BioProject

The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. More...

The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from four patients who experienced severe acute rejection leading to graft loss. Alloreactive T-cell receptors (TCRs) clones were identified in pre-transplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of TRM cells expressing an alloreactive TCR in the four kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, alloreactive clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype. Overall design: Mixed lymphocyte reactions (MLRs) were performed to identify alloreactive T cells. In brief, frozen donor splenocytes and recipient PBMCs were thawed on the day of the experiment. Donor splenocytes were labeled with CellTrace™ Violet Cell Proliferation dye (VPD; Thermo Fisher Scientific, Waltham, MA, USA) and subsequently exposed to gamma irradiation (40 Gy). Recipient PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific). Subsequently, irradiated VPD-labeled donor cells and CFSE-labeled recipient cells were cocultured for 6 days. After 6 days of coculture, cells were harvested and stained with 7-AAD (BD Biosciences, Franklin Lakes, NJ, USA) and CD3 (Biolegend, San Diego, CA, USA). Viable CD3+Violet-lowCFSE-low cells were then sorted using a FACS Aria II cell sorter (BD Biosciences). To verify the purity of the gated population, a fraction of the sorted population was subjected to flow cytometry analysis. This assessment demonstrated that all samples had a purity level above 97%. Less...