Fig. 5

FFAR2 deletion decreases the immune suppressive activity of MDSCs. A–C Ffar2^+/+ and Ffar2^−/− bone marrow-derived MDSCs (1 × 10⁶ cells/well) were seeded in a 6-well plate and cultured in complete RPMI 1640 medium overnight. After overnight resting, BM-MDSCs were re-stimulated by combination GM-CSF (40 ng/ml) with IL-6 (40 ng/ml) or by in the presence of LLC-TES (30%) for 24 h. A Relative mRNA levels of Arg1 and iNOS in bone marrow-derived Ffar2^−/− MDSCs compared to Ffar2^+/+ MDSCs, determined by real-time RT-PCR (n = 3, biological replicates). B Relative mRNA and protein levels of IL12 and TNF-α in bone marrow-derived Ffar2^−/− MDSCs compared to Ffar2^+/+ MDSCs, determined by real-time RT-qPCR or ELISA (n = 3, biological replicates). C Representative gating strategy and the percentage of Arg1⁺ and iNOS⁺ cells in bone marrow-derived MDSCs (n = 3) were analyzed by flow cytometry (n = 3, biological replicates). D Representative gating strategy and the percentage of Arg1⁺ cells in LLC tumor-infiltrating MDSC was analyzed by flow cytometry (n = 4, biological replicates). E and F Suppression of T cell proliferation in MDSCs isolated from Ffar2^+/+ or Ffar2^−/− tumor-bearing mice spleen. MDSC-CD8⁺ T cell (CFSE labelled T cells) suppression assay was analyzed by flow cytometry (E), and quantified (F) (n = 3, biological replicates). G and H, IFNγ expression of CD8⁺ T cell in co-culture with MDSCs from Ffar2^+/+ or Ffar2^−/− tumor-bearing mice spleen was shown by flow cytometry (G), and the percentage of CD8⁺IFNγ⁺ T-cells cells in total CD8⁺ T cells was quantified (H) (n = 4, biological replicates). A–D, F and H are shown as mean ± SEM, and the experiment was performed three times and a representative example is shown. A–D, F and H were analyzed by unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and NS, not significant)