Legendplex software version 7

Supernatants from various T cell proliferation cultures were collected on day 1 and day 2. Cytokines within the supernatants, including IL-4, IL-6, IL-1β, IL-10, IL-12p70, IL-17, TNFα, IFNγ, IFNβ and GM-CSF were quantified using LEGENDplex^™ kits according to the manufacturer’s instructions. The data were collected on BD LSR Fortessa (BD Biosciences) and analyzed using LEGENDplex^™ software version 7.0 (Biolegend). To test which cytokine(s) were involved in inducing arginase-1 expression in MDSC, Fr. III MDSC isolated from the bone marrow of tumor-bearing mice were cultured in RPMI 1640 medium and treated with cell culture supernatants from CD3/CD28 ligated T cells (50% of the volume) for 18h, in the presence of blocking antibodies against IL-4, IL-6, IL-10, IL-17, IFNγ and GM-CSF alone or in different combinations (20 μg/ml for each antibody). To induce arginase-1 expression, MDSC were treated with IL-4, IL-6, GM-CSF and IL-10 alone or in different combinations for 18 h (50 ng/ml each). After treatment, MDSC were lysed and tested for arginase-1 expression by western blot (WB).