CD4⁺ T-cell–derived GM-CSF drives inflammation in the colon during GVHD independent of IL-6 and IL-23. (A-B) Lethally irradiated Balb/c mice were transplanted with B6 BM and B6 spleen cells (adjusted to yield an αβ⁺ T cell dose of 0.8 × 10⁶). Animals were treated intraperitoneally on a weekly basis with an anti-IL-6R (panel A) or anti-IL-23R (panel B) antibody or an appropriate isotype control (n = 8-11/group). The frequency and absolute number of CD4⁺ GM-CSF⁺ and CD8⁺ GM-CSF⁺ T cells in the colon are shown for each antibody-treated cohort 3 weeks post transplantation. Data are from 2 experiments for anti-IL-6R and 3 experiments for anti-IL-23R. (C) Percentage of GM-CSF expressing donor-derived CD4⁺ and CD8⁺ T cells in the colon of mice transplanted as in panel A that were treated with an isotype control antibody. Data were compared using a paired t test. (D) Balb/c mice were transplanted with Rag-1^−/− BM alone (5 × 10⁶; n = 6) or together with purified CD4⁺ T cells (1.1 × 10⁶) from WT (n = 10) or GM-CSF^−/− (n = 10) mice. Results are from 2 experiments. (E-F) Magnetically purified CD4⁺ T cells (1 × 10⁵) from WT or GM-CSF^−/− mice were labeled with Cell Trace Violet and cultured with allogeneic Balb/c CD11c-enriched dendritic cells (DCs; 5 × 10⁴) for 3 days. Representative flow plots gated on CD4⁺ H2-K^b+ cells showing cell trace dye dilution is shown in panel E. The percentage of Cell Trace^lo CD4⁺ T cells in the presence or absence of DCs from replicate experiments (n = 4) is depicted in panel F. (G-J) Balb/c mice were transplanted with B6 Rag-1^−/− BM (5 × 10⁶) plus 0.4 × 10⁶ CD4⁺ T cells in a 1:1 ratio from B6.PL GM-CSF^+/+ and B6 GM-CSF^−/− animals (WT/ GM-CSF^−/−), or B6 Rag-1^−/− BM and a mixture of CD4⁺ T cells from B6.PL GM-CSF^+/+ and B6 GM-CSF^+/+ mice (WT/WT). Ratio of CD4⁺ Thy1.1/Thy1.2⁺ T cells in the colons of mice 21 days posttransplantation is depicted in panel G, and the total number of donor-derived T cells (Thy1.1⁺ and Thy1.2⁺) is shown in H. The percentage of CD4⁺ Thy1.1⁺ and Thy1.2⁺ T cells that produced IFN-γ, TNF-α, and GM-CSF is shown in panels I and J, respectively. Data are from 3 experiments with 13 to 15 mice/group. **P < .01; ****P < .0001.