Abstract

Background: Conventional therapy for late T cell mediated acute cellular rejection (ACR) in liver transplant includes corticosteroids and anti-thymocyte globulin, has remained unchanged for six decades, and is not infrequently met with treatment failure. Here, we used single cell RNAseq with TCR V(D)J profiling to identify expanded (ie, alloreactive) T cell clones and their gene expression profiles in response to anti-rejection treatment.

Methods: Single cell suspensions were generated from cryopreserved 16G liver biopsy tissue using cold active proteases. Whole cell and TCR libraries were constructed using 10X Chromium 5’v1.1 and 5’V(D)J TCR sequencing kits. Cells were clustered based on cell type specific gene expression profiles by unsupervised analysis in Seurat and TCR libraries were superimposed into the cell clusters. TCR clonotypes were defined as expanded if the CDR3α/ß sequences were expressed in >2 cells. Cellular communication between Kupffer cell (KC) and T cell populations was analyzed using CellPhoneDB.

Results: Overall, 10,896 cells isolated from 30 biopsies obtained from 14 patients (5 normal or non-ACR allograft dysfunction, 9 ACR) were analyzed. Consistent with their role in mediating ACR, all expanded T cell clones were CD8⁺ (CD8_EXP) according to transcriptional analysis and were significantly expanded in ACR biopsies (p <0.05, Student’s t-test). CD8_EXP bore markers of activated tissue resident memory cells (Trm; CD69, CXCR6, HLA-DR, GZMB), and retained their clonotype identity and phenotype in subsequent biopsies from the same patients despite histologic ACR resolution. In contrast, CD4⁺ T cells were only found in the unexpanded T cell pool and had a gene expression profile consistent with naïve T cells (TCF7, CCR7, SELL). CellPhoneDB analysis revealed differential crosstalk between KC and CD8_EXP T cell populations, with activation of the CD2-CD58 proliferative pathway and downregulation of TGFß signaling.

Conclusion: Despite 6-antigen HLA mismatches corresponding to a massive number of potential allogeneic epitopes, we uncovered a remarkably limited number of CD8_EXP clones. Sequential biopsies demonstrated persistence of these clones up to several months, suggesting that the restricted clonal expansion is not due to sampling bias, but rather to a consistently focused allo-response. Transcriptomic analysis revealed differential gene expression and crosstalk between KC-T cell populations which may facilitate T cell retention in allograft liver tissue.