OBJECTIVE: The objective of this study was to determine whether tolerance to neonatal porcine islet (NPI) xenografts could be achieved by short-term administrations of anti–LFA-1 and anti-CD154 monoclonal antibodies (mAbs).RESEARCH DESIGN AND METHODS: Diabetic B6 mice received NPI transplants and short-term injections of combined anti–LFA-1 and anti-CD154 mAbs. Mice with long-term islet graft function were treated with depleting anti-CD25 mAb or re-transplanted with a second-party NPI. At the end of the study, grafts from mice with long-term islet function were examined. Their spleen cells were characterized and used for in vitro proliferation and adoptive transfer studies.RESULTS: All mAb-treated NPI recipients maintained normoglycemia for >100 days post-transplantation. Only 5 of 50 mice rejected their grafts before 300 days post-transplantation. Intact islets, foxp3+ immune cells, as well as interleukin (IL)-10 and transforming growth factor (TGF)-β regulatory cytokine transcripts were detected in the NPI xenografts from tolerant mice. A higher percentage of CD4+ T-cell population from these mice expressed regulatory markers, suggesting that tolerance to NPI xenografts may be mediated by T regulatory cells. This was confirmed when tolerant mice treated with depleting anti-CD25 mAb became diabetic. Lymphocytes from tolerant mice inhibited the proliferation of lymphocytes from B6 mice immunized with porcine cells and they displayed limited proliferation when adoptively transferred. All protected B6 mice transplanted with a second-party NPI xenograft maintained long-term normoglycemia even after removal of the first NPI graft-bearing kidney.CONCLUSIONS: These results demonstrate that tolerance to NPI xenografts can be achieved by transient administrations of combined anti–LFA-1 and anti-CD154 mAb therapy.

Oxidative stress is a major cause of islet injury and dysfunction during isolation and transplantation procedures. Cyanidin-3-O-glucoside (C3G), which is present in various fruits and vegetables especially in Chinese bayberry, shows a potent antioxidant property. In this study, we determined whether C3G could protect neonatal porcine islets (NPI) from reactive oxygen species (H₂O₂)-induced injury in vitro and promote the function of NPI in diabetic mice. We found that C3G had no deleterious effect on NPI and that C3G protected NPI from damage induced by H₂O₂. Significantly higher hemeoxygenase-1 (HO1) gene expression was detected in C3G-treated NPI compared to untreated islets before and after transplantation (P < 0.05). Western blot analysis showed a significant increase in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K/Akt) proteins in C3G-treated NPI compared to untreated islets. C3G induced the nuclear translocation of nuclear erythroid 2-related factor 2 (NRF2) and the significant elevation of HO1 protein. Recipients of C3G-treated NPI with or without C3G-supplemented drinking water achieved normoglycemia earlier compared to recipients of untreated islets. Mice that received C3G-treated islets with or without C3G-supplemented water displayed significantly lower blood glucose levels at 5–10 weeks post-transplantation compared to mice that received untreated islets. Mice that received C3G-treated NPI and C3G-supplemented drinking water had significantly (P < 0.05) lower blood glucose levels at 7 and 8 weeks post-transplantation compared to mice that received C3G-treated islets. These findings suggest that C3G has a beneficial effect on NPI through the activation of ERK1/2- and PI3K/AKT-induced NRF2-mediated HO1 signaling pathway.

Background It was previously shown that islets are susceptible to oxidative stress due to their inherent low antioxidant capacity. Therefore, in this study, we determined whether treatment of mouse islets with an antioxidant cyanidin-3-O-glucoside (C3G) could enhance their function after transplantation under the kidney capsule or into the portal vein. Methods B6 mouse islets were treated with various concentrations of C3G, their viability, and the expression of antioxidant (HO-1) and antiapoptotic (Bcl-2 and Survivin) genes were determined. The C3G-treated (1.0 μM) or untreated B6 mouse islets (100, 200, and 400 IEQ) were transplanted under the kidney capsule or into the portal vein of diabetic B6 mice, and their blood glucose levels were monitored for more than 100 days after transplantation. Results The C3G-treated islets showed higher cell viability compared to untreated control and the expression of HO-1; Bcl-2 and Survivin genes were enhanced in a concentration-dependent manner. All mice that were transplanted with C3G-treated islets achieved normoglycemia faster than recipients of untreated islets. Mice that received 400, 200, or 100 treated islets transplanted under the kidney capsule achieved normoglycemia, whereas only mice that were transplanted with 400 and 200 treated islets into the portal vein achieved normoglycemia. The mean blood glucose levels of mice that received C3G-treated islets were lower compared to those observed in mouse recipients of untreated islets transplanted under the kidney capsule or into the portal vein. Conclusions Our results show that C3G could enhance the viability of mouse islets and improve their function after transplantation.

Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted Ag did not compromise tolerance. In this study, we determined that murine T cells that failed clonal deletion were rendered functionally impaired in the thymus. Programmed cell death protein 1 (PD-1) was induced in the thymus and was required to establish cell-intrinsic tolerance to tissue-restricted Ag in CD8+ thymocytes independently of clonal deletion. In bone marrow chimeras, tolerance was not observed in PD-L1–deficient recipients, but tolerance was largely maintained following adoptive transfer of tolerant thymocytes or T cells to PD-L1–deficient recipients. However, CRISPR-mediated ablation of PD-1 in tolerant T cells resulted in broken tolerance, suggesting different PD-1 signaling requirements for establishing versus maintaining tolerance. Finally, we showed that chronic exposure to high-affinity Ag supported the long-term maintenance of tolerance. Taken together, our study identifies a critical role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion. It also sheds light on potential mechanisms of action of anti–PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.