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BackgroundTargeting immune checkpoints that inhibit antitumor immune responses has emerged as a powerful new approach to treat cancer. We recently showed that blocking the tumor necrosis factor receptor-type 2 (TNFR2) pathway induces the complete loss of the protective function of regulatory T cells (Tregs) in a model of graft-versus-host disease (GVHD) prevention that relies on Treg-based cell therapy. Here, we tested the possibility of amplifying the antitumor response by targeting TNFR2 in a model of tumor relapse following hematopoietic stem-cell transplantation, a clinical situation for which the need for efficient therapeutic options is still unmet.MethodWe developed appropriate experimental conditions that mimic patients that relapsed from their initial hematological malignancy after hematopoietic stem-cell transplantation. This consisted of defining in allogeneic bone marrow transplantation models developed in mice, the maximum number of required tumor cells and T cells to infuse into recipient mice to develop a model of tumor relapse without inducing GVHD. We next evaluated whether anti-TNFR2 treatment could trigger alloreactivity and consequently antitumor immune response. In parallel, we also studied the differential expression of TNFR2 on T cells including Treg from patients in post-transplant leukemia relapse and in patients developing GVHD.ResultsUsing experimental conditions in which neither donor T cells nor TNFR2-blocking antibody per se have any effect on tumor relapse, we observed that the coadministration of a suboptimal number of T cells and an anti-TNFR2 treatment can trigger alloreactivity and subsequently induce a significant antitumor effect. This was associated with a reduced percentage of activated CD4+ and CD8+ Tregs. Importantly, human Tregs over-expressed TNFR2 relative to conventional T cells in healthy donors and in patients experiencing leukemia relapse or cortico-resistant GVHD after hematopoietic stem cell transplantation.ConclusionsThese results highlight TNFR2 as a new target molecule for the development of immunotherapies to treat blood malignancy relapse, used either directly in grafted patients or to enhance donor lymphocyte infusion strategies. More widely, they open the door for new perspectives to amplify antitumor responses against solid cancers by directly targeting Tregs through their TNFR2 expression.

Radiation therapy (RT) for triple-negative breast cancer (TNBC) treatment is currently delivered in the adjuvant setting and is under investigation as a booster of neoadjuvant treatments. However, TNBC radioresistance remains an obstacle, so new biomarkers are needed to select patients for any integration of RT in the TNBC therapy sequence. MicroRNAs (miRs) are important regulators of gene expression, involved in cancer response to ionizing radiation (IR) and assessable by tumor tissue or liquid biopsy. This systematic review aimed to evaluate the relationships between miRs and response to radiation in TNBC, as well as their potential predictive and prognostic values.A thorough review of studies related to miRs and RT in TNBC was performed on PubMed, EMBASE, and Web of Science. We searched for original English articles that involved dysregulation of miRs in response to IR on TNBC-related preclinical and clinical studies. After a rigorous selection, 44 studies were chosen for further analysis.Thirty-five miRs were identified to be TNBC related, out of which 21 were downregulated, 13 upregulated, and 2 had a double-side expression in this cancer. Expression modulation of many of these miRs is radiosensitizing, among which miR-7, -27a, -34a, -122, and let-7 are most studied, still only in experimental models. The miRs reported as most influencing/reflecting TNBC response to IR are miR-7, -27a, -155, -205, -211, and -221, whereas miR-21, -33a, -139-5p, and -210 are associated with TNBC patient outcome after RT.miRs are emerging biomarkers and radiosensitizers in TNBC, worth further investigation. Dynamic assessment of circulating miRs could improve monitoring and TNBC RT efficacy, which are of particular interest in the neoadjuvant and the high-risk patients' settings.

There is an urgent need for therapeutic agents that target humoral alloimmunity in solid organ transplantation. This includes sensitized patients with preformed donor-specific human leukocyte antigen antibodies and patients who develop de novo donor-specific antibodies, both of which are associated with acute and chronic antibody-mediated rejection and allograft loss. In the last decade, both experimental and clinical studies highlighted the major impact of costimulation molecules in the control of immune responses both in the field of transplantation and autoimmune disease. Although these molecules have been initially developed to control the early steps of T-cell activation, recent evidence also supports their influence at several steps of the humoral response. In this review, we aim to provide an overview of the current knowledge of the effects of costimulatory blockade agents on humoral responses in both autoimmune and allogeneic contexts. We first present the effects of costimulatory molecules on the different steps of alloantibody production. We then summarize mechanisms and clinical results observed using cytotoxic T lymphocyte antigen-4 (CTLA4)-Ig molecules both in transplantation and autoimmunity. Finally, we present the potential interest and implications of other costimulatory family members as therapeutic targets, with emphasis on combinatorial approaches, for the optimal control of the alloantigen-specific humoral response.

