Bhlhe40 regulates CD4⁺ T-cell-mediated pathological damage in the colon during GVHD. (A) Representative dot plot depicting CD4⁺ CD11c⁺ and CD4⁺ CD11c⁻ T-cell populations that were sorted for subsequent RNA sequence analysis. (B) Waterfall plot showing selected genes from RNA sequencing analysis from sorted splenic CD4⁺ CD11c⁺ and CD4⁺ CD11c⁻ T cells. Data are expressed as log₂ (expression in CD4⁺ TCRβ⁺ CD11c⁺/expression in CD4⁺ TCRβ⁺ CD11c⁻) and represent the mean of 3 independent samples. (C) mRNA expression of Bhlhe40 in sorted CD4⁺ CD11c⁺ vs CD4⁺ CD11c⁻ T cells. The expression of 18s rRNA was used as an internal control. Fold change expression was normalized to the median expression of Bhlhe40 over 18s in CD11c⁻ cells. Data are from 3 independent sorted samples of pooled splenocytes from 5 mice. (D, E). Lethally irradiated Balb/c mice were transplanted with Rag-1^−/− BM alone (5 × 10⁶) or together with purified CD4⁺ T cells (1.1 × 10⁶) from bhlhe40^−/− or WT sex-matched littermate controls. Overall survival is depicted in panel D, and serial weight curves expressed as a percentage of pretransplant weight in panel E. Results are from 3 independent experiments with 9 to 15 mice per group. (F-I). Irradiated Balb/c mice were transplanted with Rag-1^−/− BM alone (5 × 10⁶) plus purified CD4⁺ T cells (0.9 × 10⁶) from bhlhe40^−/− or WT sex-matched littermate controls. (F). Representative hematoxylin and eosin-stained sections of the liver, lung, and colon of transplant recipients from each cohort. Original magnification is 100× for photomicrographs. (G) Pathological scores of liver, lung and colon 21 days posttransplantation, using a semiquantitative scoring system, are depicted. Results are from 2 experiments (n = 9-10/group). (H) Total number of CD4⁺ T cells isolated from liver, lung, and colon of recipient mice 21 days posttransplantation. (I) Absolute number of CD4⁺ T cells producing IFN-γ, GM-CSF, and IL-10 in the colon. Results are from 2 experiments for panels H-I (n = 8-9/group). *P < .05; ***P < .001; ****P < .0001.