Figure 6.

C/EBPα negatively regulates immune suppressive and angiogenic activities of MDSCs. Gr-1 +CD11b+ were magnetically purified from spleens of WT and CebpaΔ/Δ mice bearing 3LL tumor (n = 5 mice pooled together per group). RNA was isolated and Arg1, iNOS, MMP9 and VEGF expression was measured by qPCR (A). Gr-1+CD11b+ from 3LL tumor models were cultured for 3 days. Nitrate and nitrite production was measured in the culture supernatants with Nitrate/Nitrite Assay Kit and total NO production was calculated (B). Arg1, iNOS, MMP9 and VEGF expression was measured by qPCR in MDSCs isolated from B16 tumor models (C). Arginase activity of MDSCs purified from B16 tumor models were measured (D). *p < 0.05. The experiment was done in duplicate and repeated 3 times. CFSE-labeled normal CD4+T cells were stimulated with anti-CD3 mAb plus anti-CD28 mAb in the presence or absence of MDSCs isolated from WT or CebpaΔ/Δ mice for 3 days. PBS was used as a negative control. Proliferation of CD4+T cells was evaluated as CFSE dilution by flow cytometry and quantitated (E and F). A similar study as E and F was carried out using B16 tumor model (G). HUVEC cell migration in response to conditioned media collected from cultured MDSCs purified from WT and CebpaΔ/Δ mice (pooled from 3 mice per group) in the presence of control BSA or soluble VEGFR2 at 100 ng/ml were carried out using Transwell assays. Migrated endothelial cells were counted 6 hrs later (H) *p < 0.05. The experiment was done in duplicate and repeated 3 times.