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Smooth Muscle Cells (SMC) are unique amongst all muscle cells in their capacity to modulate their phenotype. Indeed, SMCs do not terminally differentiate but instead harbour a remarkable capacity to dedifferentiate, switching between a quiescent contractile state and a highly proliferative and migratory phenotype, a quality often associated to SMC dysfunction. However, phenotypic plasticity remains poorly examined in the field of gastroenterology in particular in pathologies in which gut motor activity is impaired. Here, we assessed SMC status in biopsies of infants with chronic intestinal pseudo‐obstruction (CIPO) syndrome, a life‐threatening intestinal motility disorder. We showed that CIPO‐SMCs harbour a decreased level of contractile markers. This phenotype is accompanied by an increase in Platelet‐Derived Growth Factor Receptor‐alpha (PDGFRA) expression. We showed that this modulation occurs without origin‐related differences in CIPO circular and longitudinal‐derived SMCs. As we characterized PDGFRA as a marker of digestive mesenchymal progenitors during embryogenesis, our results suggest a phenotypic switch of the CIPO‐SMC towards an undifferentiated stage. The development of CIPO‐SMC culture and the characterization of SMC phenotypic switch should enable us to design therapeutic approaches to promote SMC differentiation in CIPO.

Gastrointestinal stromal tumours (GISTs), the most common mesenchymal neoplasm of the gastrointestinal tract, result from deregulated proliferation of transformed KIT‐positive interstitial cells of Cajal that share mesenchymal progenitors with smooth muscle cells. Despite the identification of selective KIT inhibitors, primary resistance and relapse remain a major concern. Moreover, most patients develop resistance partly through reactivation of KIT and its downstream signalling pathways. We previously identified the Limb Expression 1 (LIX1) gene as a unique marker of digestive mesenchyme immaturity. We also demonstrated that LIX1 regulates mesenchymal progenitor proliferation and differentiation by controlling the Hippo effector YAP1, which is constitutively activated in many sarcomas. Therefore, we wanted to determine LIX1 role in GIST development. We found that LIX1 is strongly up‐regulated in GIST samples and this is associated with unfavourable prognosis. Moreover, LIX1 controls GIST cell proliferation in vitro and in vivo. Upon LIX1 inactivation in GIST cells, YAP1/TAZ activity is reduced, KIT (the GIST signature) is down‐regulated, and cells acquire smooth muscle lineage features. Our data highlight LIX1 role in digestive mesenchyme‐derived cell‐fate decisions and identify this novel regulator as a target for drug design for GIST treatment by influencing its differentiation status.

supplementary data : https://www.jimmunol.org/content/suppl/2015/11/19/jimmunol.1501069.DCSupplemental; International audience; Promising immunotherapeutic strategies are emerging to restore tolerance in autoimmune diseases by triggering an increase in the number and/or the function of endogenous regulatory T (Treg) cells, which actively control pathological immune responses. Evidence suggests a remarkable heterogeneity in peripheral Treg cells that warrants their better characterization in terms of phenotype and suppressive function, to determine which subset may be optimally suitable for a given clinical situation. We found that repetitive injections of immature dendritic cells expanded Foxp3-negative CD49b+ Treg cells that displayed an effector memory phenotype. These expanded Treg cells were isolated ex vivo for transcriptome analysis and found to contain multiple transcripts of the canonical Treg signature shared mainly by CD25+ but also by other subphenotypes. We characterized the CD49b+ Treg cell phenotype, underscoring its similarities with the CD25+ Treg cell phenotype and highlighting some differential expression patterns for several markers, including lymphocyte activation gene 3, KLRG1, CD103, ICOS, CTLA-4, and granzyme B. Comparison of the CD25+ and CD49b+ Treg cells' suppressive mechanisms, in vitro and in vivo, revealed the latter's potent suppressive activity, which was partly dependent on IL-10 secretion. Altogether, our results strongly suggest that expression of several canonical Treg cell markers and suppressive function could be Foxp3 independent, and underscore the therapeutic potential of IL-10–secreting CD49b+ Treg cells in arthritis.

Background Smooth muscle cell (SMC) plasticity maintains the balance between differentiated SMCs and proliferative mesenchymal progenitors, crucial for muscular tissue homeostasis. Studies on the development of mesenchymal progenitors into SMCs have proven useful in identifying molecular mechanisms involved in digestive musculature plasticity in physiological and pathological conditions. Results Here, we show that Limb Expression 1 (LIX1) molecularly defines the population of mesenchymal progenitors in the developing stomach. Using in vivo functional approaches in the chick embryo, we demonstrate that LIX1 is a key regulator of stomach SMC development. We show that LIX1 is required for stomach SMC determination to regulate the expression of the pro-proliferative gene YAP1 and mesenchymal cell proliferation. However, as stomach development proceeds, sustained LIX1 expression has a negative impact on further SMC differentiation and this is associated with a decrease in YAP1 activity. Conclusions We demonstrate that expression of LIX1 must be tightly regulated to allow fine-tuning of the transcript levels and state of activation of the pro-proliferative transcriptional coactivator YAP1 to regulate proliferation rates of stomach mesenchymal progenitors and their differentiation. Our data highlight dual roles for LIX1 and YAP1 and provide new insights into the regulation of cell density-dependent proliferation, which is essential for the development and homeostasis of organs. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0257-2) contains supplementary material, which is available to authorized users.

