targets in immunotherapy of cancer, emphasizing on the putative suppressive effect of TNF in antitumor immunity and of the interest of blocking TNFR2 to regulate the Treg compartment.

Control of GVHD by regulatory T cells depends on TNF produced by T cells and TNFR2 expressed by regulatory T cells.

Therapeutic CD4(+)Foxp3(+) natural regulatory T cells (Tregs) can control experimental graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HCT) by suppressing conventional T cells (Tconvs). Treg-based therapies are currently tested in clinical trials with promising preliminary results in allo-HCT. Here, we hypothesized that as Tregs are capable of modulating Tconv response, it is likely that the inflammatory environment and particularly donor T cells are also capable of influencing Treg function. Indeed, previous findings in autoimmune diabetes revealed a feedback mechanism that renders Tconvs able to stimulate Tregs by a mechanism that was partially dependent on tumor necrosis factor (TNF). We tested this phenomenon during alloimmune response in our previously described model of GVHD protection using antigen specific Tregs. Using different experimental approaches, we observed that control of GVHD by Tregs was fully abolished by blocking TNF receptor type 2 (TNFR2) or by using TNF-deficient donor T cells or TNFR2-deficient Tregs. Thus, our results show that Tconvs exert a powerful modulatory activity on therapeutic Tregs and clearly demonstrate that the sole defect of TNF production by donor T cells was sufficient to completely abolish the Treg suppressive effect in GVHD. Importantly, our findings expand the understanding of one of the central components of Treg action, the inflammatory context, and support that targeting TNF/TNFR2 interaction represents an opportunity to efficiently modulate alloreactivity in allo-HCT to either exacerbate it for a powerful antileukemic effect or reduce it to control GVHD.

Effector T cells boost regulatory T cell expansion by IL-2, TNF, OX40, and plasmacytoid dendritic cells depending on the immune context.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a major role in peripheral tolerance. Multiple environmental factors and cell types affect their biology. Among them, activated effector CD4(+) T cells can boost Treg cell expansion through TNF or IL-2. In this study, we further characterized this effector T (Teff) cell-dependent Treg cell boost in vivo in mice. This phenomenon was observed when both Treg and Teff cells were activated by their cognate Ag, with the latter being the same or different. Also, when Treg cells highly proliferated on their own, there was no additional Treg cell boost by Teff cells. In a condition of low inflammation, the Teff cell-mediated Treg cell boost involved TNF, OX40L, and plasmacytoid dendritic cells, whereas in a condition of high inflammation, it involved TNF and IL-2. Thus, this feedback mechanism in which Treg cells are highly activated by their Teff cell counterparts depends on the immune context for its effectiveness and mechanism. This Teff cell-dependent Treg cell boost may be crucial to limit inflammatory and autoimmune responses.

Fetal pancreas transplants are dependent on prolactin for their development and prevent type 1 diabetes in syngeneic but not allogeneic mice.

Transplantation of adult pancreatic islets has been proposed to cure type 1 diabetes (T1D). However, it is rarely considered in the clinic because of its transient effect on disease, the paucity of donors, and the requirement for strong immunosuppressive treatment to prevent allogeneic graft rejection. Transplantation of fetal pancreases (FPs) may constitute an attractive alternative because of potential abundant donor sources, possible long-term effects due to the presence of stem cells maintaining tissue integrity, and their supposed low immunogenicity. In this work, we studied the capacity of early FPs from mouse embryos to develop into functional pancreatic islets producing insulin after transplantation in syngeneic and allogeneic recipients. We found that as few as two FPs were sufficient to control T1D in syngeneic mice. Surprisingly, their development into insulin-producing cells was significantly delayed in male compared with female recipients, which may be explained by lower levels of prolactin in males. Finally, allogeneic FPs were rapidly rejected, even in the context of minor histocompatibility disparities, with massive graft infiltration with T and myeloid cells. This work suggests that FP transplantation as a therapeutic option of T1D needs to be further assessed and would require immunosuppressive treatment.

Tumor emergence is sensed by self-specific CD44hi memory Tregs that create a dominant tolerogenic environment for tumors in mice.

Early responses of Tregs and effector T cells (Teffs) to their first encounter with tumor cells have been poorly characterized. Here we have shown, in both implanted and in situ-induced mouse tumor models, that the appearance of tumor cells is immediately sensed by CD44hi memory Tregs that are specific for self antigens. The rapid response of these Tregs preceded and prevented activation of naive antitumor Teffs. The relative speed of the Treg versus the Teff response within the first 2-4 days determined the outcome of the antitumor immune response: tolerance or rejection. If antitumor memory Teffs were present at the time of tumor emergence, both Tregs and Teffs were recruited and activated with memory kinetics; however, the Tregs were unable to control the Teffs, which eradicated the tumor cells. This balance between effector and regulatory responses did not depend on the number of Tregs and Teffs, but rather on their memory status. Thus, in the natural setting, dominant tolerogenic immunosurveillance by self-specific memory Tregs protects tumors, just as it protects normal tissues. More generally, our results reveal that the timing of Treg and Teff engagement, determined by their memory status, is an important mode of regulation of immune responses.

Clinical-grade preparation of human natural regulatory T-cells encoding the thymidine kinase suicide gene as a safety gene.

BACKGROUND: Human CD4+CD25+FOXP3+ natural regulatory T-cells (nTreg) have a great therapeutic potential for the induction of tolerance in allo-transplanted patients or for the control of severe auto-immune diseases. However, clinical-grade production of nTreg remains difficult to achieve because of the absence of a truly specific surface marker and of their low frequency that implies a need for their ex vivo expansion. Furthermore, safety issues should be taken into consideration due to the risk of either uncontrolled nTreg-induced immunosuppression or uncontrolled proliferation of autoreactive contaminating T-cells particularly in an auto-immune context. METHODS: We compared different clinical-grade conditions for immuno-magnetic selection and ex vivo expansion of nTreg. For safety, expanded cells were genetically modified with retroviral vectors co-expressing human CD90 and HSV1 thymidine kinase. The CD90 surface marker and thymidine kinase allow for selection and elimination of transduced cells by ganciclovir, respectively. RESULTS: We showed that (i) nTreg could be enriched in a one step using CD25 microbeads, were functionally suppressive and mainly FOXP3+; (ii) using anti-CD28- and anti-CD3-coated beads, interleukin-2 and rapamycin, nTreg were expanded 150-200-fold after 3 weeks. Under these clinical-grade conditions, they remained suppressive, and no major alteration of the TCR repertoire was observed; (iii) after efficient retroviral transduction and CD90 selection, nTreg maintained their suppressive activity; (iv) transduced nTreg could be eliminated by ganciclovir upon activation. CONCLUSIONS: The efficient procedure reported here for the preparation of nTreg, whose safety has been ensured, is now applicable for further clinical trials.