The role of the spleen in maintaining immune tolerance to transplanted islets - Project Summary Transplantation of islets has restored tight glycemic control in patients with type 1 diabetes, but the toxicity of chronic immunosuppression used to prevent rejection has precluded the application of cell replacement in diabetes care. By inducing immune tolerance to transplanted islets, we achieved long-term, drug-free islet transplant survival in nonhuman primates (NHPs). To justify the clinical translation of the protocol, a deep understanding of the immunobiology that determines maintenance and loss of immune tolerance is required. Using highly multiplexed immunophenotyping and imaging methods customized for NHPs, our preliminary studies comparing tissues from tolerant vs. rejecting NHPs led us to hypothesize that the spleen plays a crucial role in maintaining tolerance to islet transplants in macaques. The retention of allospecific CD4+ T cells, the abundance of exhausted T cells, their spatial arrangement within the spleen, and the ongoing Treg vs. Teff cell crosstalk via the Areg-EGFR axis are key contributors to sustained tolerance. We propose two Specific Aims to address this hypothesis. AIM 1: To comprehensively profile allospecific Areg+ ST2+ Tregs and immune cell clusters in the spleen testing the hypothesis that sustained tolerance to islet allografts in NHPs is linked to an abundance of unique Tregs and Tex cell clusters in the spleen. Splenic Tregs in tolerant NHPs are enriched with elevated Areg and ST2 expression and exhibit an elevated interaction with Teff and Tex cells via Areg-EGFR signaling. AIM 2: To analyze the spatial interaction of allospecific Areg+ ST2+ Tregs with Teff, Tex, and myeloid cells clusters in the spleen testing the hypothesis that the retention of allospecific effector cells in special microdomains of the spleen and their subsequent exhaustion, caused by their proximity to Areg+ ST2+ Tregs in the spleen, plays a role in the consolidation of tolerance to islet tx The studies proposed herein will be the first to harness the capabilities of highly multiplexed immuno-phenotyping of single cells and tissues to investigate the role of Areg-EGFR signaling in splenic Teff cell retention and their subsequent exhaustion in maintaining nonchimeric transplant tolerance in NHPs. The generated dataset will represent a valuable resource for the transplant tolerance community, trigger additional studies on the anatomy of tolerance in the spleen, and promote the clinical translation of tolerance induction to donor- and stem cell- derived islet cell transplants.
