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Crohn’s disease and ulcerative colitis, the two major forms of inflammatory bowel diseases (IBDs), are characterized by high levels of IL-22 production. Rodent studies revealed that this cytokine is protective during colitis but whether this is true in IBDs is unclear. We show here that levels of the soluble inhibitor of IL-22, interleukin 22-binding protein (IL-22BP), are significantly enhanced during IBDs owing to increased numbers of IL-22BP-producing eosinophils, that we unexpectedly identify as the most abundant source of IL-22BP protein in human gut. In addition, using IL-22BP-deficient rats, we confirm that endogenous IL-22BP is effective at blocking protective actions of IL-22 during acute colitis. In conclusion, our study provides new important insights regarding the biology of IL-22 and IL-22BP in the gut and indicates that protective actions of IL-22 are likely to be suboptimal in IBDs thus making IL-22BP a new relevant therapeutic target.

Crohn's disease and ulcerative colitis, the two major forms of inflammatory bowel diseases (IBDs), are characterized by high levels of IL-22 production. Rodent studies revealed that this cytokine is protective during colitis but whether this is true in IBDs is unclear. We show here that levels of the soluble inhibitor of IL-22, interleukin 22-binding protein (IL-22BP), are significantly enhanced during IBDs owing to increased numbers of IL-22BP-producing eosinophils, that we unexpectedly identify as the most abundant source of IL-22BP protein in human gut. In addition, using IL-22BP-deficient rats, we confirm that endogenous IL-22BP is effective at blocking protective actions of IL-22 during acute colitis. In conclusion, our study provides new important insights regarding the biology of IL-22 and IL-22BP in the gut and indicates that protective actions of IL-22 are likely to be suboptimal in IBDs thus making IL-22BP a new relevant therapeutic target.

Alterations of the BMP-9/ALK1/BMPR-II pathway play a critical role in pulmonary arterial hypertension (PAH) development. Paradoxically, previous work from our team showed that the loss of BMP-9 in the C57BL/6 strain does not lead to spontaneous pulmonary hypertension (PH) and confers reduced susceptibility to the remodeling of pulmonary vessels. However, the exact protective mechanisms remain unknown. We hypothesized that loss of BMP-9/ALK1/BMPR-II signaling induces endothelial cell activation and angiogenesis.

Using a mouse skin transplant model combining anti‐CD3 treatment with autologous tolerogenic dendritic cell infusion, the authors demonstrate the importance of antigen cross‐presentation to prolong allograft survival mediated by CD8+ regulatory T cells by characterizing a novel cation channel, Tmem176b, that participates in the fine‐tuning of phagosomal pH in dendritic cells. See editorial by Morelli and Thomson on page 989.

An active role of the host humoral response in the rejection of intracerebral neural xenografts is demonstrated by the presence of elicited antibodies directed against the donor neurons, and the prolonged survival of porcine neurons in the striatum of immunoglobulin‐deficient rats.

Xenogenic fetal neuroblasts are considered as a potential source of transplantable cells for the treatment of neurodegenerative diseases, but immunological barriers limit their use in the clinic. While considerable work has been performed to decipher the role of the cellular immune response in the rejection of intracerebral xenotransplants, there is much still to learn about the humoral reaction. To this end, the IgG response to the transplantation of fetal porcine neural cells (PNC) into the rat brain was analyzed. Rat sera did not contain preformed antibodies against PNC, but elicited anti-porcine IgG was clearly detected in the host blood once the graft was rejected. Only the IgG1 and IgG2a subclasses were up-regulated, suggesting a T-helper 2 immune response. The main target of these elicited IgG antibodies was porcine neurons, as determined by double labeling in vitroand in vivo. Complement and anti-porcine IgG were present in the rejecting grafts, suggesting an active role of the host humoral response in graft rejection. This hypothesis was confirmed by the prolonged survival of fetal porcine neurons in the striatum of immunoglobulin-deficient rats. These data suggest that the prolonged survival of intracerebral xenotransplants relies on the control of both cell-mediated and humoral immune responses.

