Mouse model recapitulating human Fcγ receptor structural and functional diversity

Abstract

The in vivo biological activities of IgG antibodies result from their bifunctional nature, in which antigen recognition by the Fab is coupled to the effector and immunomodulatory diversity found in the Fc domain. This diversity, resulting from both amino acid and glycan heterogeneity, is translated into cellular responses through Fcγ receptors (FcγRs), a structurally and functionally diverse family of cell surface receptors found throughout the immune system. Although many of the overall features of this system are maintained throughout mammalian evolution, species diversity has precluded direct analysis of human antibodies in animal species, and, thus, detailed investigations into the unique features of the human IgG antibodies and their FcγRs have been limited. We now report the development of a mouse model in which all murine FcγRs have been deleted and human FcγRs, encoded as transgenes, have been inserted into the mouse genome resulting in recapitulation of the unique profile of human FcγR expression. These human FcγRs are shown to function to mediate the immunomodulatory, inflammatory, and cytotoxic activities of human IgG antibodies and Fc engineered variants and provide a platform for the detailed mechanistic analysis of therapeutic and pathogenic IgG antibodies.

Fig. 1.

Generation of FcRα^−/− mice and huFcγR expression analysis in FcγR humanized mice. (A) Conditional targeting of Fcgr2b and Fcgr3 genes (arrows indicate the coding and noncoding regions of the genes) on the murine α-chain locus and deletion by Cre recombinase of 95 kb of genomic DNA between the two most distal LoxP sites (triangles). RI and RV indicate the EcoRI and EcoRV restriction sites, respectively, used in the Southern blotting analysis. (B) Mouse FcγRIIB, FcγRIII, and FcγRIV are not detectable in FcRα^−/− mice. Spleen CD11b⁺ cells and B220⁺ B cells from C57BL/6 wild-type (upper graphs) and FcRα^−/− (lower graphs) mice were stained for surface mouse FcγR expression. Representative flow cytometric histograms are shown. (C) HuFcγR expression in FcγR humanized mice. The indicated cell types from the indicated tissues were analyzed for huFcγR expression (heavy lines) by immunofluorescence staining and flow cytometric analysis. Shaded histograms show background staining by isotype control mAbs, except for the FcγRIIIB histograms, where shaded histograms show FcγRIIIB staining on cells from FcRα null mice.

Fig. 2.

FcγR humanized mice generate normal immune responses and develop normal lymphoid structures. (A and B) Wild-type (WT), C57BL/6 (open circles), and FcγR humanized mice (filled circles) were immunized i.p. with TNP-LPS (A) or NP-OVA (B), followed by a boost with NP-OVA in PBS on day 28. Serum was harvested at the indicated time points and analyzed for TNP- or NP-specific IgM and IgG levels by ELISA. Horizontal bars indicate mean relative OD. (C) FcRα null (Upper) and FcγR humanized (Lower) mouse spleen sections were examined by immunohistochemistry with fluorescence microscopy analysis for B220 (B cells), TCRβ (T cells), huFcγRIIA, huFcγRIIB, and huFcγRIIIA/B expression. Representative images are shown.

Fig. 3.

FcγR humanized mice perform normal effector functions during in vivo models of mAb-mediated cytotoxicity. (A) B-cell depletion with CD40 mAb Fc variants. FcγR humanized mice received CD40 mAb with the indicated Fc. The frequency of blood B220⁺CD3⁻ B cells was analyzed before (day 0) and 5 d after mAb treatment by immunofluorescence staining with flow cytometric analysis. Representative flow cytometric dot plots are shown. Values in the graph represent means (± SEM) frequencies of B cells in mice receiving the indicated treatment at the indicated time point (n ≥ 2 for all groups). Significant differences between the indicated sample means are indicated: *P < 0.05; **P < 0.01. (B) T-cell depletion with CD4 mAb Fc variants. The indicated mice received CD4 mAb with the indicated Fc. FcRα null mice received huIgG1 WT mAb. The frequency of blood CD4⁺ T cells was analyzed before and 2 d after mAb treatment by immunofluorescence staining with flow cytometric analysis. Representative flow cytometric dot plots are shown. Values in the graph represent the mean (± SEM) percentage change in the frequency of CD4⁺ T cells relative to the prebleed at 0 h (n ≥ 3 for all groups). (C) Platelet depletion with 6A6 mAb Fc variants. The indicated mice received 6A6 mAb with the indicated Fc. FcRα null mice received huIgG1 WT mAb. Platelet numbers were analyzed at the indicated time points, and values represent the mean (± SEM) percentage change in platelet number relative to the prebleed at 0 h (n ≥ 3 for all groups). (B and C) Significant differences between means from the indicated sample and the N297A group are indicated: **P < 0.01. Significant differences between sample means from the GASD/ALIE group and the IgG1 WT group are indicated: ^#P < 0.05; ^##P < 0.01.

Fig. 4.

Tumor clearance, immune complex-mediated anaphylaxis, and FcγRIIB-mediated enhancement of immunization by CD40 mAb is effective in FcγR humanized mice. (A) B16 melanoma model. FcγR humanized mice or FcRα^−/− mice were injected i.v. with B16-F10 cells and received TA99 mAb with the indicated Fc (n ≥ 4 for all groups) on days 0, 2, 4, 7, 9, and 11. FcRα null mice received huIgG1 WT mAb. On day 13, lungs were harvested and metastasis foci were counted. Pictures show representative lungs from mice receiving the indicated treatment. Values in the graph represent the mean (± SEM) number of lung metastasis foci from mice receiving the indicated treatment. Significant differences between sample means and the N297A group, or between the indicated groups are shown: *P < 0.05; **P < 0.01. (B) Immune complex-mediated anaphylaxis model. Wild-type (WT) C57BL/6, FcRα null, and FcγR humanized mice (n = 3 for each group) were injected i.v. with heat-aggregated human IVIG. Values represent the mean (± SEM) mouse core body temperature measured at the indicated time points. Significant differences between sample means from the indicated time point and time 0 are shown: *P < 0.05; **P < 0.01. (C) Inhibitory FcγR-mediated enhancement of immunization by CD40 mAb. FcγR humanized mice (n = 3 per group) were treated with DEC-OVA and CD40 mAbs with the indicated Fcs. The frequencies of OVA-specific CD8⁺ T cells [identified by SIINFEKL H-2^b tetramer (Tet-OVA) staining; Left] and the ratios of CD8⁺ T cells to CD4⁺ T cells (Right) were analyzed in the peripheral blood 7 d later. Each point represents data from an individual mouse, with bars showing mean values. Significant differences between the indicated sample means are shown: ***P < 0.001.