Figure 2.

MDSCs after cryo-thermal therapy facilitate the proliferation of CD4⁺ T cells but also the immunosuppressive subset differentiation. (A) Scheme of study design. In brief, MDSCs from the spleen of tumor-bearing mice or cryo-thermal treated mice (day 21 after treatment) were separated by MACS. To detect the function of MDSCs on CD4⁺ T-cell proliferation, MDSCs were cocultured with the splenocytes from age-paired naïve mice in the indicated E/T ratio with anti-CD3 (1 ng/ml) added to stimulate the proliferation of T cells. The splenocytes were pre-labeled with CFSE. After 72 h, the proliferated CD4⁺ T cells (CFSE⁻) were tested by flow cytometry. To analyze the function of MDSCs on the differentiation of CD4⁺ T cells, MDSCs were cocultured with the splenic CD4⁺ T cells separated from the tumor-bearing mice before treatment (16 days after tumor inoculation) in the ratio of 1:5. The corresponding serums were added to mimic the cytokine environment in vivo. The subsets of CD4⁺ T cells were detected after 24 h by flow cytometry. (B, C) The proliferation (B) and subsets of CD4⁺ T cells (C) after coculture with MDSCs in vitro. **p < 0.01, ***p < 0.001, ****p < 0.0001. The symbol & in panel B means the comparison with tumor-bearing control, & p < 0.0001. n = 4 for each group. MDSCs, myeloid-derived suppressive cells; MACS, magnetic-activated cell sorting; CFSE, carboxyfluorescein succinimidyl ester.