PROJECT SUMMARY γδ T cells can recognize diverse antigens and exert disparate functions. The innate- and adaptive-like features of human γδ T cells may be driven by differential γδ T cell receptor (TCR) repertoires, which can be shaped by tissue compartmentalization, age and history of antigen exposure. Despite comprising a significant proportion of resident T cells in many organs, including gut and liver, γδ T cells and their possible role in transplantation outcomes are largely under‐researched. Our overarching goals are to elucidate the fundamental mechanisms of how γδ T cells participate in allograft rejection and host defense after human intestinal transplantation (ITx). We revealed that clinical outcomes after ITx are largely determined by the balance between graft-versus-host (GvH) and host-versus-graft (HvG) alloreactivities, using an approach that identifies alloreactive T cell clones following pre-Tx mixed lymphocyte reactions on the basis of TCRβ CDR3 high throughput sequencing (TCRβ-seq). We showed that GvH-reactive αβ T cells, expanded from preexisting donor resident memory T cells (TRM) after encountering rapidly-repopulating recipient antigen-presenting cells in the graft mucosa, acquire effector T cell (Teff) functions and migrate into circulation and bone marrow (BM), where they attenuate HvG responses, facilitate donor cell engraftment and control rejection. Using single cell RNA-seq, we found that BM-infiltrating donor γδ T cells, dominated by “public” Vδ2 clonotypes, showed cytotoxic Teff phenotypes similar to CD8 αβ T cells, suggesting their potential to also attenuate HvG reactions and make space for donor hematopoietic stem and progenitor cell engraftment. We found that recipient γδ T cells gradually populated the allograft, undergoing phenotypic changes from Teff to TRM, mainly with “private” Vδ1 clonotypes. We hypothesize that γδ T cells not only participate in host defense against pathogens, but also have the potential to modulate two-way alloresponses after human ITx. We now propose a study of phenotypic and clonal tracking of human γδ T cells locally and systemically after ITx and further investigation of their regulatory roles on alloreactivity and association with graft outcomes. We propose to pursue three Specific Aims: To determine the chimerism, phenotype and clonotype of donor- (Aim 1) and recipient- (Aim 2) derived γδ T cells in graft mucosa, circulation and BM by flow cytometry, mass cytometry and TCRγδ-seq. Bulk TCRγδ-seq of pre-Tx unstimulated and ex vivo-stimulated γδ T cell repertoires and post-Tx γδ T cell repertoires in the circulation, intestinal allograft and BM of ITx patients will be linked with single cell transcriptional profiles of γδ T cell using the iRepertoire and 10x Genomics platforms (Aim 3). Our proposed research will provide a deeper understanding of the mechanisms behind γδ T cell chimerism, maturation of TRM features, and their modulatory roles on local and systemic alloresponses, facilit...