Allogeneic bone marrow transplantation (BMT) is a widely spread treatment of many hematological diseases, but its most important side effect is graft-versus-host disease (GVHD). Despite the development of new therapies, acute GVHD (aGVHD) occurs in 30% to 50% of allogeneic BMT and is characterized by the generation of effector T (Teff) cells with production of inflammatory cytokines. We previously demonstrated that a short anti-CD45RC monoclonal antibody (mAb) treatment in a heart allograft rat model transiently decreased CD45RChigh Teff cells and increased regulatory T cell (Treg) number and function allowing long-term donor-specific tolerance. Here, we demonstrated in rat and mouse allogeneic GVHD, as well as in xenogeneic GVHD mediated by human T cells in NSG mice, that both ex vivo depletion of CD45RChigh T cells and in vivo treatment with short-course anti-CD45RC mAbs inhibited aGVHD. In the rat model, we demonstrated that long surviving animals treated with anti-CD45RC mAbs were fully engrafted with donor cells and developed a donor-specific tolerance. Finally, we validated the rejection of a human tumor in NSG mice infused with human cells and treated with anti-CD45RC mAbs. The anti-human CD45RC mAbs showed a favorable safety profile because it did not abolish human memory antiviral immune responses, nor trigger cytokine release in in vitro assays. Altogether, our results show the potential of a prophylactic treatment with anti-human CD45RC mAbs in combination with rapamycin as a new therapy to treat aGVHD without abolishing the antitumor effect.

Generation of de novo donor-specific antibodies (dnDSAs) after renal transplant is recognized as the leading cause of late transplant failure. Hence, the optimal immunosuppressive strategies to limit dnDSA development need to be defined. Recent clinical trials using the novel costimulatory blockade agent CTLA4-Ig (Belatacept) have shown that kidney transplant recipients (KTRs) treated with Belatacept have better graft survival and function and a lower proportion of dnDSAs than control-treated KTRs. Mechanisms involved in the control of humoral responses by Belatacept remain to be investigated. Here, we analyzed the effect of Belatacept on different steps of the B cell–mediated response in humans. In vitro, Belatacept reduced plasmablast differentiation, Ig production, and the expression of the major transcription factor involved in plasma cell function, Blimp-1, in a T cell–independent manner. Moreover, Belatacept induced activation of the STAT3 transcription factor in stimulated B cells and reduced the expression of CD86. Additionally, Belatacept blocked CD28-mediated activation of T follicular helper cells (Tfhs) in an autologous Tfh-memory B cells model. We then validated these observations in KTRs treated with Belatacept, who had a reduced proportion of blood effector B cells and activated Tfh (PD1(+)ICOS(+)) compared with control-treated KTRs. Our in vitro and in vivo results suggest that Belatacept modulates B cell function directly and at the level of B cell-Tfh interaction. These mechanisms likely account for the optimal control of humoral responses observed in KTRs treated with Belatacept.

IL-33 is strongly involved in several inflammatory and autoimmune disorders with both pro- and anti-inflammatory properties. However, its contribution to chronic autoimmune inflammation, such as rheumatoid arthritis, is ill defined and probably requires tight regulation. In this study, we aimed at deciphering the complex role of IL-33 in a model of rheumatoid arthritis, namely, collagen-induced arthritis (CIA). We report that repeated injections of IL-33 during induction (early) and during development (late) of CIA strongly suppressed clinical and histological signs of arthritis. In contrast, a late IL-33 injection had no effect. The cellular mechanism involved in protection was related to an enhanced type 2 immune response, including the expansion of eosinophils, Th2 cells, and type 2 innate lymphoid cells, associated with an increase in type 2 cytokine levels in the serum of IL-33–treated mice. Moreover, our work strongly highlights the interplay between IL-33 and regulatory T cells (Tregs), demonstrated by the dramatic in vivo increase in Treg frequencies after IL-33 treatment of CIA. More importantly, Tregs from IL-33–treated mice displayed enhanced capacities to suppress IFN-γ production by effector T cells, suggesting that IL-33 not only favors Treg proliferation but also enhances their immunosuppressive properties. In concordance with these observations, we found that IL-33 induced the emergence of a CD39high Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.

