Figure 1
Figure 1. MDSCs prevent GVHD in an MHC class I mismatched B6 → B6.bm1 BMT model when co-injected with the transplant. (A) MDSCs were generated in vitro from BM cells of B6 mice in the presence of GM-CSF and G-CSF. After 4 days, cells were stained for CD11b and Gr-1 expression or the distribution of MDSC subsets was defined on side scatter + CD11b+ cells by the expression of Ly-6C and Ly-6G. Data show 1 representative fluorescence-activated cell sorter (FACS) staining from 3 independent experiments performed. (B) CFSE-labeled B6.SJL-derived SCs (CD45.1+, H-2Kb) were stimulated with medium or irradiated B6.bm1 (CD45.2+, H-2Kbm1) allogeneic SCs in the presence or absence of B6-derived MDSCs. iNOS inhibitor L-NMMA or arginase-1 inhibitor nor-NOHA was added. After 4 days, cells were stained for CD45.1 CD3, CD4, and CD8. Proliferation of CD45.1+CD3+ CD8+ T cells was analyzed and suppression of proliferation was calculated. Data represents the mean of triplicates ± standard deviation (SD) of 1 representative experiment out of 3 experiments performed. ***P < .001. (C-E) Lethally irradiated B6.bm1 recipient mice were reconstituted with TCD-BM from B6 mice without or with B6-derived SCs and co-injected with 1 × 106, 5 × 106, or 1 × 107 B6-derived MDSCs on the day of transplantation. Survival (C) and GVHD scores (D) were determined. (C) TCD-BM + SC vs TCD-BM + SC + 1 × 106 MDSCs, *P ≤ .05 vs TCD-BM + SC + 5 × 106 MDSCs, *P ≤ .05 vs TCD-BM + SC + 1 × 107 MDSCs, **P ≤ .01. Surviving animals/total animals treated are indicated in brackets. (D) Error bars indicate ± standard error of the mean (SEM). (E) Paraffin sections of ileum and colon (left), liver (middle), and skin (right) of 5 to 9 animals/treatment group were analyzed for histologic signs of GVHD on the day mice were euthanized due to their moribund state or at the end of the experiment, *P ≤ .05; **P ≤ .01. (F) Lethally irradiated B6.bm1 recipient mice were reconstituted with TCD-BM and SC from B6 mice; 1 × 107 B6-derived MDSCs were injected either on the day of transplantation (day 0) or 7 days after BMT (day 7) and survival was determined. Surviving animals/total animals treated are indicated in brackets. TCD-BM + SC vs TCD-BM + SC + 1 × 107 MDSCs day 0, *P ≤ .05 vs TCD-BM + SC + 1 × 107 MDSCs day 7, P = .49 considered not significant. n.s., not significant.

MDSCs prevent GVHD in an MHC class I mismatched B6 → B6.bm1 BMT model when co-injected with the transplant. (A) MDSCs were generated in vitro from BM cells of B6 mice in the presence of GM-CSF and G-CSF. After 4 days, cells were stained for CD11b and Gr-1 expression or the distribution of MDSC subsets was defined on side scatter + CD11b+ cells by the expression of Ly-6C and Ly-6G. Data show 1 representative fluorescence-activated cell sorter (FACS) staining from 3 independent experiments performed. (B) CFSE-labeled B6.SJL-derived SCs (CD45.1+, H-2Kb) were stimulated with medium or irradiated B6.bm1 (CD45.2+, H-2Kbm1) allogeneic SCs in the presence or absence of B6-derived MDSCs. iNOS inhibitor L-NMMA or arginase-1 inhibitor nor-NOHA was added. After 4 days, cells were stained for CD45.1 CD3, CD4, and CD8. Proliferation of CD45.1+CD3+ CD8+ T cells was analyzed and suppression of proliferation was calculated. Data represents the mean of triplicates ± standard deviation (SD) of 1 representative experiment out of 3 experiments performed. ***P < .001. (C-E) Lethally irradiated B6.bm1 recipient mice were reconstituted with TCD-BM from B6 mice without or with B6-derived SCs and co-injected with 1 × 106, 5 × 106, or 1 × 107 B6-derived MDSCs on the day of transplantation. Survival (C) and GVHD scores (D) were determined. (C) TCD-BM + SC vs TCD-BM + SC + 1 × 106 MDSCs, *P ≤ .05 vs TCD-BM + SC + 5 × 106 MDSCs, *P ≤ .05 vs TCD-BM + SC + 1 × 107 MDSCs, **P ≤ .01. Surviving animals/total animals treated are indicated in brackets. (D) Error bars indicate ± standard error of the mean (SEM). (E) Paraffin sections of ileum and colon (left), liver (middle), and skin (right) of 5 to 9 animals/treatment group were analyzed for histologic signs of GVHD on the day mice were euthanized due to their moribund state or at the end of the experiment, *P ≤ .05; **P ≤ .01. (F) Lethally irradiated B6.bm1 recipient mice were reconstituted with TCD-BM and SC from B6 mice; 1 × 107 B6-derived MDSCs were injected either on the day of transplantation (day 0) or 7 days after BMT (day 7) and survival was determined. Surviving animals/total animals treated are indicated in brackets. TCD-BM + SC vs TCD-BM + SC + 1 × 107 MDSCs day 0, *P ≤ .05 vs TCD-BM + SC + 1 × 107 MDSCs day 7, P = .49 considered not significant. n.s., not significant.

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