Ierino F L, Hulett M D, McKenzie I F, Hogarth P M
Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.
J Immunol. 1993 Mar 1;150(5):1794-803.
Fc gamma RII is a low affinity FcR for IgG with two Ig-like extracellular domains (D1 gamma and D2 gamma), a transmembrane domain, and a cytoplasmic domain. The production, characterization, and epitope analysis of four anti-human Fc gamma RII 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 Fc gamma RII was demonstrated by (a) specific binding to Fc gamma RII+ L cells (produced after transfection of L cells with human Fc gamma RIIa cDNA, HFc3.0), by using flow cytometry, (b) inhibition of the binding of SRBC sensitized with rabbit antibody (EA) to Fc gamma RII+ 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 Fc gamma RII+ L cells. The mAb were able to detect different forms of Fc gamma RII, 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 Fc gamma RII (a and b1) and four allelic variants of Fc gamma RIIa 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 Fc gamma RIIa by using genetically engineered chimeric receptors. Chimeric receptors expressing either D1 gamma linked to domain 2 of Fc epsilon RI or domain 1 of Fc epsilon RI linked to D2 gamma were produced by exchanging homologous, but antigenically different, regions of Fc gamma RIIa and the high affinity receptor for IgE. Four clusters of mAb were identified, each mapping to discrete epitopes of Fc gamma RII. Cluster I (mAb 8.2 and CIKM5) defines a combinatorial epitope with determinants in D1 gamma and D2 gamma 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 Fc gamma RII. The epitopes of clusters 2 (mAb 8.26), 3 (mAb IV.3), and 4 (mAb 8.7 and 7.30) are located entirely in D2 gamma and all involve the IgG Fc binding region, because F(ab')/F(ab')2 fragments of the mAb inhibit EA binding to Fc gamma RII. Thus, all mAb that inhibit the binding of EA map totally to D2 gamma; it is likely the IgG Fc binding region is also contained in D2 gamma.
FcγRII是一种对IgG具有低亲和力的Fc受体，具有两个Ig样细胞外结构域（D1γ和D2γ）、一个跨膜结构域和一个细胞质结构域。详细介绍了四种抗人FcγRII单克隆抗体（8.2、8.7、8.26和7.30）的产生、特性及表位分析，并将这些单克隆抗体与两种已定义的CDw32单克隆抗体IV.3和CIKM5进行了比较。通过以下方法证实了所有单克隆抗体与FcγRII的反应性：(a) 使用流式细胞术特异性结合FcγRII + L细胞（用人FcγRIIa cDNA，HFc3.0转染L细胞后产生）；(b) 抑制兔抗体致敏的绵羊红细胞（EA）与FcγRII + L细胞的结合；(c) 免疫沉淀和SDS-PAGE，在K562和U937细胞上检测到一种42 kDa的蛋白质，在FcγRII + L细胞上检测到一种单一的45 kDa蛋白质。通过流式细胞术，这些单克隆抗体能够在Daudi细胞（8.7和7.30）和U937细胞（8.2、IV.3和CIKM5）上检测到不同形式的FcγRII；相对于其余单克隆抗体，8.26使Daudi细胞呈现中等荧光，使U937细胞呈现最高荧光。在COS-7细胞中，单克隆抗体与瞬时表达的FcγRII同工型（a和b1）以及FcγRIIa的四个等位基因变体的结合并未区分单克隆抗体表位。通过以下方法进一步确定单克隆抗体表位的定位：(a) EA抑制试验；(b) 单克隆抗体阻断研究；(c) 使用基因工程嵌合受体使单克隆抗体与人FcγRIIa片段结合。通过交换FcγRIIa和IgE高亲和力受体的同源但抗原性不同的区域，产生了表达与FcεRI结构域2连接的D1γ或与D2γ连接的FcεRI结构域1的嵌合受体。鉴定出四组单克隆抗体，每组对应FcγRII的离散表位。第一组（单克隆抗体8.2和CIKM5）定义了一个组合表位，其决定簇位于远离IgG Fc结合位点的D1γ和D2γ中，因为8.2和CIKM5的F(ab')2片段不抑制EA与FcγRII的结合。第二组（单克隆抗体8.26）、第三组（单克隆抗体IV.3）和第四组（单克隆抗体8.7和7.30）的表位完全位于D2γ中，并且都涉及IgG Fc结合区域，因为这些单克隆抗体的F(ab')/F(ab')2片段抑制EA与FcγRII的结合。因此，所有抑制EA结合的单克隆抗体完全定位在D2γ；IgG Fc结合区域可能也包含在D2γ中。