Background Adoptive cell transfer of Treg cells is a promising approach to restore tolerance in autoimmune disease. However the various type of Tregs, their doses of injection and their in vivo-suppressive mechanism need to be precisely define to clearly establish which Tregs will be able to dampen efficiently the immune response in the various settings. In our study, we compared the therapeutic potential of induced CD25FoxP3 and two IL10-secreting Tregs: Tr1 and CD49b-induced Tregs.

Vγ9Vδ2 T cells play an important role in the immune response to infectious agents but the mechanisms contributing to this immune process remain to be better characterized. Following their activation, Vγ9Vδ2 T cells develop cytotoxic activity against infected cells, secrete large amounts of cytokines and influence the function of other effectors of immunity, notably cells playing a key role in the initiation of the adaptive immune response such as dendritic cells. Brucella infection dramatically impairs dendritic cell maturation and their capacity to present antigens to T cells. Herein, we investigated whether V T cells have the ability to restore the full functional capacities of Brucella-infected dendritic cells. Using an in vitro multicellular infection model, we showed that: 1/Brucella-infected dendritic cells activate Vγ9Vδ2 T cells through contact-dependent mechanisms, 2/activated Vγ9Vδ2 T cells induce full differentiation into IL-12 producing cells of Brucella-infected dendritic cells with functional antigen presentation activity. Furthermore, phosphoantigen-activated Vγ9Vδ2 T cells also play a role in triggering the maturation process of dendritic cells already infected for 24 h. This suggests that activated Vγ9Vδ2 T cells could be used to modulate the outcome of infectious diseases by promoting an adjuvant effect in dendritic cell-based cellular therapies.

International audience; For many years, a great deal of interest has been focusing on the optimization of peptide presentation by dendritic cells (DCs) using peptide-encapsulated particles, in order to enhance the immune response. Nowadays, DCs are also known to be involved in peripheral tolerance, inducing anergy or regulatory T lymphocytes. To preserve the plasticity of DCs, we formulated non-cytotoxic pH-sensitive polyion complex micelles based on an original tripartite association of polymethacrylic acid-b-polyethylene oxide, poly-Llysine and fluorescent-peptide: OVAFITC peptide, as a model drug. We demonstrated that the OVAFITC peptide was successfully entrapped into the micelles, released into DC endosomes thanks to the pHsensitivity property of themicelles, and efficiently loaded onto MHC class II molecules. The phenotype as well as the cytokinic secretion profile of the mature and immature DCs loaded with peptide-encapsulated micelles was unaltered by the tripartite polyion micelles. The efficient loading of the peptide by immature and mature DCs was shown by the in vitro proliferation of OVA-specific transgenic T cells. Therefore, the present results show that the tripartite polyion complex micelles can be used as efficient peptide vectors immunogically inert for ex vivo DCs engineering without modifying their intrinsic immune plasticity.

International audience; Introduction: Regulatory T (Treg) cells play a crucial role in preventing autoimmune diseases and are an idealtarget for the development of therapies designed to suppress inflammation in an antigen-specific manner.Type 1 regulatory T (Tr1) cells are defined by their capacity to produce high levels of interleukin 10 (IL-10), whichcontributes to their ability to suppress pathological immune responses in several settings. The aim of this study wasto evaluate the therapeutic potential of collagen type II–specific Tr1 (Col-Treg) cells in two models of rheumatoidarthritis (RA) in mice.Methods: Col-Treg clones were isolated and expanded from collagen-specific TCR transgenic mice. Their cytokinesecretion profile and phenotype characterization were studied. The therapeutic potential of Col-Treg cells wasevaluated after adoptive transfer in collagen-antibody– and collagen-induced arthritis models. The in vivosuppressive mechanism of Col-Treg clones on effector T-cell proliferation was also investigated.Results: Col-Treg clones are characterized by their specific cytokine profile (IL-10highIL-4negIFN-γint) and mediatecontact-independent immune suppression. They also share with natural Tregs high expression of GITR, CD39 andgranzyme B. A single infusion of Col-Treg cells reduced the incidence and clinical symptoms of arthritis in bothpreventive and curative settings, with a significant impact on collagen type II antibodies. Importantly, injection ofantigen-specific Tr1 cells decreased the proliferation of antigen-specific effector T cells in vivo significantly.Conclusions: Our results demonstrate the therapeutic potential of Col-Treg cells in two models of RA, providingevidence that Col-Treg could be an efficient cell-based therapy for RA patients whose disease is refractory to currenttreatments.

