Deep Immune Profiling of Nonchimeric Tolerance of Transplants in Nonhuman Primates

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Abstract Transplant (tx) tolerance would profoundly improve the lives of thousands of people who will receive tx in the years ahead. Recently, we reported safe and consistent induction of long-term (>1 year) tolerance to islet allografts in nonhuman primates (NHPs) with two peritx infusions of apoptotic donor leukocytes (ADLs) under induction immunosuppression (IIS) (in part supported by U01-AI102463). While these findings are unprecedented and suggest the possibility of nonchimeric tx tolerance in humans, further insight into the mechanisms by which tolerance is induced and maintained will be required before clinical translation of the protocol can be initiated. Owing to the progress made in the epigenetic, transcriptomic and cytometric profiling of single cells and computational analyses of high-dimensional datasets, the contributions of distinct immune cell subsets and the graft to tolerance can now be investigated with previously unimaginable explicitness. Accordingly, the goal of the proposed studies is to harness these novel tools and existing samples to delineate the critical factors and mechanisms responsible for tx tolerance induced by the ADLs+IIS regimen. To this end, we have established techniques for i) tracking and sorting allospecific CD4+ T cells, ii) single-cell ATAC- & RNA- sequencing of these sorted cells, iii) digital spatial profiling (DSP) of grafts and graft microenvironments, and iv) mass cytometry profiling of circulating mononuclear cells with four macaque-validated CyTOF panels. In Aim 1, we will dissect the molecular diversity of circulating allospecific CD4+ T cells from tolerant and nontolerant tx recipients in an unbiased manner by single-cell ATAC & RNA sequencing, testing the hypothesis that the ADLs+IIS regimen causes i) exhaustion, ii) failure to acquire a memory phenotype and iii) expansion and activation of regulatory subsets in CD4+ T cells via epigenetic and transcriptomic alterations. In Aim 2, we will leverage DSP to investigate the role of the graft in the maintenance of tx tolerance, testing the hypothesis that ADLs+IIS alters intragraft transcriptomes of co-inhibitory and protective molecules that determine immune cell recruitment to islet and kidney grafts and their survival. In Aim 3, we will utilize high-dimensional CyTOF profiling of circulating mononuclear cells (MNCs) with existing as well as novel multiomic-guided panels focused on exhausted/memory (Tex/mem) and regulatory (Treg and Tr1) T cells, testing the hypothesis that the same key heterogeneous clusters among Tex, Treg and Tr1 cell populations are associated with islet and kidney tx tolerance induced by ADLs+IIS. The studies proposed herein will be the first to leverage the power of epigenetic, transcriptomic and cytometric analyses of single cells for investigation of mechanisms of peripheral tx tolerance in NHPs. The datasets generated and analyzed will present a resource for investigating the immunobiology of nonchimeric tolerance, with implications for biomarker discovery and clinical translation of the ADLs+IIS regimen.