Fig. 4. aPD-1 mAb transfer to macrophages is mediated by FcγRs.

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Fig. 4. aPD-1 mAb transfer to macrophages is mediated by FcγRs. aPD-1 mAb transfer to macrophages is mediated by FcγRs. (A) Ex vivo flow cytometry histograms of MC38 tumors stained with phycoerythrin (PE)–aPD-1 show that CD8+ T cells but not TAMs express cell surface PD-1. (B) Coculture of bone marrow–derived macrophages (Mø) and AF647–aPD-1–coated EL4 lymphocytes was used to quantify the AF647–aPD-1 puncta in macrophages preblocked with Fcγ receptor (FcγR) inhibitors or the phagocytosis inhibitor dynasore. *P < 0.05; ****P < 0.0001, one-way analysis of variance (ANOVA). n.s., not significant. (C) Representative images of macrophages labeled with PKH red and observed by time-lapse imaging for aPD-1 mAb (yellow) transfer from neighboring lymphocytes (cerulean) after 30 min. FcγRII/III inhibition blocked antibody transfer (insets). Scale bars, 10 μm. (D and E) Flow cytometry was used to estimate AF647–aPD-1 transfer to F4/80+ bone marrow–derived macrophages in coculture assays when the mAb was added directly (green), bound to EL4 cells (red), or bound to EL4 cells in the presence of FcγRII/III neutralizing antibody (blue). Results in (D) are a representative histogram of AF647 signal in macrophages, and (E) is the geometric mean fluorescence intensity (G-MFI) value of conditions presented in (D). Data are from three independent experiments. *P < 0.05; ** P < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. Values represent SEM for three separate experiments. Sean P. Arlauckas et al., Sci Transl Med 2017;9:eaal3604 Published by AAAS