IFN-γ induces TNF family ligand protein expression in vitro and in vivo. IFN-γ induces TNF family ligand protein expression in vitro and in vivo. (A and B) TG macrophages (TG MF) were harvested from mouse peritoneal cavity for in vitro culture. (A) TG MF were treated with IFN-γ at 0–100 U/ml for 24 h and analyzed for 4-1BBL, CD70, and OX40L expression by flow cytometry with summary data and representative histograms shown. Results represent mean ± SEM of six independent cultures harvested from separate mice and two independent experiments. Two-tailed, nonparametric unpaired t test were performed for statistical analyses. *p ≤ 0.05, **p ≤ 0.01. (B) TG MF were pretreated with 200 μg/ml of IFN-γR blocking Ab or isotype control 3 h prior to culturing with 0 or 50 U/ml of IFN-γ followed by flow cytometry analysis for TNF family ligand expression. Results represent mean ± SEM of four experiments using independent cultures of TG MF harvested from three to four separate mice per experiment. (C) Mice were injected i.p. with IFN-γR blocking or isotype control Abs at day (D) 1, 2, and 3 p.i. (500 μg each injection). Splenic APC subsets were analyzed by flow cytometry at D5 p.i. using the gating strategy defined in Supplemental Fig. 1 for 4-1BBL surface expression. Bars show mean ± SEM of eight mice pooled from two independent experiments. Two-tailed, nonparametric unpaired t test were performed for statistical analyses (B and C). *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. Kuan C. Wang et al. ImmunoHorizons 2018;2:407-417 Copyright © 2018 The Authors