Quantifying MDSC-Mediated T Cell Suppression

MDSC-mediated suppression was assessed using previously established methodology^{7 (link)}. Briefly, splenic MDSCs were isolated from the spleens of mice bearing B16-F10 melanoma or MC17-51 fibrosarcoma, by using the EasySep Mouse (CD11b⁺GR1⁺) isolation kit (Stemcell Technologies, 19867), or the MDSC isolation kit (Miltenyi Biotech, 130-094-538) to separate GR1^hiLy6G⁺Ly6C⁻ (PMN-MDSC) and GR1^dimLy6G⁻Ly6C⁺ (M-MDSC) cells. Serial dilutions of MDSCs (2 × 10⁵, 1 × 10⁵, 0.5 × 10⁵, 0.25 × 10⁵ and 0.125 × 10⁵) were plated in flat bottom 96-well plates with 2 × 10⁵ splenocytes per well isolated from OTI-TCR transgenic mice and 250 ng ml^-1 of ovalbumin peptide (OVA_257-264) for 72 h. As a control, OTI splenocytes were incubated with OVA peptide (OVA_257–264) without MDSC. ³H-thymidine was added for the last 16 h of a 72 h culture, and thymidine incorporation was measured by MicroBeta plate counter (Perkin Elmer).