An in vitro T-cell assay was performed as described to evaluate the MDSC’s activity [22 (link)]. Briefly, 1 × 105 carboxy fluorescein diacetate succinimidyl ester (Invitrogen, California, USA) labeled T-cells were activated with a 1/800 dilution of anti-CD3/CD28 antibody coated beads (Invitrogen, California, USA) and cultured with or without 5 × 104 WT or KO MDSCs. When indicated, the GzmB inhibitor, Z-AAD-CMK (Enzo Life Sciences, Antwerpen, Belgium), was added (10 μM). T-cell proliferation and viability were determined in flow cytometry.
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