Th64 - Renal Allograft Rejection: New Paradigms in T Cell Reactivity Revealed by Single Cell RNAseq Analysis

BACKGROUND: Transcriptomic definition of the T-cell landscape in renal allograft rejection (RAR) is complicated by the inability of CDR3 sequence data to distinguish allospecificity of TCRs in graft infiltration. Here, we have begun functional analysis of infiltrating TCRs in kidney allografts.

METHODS: Following 5’ single cell RNAseq with paired TCRα/β sequencing of RAR biopsies, Seurat and Loupe V(D)J analysis identified individual T-cell clonotypes. Expanded clonotypes were defined as >2 cells and >0.5% of T-cell barcodes identified with identical CDR3a/b sequences. To further understand the alloresponse and potential cross-reactivity, 5 expanded clonotypes randomly selected from 1 patient were transfected into Jurkat76 cells, with 1 identified as having a TCRα chain with EBV reactivity via VDJdb. Jurkat clones were co-cultured overnight with donor vs. third-party APCs and IL-2 ELISA was performed to determine T-cell activation.

RESULTS: Samples obtained at the time of RAR diagnosis revealed an impressive degree of TCR restriction (avg. 14 expanded CD8+ clones/patient). In patients who had subsequent biopsies following RAR diagnosis, >80% of the original clones were present in subsequent biopsies, suggesting a limited number of clonally expanded T-cells during rejection. However, expanded T cell clonotypes are eliminated following rejection resolution. Strikingly, all 5 TCRs subcloned into Jurkats displayed evidence of alloreactivity through production of IL-2 in response to donor but not third-party APCs.

CONCLUSIONS: 1) RAR is characterized by a remarkably limited number of distinct CD8+ T-cell clones and 2) TCR subcloning can allow determination of TCR specificity and link alloreactive T-cells to their transcriptomes.