In vitro-generated MDSCs proliferate in vivo, reduce alloantigen-specific T-cell proliferation only early after transplantation, and do not interfere with allogeneic T-cell homing. (A) Lethally irradiated B6.bm1 recipient mice (H-2K^bm1, CD45.2⁺) were reconstituted with BM and SCs from B6 mice (H-2K^b, CD45.2⁺) and co-injected with 1 × 10⁷ CFSE labeled B6.SJL-derived MDSCs (H-2K^b, CD45.1⁺). Different days after transplantation, mice were euthanized, stained for CD45.1, and proliferation of transplanted CD45.1⁺ MDSCs was analyzed by CFSE dilution in BM (top), spleen (middle), and liver (bottom) cells. (B-D) Lethally irradiated B6.bm1 recipient mice (H-2K^bm1, CD45.2⁺) were reconstituted with BM from B6 mice (H-2K^b, CD45.2⁺) plus B6.SJL-derived SCs (H-2K^b, CD45.1⁺) in the presence or absence of 1 × 10⁷ B6-derived MDSCs (H-2K^b, CD45.2⁺). One, 3, and 11 days after BMT, mice were euthanized, SCs stained for CD45.1 and CD3, and numbers of CD45.1⁺CD3⁺ T cells were determined (B). Different days after transplantation, spleen, blood, liver, and colon cells were stained for CD45.1 and CD3, and numbers of infiltrating CD45.1⁺CD3⁺ T cells were determined in spleen and liver, whereas the percentage of CD45.1⁺CD3⁺ T cells was calculated in the blood and colon (C). SCs of mice receiving allogeneic BM and SCs in the presence or absence of MDSCs were isolated 7 days after transplantation and re-stimulated in vitro with medium, PMA/iono or allogeneic B6.bm1 SCs (allo). After 5 hours, the percentage of IL-2–secreting CD45.1⁺ T cells was determined (D). FACS diagrams are representative for 1 mouse out of 5 analyzed (A). Data represent the mean value ± SD of 5 mice analyzed in (B), of 3 mice analyzed in (C), and of triplicates from SCs, which were pooled from spleens of 7 mice treated in (D); *P ≤ .05; **P ≤ .01. n.s., not significant. No significant statistical differences were detected in T-cell numbers between TCD-BM + SC vs TCD-BM + SC + 1 × 10⁷ MDSCs in (C).