Background Adoptive transfer of regulatory T cells (Tregs) is a promising approach to restore tolerance in autoimmune diseases. However based on the heterogeneity of the Tregs, we need to precisely establish which Tregs will be able to dampen efficiently the immune response in the various settings. We previously showed the potential of CD49b + Treg cells to protect and prevent an experimental model of arthritis. Nevertheless the optimal injection dose, the phenotype and the in vivo -suppressive mechanism of these Treg cells remain unknown. In our study, we investigated and compared the therapeutic potential of CD25 + FoxP3 + and induced IL10-secreting CD49b + Treg cells in an experimental model of arthritis, the collagen-induced arthritis (CIA). Materials and Methods IL-10 secreting CD49b + Treg cells were generated in naive mice following repetitive injections of immature DCs (iDCs). Treg purification was based on the negative selection of CD4 T cells isolated from spleen and liver of the iDC-vaccinated mice. Cell sorting was performed to obtain 98% pure CD49b + or CD25 + Treg cells. Several doses of CD49b + were intravenously (i.v.) injected at day 28 in established CIA. Clinical signs of arthritis were scored, as well as biological parameters such as the level of anti-bCII antibodies in sera and the cytokine profile of bCII specific T cells. Phenotypes of both Treg cells were compared as well as their suppressive activity in vitro and in vivo. Results Several doses of CD49 + Treg cells were tested in curative settings experiments. The dose of 10 5 CD49b + or CD25 + cells reverse clinical symptoms of arthritis while interestingly, a lack of efficacy was observed after higher doses. In vitro suppressive experiments confirmed the similar efficiency of both populations and phenotype analyses of CD49b + Treg cells showed expression profile of several Treg specific markers (LAP + , LAG + , CTLA-4 high ). Moreover, in an OVA-specific model of inflammation, we demonstrated the high impact of the CD49b Treg cells on the proliferation of effector cells in vivo. Conclusions Altogether, our results confirm the therapeutic potential of IL-10 secreting T cells in experimental model of arthritis in curative settings and unravel their mechanism of suppression.

Backgroundand objectives Adoptive cell transfer of T regulatory (Treg) cells is a promising approach to restore tolerance in autoimmune disease. However the various type of Tregs, their doses of injection and their in vivo-suppressive mechanism need to be precisely define to clearly establish which Tregs will be able to dampen efficiently the immune response in the various settings. In this study, the authors compared the therapeutic potential of two IL10-secreting Tregs: Tr1 and CD49b-induced Tregs. The authors previously demonstrated that a single injection of iDC-induced CD49b T cells reversed clinical symptoms of arthritis. This inducible CD49b Treg population shares several phenotypic markers as well as immunosuppressive properties compared with the in vitro-expanded IL-10-secreting Tr1 cells. More recently, the authors provide evidence that injections of Tr1 cells, after the onset of the disease, could protect mice from severe arthritis. In the present study the authors perform adoptive cell transfer experiments of these two inducible Treg cells in order to compare their impact on the immune response. Materials and methods CD49b Treg cells were generated in naive mice following repetitive injections of iDC. The purification was based on the negative selection of CD4 T cells isolated from the spleen and liver of the iDC-vaccinated mice. Cell sorting experiments were realised to obtain 98% pure CD49b T cells. Collagen type II (bCII) specific Tr1 clones were obtained from TCR transgenic mice and expanded in vitro. Selected clones showed in vitro antigen specificity, Tr1 cytokine profile and IL10- and tumour necrosis factor β-dependent suppressive activity. Results Several doses of CD49b or Tr1 cells were injected intravenously at day 28 in established collagen-induced arthritis. Clinical signs of arthritis were scored, as well as biological parameters such as the level of anti-bCII antibodies in sera and the cytokine profile of bCII specific T cells. The authors defined for both Treg cell populations the dose effect in curative settings experiments. One single dose of 3×106 or 1×106 of Tr1 cell administration could reduce the incidence and severity of CIA. Interestingly, higher dose of 10M of Tr1 cells did not improve the disease. In the same manner, the dose of 105 CD4CD49b + cells reverse clinical symptom with a lack of efficacy of higher doses. The homing ability and plasticity of Tr1 cells was also investigated. Conclusions These results suggest that even if the Treg cells present some similarities, one should precisely define the dose and type of Treg that will be efficient in each experimental setting.

