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Background: Xenogeneic islet transplantation is an emerging therapeutic option for diabetic patients. However, immunological tolerance to xenogeneic islets remains a challenge., Methods: The current study used a pig-to-mouse discordant xenogeneic islet transplant model to examine antidonor xenogeneic immune responses during early and late rejection and to determine experimental therapeutic interventions that promote durable pig islet xenograft survival., Results: We found that during early acute rejection of pig islet xenografts, the rejecting hosts exhibited a heavy graft infiltration with B220 B cells and a robust antipig antibody production. In addition, early donor-stimulated IL-17 production, but not IFN-γ production, dominated during early acute rejection. Recipient treatment with donor apoptotic 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide-treated splenocytes significantly inhibited antidonor IL-17 response, and when combined with B cell depletion and a short course of rapamycin led to survival of pig islet xenografts beyond 100 days in approximately 65% recipients. Interestingly, treated recipients in this model experienced late rejection between 100 and 200 days posttransplant, which coincided with B cell reconstitution and an ensuing emergence of a robust antidonor IFN-γ, but not IL-17, response., Conclusions: These findings reveal that early and late rejection of pig islet xenografts may be dominated by different immune responses and that maintenance of long-term xenogeneic tolerance will require strategies that target the temporal sequence of antixenogeneic immune responses.

Toll-like receptor 5 (TLR5) signaling in response to flagellin is dispensable for inducing humoral immunity, but alterations of aa 89-96, the TLR5 binding site, significantly reduced the adjuvanticity of flagellin. These observations indicate that the underlying mechanism remains incompletely understood. Here, we found that the native form of Salmonella typhimurium aa 89-96-mutant flagellin extracted from flagella retains some TLR5 recognition activity, indicating that aa 89-96 is the primary, but not the only site that imparts TLR5 activity. Additionally, this mutation impaired the production of IL-1β and IL-18. Using TLR5KO mice, we found that aa 89-96 is critical for the humoral adjuvant effect, but this effect was independent of TLR5 activation triggered by this region of flagellin. In summary, our findings suggest that aa 89-96 of flagellin is not only the crucial site responsible for TLR5 recognition, but is also important for humoral immune adjuvanticity through a TLR5-independent pathway.