Interleukin (IL)-35 was initially described as an immunosuppressive cytokine specifically produced by CD4(+)FoxP3(+) regulatory T cells (Treg). Since Treg play a major role in autoimmunity control and protect from inflammation, we aimed at evaluating the role of IL-35 in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), using a non-viral gene transfer strategy. The clinical and histological effect of IL-35 was assessed in mice with CIA receiving an injection of two distinct plasmids encoding IL-35 gene (pIGneo-mIL-35 or pORF-mIL-35) 3 and 18 days after CIA induction. Treg and Th17 were characterized by flow cytometry in the spleen and lymph nodes of treated mice. Our results showed that whatever the plasmid used, IL-35 gene transfer resulted in a statistically significant increase in clinical scores of CIA compared to results with empty plasmid. The underlying cellular mechanisms of this effect were shown to be related to an increased Th17/Treg ratio in the spleen of pORF-mIL-35 treated mice. In conclusion, we show an unexpected but clear exacerbating effect of IL-35 gene transfer in an autoimmune and inflammatory RA model, associated with a modification of the Th17/Treg balance. Altogether, these result shows that this cytokine can promote chronic inflammation.

IL-6 is a key cytokine in the pathogenesis of rheumatoid arthritis (RA). The clinical efficacy of tocilizumab (TCZ), a humanized anti-IL6-receptor mAb, confirmed the value of IL-6 blockade in this disease. A number of new anti-IL-6 biologics are currently in Phase I - III of clinical development for RA.This article reviews the available results from Phase II trials of investigational anti-IL-6 agents in RA. The authors discuss the potential relevance of alternative IL-6-blocking agents, with regard to their specific molecular targets in IL-6 signaling pathways and to the main open questions in the clinical research agenda for anti-IL-6 biologics.The results of Phase II trials of new anti-IL-6 biologics show promising results in terms of efficacy. The most frequently reported adverse events were not unexpected based on previous experience with TCZ. Further evidence is needed to appraise whether the difference in molecular structure or in the specific target of new anti-IL-6 biologics might result in added therapeutic value over TCZ. New data from Phase III trials that provides a head-to-head comparison against TCZ and anti-TNF agents with or without methotrexate background treatment are expected in the future.

Objective The rationale for blocking interleukin-6 (IL-6) in rheumatoid arthritis (RA) lies chiefly in the proinflammatory effect of this cytokine. Few studies have evaluated the consequences of anti–IL-6 receptor (IL-6R) antibody treatment on Treg cells. This study was undertaken to elucidate the mechanism of action of anti–IL-6R antibody treatment by studying the effects on Treg cells in an experimental arthritis model and in patients with RA. Methods Mice with collagen-induced arthritis (CIA) were treated with a mouse anti–IL-6R antibody (MR16-1), and changes in Treg, Th1, and Th17 cells were assessed at key time points during the course of the disease. Peripheral blood from 15 RA patients was collected on day 0 and after 3 months of tocilizumab treatment for flow cytometry analysis of Th17 and Treg cells. Results In MR16-1–treated mice, Th17 cell frequencies were unchanged, whereas Treg cell frequencies were increased. The Treg cell phenotype showed marked changes, with an increase in the frequency of CD39+ Treg cells in the lymph nodes and spleen. Interestingly, similar CD39+ Treg cell expansion was observed in RA patients who were tocilizumab responders at 3 months, with no change in Th17 cell frequency. Moreover, fluorescence-activated cell–sorted CD39+ Treg cells from responder RA patients were functionally able to suppress the proliferation of conventional T cells. Conclusion In both CIA and RA, the frequency of functionally suppressive CD39+ Treg cells is increased as a result of anti–IL-6R treatment, whereas Th17 cells are unaffected. The modification of Treg cell frequency and phenotype may be one of the mechanisms involved in the therapeutic effect of IL-6 blockade in RA.

