Intragraft clonal expansion of cytotoxic CD8⁺ and CD4⁺ T cells in antibody-mediated kidney transplant rejection.

Abstract

The traditional dichotomy between antibody-mediated rejection (AMR) and T cell- mediated rejection (TCMR) after kidney transplantation is increasingly challenged. While T cell populations and their clonal expansion have been demonstrated in TCMR, the involvement and clonality of T cells remains unexplored in AMR. Kidney transplant biopsy datasets from four independent cohorts (192 cases bulk transcriptomics, 16 cases of single-cell RNA/TCR sequencing, along with 18 and 75 cases of multiplex immunostaining) were analyzed to characterize CD4⁺ and CD8⁺ T cell enrichment, clonal expansion and spatial organization in AMR and microvascular inflammation (MVI). Deconvolution of bulk transcriptomes showed a significant enrichment of CD8⁺ T cells in 33 AMR/MVI samples versus 139 no rejection, with comparable levels between AMR/MVI and 20 (borderline)TCMR cases. Unsupervised clustering of scRNA-Seq data identified five major T cell clusters. Clonal expansion was mostly present in CD8⁺ Tem/Temra cells, CD4⁺ cytotoxic T cells and CD8⁺ HLA-DR⁺ CD27⁺ T cells. The CD4⁺ Naïve/Tcm cluster was mostly not expanded. The last CD8⁺ cluster was characterized by early activation markers (CD69⁺, FOS⁺, JUN⁺) and intermediate clonality. The four AMR biopsies had the highest proportion of clonally expanded T cells, most notably in early AMR cases in patients with pre-existing donor-specific antibodies without histological diagnosis of TCMR in three cases. Both the fraction of expanded T cells and the number of distinct expanded clones correlated with the PIRCHE-II score, which estimates the number of donor HLA-derived epitopes presented by the recipient's HLA molecules. Finally, spatial proteomics confirmed the T-cell enrichment in the intravascular and glomerular compartments of AMR/MVI biopsies. We demonstrated the presence of clonally expanded CD4⁺ and CD8⁺ cytotoxic T cells in early AMR biopsies with pre-existing donor-specific antibodies without histological diagnosis of TCMR. These findings highlight a level of complexity in the cellular mechanisms of rejection not currently captured by routine histology, suggesting that the dichotomy between AMR and TCMR needs reconsidering.