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. 2014 May 5;211(5):781–790. doi: 10.1084/jem.20131916

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© 2014 Noman et al.

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Figure 4. — Blockade of PD-L1 under hypoxia down-regulates MDSC IL-6 and IL-10 and enhances T cell proliferation and function. MDSCs isolated from spleens of B16-F10 tumor-bearing mice were pretreated for 30 min on ice with 5 µg/ml control antibody (IgG) or antibody against PD-L1 (PDL1 Block) and co-cultured with splenocytes under normoxia and hypoxia for 72 h. (A and B) Effect of MDSC on proliferation of splenocytes stimulated with (A) anti-CD3/CD28 coated beads or (B) TRP-2_(180–88) peptide under the indicated conditions. Cell proliferation was measured in triplicates by [³H]thymidine incorporation and expressed as counts per minute (CPM). (C and D) MDSCs were cultured with splenocytes from B16-F10 mice stimulated with anti-CD3/CD28. Intracellular IFN-γ production was evaluated by flow cytometry by gating on (C) CD3⁺CD8⁺ IFN-γ⁺ and (D) CD3⁺CD4⁺ IFN-γ⁺ populations. Statistically significant differences (indicated by asterisks) are shown (**, P < 0.005; ***, P < 0.0005). Three separate experiments (in triplicates) with the same results were performed. Error bars indicate SD. (E) SYBR-GREEN RT-qPCR was performed to evaluate the mRNA expression levels of Ldha, Car-9, Arg-1, Nos2, Ncf-1, and Cybb-1. (F) Arginase enzymatic activity measured in MDSCs under indicated conditions. (G) After 72 h of MDSC co-culture with splenocytes, supernatants were collected and assayed for nitrites. Data represents three independent experiments with SD. (H) SYBR Green RT-qPCR was performed for expression levels of IL6, IL10, Il12p70, and Tgfb1 under the indicated conditions. IL-6, IL-10, IL-12p70, and TGF-β1 cytokine production and secretion was detected by (I and J) intracellular FACS staining (isotype control is gray-shaded histogram) and (K) ELISA, respectively. Statistically significant differences (indicated by asterisks) are shown (*, P < 0.05; **, P < 0.005; ***, P < 0.0005). The experiment was performed in triplicates and repeated three times with the same results. Error bars indicate SD. (L–O) MDSCs isolated from spleens of B16-F10 tumor-bearing mice were co-cultured with splenocytes under normoxia and hypoxia for 72 h in the presence of either 10 µg/ml control antibody (IgG), anti–mouse IL-6 Functional Grade Purified neutralizing antibody (IL-6 Block) or anti–mouse IL-10 Functional Grade Purified neutralizing antibody (IL-10 Block). Effect of MDSCs on proliferation of splenocytes stimulated with (L) anti-CD3/CD28–coated beads or (M) TRP-2_(180–88) peptide under the indicated conditions. Cell proliferation was measured as indicated above. MDSCs were cultured with splenocytes from B16-F10 mice stimulated with anti-CD3/CD28. Intracellular IFN-γ production was evaluated by flow cytometry by gating on (N) CD3⁺CD8⁺ IFN-γ⁺ and (O) CD3⁺CD4⁺ IFN-γ⁺ populations. Statistically significant differences (indicated by asterisks) are shown (*, P < 0.05; **, P < 0.005). Two separate experiments (in triplicates) with the same results were performed. Error bars indicate SD.