Single-cell RNAseq of AHSCT patient-derived PBMCs. (A) Diagram depicting sample selection. PBMCs of 2 patients, one of which developed GVHD shortly after sample collection (pre-GVHD) and one who did not (no GVHD), were thawed; red blood cells were lysed, and cell viability was assessed. Single-cell preparation was performed using the Chromium Next GEM Single Cell 5′ Library & Gel Bead Kit (10× Genomics). Sequenced reads were aligned to the human GRCh38 reference sequence provided by 10× Genomics. Clustering and visualization were performed in R using the Seurat package (v3.1.1) with integrated datasets. (B) Volcano plot of the differential gene expression within 1 cluster comparing the no GVHD vs pre-GVHD sample for the CD4 cluster (left) and T_proliferating cluster (right). Genes with a fold change >1.5 and adjusted P value <.01 are highlighted in red. (C) Dot plot visualizing the scaled gene expression level of key glycolysis enzymes for the CD4, CD8, and T_proliferating cluster. (D) Comparison of the module scores for Glycolysis (left) and TCA (right) between both patients for CD4, CD8, and proliferating T cells. The analyzed cell numbers for each cluster are indicated in parentheses. Data displayed as mean ± SEM. FC, fold change; GI, gastrointestinal; MUD, matched unrelated donor; NS, not significant; PBHSCT, peripheral blood hematopoietic stem cell transplantation.