Fig. 5: An anti-FcγRIIIB-antigen conjugate converts murine and human neutrophils to nAPCs that is dependent on FcγRIIIB endocytosis. | Nature Communications

Fig. 5: An anti-FcγRIIIB-antigen conjugate converts murine and human neutrophils to nAPCs that is dependent on FcγRIIIB endocytosis.

From: FcγR engagement reprograms neutrophils into antigen cross-presenting cells that elicit acquired anti-tumor immunity

Fig. 5

a Blood neutrophils from 2A3B/γ−/− and γ−/− mice incubated with isotype (Iso) or 3G8-fOva, cultured with GM-CSF and analyzed after 3 days for CD11c, MHCII on Ly6G positive cells. CD11c+MHCII+Ly6G+ cells in 2A3B/γ−/− + 3G8-fOva sample further analyzed for CD80, CD86, and CCR7. b CD11c and MHCII acquisition after 3G8-fOva or isotype treatment of murine neutrophils from indicated mouse strains and culture with GM-CSF for 3 days. c, d 3B/γ−/− bone marrow neutrophils pretreated with vehicle, methyl-beta cyclodextrin (MβCD) or Cytochalasin D (Cyto D) and treated with isotype, 3G8-fOva or 3G8 antibody. FcγRIIIB on the cell surface evaluated using anti-FcγRIIIB, clone REA589 (c). Cells from c analyzed for the percent of cells acquiring CD11c and MHCII after 3 days in culture with GM-CSF (d). e, f Cells treated with Ova, Ova-IC, or SLE-IC and analyzed for surface FcγRIIIB (e) and acquisition of CD11c and MHCII on Ly6G+ cells (f) as in (c, d). g, h Human blood neutrophils treated with 3G8-fOva or isotype control. Evaluation of surface FcγRIIIB (assessed with REA589 antibody), FcγRIIA (assessed with IV.3 antibody) and FITC (fOva) positivity over time (g). The percent of CD11c and HLA-DR positive cells with neutrophil markers (CD15, CD10) after 2 days in culture with GM-CSF. CD11c and HLA-DR double-positive cells further examined for CD80, CD86, and CCR7 (h). i Flow plots assessing the purity of human neutrophils at day 0 (upper panels) using markers for neutrophils (CD15, CD66b), DCs (CD11c), monocytes (CD14), B cells (B220), NK cells (NK1.1), and T cells (CD3) (Lin−). Gating strategy for nAPC markers at day 2 after 3G8-fOva treatment (lower panels). j Images of purified human neutrophils, treated as in (g, h), FACS sorted on day 1 and stained with Wright-Giemsa. FACS profiles of day 0 human blood neutrophils stained for markers of neutrophils (left), monocytes (middle) and NK, T and B cells (right). Data are mean ± s.e.m. c–g One-way analysis of variance and Dunnett’s multiple comparison test; a, b, h Multiple t-tests between pairs of samples. **p < 0.005.

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