Antigen-specific T cell proliferation assay

CD8⁺ T cells were isolated by FACS from the spleen and lymph nodes of OT-I mice and labeled with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE; Sigma) according to the manufacturer’s instructions. For co-culture, 1x10⁵ CFSE-labeled CD8⁺ T cells were cultured with 1x10⁵ irradiated splenocytes (50 Gy for 30 minutes) from naïve C57Bl/6 mice pulsed with SIINFEKL peptide (f.c.1 μM; Sigma) for 2 hours at room temperature. SIINFEKL is a MHC class I-restricted peptide epitope of ovalbumin (OVA) that can be used to detect an OVA-specific CD8+ cytolytic T cell response in cells derived from OT-I mice. BM-MDSCs (generated as above) were added at various ratios as indicated and incubated for 72 hours at 37°C. Proliferation of CD8⁺ T cells was assessed by flow cytometry as described below.