The administration of autologous (recipient-derived) tolerogenic dendritic cells (ATDCs) is under clinical evaluation. However, the molecular mechanisms by which these cells prolong graft survival in a donor-specific manner is unknown. Here, we tested mouse ATDCs for their therapeutic potential in a skin transplantation model. ATDC injection in combination with anti-CD3 treatment induced the accumulation of CD8+CD11c+T cells and significantly prolonged allograft survival. TMEM176B is an intracellular protein expressed in ATDCs and initially identified in allograft tolerance. We show that Tmem176b−/−ATDCs completely failed to trigger both phenomena but recovered their effect when loaded with donor peptides before injection. These results strongly suggested that ATDCs require TMEM176B to cross-present antigens in a tolerogenic fashion. In agreement with this, Tmem176b−/−ATDCs specifically failed to cross-present male antigens or ovalbumin to CD8+T cells. Finally, we observed that a Tmem176b-dependent cation current controls phagosomal pH, a critical parameter in cross-presentation. Thus, ATDCs require TMEM176B to cross-present donor antigens to induce donor-specific CD8+CD11c+T cells with regulatory properties and prolong graft survival.

Innovative therapeutic strategies are needed to diminish the impact of harmful immunosuppression in transplantation. Dendritic cell (DC)‐based therapy is a promising approach for induction of antigen‐specific tolerance. Using a heart allograft model in rats, we analyzed the immunoregulatory mechanisms by which injection of autologous tolerogenic DCs (ATDCs) plus suboptimal immunosuppression promotes indefinite graft survival. Surprisingly, we determined that Interferon‐gamma (IFNG), a cytokine expected to be propathogenic, was threefold increased in the spleen of tolerant rats. Importantly, its blockade led to allograft rejection [Mean Survival Time (MST) = 25.6 ± 4 days], showing that IFNG plays a critical role in immunoregulatory mechanisms triggered by ATDCs. IFNG was expressed by TCRαβ+CD3+CD4−CD8−NKRP1−cells (double negative T cells, DNT), which accumulated in the spleen of tolerant rats. Interestingly, ATDCs specifically induced IFNG production by DNT cells. ATDCs expressed the cytokinic chain Epstein‐Barr virus‐induced gene 3 (EBI3), an IL‐12 family member. EBI3 blockade or knock‐down through siRNA completely abolished IFNG expression in DNT cells. Finally, EBI3 blockade in vivoled to allograft rejection (MST = 36.8 ± 19.7 days), demonstrating for the first time a role for EBI3 in transplantation tolerance. Taken together our results have important implications in the rationalization of DC‐based therapy in transplantation as well as in the patient immunomonitoring follow‐up.

Innovative therapeutic strategies are needed to diminish the impact of harmful immunosuppression in transplantation. Dendritic cell (DC)-based therapy is a promising approach for induction of antigen-specific tolerance. Using a heart allograft model in rats, we analyzed the immunoregulatory mechanisms by which injection of autologous tolerogenic DCs (ATDCs) plus suboptimal immunosuppression promotes indefinite graft survival. Surprisingly, we determined that Interferon-gamma (IFNG), a cytokine expected to be propathogenic, was threefold increased in the spleen of tolerant rats. Importantly, its blockade led to allograft rejection [Mean Survival Time (MST) = 25.6 ± 4 days], showing that IFNG plays a critical role in immunoregulatory mechanisms triggered by ATDCs. IFNG was expressed by TCRαβ+CD3+CD4−CD8−NKRP1−cells (double negative T cells, DNT), which accumulated in the spleen of tolerant rats. Interestingly, ATDCs specifically induced IFNG production by DNT cells. ATDCs expressed the cytokinic chain Epstein-Barr virus-induced gene 3 (EBI3), an IL-12 family member. EBI3 blockade or knock-down through siRNA completely abolished IFNG expression in DNT cells. Finally, EBI3 blockade in vivoled to allograft rejection (MST = 36.8 ± 19.7 days), demonstrating for the first time a role for EBI3 in transplantation tolerance. Taken together our results have important implications in the rationalization of DC-based therapy in transplantation as well as in the patient immunomonitoring follow-up.

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4CD25T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4T cells to a secondary irradiated recipient, regulatory CD25Foxp3and CD25Foxp3?CD4T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFN?-dependent mechanism. Moreover, in vivotransfer of tolerance can be abrogated by blocking IFN? or IDO, and anti-IFN? reduces the survivalexpansion of alloantigen-induced regulatory Foxp3CD4T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4CD25T cells and the graft endothelial cells in this local immune privilege, and a key role for IFN? and IDO in this process.