Resume Le developpement de technologies innovantes permet l’identification et la caracterisation reguliere de nombreuses molecules. C’est ainsi qu’un certain nombre d’interleukines a pu etre identifie et caracterise ces dernieres annees. Nous detaillons dans cet article les caracteristiques structurales et fonctionnelles des interleukines IL-34 a IL-38 et en particulier leur role dans les rhumatismes inflammatoires. L’IL-34 a principalement une fonction de stimulation et de proliferation des osteoclastes et intervient donc directement dans la destruction osseuse au cours de la polyarthrite rhumatoide. L’IL-35 est exprimee par les cellules T regulatrices et a ce titre possede une action anti-inflammatoire en restaurant les capacites suppressives de ces cellules et en inhibant la voie TH17. L’IL-37 a pour sa part une action anti-inflammatoire en exercant un retro-controle sur la secretion des cytokines pro-inflammatoires. L’IL-36 fait partie de la famille de l’IL-1 et est presente sous trois formes differentes. Elle a principalement ete etudiee dans le psoriasis et le rhumatisme psoriasique, mais son role pro-inflammatoire s’exerce aussi dans la polyarthrite rhumatoide. L’antagoniste specifique de l’IL-36, l’IL-36Ra se fixe sur son recepteur et empeche la transduction du signal. L’IL-38 a ete identifiee recemment et aurait une action proche de l’IL-36Ra puisqu’elle se fixe sur le recepteur del’IL-36 et inhibe son action en particulier la reponse TH17. Si les fonctions exactes de ces differentes cytokines demandent encore a etre precisees, l’amelioration de nos connaissances des mecanismes de regulation d’un phenomene inflammatoire chronique comme la polyarthrite rhumatoide, peut permettre le developpement de nouvelles cibles therapeutiques.

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.

The development of innovative technologies is steadily increasing the body of knowledge on molecules involved in physiological functions. Thus, several interleukins (ILs) have been identified and characterized in the past few years. Here, we detail the structural and functional characteristics of IL-34 to IL-38 with special attention to their involvement in inflammatory joint disease. IL-34 chiefly increases osteoclast activation and proliferation and therefore, it plays a direct role in bone destruction as seen in rheumatoid arthritis (RA). Regulatory T-cells (Tregs) express IL-35, which therefore exerts anti-inflammatory effects by restoring Treg suppressive capabilities and by inhibiting the Th17 pathway. IL-37 has anti-inflammatory effects mediated by a negative feedback loop that decreases the release of pro-inflammatory cytokines. IL-36 belongs to the IL-1 family and has three different forms. Although this cytokine has been chiefly studied in psoriasis and psoriatic arthritis, it also exerts pro-inflammatory effects in RA. The specific IL-36 antagonist, IL-36Ra binds to the IL-36 receptor, thereby, preventing signal transduction. Finally, IL-38 is a recently identified cytokine whose effect may resemble that of IL-36Ra as it binds to the IL-36 receptor and inhibits its effects, particularly the Th17-response. Although the exact roles for these cytokines awaits elucidation, the current improvements in our knowledge of the mechanisms that regulate chronic inflammatory conditions, such as RA may lead to the identification of new treatment targets.

Dendritic cells (DC) play a key role in regulating immune responses and are the best professional antigen-presenting cells. Two major DC populations are defined in part according to cell surface CD11c expression levels. Unexpectedly, we observed that mouse DC strongly down-regulate the typical DC marker CD11c upon activation. To better characterize DC responses, we have analyzed CD11c expression on mouse and human myeloid DC after Toll-like receptor (TLR) triggering. Here we show that mouse bone marrow-derived DC (BMDC) as well as spleen DC down-regulate cell surface CD11c upon activation by TLR3/4/9 agonists. In all cases, full DC activation was reached, as determined by cytokine secretion, cell stimulation in mixed leukocyte reactions (MLR), and CD40/CD86/major histocompatibility complex (MHC) up-regulation. Interestingly, membrane CD11c down-regulation correlated with increased cytoplasmic pools of CD11c. In contrast to the up-regulation of CD40 and MHC class II molecules, lipopolysaccharide (LPS)-induced CD11c down-regulation was MyD88-dependent. Polyinosinic-polycytidylic acid (poly I:C), which does not signal through MyD88, also induced cell surface CD11c down-regulation. Notably, CD11c down-regulation was not observed upon activation of human DC, either through TLR-dependent or -independent cell activation. Thus, activated mouse DC may be transiently CD11c-negative in vivo, hampering the identification of those cells. On the other hand, cell surface CD11c down-regulation may serve as a new activation marker for mouse DC.

