Autologous NK cells labeled with CTDR were cultured alone or co-cultured with macrophages with the indicated treatments (2:1) in complete medium (RPMI-1640 supplemented with 10% FBS) and stimulated with 100 U/mL IL-2 and 50 U/mL IL-15 (Cat# 200-15; PeproTech) for 4 days. The proliferation rate was then evaluated by flow cytometry for Ki-67 staining (Cat# 350503; BioLegend). In some experiments, 5 μg/mL anti-human B7-H4 neutralizing Ab (eBioscience) or 5 μg/mL mouse IgG2b control (Cat# 400301; BioLegend) was added to the co-culture.
For the T cell proliferation assay, autologous CD8+ T cells were labeled with 0.5 μM CFSE (Cat# C34554; Thermo Fisher Scientific) for 15 min at room temperature and incubated with mature dendritic cells (DCs; 5:1) and the indicated labeled macrophages (2:1) in RPMI-1640 medium supplemented with 5 μg/mL IL-12, 25 mM HEPES, 4 mM L-glutamine, 25 μM 2-mercaptoethanol, and 10% FBS. Proliferation of CD8+ T cells was measured by CFSE staining and flow cytometry after 4 days. In some experiments, 5 μg/mL anti-human B7-H4 neutralizing Ab or 5 μg/mL mouse IgG2b control (Cat# 400301; BioLegend) was added to the co-culture.
For the T cell proliferation assay, autologous CD8+ T cells were labeled with 0.5 μM CFSE (Cat# C34554; Thermo Fisher Scientific) for 15 min at room temperature and incubated with mature dendritic cells (DCs; 5:1) and the indicated labeled macrophages (2:1) in RPMI-1640 medium supplemented with 5 μg/mL IL-12, 25 mM HEPES, 4 mM L-glutamine, 25 μM 2-mercaptoethanol, and 10% FBS. Proliferation of CD8+ T cells was measured by CFSE staining and flow cytometry after 4 days. In some experiments, 5 μg/mL anti-human B7-H4 neutralizing Ab or 5 μg/mL mouse IgG2b control (Cat# 400301; BioLegend) was added to the co-culture.