Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance.

Tolerance of mouse kidney allografts arises in grafts that develop regulatory tertiary lymphoid organs (rTLOs). Single-cell RNA-seq (scRNA-seq) data and adoptive transfer of alloreactive T cells after transplantation showed that cytotoxic CD8+ T cells are reprogrammed within the accepted graft to an exhausted/regulatory-like phenotype mediated by IFN-γ. Establishment of rTLOs was required because adoptive transfer of alloreactive T cells prior to transplantation results in kidney allograft rejection. Despite the presence of intragraft CD8+ cells with a regulatory phenotype, they were not essential for the induction and maintenance of kidney allograft tolerance since renal allotransplantation into CD8-KO recipients resulted in acceptance and not rejection. Analysis of scRNA-seq data from allograft kidneys and malignant tumors identified similar regulatory-like cell types within the T cell clusters and trajectory analysis showed that cytotoxic CD8+ T cells are reprogrammed into an exhausted/regulatory-like phenotype intratumorally. Induction of cytotoxic CD8+ T cell dysfunction of infiltrating cells appears to be a beneficial mechanistic pathway that protects the kidney allotransplant from rejection through a process we call "defensive tolerance." This pathway has implications for our understanding of allotransplant tolerance and tumor resistance to host immunity.

Intragraft B cell differentiation during the development of tolerance to kidney allografts is associated with a regulatory B cell signature revealed by single cell transcriptomics.

Mouse kidney allografts are spontaneously accepted in select, fully mismatched donor-recipient strain combinations, like DBA/2J to C57BL/6 (B6), by natural tolerance. We previously showed accepted renal grafts form aggregates containing various immune cells within 2 weeks posttransplant, referred to as regulatory T cell-rich organized lymphoid structures, which are a novel regulatory tertiary lymphoid organ. To characterize the cells within T cell-rich organized lymphoid structures, we performed single-cell RNA sequencing on CD45+ sorted cells from accepted and rejected renal grafts from 1-week to 6-months posttransplant. Analysis of single-cell RNA sequencing data revealed a shifting from a T cell-dominant to a B cell-rich population by 6 months with an increased regulatory B cell signature. Furthermore, B cells were a greater proportion of the early infiltrating cells in accepted vs rejecting grafts. Flow cytometry of B cells at 20 weeks posttransplant revealed T cell, immunoglobulin domain and mucin domain-1+ B cells, potentially implicating a regulatory role in the maintenance of allograft tolerance. Lastly, B cell trajectory analysis revealed intragraft differentiation from precursor B cells to memory B cells in accepted allografts. In summary, we show a shifting T cell- to B cell-rich environment and a differential cellular pattern among accepted vs rejecting kidney allografts, possibly implicating B cells in the maintenance of kidney allograft acceptance.