Fig. 5. Direct and Ab-dependent cellular cytotoxicity (ADCC) activity of anti-PD-1 Ab on PD-1 expressing macrophage/microglia.

A, PD-1 expression of EOC-20 cells, gated on CD45+ CD11b+ cells. B, PD-1 expression on myeloid cells (CD45+, CD11b+) isolated from tumor bearing Ntv-a mice. C, Coincubation of only the anti-PD-1 Ab at the designated concentrations with PD-1 expressing EOC-20 microglia resulted in diminished cellular viability starting one day after exposure, which was further enhanced with increased exposure time. D, Coincubation of BrdU-labeled EOC-20 microglia with increasing concentrations of anti-PD-1 relative to the IgG control demonstrated decreased proliferative capacity. E, Cell cycle analysis of EOC-20 cells exposed to IgG control or anti-PD-1 demonstrating that these antibodies do not impact cellular proliferation. F, Ab-dependent cellular cytotoxicity (ADCC) assay detecting lactic dehydrogenase leakage (luminescence; relative light units [RLU]) from target EOC-20 microglia cells upon exposure to anti-PD-1 and in the presence of effector cells capable of mediating ADCC. Mouse microglial target cells, EOC20, were incubated with control Ab or anti-PD-1 Ab at a concentration of 125 μg/ml or 12.5 μg/ml, followed by the addition of effector cells. The E:T ratio was 20:1. After 8h of induction at 37^o C, Bio-Glo luciferase reagent was added, and luminescence (RLU) was determined. The ADCC was further potentiated by the presence of a secondary Ab (mouse anti-rat) that could be generated by repeat administration of a rat anti-mouse Ab (anti-PD-1) in vivo. M = Media; T = Target; E = Effector Cell. When the anti-PD-1 Ab was decreased to 12.5 μg/ml, similar results were obtained.