FcγRII is a low affinity FcR for IgG with two Ig-like extracellular domains (D1γ and D2γ), a transmembrane domain, and a cytoplasmic domain. The production, characterization, and epitope analysis of four anti-human FcγRII mAb (8.2, 8.7, 8.26, and 7.30) are detailed, and the mAb are compared with two defined CDw32 mAb, IV.3 and CIKM5. Reactivity of all mAb with FcγRII was demonstrated by (a) specific binding to FcγRII+ L cells (produced after transfection of L cells with human FcγRIIa cDNA, HFc3.0), by using flow cytometry, (b) inhibition of the binding of SRBC sensitized with rabbit antibody (EA) to FcγRII+ L cells, and (c) immunoprecipitation and SDS-PAGE, which detected a 42-kDa protein on K562 and U937 cells and a single 45-kDa protein on FcγRII+ L cells. The mAb were able to detect different forms of FcγRII, by flow cytometry, on Daudi cells (8.7 and 7.30) and U937 cells (8.2, IV.3, and CIKM5); 8.26 stained Daudi cells with intermediate fluorescence and U937 cells with the highest fluorescence, relative to the remaining mAb. Binding to transiently expressed isoforms of FcγRII (a and b1) and four allelic variants of FcγRIIa in COS-7 cells did not distinguish the mAb epitopes. Further mapping of the mAb epitopes was determined by (a) EA inhibition assays, (b) mAb blocking studies, and (c) the binding of the mAb to segments of human FcγRIIa by using genetically engineered chimeric receptors. Chimeric receptors expressing either D1γ linked to domain 2 of FcεRI or domain 1 of FcεRI linked to D2γ were produced by exchanging homologous, but antigenically different, regions of FcγRIIa and the high affinity receptor for IgE. Four clusters of mAb were identified, each mapping to discrete epitopes of FcγRII. Cluster I (mAb 8.2 and CIKM5) defines a combinatorial epitope with determinants in D1γ and D2γ distant from the IgG Fc binding site, inasmuch as F(ab')2 fragments of 8.2 and CIKM5 do not inhibit the binding of EA to FcγRII. The epitopes of clusters 2 (mAb 8.26), 3 (mAb IV.3), and 4 (mAb 8.7 and 7.30) are located entirely in D2γ and all involve the IgG Fc binding region, because F(ab')/F(ab')2 fragments of the mAb inhibit EA binding to FcγRII. Thus, all mAb that inhibit the binding of EA map totally to D2γ; it is likely the IgG Fc binding region is also contained in D2γ.
|Number of pages||10|
|Journal||Journal of Immunology|
|Publication status||Published - 1 Jan 1993|