*Purpose: Development of T-cell hyporesponsiveness to donor antigen may explain the substantial decrease in the risk for acute rejection in the years following kidney transplantation. The underlying mechanisms of donor-specific hyporesponsiveness (DSH) are largely unknown but may allow for lowering of immunosuppressive medication. This study aimed to test the hypothesis that donor-specific recipient T cells become hyporesponsive due to exhaustion from continuous stimulation by donor antigen.

*Methods: Peripheral blood mononuclear cells (PBMCs) of stable kidney transplant recipients (N=17) before and 3-5 years after kidney transplantation were stimulated with CD3-negative donor cells for 18-24 hours. Donor-specific T lymphocytes were identified at the single-cell level by CD137 (marker for antigen-specific T cells) and characterized for exhaustion marker expression by multi-parameter flow cytometry. Analysis was performed through unsupervised and unbiased clustering using Flow Self-Organizing Map (FlowSOM), a dimensionality reduction technique. The FlowSOM clusters containing cells of a particular expression profile with significant differential abundance after transplantation were identified using the statistical method, edgeR, via diffcyt.

*Results: Unexpectedly, our results do not demonstrate an increase in exhausted donor antigen-specific T cells post transplantation. Instead, the study shows a significant decrease in donor antigen-specific CD4+ T cells expressing T cell immunoglobulin and ITIM domain (TIGIT) long after transplantation. Further analysis at earlier timepoints indicated that this decrease is already present at six months post transplantation. Although TIGIT is often thought of as a marker for exhausted T cells, characterization of CD4+ T cells expressing TIGIT revealed these cells to have a predominantly central and effector memory T cell phenotype and a highly polyfunctional cytokine expression profile.

*Conclusions: This study has identified TIGIT as a marker for a previously undescribed polyfunctional donor-specific CD4+ T cell population whose decline after kidney transplantation may underlie the phenomenon of DSH. Prospective clinical studies could help determine whether a low frequency of donor-specific TIGIT-expressing CD4+ T cells could guide lowering of immunosuppressive drugs.

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