We created a physiological model of islet injury by transplanting islet preparations with 50% purity (by adding exocrine debris). It is worth noting that our standard islet purity after isolation is >90%. We observed that WT islets of 50% purity did not restore euglycemia, whereas transplantation of TLR2/4−/− islets cured diabetes despite the presence of exocrine debris (Fig. 3A). WT islets of 50% purity expressed more intragraft proinflammatory cytokines, macrophages and T cells compared with TLR2/4−/− islets (Fig. 3B), showing that debris activated islet TLR2/4, and exaggerated the inflammatory response synergistically. By day 7 post-transplant,
the inflammatory response had subsided. We and others have recently shown that purified islets deficient in TLR2, TLR4, or MyD88 were rejected at the same tempo as WT controls when transplanted into untreated check details allogeneic recipients 16, 17. We also found increased endogenous TLR ligands in allografts, including HMGB1 16. Thus, we determined whether TLR2/4−/− islets allografts resulted in improved glucose reduction and lower intragraft see more inflammation. A marginal mass of untreated allogeneic TLR2/4−/−
islets produced only a modestly better glucose reduction in contrast to WT islets (Fig. 4A) but the absence of TLR2/4 signaling was linked with lower levels of TNF-α, IP-10, and IL-1β, and decreased macrophage and T-cell recruitment (Fig. 4B). These experiments support a role for TLR2/4 in sensing islet injury. It is currently unknown whether the reduced inflammatory state affects allograft survival in the context of subtherapeutic immunosuppression. Since early
islet dysfunction is associated with mononuclear cell chemoattractants and mononuclear cell infiltrates, we tested whether after TLR stimulation T cells are requisite pathogenic mediators of impaired islet engraftment. Syngeneic transplants were placed into T-cell-deficient nude mice. In striking contrast to the observed effects of TLR stimulation on engraftment in WT recipients, LPS- or PGN-stimulated islets engrafted in all nude recipients, rapidly normalizing serum glucose levels (Fig. 5A). To identify which T-cell subset was responsible for preventing engraftment, additional transplants into CD4- or CD8-deficient recipients were performed. TLR-stimulated DOK2 islets did not engraft in CD4−/− mice (all animals remained hyperglycemic), indicating that CD8+ T cells were sufficient to prevent engraftment. On the contrary, TLR-stimulated islets normalized serum glucose values following transplantation into diabetic CD8−/− recipients, albeit with slightly delayed kinetics (Fig. 5B). Both TLR2 and TLR4 stimulated islets resulted in euglycemia when transplanted into CD8-deficient mice, but had higher area under the curve (AUC) on day 7 compared with nude mice, indicating some effects of CD4+ T cells (Fig. 5C).