Abstract

Acute gastrointestinal Graft-versus-Host-Disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem-cell transplantation (alloSCT). It is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFN{gamma}, IL-17A or GM-CSF, and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between T-helper states during priming in mesenteric lymph nodes (mLN) and effector function in the GI tract remain undefined at genome-scale. We used scRNA-seq and computational modelling to create an atlas of putative differentiation pathways during GVHD. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLN. Importantly, we identified an unexpected second trajectory, categorised by little proliferation or cytokine expression, reduced glycolysis, and high TCF1 expression. TCF1hi cells upregulated 4{beta}7 prior to gut migration and failed to express cytokines therein. Nevertheless, they demonstrated recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced TCF1. Thus, scRNA-seq revealed divergence of allo-reactive CD4+ T cells into quiescent and effector states during gut GVHD, reflecting putative heterogenous priming in vivo. These findings, the first at a single-cell level during GVHD over time, can now be used to interrogate T cell differentiation in patients undergoing alloSCT.