Single-cell transcriptomics of allo-reactive CD4+ T cells over time reveals divergent fates during gut GVHD.

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 IFNγ, 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 applied scRNA-seq and computational modelling to a mouse model of donor DC-mediated GVHD exacerbation, creating an atlas of putative CD4+ T-cell differentiation pathways in vivo. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLN. Importantly, we inferred an unexpected second trajectory, categorised by little proliferation or cytokine expression, reduced glycolysis, and high tcf7 expression. TCF1hi cells upregulated α4β7 prior to gut migration and failed to express cytokines therein. Nevertheless, they exhibited recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced TCF1. Thus, scRNA-seq suggested divergence of allo-reactive CD4+ T cells into quiescent and effector states during gut GVHD exacerbation by donor DC, reflecting putative heterogenous priming in vivo. These findings, the first at a single-cell level during GVHD over time, may assist in examination of T cell differentiation in patients undergoing alloSCT.