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

Introduction 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. Methods 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). Results 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. Conclusions 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.