Introduction: Although CD8+ T cells are the major drivers of acute cellular rejection (ACR) across multiple organs, little is known about their biology and persistence during rejection.
Methods: We performed single cell RNA + TCR-CDR3αβ sequencing (scRNAseq, scTCRseq) of renal allograft biopsies from 10 patients undergoing ACR while on 3 different types of maintenance immunosuppression (tacrolimus, iscalimab (anti-CD40 mAb), or belatacept (CTLA4-Ig)).  In addition, we performed temporal scRNAseq/scTCRseq on biopsies and urine from 3 patients undergoing anti-rejection therapy.
Results: We observed a stunning degree of TCR restriction (~20 expanded CD8+ clonotypes [CD8_EXP]/patient).  This clonal restriction was not due to sampling bias, as clonotypes observed at initial ACR were observed in subsequent persistent rejection biopsies (Fig. 1) as well as in paired urine samples. Further, unexpanded clones at one timepoint were rarely observed as expanded at a later timepoint. Transcriptomic analysis of expanded clonotypes (CD8_EXP) demonstrated a variety of cellular states/phenotypes, including circulating memory, resident memory, and varying degrees of exhaustion. Intriguingly, a resident memory phenotype was found in the CD8_EXP in the 2 patients in this cohort with persistent rejection and eventual graft loss. In contrast, rejection resolution was associated with a near elimination of CD8_EXP in the biopsy.  Next, we found unique DEG signatures of the CD8_EXP for each IS therapy, suggesting that IS therapy determines the gene expression of CD8_EXP (Fig.2).  Our data suggest that these transcriptomic profiles may assist in the prediction of responsiveness to particular anti-rejection therapies.  Finally, we found that CD8_EXP that persist after anti-rejection therapy often change their transcriptomic phenotype, which may contribute to their lack of responsiveness to therapy.
Conclusion: Together our data show that ACR is characterized by: 1) a remarkably limited number of distinct CD8_EXP, 2) CD8_EXP that persist in ongoing rejection and disappear with rejection resolution, 3) the presence of clonally identical CD8_EXP in the urine and kidney biopsies from the same patients, 4) a persistence of CD8_EXP with a resident memory phenotype which is associated with later graft loss, 5) distinct transcriptional profiles of CD8_EXP depending on the type of IS therapy, 6) transcriptomic adaptation of  CD8_EXP in response to anti-rejection therapy.  Combined, our data provide several novel insights into the biology of ACR and strongly argue for a personalized approach to understanding ACR to guide anti-rejection therapies.