Plasticity of intragraft alloreactive T cell clones in human gut correlates with transplant outcomes

The site of transition between tissue resident memory (TRM) and circulating phenotypes of T cells is unknown. We integrated clonotype, alloreactivity and gene expression profiles of graft-repopulating recipient T cells in the intestinal mucosa at the single cell level after human intestinal transplantation. Host-versus-graft (HvG)-reactive T cells mainly distributed to TRM, Teff/TRM and T follicular helper compartments. RNA velocity analysis demonstrated a trajectory from TRM to effector T (Teff)/TRM clusters in association with rejection. By integrating pre- and post-Tx mixed lymphocyte reaction-determined alloreactive repertoires, we observed that pre-existing HvG-reactive T cells that demonstrated tolerance in the circulation were dominated by TRM profiles in quiescent allografts. Putative de novo HvG-reactive clones showed a transcriptional profile skewed to cytotoxic effectors in rejecting grafts. Inferred protein regulon network analysis revealed upstream regulators that accounted for the effector and tolerant T cell states. We demonstrate Teff/TRM interchangeability for individual T cell clones with known (allo)recognition in human gut, providing novel insight into TRM biology. ScRNA-seq data were generated from FACS-sorted recipient HLA+ CD45+ CD3+ T cells from fresh or frozen/thawed suspensions of intraepithelial lymphocytes (IEL) and/or lamina propria lymphocytes (LPL), isolated from ileal biopsies or tissue resections collected during the late post-Tx (POD626-1764) period from a total of six pediatric ITx patients, including 6 quiescent and 5 rejecting allograft specimens. Samples from Pt15 MVTx POD1194 (IEL + LPL mixed), Pt13 iITx POD1032 IEL and LPL, Pt16” (secondary Tx of Pt16) MVTx POD1004 IEL and LPL, and Pt21 MVTx POD626 (IEL+LPL mixed) were quiescent and free of rejection. All rejecting allograft specimens used in the scRNA-seq study were collected during ileal graft explantation, providing a snapshot of immunological events during graft loss.