Introduction Tr1 cells have been characterised as induced T regulatory lymphocytes (Treg) inhibiting inflammation in various chronic inflammatory models. Based on these data, a phase I/II clinical trial is currently under investigation in Crohn9s disease (TxCell). However, the therapeutic potential of these cells has not yet been evaluated in rheumatoid arthritis. In this study, the authors investigated the therapeutic potential of bovine type II collagen (bCII) specific Tr1 cells, isolated from TBC mice, in the experimental model of collagen-induced arthritis (CIA). Methods Collagen type II specific Tr1 clones were obtained from T cell receptor transgenic mice and expanded in vitro. Selected clones showed in vitro antigen specificity, Tr1 cytokine profile (IL-10 high /IL-4 neg ) and IL-10- and transforming growth factor β-dependent suppressive activity. Male DBA/one mice were immunised with bCII and 10×10 6 , 3×10 6 , 1×10 6 , 0.3×10 6 of Tr1 cells were injected (intravenously) 28 days postimmunisation. Hind paws swelling and clinical signs of arthritis were scored, as well as biological parameters such as the level of anti-bCII antibodies in the sera of treated mice and the cytokine profile of bCII specific T cells. Results One single injection of 3×10 6 or 1×10 6 of Tr1 cells at day 28, in ongoing arthritis, significantly inhibits the development of arthritic disease, shown by reduction of disease severity and incidence. In contrast the injection of 0.3×10 6 and 10 M of Tr1 cells did not improve the clinical signs of arthritis. The analysis of the bCII specific T cell responses following euthanasia of the mice injected with 3×10 6 and 1×10 6 of Tr1 cells revealed a decrease of anti-bCII specific antibodies in the sera, a decrease of proliferation of bCII specific T cells and a slight increase of IL-10 secreted by activated splenocytes. Importantly, these preliminary data indicate that a single injection of Tr1 cells at disease onset could reduce disease severity and incidence in experimental arthritis. Conclusions Single dose 3×10 6 , 1×10 6 of Tr1 cell administration showed a reduction of disease incidence and severity in CIA demonstrating the therapeutic potential of Tr1 cells in arthritis and confirming the clinical potential of these induced Treg.

Background Dendritic cells (DC) are professional antigen-presenting cells which have unique properties to steer the outcome of immune responses. The authors previously demonstrated that repetitive injection of immature DC (iDC) was able to protect mice from collagen-induced arthritis (CIA). Rapamycin (Rapa) is a macrolide antibiotic with potent immunosuppressive properties that have been ascribed primarily to its inhibitory effects on T cells. More recently the effect of rapamycin on differentiation, antigen uptake and the immunostimulatory capacity of human DC were examined. In the present study the authors investigated the effect of rapamycin on the phenotype as well as the immunogenicity of iDC, and evaluated the tolerogenic potential of such rapamycin-treated DC in CIA. Methods Bone marrow-derived DCs were generated in the presence of 10 ng/ml of rapamycin during 6 days. Maturation of DCs was induced or not by addition of lipopolysaccharides ± interferon or interleukin 12 (IL-12). Phenotype of iDC and mature DC were characterised by FACS analysis and their cytokinic secretions were quantified by ELISA. Repetitive injections of 5×10 5 rapamycin-treated DC were performed in naive mice and regulatory populations were monitored in various lymphoid organs. In the CIA mouse model, injections of DCs were performed at days 21, 23 and 25 after immunisation. Results The addition of rapamycin has an impact on the basal and induced expression of major histocompatibility complex class II and co-stimulatory molecules, with a major impact on CD86 expression. These results suggest that the rapamycin alters the maturation process of murine DC. The secretion profiles of the mDCs were also altered by the addition of rapamycin. The decrease of the immunogenicity and antigen-presentation abilities of rapamycin-treated DCs were confirmed in mixed lymphocyte reaction and antigen presentation assays respectively. In vivo, the repetitive injections of rapamycin-treated or control DCs were able to induce the expansion of IL-10-secreting CD4 CD49b regulatory T cells, but not FoxP3 + CD25 Treg cells in naive mice. To evaluate the tolerogenic potential of rapamycin-treated DCs, they were repetitively injected in arthritic mice. The authors observed that mice vaccinated only with Rapa-iDC were protected from arthritis whereas those that received CTL-DC showed highest severity. A specific decrease of IgG2a antibody response was observed in the sera as well as an expansion of IL-10 secreting CD4 CD49b regulatory T cells in the liver of the protected mice. Conclusion This results suggest that the DC maturation is altered by Rapamycin, improving their tolerogenic potential in vivo. The therapeutic potential of such a cell-based immuno-modulatory strategy might be assessed in rheumatoid arthritis.