Single Cell Transcriptomic Analysis of Renal Allograft Rejection Reveals Novel Insights into Intra-graft TCR Clonality and Rejection Mechanisms

Abstract Text: Alloreactive expansion and transcriptomic heterogeneity of intragraft T-cells is poorly understood. Here, we characterize clonally expanded CD8+ T-cell populations (CD8EXP) during acute renal allograft rejection (AR) under various immunosuppression (IS) regimens. Biopsy samples were obtained at the time of initial AR diagnosis from 10 patients on either iscalimab (anti-CD40 mAb), belatacept (CTLA4-Ig), or tacrolimus maintenance IS and subjected to 5’ single-cell RNA + TCRα/β sequencing. Seurat and Loupe V(D)J analysis identified differentially expressed genes (DEGs) between individual CD8EXP clonotypes. Expanded clonotypes were defined as >2 cells with identical TCRα/β.

Initial AR samples revealed a stunning degree of TCR restriction with original CD8EXP observed at initial AR remaining present in ongoing rejection biopsies as well as paired urine samples. In contrast, CD8EXP were largely eliminated upon rejection resolution. Transcriptomes of CD8EXP demonstrated a variety of effector states, and co-culture of Jurkat76 cell lines expressing CD8EXP-specific TCRs with donor PBMCs revealed alloreactivity of CD8EXP. Intriguingly, a resident memory phenotype was found in patients with persistent rejection and eventual graft loss. Finally, DEGs suggested that the type of IS significantly affected gene expression of CD8EXP, which was further analyzed through immunostaining.

In conclusion, AR is characterized by a remarkably limited number of distinct CD8EXP that persist in ongoing rejection and disappear with rejection resolution, which is also reflected in paired urine samples. Persistent CD8EXP develop a resident memory phenotype that may be associated with allograft loss, and transcriptional profiles of the CD8EXP are distinct and dependent on the type of IS therapy.