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. 2021 Oct 14;12:754316. doi: 10.3389/fimmu.2021.754316

Figure 2.

Figure 2

CD11b+CD11c+ MDSCs exhibit increased immunosuppressive capacity than CD11b+CD11c− MDSCs and induce type 2 immunity in vitro. CD11b+CD11c+ and CD11b+CD11c− MDSCs were isolated from B6-derived (H-2b, CD45.2+) in vitro-generated MDSCs. (A) CD11b+CD11c+ or CD11b+CD11c− MDSCs were cocultivated with B6.SJL-derived (H-2b, CD45.1+) CFSE-labeled spleen cells stimulated by irradiated allogeneic DBA/2-derived (H-2d, CD45.2+) spleen cells. After 4 days, CD45.1+ T cells were stained for CD3, CD4, and CD8 and suppression of CD4+ and CD8+ T-cell proliferation was calculated. (B) CD11b+CD11c+ and CD11b+CD11c− MDSC subpopulations were analyzed for relative expression of immunosuppressive molecules by qRT-PCRs. (C) CD11b+CD11c+ or CD11b+CD11c− MDSCs were cocultivated with B6.SJL-derived CFSE-labeled spleen cells stimulated by irradiated allogeneic DBA/2-derived spleen cells in the absence or presence of iNOS inhibitor L-NMMA (500 µM). After 4 days, suppression of CD4+ and CD8+ T-cell proliferation was determined. (D) B6-derived wildtype (WT) and PD-L1−/− CD11b+CD11c+ or CD11b+CD11c− MDSCs were cocultured with CFSE-labeled B6.SJL-derived spleen cells stimulated with irradiated allogeneic DBA/2 spleen cells. (E) To block PD-L2, antagonistic PD-L2 antibodies or recombinant isotype control were added to MLRs, in which CFSE-labeled B6.SJL-derived spleen cells were stimulated with irradiated allogeneic DBA/2 spleen cells in the presence of CD11b+CD11c+ MDSCs. (F) CD11b+CD11c+ or CD11b+CD11c− MDSCs were cocultivated with B6.SJL-derived CFSE-labeled spleen cells stimulated by irradiated allogeneic DBA/2-derived spleen cells. After 4 days, secretion of cytokines associated with type 2 T-cell immunity (IL-4, IL-5, and IL-13) or type 1 T-cell immunity (IFN-γ) were determined in the supernatants. (A) Data represent the mean value ± SD of triplicates of one representative experiment out of four experiments performed. (B) Data represent the mean value ± SD of six to nine samples. (C) Data represent the mean value ± SD of triplicates of one representative experiment out of three experiments performed. (D) Data represent the mean value ± SD of n = 3 PD-L1−/− and WT mice. (E) One experiment out of two experiments performed. Values present the mean value ± SD of triplicates. (F) Data represent the mean value ± SD of n = 3 experiments. (A, C–F) Student’s t-test. (B) Mann-Whitney U test. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001. n.s., not significant.