A CD11b⁺CD11c⁺Gr-1^medCD301b⁺I-A^b+ MDSC subpopulation expands under GVHD conditions in vitro. (A-C) B6-derived MDSCs were generated in vitro in the presence of GM-CSF and G-CSF. After 4 days, in vitro-generated MDSCs (day4-MDSCs) were incubated for 3 days with medium supplemented with 2.5% serum from mice receiving either TCD-BM alone (BM-MDSCs) or TCD-BM plus SCs (GVHD-MDSCs). Cells were stained for CD11b and CD11c expression before and after serum incubation, and percentage and absolute numbers of CD11b⁺CD11c⁻ and CD11b⁺CD11c⁺ cells were determined. Data represent the mean of triplicates ± SD of 1 representative experiment out of 3 experiments performed (A). Cells were stained for CD11b and CD11c, and the expression of CD301b, F4/80, I-A^b, and Gr-1 was determined on CD11b⁺CD11c⁻ and CD11b⁺CD11c⁺ subpopulations 3 days after serum incubation. Data show 1 representative FACS staining from 3 independent experiments performed. Isotype staining is shown for the CD11b⁺CD11c⁺ population but was identical in the CD11b⁺CD11c⁻ population (B). CD11b⁺CD11c⁻ and CD11b⁺CD11c⁺ cells were isolated from day 4 MDSCs and GVHD-MDSCs, and mRNA expression of Klf4, IRF4, and Batf3 was determined, and relative expression to AIP was calculated. Data represent the mean ± SD of 3 independent experiments performed (C). *P ≤ .05; **P ≤ .01; ***P ≤ .001. n.s., not significant.