Mouse model recapitulating human Fcγ receptor structural and functional diversity Patrick Smith1, David J. DiLillo1, Stylianos Bournazos, Fubin Li, and Jeffrey V. Ravetch2 Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, NY 10021 Contributed by Jeffrey V. Ravetch, March 7, 2012 (sent for review March 2, 2012) The in vivo biological activities of IgG antibodies result from their a particular species, such that the absolute affinities of IgG sub- bifunctional nature, in which antigen recognition by the Fab is classes for their cognate FcγRs cannot be extrapolated between coupled to the effector and immunomodulatory diversity found in species, even for recently diverged human and primate species (1, the Fc domain. This diversity, resulting from both amino acid and 12). This situation is further complicated by the existence of poly- γ γ glycan heterogeneity, is translated into cellular responses through morphisms in the human population for Fc RIIA and Fc RIIIA γ γ that result in different affinities for huIgGs (13–16), as well as Fc receptors (Fc Rs), a structurally and functionally diverse family γ of cell surface receptors found throughout the immune system. polymorphisms in Fc RIIB regulating its level of expression or Although many of the overall features of this system are main- signaling (17). Attempts to model huIgG interactions with human FcγR-expressing cells in vitro fail to mirror the diversity of cellular tained throughout mammalian evolution, species diversity has pre- populations that may be required for an in vivo response. There- cluded direct analysis of human antibodies in animal species, and, fore, new systems to study the in vivo function of the huFcγRsystem thus, detailed investigations into the unique features of the human γ and the biological effects of engaging the activating and inhibitory IgG antibodies and their Fc Rs have been limited. We now report huFcγRs by IgG are required. Furthermore, the increasing number γ the development of a mouse model in which all murine Fc Rs have of Ab-based therapeutics being developed for the treatment of been deleted and human FcγRs, encoded as transgenes, have been neoplastic, infectious, and autoimmune diseases requires a system inserted into the mouse genome resulting in recapitulation of the in which evaluation of the consequences of huFcγR interactions be unique profile of human FcγR expression. These human FcγRs are addressed. We describe the generation and characterization of shown to function to mediate the immunomodulatory, inflamma- an FcγR humanized mouse generated through the transgenic ex- tory, and cytotoxic activities of human IgG antibodies and Fc engi- pression of the entire huFcγR family, under the control of their neered variants and provide a platform for the detailed mechanistic human regulatory elements, on a genetic background lacking all analysis of therapeutic and pathogenic IgG antibodies. mouse FcγRs. These mice are viable, breed normally, demonstrate normal lymphoid tissue development, and generate normal im- γ γ he IgG Fc receptor (FcγR) system is composed of both acti- mune responses. The Fc R humanized mice recapitulate huFc R expression patterns and expression levels and are functional in a Tvating and inhibitory receptors expressed on a diversity of im- fl mune cells whose signals must be appropriately integrated to variety of huIgG-mediated models of in ammation, cytotoxicity, regulate the outcome of inflammation and immunity and to main- and tumor clearance. HuIgG1 mAbs engineered for selectively enhanced Fc-huFcγRaffinities demonstrate enhanced in vivo tain tolerance (1). Defects in the correct functioning of this complex γ system can result in a variety of autoimmune and inflammatory responses in the Fc R humanized mouse. This mouse model, thus, provides an in vivo system to address the functional contributions conditions, on the one hand, or defective host defense responses on γ the other. Importantly, the biological activities of the various IgG Fc of huFc Rs to Ab-mediated biological responses. fi subclasses are critically dependent on their relative af nities for Results activating and inhibitory FcγRs (2). Thus, the in vivo activity of an γ γ γ α−/− Ab in mediating antitumor responses or conventional neutraliza- huFc R Expression in Fc R Humanized Mice. Fc R mice lacking mouse FcγRIIB, FcγRIII, and FcγRIV were generated by condi- tion of a virus or toxin, for example, requires Ab engagement of fi γ α both the appropriate epitope and FcγR. For example, activating tionally targeting the low and intermediate af nity mouse Fc R FcγRs are vital during cancer immunotherapy using mAbs such as locus found on chromosome 1 (Fig. 1A) using a sequential strategy – of integration of loxP sites into the mouse Fcgr3 and Fcgr2b genes anti-CD20 mAb, anti-Her2neu, and anti-EGFR (3 6). Fc inter- fl actions with activating FcγRs are also critical for the neutralization which ank this locus. Cre recombinase-mediated deletion in the of bacterial toxins by mAb (7) and during Ab-mediated neutrali- C57BL/6 ES cells harboring this dual loxP integration resulted in zation of viral pathogens (8–10). By contrast, inhibitory FcγRen- an intragenic deletion of 95 kb with resulting inactivation of the gagement by passively-administered agonistic anti-CD40 mAb or murine Fcgr2b, Fcgr3,andFcgr4 genes. Flow cytometric analysis confirmed that mouse FcγRIIB, FcγRIII, and FcγRIV were un- similar antibodies reactive with other members of the TNFR su- + + detectable on the surface of CD11b cells or B220 Bcellsin IMMUNOLOGY perfamily are required for their ability to enhance cellular immune − − FcRα / mice (Fig. 1B). To generate a strain lacking all mouse responses by inducing adjuvant or apoptotic effects (11). − − FcγR α-chain genes, the FcRα / mice were crossed to a strain It has not been possible to accurately predict the consequences fi γ α of engaging activating or inhibitory human (hu)FcγRs by IgGs for deleted for the mouse high af nity Fc RI -chain found on mouse chromosome 4, and the resulting mice were bred to homozygosity a particular biological response, either for its therapeutic or ad- α α verse effects, using currently available in vitro or in vivo model and named FcR null mice. These FcR null mice were than bred to a series of human transgenic lines expressing either huFCGR1A systems. Although murine and nonhuman primate models can be R131 I232 informative, they do not mirror the structural diversity or unique (18), huFCGR2A (19), huFCGR2B (generated for the expression patterns observed for huFcγRs on human cells (12). purposes of this current study; see materials and methods), For example, humans express a single-chain activation receptor, FcγRIIA, on dendritic cells (DCs), monocytes, and neutrophils, as well as a glycosylphosphatidylinositol (GPI)-anchored FcγRIIIB Author contributions: P.S., D.J.D., S.B., F.L., and J.V.R. designed research; P.S., D.J.D., S.B., exclusively on neutrophils. Both of these receptors are lacking in and F.L. performed research; and P.S., D.J.D., and J.V.R. wrote the paper. mice (1). Expression patterns of FcγRs also differ between mice The authors declare no conflict of interest. and humans; for example, monocyte-derived DCs from mice ex- 1P.S. and D.J.D. contributed equally to this work. press FcγRI, FcγRIIB, FcγRIII, and FcγRIV, whereas human 2To whom correspondence should be addressed. E-mail: [email protected]. monocyte-derived DCs express only FcγRIIA and FcγRIIB. It is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. also apparent that the IgG subclasses and FcγRs has coevolved for 1073/pnas.1203954109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1203954109 PNAS | April 17, 2012 | vol. 109 | no. 16 | 6181–6186 Downloaded by guest on September 25, 2021 Fcgr3 Fcgr4 Fcgr2b 1 4 A 5 1 7 3 6 Exon 5 Exon Exon 2 Exon Exon 2 Exon Exon 4 Exon 3 Exon Exon 1 Exon 3 Exon 5 Exon Exon 8 Exon Exon 4 Exon Exon 2 Exon Exon Exon R2,R3,R4 α chain locus 22.5 kb 40 kb 10 kb 10.5 kb 16 kb Fcgr2b Fcgr3 floxed RI RV RV RI RI RI Fcgr2 targeted S1S2 EC1EC2 TM/IC S1 S2 EC1EC2 TM IC1 IC2IC3 Fcgr3 Fcgr2b floxed RV RV targeted RI RI RI RI Fcgr3 NEO S1 S2 EC1EC2 TM IC1 IC2IC3 targeted S1S2 EC1 EC2TM/IC + cre recombinase R3 loxp -5primer RI RI RI Δ R2,R3,R4 α Chain S1S2 EC1 IC1 IC2IC3 R2Δ4.2primer B CD11b- CD11b+ CD11b- CD11b+ B Cells C57Bl/6 WT C57Bl/6 WT FcRα-/- FcRα-/- CD11b FSC msFcγRIIB/III msFcγRIV FSCB220 msFcγRIIB/III huFc R huFc R huFc RI huFc RIIA huFc RIIB C IIIA/B IIIB − − Fcγ α / γ Null Fig. 1. Generation of FcR mice and huFc R γ R expression analysis in Fc R humanized mice. (A) α Conditional targeting of Fcgr2b and Fcgr3 genes F4/80 + (arrows indicate the coding and noncoding regions of the genes) on the murine α-chain locus and Spleen Monocytes deletion by Cre recombinase of 95 kb of genomic + CD11b DNA between the two most distal LoxP sites (tri- angles). RI and RV indicate the EcoRI and EcoRV restriction sites, respectively, used in the Southern blotting analysis. (B) Mouse FcγRIIB, FcγRIII, and Fc DC BM- γ α−/− γ Fc RIV are not detectable in FcR mice. Spleen Derived Derived 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 R Humanized CD11b+ cells and B220+ B cells from C57BL/6 wild- −/− + + type (upper graphs) and FcRα (lower graphs) mice were stained for surface mouse FcγR expres- B220 Spleen B Cells sion. Representative flow cytometric histograms are shown. (C) HuFcγR expression in FcγR human- + + ized mice. The indicated cell types from the in- dicated tissues were analyzed for huFcγR NK1.1 Spleen CD49b fl NK Cells expression (heavy lines) by immuno uorescence hi staining and flow cytometric analysis.