Integrated Analysis of T-cell Repertoire and Transcriptome Shed Light into the Mechanisms of Regulatory T cell (Treg) Suppression of Acute Graft-versus-Host-Disease

CD4+ FOXP3+ regulatory T (Treg) cells are currently under clinical evaluation as an immunoregulatory cellular therapy for graft-versus-host disease (GvHD) prevention and treatment. Preclinical and clinical studies indicate that Treg are able to protect from GvHD without interfering with the graft-versus-tumor (GvT) effect of hematopoietic cell transplantation (HCT), although the underlying molecular mechanisms are largely unknown. To elucidate Treg suppressive function during in vivo suppression of acute GvHD, we performed paired T cell receptor (TCR) sequencing and RNA sequencing analysis on conventional T cells (Tcon) and Treg before and after transplantation in an MHC major-mismatch mouse model of HCT. We show that both Treg and Tcon underwent clonal expansion and that Treg did not interfere with the activation of alloreactive Tcon clones. Transcriptomic analysis revealed that Treg predominantly affected CD4 Tcon and to a lesser extent CD8 Tcon transcriptome, modulating the transcription of genes encoding pro- and anti-inflammatory molecules as well as enzymes involved in metabolic processes, inducing a switch from glycolysis to oxidative phosphorylation. Finally, Treg did not interfere with the induction of gene sets involved in the GvT effect. Our results shed light into the mechanisms of acute GvHD suppression by Treg and will support the clinical translation of this immunoregulatory approach. Donor CD4 and CD8 conventional T cells and Treg from C57Bl6 mice before and 8 days post transplant in a major mismatch GVHD model (Bl6 --> Balb/c) treated with Treg on day 0 or not were sorted and analyzed by bulkRNA and TCR seq