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and destruction of the joints. In the collagen-induced arthritis mouse model of RA, we developed a nonviral gene therapy method designed to block in situ the main cytokine tumour necrosis factor (TNF)-α Methods Electrotransfer was used to deliver a plasmid encoding extracellular domain of mouse soluble TNF-α receptor type I fused to the Fc fragment of mouse immunoglobulin (Ig)G1 (pTNFR-Is) corresponding to a dimeric TNF-α soluble receptor fusion protein (mTNFR-Is/Ig). Results Delivery of the plasmid into the knees at symptom onset improved the histological inflammation and destruction not only at the knees, but also at the ankles, indicating a local and a regional therapeutic effect. The plasmid was detected in synovial membrane and meniscus specimens from the injected joints. In the synovial membrane, 15 days post-injection, interleukin (IL)-17 and TNF-α mRNAs expression were increased, whereas IL-10 mRNA was unchanged. However, the empty plasmid exerted a pro-inflammatory effect 30 days post-injection. Conclusions These data indicate that local nonviral gene therapy against TNF-α is effective, although further work is needed to decrease plasmid induced inflammation. Copyright © 2010 John Wiley & Sons, Ltd.

Background and Objectives Interleukin (IL)-33 is a new member of the IL-1 family that exerts pleiotropic activities in innate and adaptive immunity. With its receptor ST2, they have newly emerged as key molecules strongly involved in several inflammatory and autoimmune disorders. Recent evidence suggests that the IL-33/ST2 axis is strongly involved in the pathophysiology of rheumatoid arthritis (RA). However in RA models, the role of IL-33 and its receptor is still controversial. We aimed at deciphering IL-33 mode of action after administration in an experimental model of RA, namely collagen-induced arthritis (CIA). Material and Methods CIA was induced by immunisation of C57Bl/6 mice with type 2 collagen. IL‑33 was ip administrated in CIA mice and cells were analysed by flow cytometry on day 28 after CIA induction. Results We show a previously unshown dramatic inhibition of mouse collagen-induced arthritis (CIA) development after repeated administration of IL-33. This therapeutic effect was related to an enhanced type-2 immune response, including the expansion of eosinophils, Th2 cells, innate type 2 lymphoid cells (ILC2, defined as CD25 + c-Kit + Lin - Sca-1 + ST2L + ) and an increase in Th2 cytokines levels in the serum of treated mice. Moreover, our work brings out the interplay between Treg and IL-33. Since IL-33 acts directly on Treg via ST2L, we showed that IL-33 treatment of CIA majors Treg frequency and increases the suppressive capacities of those cells. IL-33 also induces the emergence of a CD39 +/high Treg population in a ST2L dependant manner. Conclusions In the light of our present study, IL-33 can exert powerful anti-inflammatory properties in CIA, integrating the establishment of a type-2 immune response, the expansion and the activation of Treg. Our study reveals an undescribed mechanism by which IL-33 inhibits arthritis development, thus updating and strengthening the crucial role of IL-33 in RA.

