Single cell transcriptomic analysis of renal allograft rejection reveals novel insights into intragraft TCR clonality

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCRα/β sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8 ⁺ T cell clonal expansion (CD8 _EXP ), independent of HLA mismatch or IS type. Subcloning of TCRα/β cDNAs from CD8 _EXP into Jurkat76 cells (TCR ^-/- ) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8 _EXP revealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8 _EXP , while CD8 _EXP were maintained during treatment-refractory rejection. Finally, most rBx-derived CD8 _EXP were also observed in matching urine samples. Overall, our data define the clonal CD8 ⁺ T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.