TLI/ATS/CTX compared with TLI/ATS conditioning results in enhanced generation of recipient Gr-1^highCD11b⁺CD11c^negMDSCs and augments recovery of donor Foxp3⁺Treg after BMT. (A) Representative 3-dimensional plot of FLOCK centroid analysis (top) on gated H-2K^d-negB220^neg cells for PBMC obtained at day 7 post-BMT from a β-thal^+/− HW-80 recipient of TLI/ATS or TLI/ATS/CTX and BALB/c BMT, showing recipient MDSCs (blue, pink) and MDC (red, gold). The MDSC (lower left) and MDC tables (lower right) show frequency (%) of the indicated color plots among total analyzed cells as well as percentages for analysis of a control nonincreased population (MDSC, teal; MDC, violet). T/A, TLI/ATS; T/A/C, TLI/ATS/CTX. (B) Representative 3-dimensional plot of FLOCK centroid analysis (top) for PBMC at day 7 post-BMT from a β-thal^+/− HW-80 recipient of TLI/ATS or TLI/ATS/CTX and BALB/c BMT. Percentage nTreg populations (green, gold) and 1 control nonincreased population (violet) among total analyzed cells is shown in the Treg table (bottom). (C) Mean ± SEM absolute number H-2K^d-negB220^negCD11b⁺Gr-1^highCD11c^neg cells (MDSC) (left) and H-2K^d-negB220^negCD11b⁺Gr-1^lowCD11c⁺ cells (MDC) (right) at day 7 in spleen of recipients of TLI/ATS or TLI/ATS/CTX and WT BALB/c donor BMT. (D) Protocol for MDSC depletion during TLI/ATS/CTX conditioning. WT B6 mice received TLI/ATS/CTX and IP injection of depletive anti-Gr-1/Ly6 clone RB6-8C5 or rat IgG2a isotype control antibody, followed by WT BALB/c BMT. At day 7 after BMT, recipients were euthanized for FACS analysis of MDSC, MDC, and Foxp3⁺ Treg recovery and histopathologic assessment of GVHD target organs. BMT = 50 × 10⁶ bone marrow cells + 60 × 10⁶ spleen cells from WT BALB/c donors. (E) Mean ± SEM absolute number recipient MDSCs (left), recipient MDCs (middle), and donor Treg (right) at day 7 post-BMT in spleens of recipients of IgG2a isotype control antibody or RB6-8C5 during TLI/ATS/CTX conditioning. (F) Mean ± SEM cumulative GVHD scores for mice represented in panel E. Data are cumulative (n = 3 experiments). Representative photomicrographs of hematoxylin and eosin–stained sections of colon (×40) (left) and liver (×40) (right) from each group.