Host-versus-graft (HvG) T cell clones are enriched in the graft mucosa during early rejection after human intestinal transplantation and persist despite rejection resolution. Recipient T cells in the mucosa eventually took on resident memory T cell (TRM) features, likely posing a constant threat of late rejection.
To directly address this hypothesis, we integrated clonotype, alloreactivity and gene expression profiles of FACS sorted allograft recipient T cells at the single cell level to assess the functional phenotype of alloreactive T cells in association with graft outcomes using the 10x Genomics single cell RNA sequencing and our published method^1,2 for identifying alloreactive TCR repertoires from pre-transplant mixed lymphocyte reactions (pre-Tx MLRs) (Fig. 1A).
Recipient T cells sorted from graft mucosa of three quiescent and two chronically rejecting allografts and a deceased donor intestine (Fig. 1B) shared at least four clusters (Fig. 2): multifunctional (IL17A⁺, IL22⁺, TNF⁺) TRMs (CD69⁺, ITGAE⁺, CXCR6⁺, PRDM1⁺) that were enriched for HvG clones (cluster 0); cytotoxic γδ and CD8 TRMs (GZMA⁺, GNLY⁺) (cluster 2); nonTRMs (CCR7⁺, KLF2⁺, S1PR1⁺, SELL⁺) (cluster 4); and regulatory T cells (Foxp3⁺, CTLA4⁺) (cluster 13). Cytotoxic (EOMES⁺, GZMB⁺, PRF1⁺) effector T cells (Teff) that were enriched for CD8 HvG clones were identified only in a graft with chronic rejection (Pt14 cluster 1). γδ TRMs that highly express TCF7, CD28H, PRF1 and IL-2 (cluster 11) and CD4 Teff cells enriched for HvG clones expressing RUNX3, RORA, AHR, IL23 and IFNG (cluster 6) were identified only in a graft with chronic rejection (Pt4). T follicular helper (Tfh) cells (BCL6⁺, CXCR5⁺, PDCD1⁺, IL21⁺) lacking a TRM signature (cluster 3) that were enriched for CD4 HvG clones were identified in a quiescent graft (Pt21) with mucosal lymphoid follicles. Hyporesponsiveness of circulating recipient T cells to donor versus third-party antigens in post-Tx MLR indicated partial tolerance of circulating recipient T cells to donor antigens post-Tx. In line with these observations in circulation, there was a significantly decreased likelihood of detecting HvG clones defined by post- compared to pre-Tx MLR in late quiescent (but not chronically rejecting) allografts (Fig. 3A). Furthermore, quiescent allografts contained a significantly greater percentage of pre-Tx HvG clones that became tolerant in post-Tx MLR compared to chronically rejecting allografts (Fig. 3B).
Our data integrate T cell alloreactivity with clonotype and gene expression profiles at the single cell level. We have demonstrated heterogeneous contributions of pre-existing HvG-reactive T cells to TRM and Tfh compartments in quiescent allografts and evidence for donor-specific tolerance locally and systemically. Chronic rejection is associated with unique CD4 and CD8 Teff cells that are enriched for pre-existing HvG clones and cytotoxic γδ TRMs.