RARαhi effector T cells are also selectively expanded in the peripheral blood of GI-GVHD patients. (A) Representative dot plots and histograms of RARα, IL-23R, and T-bet expression on live CD8 T cells in peripheral blood of an allo-HSCT patient at onset of GI-GVHD and an allo-HSCT patient without GVHD from a similar time point. CD8 T cells coexpressing high RARα and IL-23R in boxed region R1 (left) expressed high levels of T-bet (right), whereas RARαdim/neg and IL-23Rneg CD8 T cells in boxed region R2 expressed low levels of T-bet. Percentage of live TP (B) CD8 and (C) CD4 T cells (coexpressing high RARα, T-bet, and IL-23R) in peripheral blood from healthy controls (n = 4) and allo-SCT patients with GI-GVHD (n = 7) or skin GVHD (n = 5) or without GVHD (n = 8) at matched time points. (D) Unsupervised phenotypic clustering of live mononuclear cells from samples depicted in panel B displayed as phenograph-generated t-distributed stochastic neighbor embedding (t-SNE) plots. Each cluster represents 1 of 24 phenotypically distinct T-cell subsets. (E) Heat map of relative expression levels of RARα, T-bet, IL-23R, and the GI-tropic molecules β7 and CCR9 of distinct CD3 T-cell clusters displayed in panel D. (F-G) Volcano plots depicting fold-change and statistical significance of abundance of distinct clusters within the peripheral blood live CD3 T-cell compartment from allo-HSCT patients with or without (F) GI-GVHD and with (G) GI- or skin GVHD. (H) Percentage of live mononuclear cells in cluster 4 in peripheral blood of allo-HSCT patients. Horizontal lines are medians. P > .10 indicates nonsignificant (ns) values. *P < .05; **P < .01, analysis of variance (ANOVA) with posttest correction for multiple comparisons (B,C,H) or Mann-Whitney U tests (F-G) .