BM-derived MDSC-IL13 enhance GVHD survival, but suppression is compromised after 5 days in vivo. (A) Lethally irradiated BALB/c recipients were given 1 × 107 C57Bl/6 BM (BM only), BM plus 2 × 106 CD25-depleted T-cells (GVHD), or BM, T cells, and 6 × 106 MDSC-IL13 (GVHD + MDSC) as indicated. Kaplan-Meier survival curve represents 4 pooled and independent experiments (n = 40 animals/group). GVHD vs GVHD + MDSC, P < .0001. (B-C) Surface expression of congenic (CD45.2+) MDSC-IL13 recovered from spleens 5 days after transfer to irradiated animals receiving BM only (no GVHD) or BM plus T cells (GVHD). Data represent 3 replicates per group with P < .001 for all markers shown. (D) Representative histograms indicating responding T-cell proliferation as denoted by CFSE dilution. Purified MDSC-IL13 from pooled spleens 5 days after transplant were plated at 5 × 105/mL with an equal number of CFSE-labeled responder T cells, 0.25 μg/mL anti-CD3ε mAb, and 2.5 × 105/mL irradiated T-cell–depleted splenocytes in specially formulated 150 μM l-arginine RPMI media. Shaded histogram indicates proliferation of unstimulated controls. Data are representative of 3 samples per group and a total of 3 independent experiments. (E) Summary data of recovered MDSCs showing viability and total cell numbers recovered, gated CD11b+ CD45.2+. Data represent 3 samples per group and are representative of 3 independent experiments. (F) Lethally irradiated BALB/c recipients transplanted as above or given 3 consecutive infusions of MDSC-IL13 as indicated on days 0, 3, and 6. All mice receiving MDSCs demonstrated increased survival vs GVHD (P < .001). MDSCs vs MDSCs on days 0, 3, and 6 (P < .0001). Survival curve represents 20 animals per group from 2 independent experiments and is representative of an additional experiment giving multiple infusions on days 0, 7, and 14. ns, not significant.
