A major challenge for human allogeneic islet transplantation is the development of effective methods to induce donor-specific tolerance to obviate the need for life-long immunosuppression that is toxic to the insulin-producing ␤ cells and detrimental to the host. We developed an efficient donor-specific tolerance therapy that utilizes infusions of ethylene carbodiimide (ECDI)-treated donor splenic antigen-presenting cells that results in indefinite survival of allogeneic islet grafts in the absence of immunosuppression. Furthermore, we show that induction of tolerance is critically dependent on synergistic effects between an intact programmed death 1 receptorprogrammed death ligand 1 signaling pathway and CD4 ؉ CD25 ؉ Foxp3 ؉ regulatory T cells. This highly efficient antigen-specific therapy with a complete avoidance of immunosuppression has significant therapeutic potential in human islet cell transplantation.anergy ͉ programmed death-1 ͉ regulatory T cells ͉ transplantation ͉ islet transplantation

CD4 ؉ CD25 ؉ Foxp3 ؉ regulatory T cells (T regs) are important for preventing autoimmune diabetes and are either thymic-derived (natural) or differentiated in the periphery outside the thymus (induced). Here we show that ␤-cell peptide-pulsed dendritic cells (DCs) from nonobese diabetic (NOD) mice can effectively induce CD4 ؉ CD25 ؉ Foxp3 ؉ T cells from naïve islet-specific CD4 ؉ CD25 ؊ T cells in the presence of TGF-␤1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro. When cotransferred with diabetogenic cells into NOD scid recipients, T regs induced with DCs and TGF-␤1 prevent the development of diabetes. Furthermore, in overtly NOD mice, these cells are able to significantly protect syngeneic islet grafts from established destructive autoimmunity. These results indicate a role for DCs in the induction of antigen-specific CD4 ؉ CD25 ؉ Foxp3 ؉ T cells that can inhibit fully developed autoimmunity in a nonlymphopoenic host, providing an important potential strategy for immunotherapy in patients with autoimmune diabetes.antigen-presenting cells ͉ autoimmunity ͉ type 1 diabetes ͉ nonobese diabetic (NOD) mice T he nonobese diabetic (NOD) mouse models the pathogenesis of human type 1 diabetes and allows the study of potential therapeutics (1). In NOD mice, the thymic-derived CD4 ϩ CD25 ϩ regulatory T cells (T regs) expressing the transcription factor Foxp3 suppress autoimmunity and delay the development of diabetes (2). Considerable effort has focused on expanding the small numbers of these so-called ''natural'' CD4 ϩ CD25 ϩ T regs in the NOD and other models (3-6). Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that can effectively expand and sustain antigen-specific CD4 ϩ CD25 ϩ T regs (3, 4). However, in humans, only the infrequent cells with high CD25 expression have regulatory function (7). Alternatively, the more abundant CD4 ϩ CD25 Ϫ T cells can be differentiated into CD4 ϩ CD25 ϩ T regs that express Foxp3 by stimulation with mitogenic antibodies in the presence of TGF-␤1, although it is not known whether these induced cells are functionally identical to T regs that develop in the thymus (8). Such ''induced T regs'' with islet specificity can prevent diabetes in lymphopoenic models (9), but their ability to induce tolerance at late pathogenic stages of autoimmunity, such as in already-diabetic NOD mice, has not been fully addressed.A separate issue that remains to be addressed is the requirement for APCs in the induction of T regs with TGF-␤1. The use of DCs instead of mitogenic stimuli to differentiate T regs de novo from CD4 ϩ CD25 Ϫ T cells has numerous potential advantages, including selection and maintenance of antigen specificity (10, 11); provision of paracrine TGF-␤1 (12-14), which is known to play an important role in T reg homeostasis by its ability to induce Foxp3 (8, 15); and/or provision of costimulatory signals such as CD80/86 for CTLA-4 ligation, which is necessary f...

Strategic exposure to donor antigens prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pre-transplant infusion of donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allo-specificities. To target indirect allo-specific T cells, ECDI-SPs induce up-regulation of negative, but not positive, co-stimulatory molecules on recipient splenic CD11c+ DCs phagocytosing the injected ECDI-SPs. Indirect allo-specific T cells activated by such CD11c+ DCs undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allo-specific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4+CD25+Foxp3+ T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SPs infusions target host allogeneic responses via a multitude of mechanisms including clonal depletion, anergy and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.

Background-Epithelial barrier dysfunction is thought to play a role in many mucosal diseases including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis (EoE).

Background We have previously shown that Oncostatin M (OSM) is elevated in nasal polyps of chronic rhinosinusitis (CRS) patients, as well as in bronchoalveolar lavage (BAL) fluids after segmental allergen challenge in allergic asthmatics. We also showed in vitro that physiological levels of OSM impair barrier function in differentiated airway epithelium. Objective We sought to determine which hematopoietic or resident cell type(s) were the source of the OSM expressed in mucosal airways disease. Methods Paraffin-embedded NP sections were stained with fluorescence-labeled specific antibodies against OSM, GM-CSF and hematopoietic cell specific markers. Live cells were isolated from NP and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood, cultured with the known OSM inducers GM-CSF and FSTL1, and levels of OSM were measured in the supernatants. Bronchial biopsy sections from controls, moderate asthmatics and severe asthmatics were stained for OSM and neutrophil elastase. Results OSM staining was observed in NP, showed co-localization with neutrophil elastase (n=10), and did not co-localize with markers for eosinophils, macrophages, T cells or B cells (n=3–5). Flow cytometric analysis of NP (n=9) showed that 5.1±2% of CD45+ cells were OSM+, and of the OSM+ cells, 56±7% were CD16+Siglec8−, indicating neutrophil lineage. Only.6±.4% of CD45+ events from matched blood samples (n=5) were OSM+, suggesting that elevated OSM in CRS was locally stimulated and produced. A majority of OSM+ neutrophils expressed Arginase 1 (72.5±12%), suggesting a N2 phenotype. GM-CSF was elevated in nasal polyp tissue compared to control, and was sufficient to induce OSM production (p<.001) in peripheral blood neutrophils in vitro. OSM+ neutrophils were also observed at elevated levels in biopsies from patients with severe asthma. Additionally, OSM protein was elevated in induced sputum from asthmatic patients compared to controls (p<.05). Conclusions Neutrophils are a major source of OSM producing cells in CRS and severe asthma.