Discovering molecular signatures in kidney transplant biopsies with borderline changes and isolated V-lesions: single-cell RNA-sequencing analysis of human blood and tissue Spatial transcriptomics

Acute T cell-mediated rejection (TCMR) is one of the most important causes of kidney graft injury and loss; however, in-depth transcriptomic analyses of relevant tissues and circulating immune cells are limited. In this study, we conducted single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells and spatial transcriptomics using kidney allograft biopsy samples. The scRNA-seq study included four patients with borderline rejection or biopsy-proven TCMR and two with no evidence of TCMR. For spatial analysis, we compared tissue specimens from one patient diagnosed with borderline rejection and another patient with TCMR to their time-zero protocol biopsies. Six regions of interest per biopsy section were selected, focusing on the evident T-cell infiltration area in the tubulointerstitium and glomeruli. Integrating two methodologies, we pinpointed CD8⁺ effector memory T cell (T_EM) expression profiles and key upregulated genes, including LTB, GZMK, PSME2, UBE2L6, and STAT1. Among them, STAT1 was confirmed as a hub gene through network and pathway analysis. Immunohistochemistry and immunofluorescence on kidney allograft tissue validated the co-expression of STAT1 with CD8, indicating an active inflammatory response. Our findings suggest the presence of a CD8⁺ STAT1⁺ T_EM subset with features of clonal expansion, providing additional insight into the immunological processes associated with TCMR.