Myeloid Cell-Mediated T Cell Suppression Assay

For ex vivo and in vitro T cell suppression assays, T cells were purified from spleens of naive C57BL/6 J mice using the MagniSort™ Mouse T cell Enrichment Kit (eBioscience; 8802-6820), labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher; C34570) and pre-activated prior to myeloid cell co-culture with plate-bound 0.1 µg ml⁻¹ anti-CD3 (145-2C11; eBioscience;) and 1 µg ml⁻¹ anti-CD28 (37.51; Biolegend) at 37 °C, 5% CO₂ for 16–18 h. Ex vivo T cell suppression assay with tumor-associated myeloid cells was adapted from De Henau et al.^{18 (link)}. In brief, Gl261-associated myeloid cells were isolated of from ICB R, ICB NR, and C mice. To this end, single-cell suspensions of tumor-bearing hemispheres were subjected to myelin removal (Myelin removal beads II; Miltenyi Biotec; 130-096) and CD11b⁺ cells were purified by MACS using the MagniSort™ Mouse CD11b Positive Selection Kit (eBioscience; 8802-6860). Gl261-associated CD11b⁺ cells were co-cultured with pre-activated T cells at a ratio of 1:1 (2.5 × 10⁴ CD3⁺T cells and 2.5 × 10⁴ CD11b⁺ myeloid cells) in murine T cell proliferation medium at 37 °C, 5% CO₂ for 72 h. T cell proliferation was examined by CFSE mean fluorescence intensity of living CD3⁺ CD8⁺ and living CD3⁺ CD4⁺ T cells, and percentage of cells per cell division.