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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.

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.

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.

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.

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 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.