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. 2020 Jul 13;217(10):e20200359. doi: 10.1084/jem.20200359

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© 2020 Hubbard et al.

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Figure 2. — FcRn regulates CD32a-induced responses to IgG IC. (a–c) Absolute TNF-α (a), IL-12/23p40 (b), and IL-6 (c) production by primary splenic CD11c⁺MHCII⁺ DCs from CD32a^R-Tg (R) or CD32a^H-Tg (H) mice after 24 h of exposure to antigen (OVA^NIP) alone (1 µg/ml) or 100 µg/ml anti-NIP hIgG1^WT (WT), hIgG1^IHH (IHH), or hIgG1^N297A (N297A) in monomeric (mono) or OVA^NIP-IC form. Black-filled circles, OVA^NIP; white-filled circles, monomeric or hIgG1^WT ICs; gray-filled squares, monomeric or hIgG1^IHH ICs; white-filled diamonds, monomeric or hIgG1^N297A ICs. (d and e) IL-2 production by MHCII-restricted, OVA-specific CD4⁺ T cells after 24 h of co-culture with CD32a^R- or CD32a^H- or vector control plasmid–transfected RAW 264.7 cells that were treated with hIgG1^WT ICs prepared with increasing concentrations of OVA^NIP (d) or treated with hIgG1 ICs composed of hIgG1^WT, hIgG1^IHH, or hIgG1^N297A mutants and 10 µg/ml OVA^NIP (e). (f) IL-2 production by OVA-specific CD8⁺ OT-I T cells after 48 h of co-culture with CD32a^R- or CD32a^H- or vector control plasmid–transfected HEK 293^H2-Kb cells loaded with OVA^NIP-containing hIgG1^WT ICs as in Fig. 1 d. (g) IFN-γ production by CD8⁺ OT-I T cells after 48 h of co-culture with primary CD11c⁺MHCII⁺ CD32a^R-Tg or CD32a^H-Tg DCs loaded with hIgG1^WT ICs, hIgG1^IHH ICs, or hIgG1^N297A ICs. (h and i) IFN-γ production by CD8⁺ OT-I T cells co-cultured for 48 h with CD11c⁺MHCII⁺ FcγR^KO DCs loaded with hIgG1^WT ICs or hIgG1^IHH ICs at pH 7.4 or pH 5.6 (h) or pretreated with an isotype IgG2a control antibody or anti-m/hFcRn mAb DVN24 for 30 min before treatment with OVA^NIP only, or hIgG1^WT, hIgG1^IHH, or hIgG1^MST/HN ICs (white-filled triangles) at pH 7.4 (i). All data represent arithmetic mean ± SEM of duplicate or triplicate technical replicates from three independent experiments (a–c), or are representative of three independent experiments (d–i), with triplicate technical replicates shown. All data were analyzed by two-way ANOVA followed by the two-stage linear step-up procedure of Benjamin, Krieger, and Yekutieli with FDR controlled at <0.05. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.