Blockade of IL-7R signaling significantly reduces the emergence of CD4⁺ GM-CSF⁺ IFN-γ^– T cells and attenuates pathologic damage in the colon during GVHD. (A) Gene set enrichment analysis comparing expression of IL-2/STAT5 signaling-related genes between cluster 4 and other CD4⁺ T-cell clusters in the scRNAseq data set presented in Figure 2. (B) Violin plots showing log-normalized expression of Il2ra and Il7r in CD4⁺ T-cell clusters. (C-D) Lethally irradiated Balb/c mice were transplanted with B6 bone marrow (BM) (5 × 10⁶) plus spleen cells (adjusted to yield an αβ T-cell dose of 8 × 10⁵). Representative flow cytometry dot plots (C) and summary data (D) depicting IL-7R 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 = 8 mice per group). (E-L) Lethally irradiated Balb/c mice were transplanted with B6 BM (5 × 10⁶) plus spleen cells (adjusted to yield an αβ T-cell dose of 8 × 10⁵). Recipient mice were treated 3 times weekly intraperitoneally with an anti–IL-7R or isotype control antibody. Colons were harvested 21 days posttransplantation. (E) The total number of donor-derived CD4⁺ and CD8⁺ in the colons of mice receiving the indicated treatment. (F) Representative dot plots gated on donor-derived CD4⁺ cells from the colon showing expression of GM-CSF and IFN-γ. (G) The proportion of donor CD4⁺ cells isolated from mice in the indicated groups producing IFN-γ (left) and GM-CSF (right). (H-I) The percentage (H) and total number (I) of donor CD4⁺ T cells isolated from the colon based on expression IFN-γ and/or GM-CSF. (J) The percentage of donor CD8⁺ T cells isolated from the colon based on expression of IFN-γ and/or GM-CSF. Data in panels E, G, H, I, and J are from 3 experiments (n = 13-14 mice per group). (K) Representative hematoxylin and eosin–stained images of colons 21 days’ posttransplantation. Original magnification is ×100. (L) Pathologic scores of colon tissue from mice transplanted with BM alone or BM and spleen cells and then treated with an isotype control or anti–IL-7R antibody using a semi-quantitative scoring system. Data are from 2 experiments (n = 8-10 mice per group). *P < .05, **P < .01, ***P < .001. FSC-A, forward scatter area; Log Norm. Expr., log-normalized expression; NES, normalized enrichment score.