GM-CSF does not adversely affect Treg reconstitution during GVHD. (A) Representative dot plot depicting the percentage of CD4⁺Foxp3⁺ (GFP⁺) Tregs in the spleen of Foxp3^EGFP vs GM-CSF^−/− Foxp3^EGFP mice. (B) Frequency and absolute number of CD4⁺ Tregs in spleens of Foxp3^EGFP vs GM-CSF^−/− Foxp3^EGFP animals (n = 6 for each genotype). (C-D) TCR-β⁺ CD4⁺ GFP⁻ cells (5 × 10⁵) from WT or GM-CSF^−/− FoxP3^EGFP reporter animals were sort purified and transplanted along with 5 × 10⁶Rag-1^−/− BM cells into lethally irradiated Balb/c mice. (C) Experimental design. (D) The frequency and absolute number of H2-K^b+ TCR-β⁺ CD4⁺ EGFP⁺ induced Tregs in the liver, lung, and colon of mice 13 days posttransplantation. Data are from 3 independent experiments (n = 9-14 mice/group). (E-H) Lethally irradiated Balb/c mice were transplanted with 5 × 10⁶ BM cells plus spleen cells (adjusted to yield an αβ⁺ T cell dose of 0.8 × 10⁶) from WT or GM-CSF^−/− 10bit/FoxP3^EGFP double reporter mice. (E) Experimental design. (F) Representative dot plot depicting donor-derived CD4⁺ T cells in the colon that expressed FoxP3 (GFP) and/or IL-10 (Thy1.1). (G) The absolute number of CD4⁺ FoxP3⁺ Tregs in the liver, lung, and colon of mice. (H) The absolute number of CD4⁺ FoxP3⁻ cells which expressed IL-10 (Tr1 cells) in these same tissues. Data in panels G and H are from 2 independent experiments (n = 9-10 per group). *P < .05.