Background and Objectives Studies have demonstrated the clinical efficacy of tocilizumab, a humanised anti-IL-6 receptor (R) antibody (Ab), in patients with rheumatoid arthritis (RA). The rational for blocking IL-6 in this disease mainly lays on the pro-inflammatory role of this cytokine in the disease. However, only few works have studied the consequences of anti-IL-6R treatment on Tregs cells and mainly focuses on their frequency. Our objective was to elucidate anti-IL-6R mode of action on Tregs in RA patients treated with tocilizumab and in a RA model. Methods Mice with collagen-induced arthritis (CIA) were treated at day 0 by MR16–1 (a rat anti-mouse IL-6 receptor monoclonal Ab provided by Chugai Pharmaceutical Co. LTD, Japan) and the evolution of CD4 + FoxP3 + Tregs during arthritis course was assessed at key time points (day 8–18–28 and 42 after CIA induction) by studying their number, frequency and phenotype (expression of GITR, ICOS, Helios, CD62L, CTLA-4 and CD39) in lymph nodes (LN), thymus and spleen by flow cytometry. Numerical analysis of Th17 and Th1 cells was also performed by flow cytometry. Twenty patients with severe and active RA were recruited and treated with 8 mg/kg of tocilizumab monthly. Peripheral blood was recovered at day 0, as well as 1 and 3 month, and Th17and Tregs were analysed by flow cytometry. Results Clinical and histological evaluation of arthritides in mice treated with anti-mouse IL-6R mAb showed, as expected, a less severe disease as compared to control Ig treated mice. Th17 frequency was unchanged, but Tregs frequency was enhanced in the LN of MR16–1 treated mice. In the thymus, we observed an enhanced frequency of Tregs CD4 + CD8 − FoxP3 + . Tregs phenotype was also modified in treated mice, with an increased frequency of CD39 + Tregs (LN and spleen), suggesting an enhanced ATP hydrolysis immunosuppressive activity of Tregs. In RA patients, Th17 frequencies were not modified by tocilizumab therapy and did not differ between responders and non-responders. Interestingly, CD39 + Treg cell among CD4 + cells frequencies were significantly higher in responders than in non-responders after 3 months of tocilizumab therapy. Conclusions Tregs, but not Th17, are modified by anti-IL-6R treatment in both CIA and RA. These results support a beneficial effect in RA of treatments responsible for CD39 + Tregs enhancement and emphasise the relevance of the monitoring cell populations after cytokine blockade used to treat arthritis.

Introduction Studies have demonstrated the clinical efficacy of tocilizumab, a humanized anti-IL-6 receptor (R) antibody (Ab), in patients with rheumatoid arthritis (RA). The rational for blocking IL-6 in this disease mainly lays on the pro-inflammatory role of this cytokine in the disease. However, only few works have studied the consequences of anti-IL-6R treatment on Tregs cells and mainly focuses on their frequency.In this study, we hypothesized that IL-6 inhibition leads to Tregs modifications in RA models given that (i) anti-IL6R is therapeutic in RA and its models by inhibiting Th17 cells, and (ii) IL-6 plays a determining role in inducing Th17 when present, or Tregs when it is absent. Our objective was thus to elucidate anti-IL-6R mode of action in RA models by studying the consequences of this treatment on Tregs phenotype and biological activity. Methods Mice with collagen-induced arthritis were treated at day 0 by MR16-1 (a rat anti-mouse IL-6 receptor monoclonal Ab provided by Chugai Pharmaceutical Co. LTD, Japan) and the evolution of Tregs (defined as CD4+ FoxP3+) during arthritis course was assessed at key time points (day 9-17-28 and 43 after CIA induction) by studying their number, frequency and phenotype (expression of GITR, ICOS, Helios, CD62L, CTLA-4 and CD39) in lymph nodes (LN), thymus, spleen by flow cytometry. Finally, the immunosuppressive activity of the Treg cells on CFSE-labeled CD4+ CD25-T effector (Teff) cells proliferation was also studied. Numerical analysis of Th17 and Th1 cells was also performed at the same times by flow cytometry. Results Clinical and histological evaluation of arthritides in mice treated with anti-mouse IL-6R mAb showed, as expected, a less severe disease as compared to control Ig treated mice. A decreased Teff/Tregs ratio, together with an increased Treg/Th17 overtime were observed in the LN of MR16–1 treated mice. In the thymus, we observed an enhanced frequency of Tregs CD4+ CD8-FoxP3+. Tregs phenotype was modified in treated mice, with a decreased frequency of Tregs expressing Helios (spleen) but an increased frequency of CD39+ Tregs (LN and spleen). Lastly, the immunosuppressive activity of the Treg cells on Teff cells proliferation was not modified, suggesting that Tregs immunosuppression activity was probably due to another mechanism. Conclusion Tregs are modified by anti-IL-6R treatment in CIA, that could result in an enhanced central and peripheral generation of Tregs. The increased CD39 expression may reflect the generation of a more suppressive Tregs population, acting through ATP hydrolysis. This hypothesis should be verified in the future. All together, this study shows that inhibition of IL-6 in CIA also acts by enhancing Tregs/Th17 balance and by Tregs phenotype.

