CD4⁺ GM-CSF⁺ IFN-γ^– T cells possess a distinctive transcriptional signature. (A) Volcano plot showing over/underexpressed genes in CD4⁺ GM-CSF⁺ IFN-γ⁻ T cells (cluster 4) vs CD4⁺ GM-CSF⁺ IFN-γ⁺ T cells (cluster 3) from the colons of GVHD mice 21 days’ posttransplantation. Cutoff parameters were |ln(fold change)| > 0.5 and p_adjusted < .0001. (B) Violin plots showing log-normalized expression of the indicated T-cell activation markers and immune checkpoint inhibitors in clusters 3 and 4. P values are depicted. (C) Summary data of PD-1, Lag3, and CD81 surface expression by flow cytometry on CD4⁺ GM-CSF⁺ IFN-γ⁺ and GM-CSF⁺ IFN-γ^– cells in the colon at 21 days’ posttransplantation. Data are from 2 experiments (n = 7 mice per group). (D) Violin plots showing log-normalized expression of CD69 and CD103 on cells in clusters 3 and 4. (E-F) Representative flow cytometry dot plots (left) and summary data (right) depicting CD69 and CD103 expression on donor CD4⁺ GM-CSF⁺ IFN-γ⁺ or CD4⁺ GM-CSF⁺ IFN-γ^– T cells in the colon at 21 days posttransplantation. Data are from 2 experiments (n = 9 mice per group). (G) Violin plots showing log-normalized expression of Il1r1 (ST2) in clusters 3 and 4. (H-I) Representative flow cytometry dot plots (left) and summary data (right) depicting ST2 expression on donor CD4⁺ GM-CSF⁺ IFN-γ⁺ or CD4⁺ GM-CSF⁺ IFN-γ⁻ T cells in the colon at 21 days’ posttransplantation. Data are from 2 experiments (n = 9 mice per group). In violin plots, each dot represents a single cell. ***P < .001, ****P < .0001. Geom. MFI, geometric mean fluorescence intensity; Log. Norm. Expr., log-normalized expression.