Figure 5.

Soluble PD-1 and PD-L1 ectodomain co-expression is not inducing an autologous antibody response. 293T cells were transfected with plasmids encoding for Env, Gag, sPD-1, or sPD-L1. Twenty-four hours after transfection, cells were treated with Brefeldin A for 6 h in order to inhibit protein transport. Subsequently, cells were fixed, permeabilized, and incubated with sera from mice immunized with Env- and Gag-DNA either with empty vector (mock) or with corresponding checkpoint ectodomain DNA (sPD-1, sPD-L1). Murine antibodies were detected using an APC-conjugated anti-mouse IgG1 antibody. Shown are the histograms of transfected cells (A) as well as the Geometric Mean Fluorescence Intensity of Env- (B), Gag- (C), sPD-1 (D), and sPD-L1 transfected cells (E) after incubation with immunized mouse sera and the respective secondary antibody. (A) Data are representative of three independent experiments. (B–E) Data represent the mean with SEM of one out of three representative experiments with three sera samples from each group.