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen‐presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short‐term treatment with a deoxyspergualine analogue, LF15‐0195, induces long‐term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+and CD25+Foxp3−CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3‐dioxygenase (IDO) by an IFNγ‐dependent mechanism. Moreover, in vivotransfer of tolerance can be abrogated by blocking IFNγ or IDO, and anti‐IFNγ reduces the survival/expansion of alloantigen‐induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNγ and IDO in this process.

Heme oxygenase-1 (HO-1) expression protects cells from a variety of cellular insults and inhibits inflammation. However, its role in the regulation of immune responses has not yet been clearly established. We generated HO-1 transgenic rats to directly test the impact of HO-1 on the different immune mechanisms. To temporally control the expression of HO-1, we used a one-plasmid tetracycline (tet)-inducible system. This plasmid contains the H-2Kbpromoter, which transcribes the tet transactivator (tTA) and expression of a human HO-1 cDNA is obtained in the absence of tetracycline. The DNA construct was microinjected into one-cell rat embryos and mothers and pups were maintained with tetracycline. Eight transgenic founders were obtained. Analysis of transgene expression in the absence of tet showed that 2 lines (12.4 and 12.6) expressed HO-1 mRNA in several organs (as detected by reverse transcription polymerase chain reaction) and at the protein level only in the thymus. Expression levels of transgene-derived HO-1 increased after withdrawal of tet compared with transgenic rats maintained with tet, as detected by analysis of mRNA levels by quantitative real-time reverse transcription polymerase chain reaction. Gross examination and histopathological analysis of several organs in both lines showed no anomalies. Thymocytes and splenocytes of both lines showed normal cell subpopulations and allogeneic proliferation compared with controls. Systemic immune responses against cognate antigens were normal in both lines, as evaluated by the proliferation of lymph node cells and the production of antibodies against keyhole limpet hemocyanin after immunization. Animals from line 12.6 rejected transplanted allogeneic hearts with the same kinetics as controls. In conclusion, short-term induction of HO-1 overexpression did not modify immune responses compared to those of control non-transgenic animals.

The hallmark of chronic rejection is the occlusion of the artery lumen by intima hyperplasia as a consequence of leukocyte infiltration and vascular smooth muscle cell (VSMC) migration and proliferation. Heme oxygenase-1 (HO-1) is a tissue protective molecule which degrades heme into carbon monoxide (CO), free iron and biliverdin. We analyzed the effects of HO-1 gene transfer into the vessel wall using an adenoviral vector (AdHO-1) and of CO delivery in a model of chronic allogeneic aorta rejection in rats. Carbon monoxide treatment was achieved by a new pharmacological approach in transplantation using methylene chloride (MC), which releases CO after degradation. AdHO-1-mediated gene transfer into aorta endothelial cells (ECs) or CO delivery resulted in a significant reduction in intimal thickness compared to untreated or noncoding adenovirus-treated controls. Aortas transduced with AdHO-1 or treated with CO showed a reduction in the number of leukocytes as well as in the expression of adhesion molecules, costimulatory molecules and cytokines, with the gene transfer treatment displaying a more pronounced effect than the CO treatment. Conversely, CO inhibited VSMC accumulation in the intima more efficiently than AdHO-1 treatment. Gene transfer of HO-1 and pharmacological manipulation of CO are novel approaches to the analysis and treatment of chronic rejection.

We have tested Leukaemia Inhibitory Factor (LIF) production by 12 rat colon tumour clones isolated from a single cell line that display various degrees of tumorigenicity A highly significant relationship was found between levels of soluble LIF produced by the clones and their in vivo tumorigenicity. Such results suggested a role for LIF as a tumour facilitating agent. To test this hypothesis, the highly tumorigenic and LIF producing PROb clone was transfected with the LIF cDNA in antisense orientation in order to decrease LIF production. Conversely, REGb, a low LIF producer that is rejected by syngeneic animals, as well as nude mice, was transfected with the LIF cDNA to increase its production. PROb cells transfected with antisense cDNA were shown to have decreased LIF production along with decreased tumorigenicity. LlF-transfected REGb cells expressing high LIF levels still regressed in syngeneic rats, but could form progressive tumours in nude mice. We did not detect LIF receptors on PROb or REGb cells and their in vitro proliferation was not modified by the addition of exogenous LIF. Therefore, LIF was not an autocrine growth regulator for PROb and REGb cells. Instead, LIF appears to facilitate in vivo tumour growth, without being an immunosuppressive factor sufficient on its own to allow growth of immunogenic cells in fully immunocompetent hosts.