Introduction Interleukin (IL)-35 is a novel interleukin described as an immunosuppressive cytokine specifically produced by CD4+ FoxP3+ regulatory T (Tregs) cells but not by conventional T cells. Since Tregs play a major role in autoimmunity maintenance and protects from inflammation, we aimed at evaluating the role of IL-35 in a model of rheumatoid arthritis in mice using a non viral gene transfer strategy. Methods Two plasmids were used and compared: pIGneo-mouse (m) IL-35-GFP (gift from Dario Vignali) and pORF-mIL-35, where IL-35 is expressed under CMV or HTLV promoter, respectively. pIGneo-mouse (m) IL-35-GFP contains a “native” form of IL-35 made of Ebi3 and p35 linked subunits, while pORF-mIL-35contains a “single chain” construct. In vitro, IL-35 expression was detected in lysate of transfected HEK cells by western blot (p35 or Ebi3) and immunoprecipitation (IP). The in vivo trangene expression was detected by fluorescence analysis of GFP on muscle slides after intra-muscular electrotransfer (ET) of 60 μg pIGneo-mIL-35-GFP plasmid. The clinical effect of IL-35 was assessed in collagen-induced arthritis in mice receiving two i.m. ET of 60 μg pIGneo-mIL-35-GFP or pORF-IL-35, 3 and 18 days after CIA induction. Control groups received the corresponding empty plasmids or PBS. Tregs and Th17 frequencies and phenotypes were performed by flow cytometry in the spleen and lymph nodes (LN) of pORF-IL-35 treated mice at day 34. Results Both subunits p35 and Ebi3 were expressed in lysates from either pIGneo-mIL-35-GFP or pORF-IL-35. HEK transfected cells, and formed the IL-35 heterodimer as shown by IP. At least 3 days after im ET of pIGneo-mIL-35-GFP plasmid, we were able to detect GFP expression, revealing in vivo IL-35 expression, on muscle histological slides. Surprisingly, whatever the plasmid used, IL-35 gene transfer resulted in a statistically significant increase in clinical scores of collagen-induced arthritis as compared to empty plasmid. The underlying cellular mechanisms of this effect were shown to be related to an increased in number and frequency of Th17 cells in the spleen of pORF-mIL-35 treated mice, leading to an increased Th17/Tregs ratio. Interestingly, CD39 expression on Tregs increased (frequency and intensity) in the LN of pORF-mIL-35 treated mice as compared to control groups, but CD62L expression also increased (spleen and LN) on these cells. Conclusion We show an unexpected but clear exacerbating effect of IL-35 gene transfer in an autoimmune and inflammatory model of RA. We can hypothesize that IL-35, as already known, induced an activation of Tregs by up-regulating CD39 expression on these cells, but that the concomitant up-regulation of CD62L prevent their migration into the inflammatory joint by retaining them in secondary lymphoid organs, thus impeding them from exerting their immunosuppressive role.

Backgroundand objectives Interleukin (IL)-35 is a novel interleukin described as an immunosuppressive cytokine specifically produced by CD4+FoxP3+ regulatory T (Tregs) cells but not by conventional T cells. Since Tregs play a major role in autoimmunity maintenance and protects from inflammation, the authors aimed at evaluating the role of IL-35 in a model of rheumatoid arthritis in mice using a non viral gene transfer strategy. Methods Two plasmids were used and compared: pIGneo-mouse (m)IL-35-GFP (gift from Dario Vignali) and pORF-mIL-35, where IL-35 is expressed under CMV or HTLV promoter, respectively. pIGneo-mouse (m)IL-35-GFP contains a ‘native’ form of IL-35 made of Ebi3 and p35 linked subunits, while pORF-mIL-35contains a ‘single chain’ construct. In vitro, IL-35 expression was detected in lysate of transfected HEK cells by western blot (p35 or Ebi3) and immunoprecipitation (IP). The in vivo trangene expression was detected by fluorescence analysis of GFP on muscle slides after intra-muscular electrotransfer (ET) of 60 µg pIGneo-mIL-35-GFP plasmid. The clinical effect of IL-35 was assessed in collagen-induced arthritis in mice receiving two im ET of 60 µg pIGneo-mIL-35-GFP or pORF-IL-35, 3 and 18 days after CIA induction. Control groups received the corresponding empty plasmids or PBS. Tregs and Th17 frequencies and phenotypes were performed by flow cytometry in the spleen and lymph nodes (LN) of pORF-IL-35 treated mice at day 34. Results Both subunits p35 and Ebi3 were expressed in lysates from either pIGneo-mIL-35-GFP or pORF-IL-35 HEK transfected cells, and formed the IL-35 heterodimer as shown by IP. At least 3 days after intramuscular ET of pIGneo-mIL-35-GFP plasmid, the authors were able to detect GFP expression, revealing in vivo IL-35 expression, on muscle histological slides. Surprisingly, whatever the plasmid used, IL-35 gene transfer resulted in a statistically significant increase in clinical scores of collagen-induced arthritis as compared to empty plasmid. The underlying cellular mechanisms of this effect were shown to be related to an increased in number and frequency of Th17 cells in the spleen of pORF-mIL-35 treated mice, leading to an increased Th17/Tregs ratio. Interestingly, CD39 expression on Tregs increased (frequency and intensity) in the LN of pORF-mIL-35 treated mice as compared to control groups, but CD62L expression also increased (spleen and LN) on these cells. Conclusion The authors show an unexpected but clear exacerbating effect of IL-35 gene transfer in an autoimmune and inflammatory model of RA. The authors can hypothesise that IL-35, as already known, induced an activation of Tregs by up-regulating CD39 expression on these cells, but that the concomitant up-regulation of CD62L prevent their migration into the inflammatory joint by retaining them in secondary lymphoid organs, thus impeding them from exerting their immunosuppressive role.

