Fig. 1.

Divergent hIgG agonism and specific FcγR-binding requirement. a Diagram showing the OVA-specific CD8⁺ T-cell response model. In brief, FcγR-humanized (hFCGR^Tg) or -deficient (FcγRα^−/−) mice were adoptively transferred with OT-I cells on day-1, immunized intraperitoneally (i.p.) with 2 μg of DEC-OVA in the presence of control or anti-CD40 antibodies on day 0. Splenocytes were harvested to quantify OVA-specific CD8⁺ T cells on day 6. b, c Representative FACS profile (b) and quantification (c) showing the percentage of OT-I cells (CD45.1⁺TCRVα2⁺) among CD8⁺ T cells in mice treated and analyzed as in (a) together with 30 μg of indicated control or anti-mCD40 antibodies. Numbers of mice: b, c three hFCGR^Tg mice for Ctrl IgG, five hFCGR^Tg, and three FcγRα^−/− mice for other groups. d–f Quantification of OT-I cells as the percentage of OT-I cells among CD8⁺ T cells (d, e) or cell count (f) in FcγR-humanized mice treated and analyzed as in (a) together with 10 μg (d, e) or 30 μg (f) of indicated control or anti-mCD40 antibodies (the N297A mutation abrogates Fc–FcγR binding) and with/without FcγRIIB-blocking antibody 2B6 (150 μg per mouse) (f). Numbers of mice: d four mice per group; e five mice per group; f two mice for Ctrl IgG, five mice for αmCD40:G2, six mice for αmCD40:G2 + 2B6. g Quantification of OT-I cells as the percentage of OT-I cells among CD8⁺ T cells in mice of indicated genotypes (FcγR-deficient (FcgRα^−/−, five mice per group), FcγRIIB-deficient (Fcgr2b^−/−, five mice per group) or humanized (Fcgr2b^−/−hFCGR2B^Tg, four mice per group)) treated and analyzed as in (a) together with 10 μg of indicated control or IgG2 anti-mCD40 antibodies. Each symbol represents an individual mouse. Bars represent the mean ± SEM. ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001; unpaired two-tailed t test (c, g), one-way ANOVA with Holm–Sidak’s post hoc (d–f). Source data (c–g) are provided as a Source Data file. A representative of two independent experiments is shown