BALB/c mice were immunized with low doses c-di-GMP and LM-Mb and challenged with 4T1 tumor cells according to Immunization protocol C. Nineteen days after tumor challenge mice were euthanized and the percentage of MDSCs (CD11b+Gr1+) was determined in blood in vivo by flow cytometry (A). An example of gating MDSC in blood is shown in Figure S10. In addition, gMDSCs (B) and mMDSCs (C) were isolated from 4T1 tumor-bearing mice, incubated with various concentrations of c-di-GMP in vitro, and then analyzed for IL-12 secretion in the culture supernatant by ELISA (C). The results shown here are the averages of three independent experiments (n=5 mice per group). In A, all groups were compared to LM-Mb + c-di-GMP. The error bars represent the SEM. Mann-Whitney test. *p<0.05, **<0.01, ***<0.001, ****<0.0001. Values p<0.05 were considered statistically significant. Also the expression of STING was analyzed in MDSCs by western blotting. Bone marrow (BM) cells and HEK293 cells were used as positive and negative controls, respectively.