Backgroundand objectives Studies have demonstrated the clinical efficacy of tocilizumab, a humanised anti-IL-6 receptor (R) antibody (Ab), in patients with rheumatoid arthritis (RA). The rational for blocking IL-6 in this disease mainly lays on the pro-inflammatory role of this cytokine in the disease. Moreover, IL-6 acts as a ‘pivot’, determining whether an immune response is dominated by regulatory CD4 + Foxp3 + T cells or Th17 effector T cells. However, only few works have studied the consequences of anti-IL-6R treatment on Tregs cells and only focuses on their frequency. In this study, the authors hypothesised that IL-6 inhibition leads to Tregs modifications in RA models given that (i) anti-IL6R is therapeutic in RA and its models by inhibiting Th17 cells, and (ii) IL-6 plays a determining role in inducing Th17 when present, or Tregs when it is absent. Our objective was thus to elucidate anti-IL-6R mode of action in RA models by studying the consequences of this treatment on Tregs phenotype and biological activity. Methods Mice with collagen-induced arthritis were treated by MR16-1 (a rat antimouse IL-6 receptor monoclonal Ab provided by Chugai Pharmaceutical Co LTD, Japan) and the evolution of Tregs (defined as CD4 + FoxP3 + ) during arthritis course was assessed at key time points (day 9–17–28 and 43 after CIA induction) by studying their number, frequency and phenotype (expression of GITR, ICOS, Helios, CD62L, CTLA-4 and CD39) in lymph nodes (LN) and spleen by flow cytometry. Finally, the immunosuppressive activity of the Treg cells on CFSE-labeled CD4+ CD25- T effector (Teff) cells proliferation was also studied. Numerical analysis of Th17 and Th1 cells was also performed at the same times by flow cytometry. Results Clinical evaluation of arthritides in mice treated with anti-mouse IL-6R mAb showed, as expected, a less severe disease as compared to control Ig treated mice. A decreased Teff/Tregs ratio overtime were observed in the LN of MR16-1 treated mice. Tregs phenotype was modified in treated mice mostly at day 17, with a decreased frequency of Tregs expressing CD62L or Helios (spleen) but an enhanced intensity of CD39 expression on Tregs (spleen and LN) and increased frequency of CD39+ Tregs (LN). No modification in ICOS, GITR and CTLA-4 were observed on Tregs at any time in any compartments. Lastly, the immunosuppressive activity of the Treg cells on Teff cells proliferation was not modified, suggesting that Tregs immunosuppression activity was probably due to another mechanism.. Conclusion Tregs are modified by anti-IL-6R treatment in CIA, that could result in an enhanced peripheral generation of Tregs (Helios down regulation), an increased Tregs activation (CD39+ upregulation) and a better capacity to leave the secondary lymphoid organs and to migrate within the inflammatory joint (decreased CD62L down regulation). Altogether, this study shows that inhibition of IL-6 in CIA also acts by modifying Tregs state.