Gharagozlou S, Ghods R, Bahrami Z Samadi, Roohi A, Jeddi-Tehrani M, Conti-Fine B M, Sharifian R A, Rabbani H, Shokri F
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Hum Antibodies. 2003;12(3):67-76.
Hemophilia A patients treated with human coagulating factor VIII (FVIII) may develop inhibitory antibodies (inhibitors). Characterization of the inhibitors at the clonal level may help exploring new therapeutic strategies. We have generated lymphoblastoid cell lines (LCLs) producing anti-FVIII antibodies from peripheral blood lymphocytes of hemophilia A patients with high inhibitor titers. We fused the anti-FVIII-positive LCLs with a heteromyeloma, to produce FVIII specific hybridomas. We determined the specificity, isotype, idiotypic and immunoglobulin (Ig) variable region heavy (VH) chain gene family profiles of the secreted antibodies (Ab) by ELISA, immunoblotting and RT-PCR. We established eight hybridomas which produced high titers of anti-FVIII Ab. All hybridomas secreted IgM Ab, associated with either kappa(5/8) or lambda(3/8) light chain. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized only the VH1 (63%) or the VH3 (37%) gene families. Among the cross-reactive idiotypes (CRIs) we tested, only the VH1 and VK3b-associated CRIs were expressed by 3 hybridomas. Immunoblotting of thrombin-digested FVIII demonstrated distinct patterns of reactivity of the monoclonal Ab (MAb) secreted by the hybridomas, which recognized either the A2 domain of the Fvm heavy chain, or the light chain, or both. Our findings suggest that: a) the isotype of the anti-FVIII Ab secreted by LCLs and hybridoma clones (IgM) differs from that of anti-FVIII Ab in vivo, which are predominantly IgG4: this suggests a negative selection of the isotype-switched FVIII-specific B-cells in the periphery of these patients; b) the anti-FVIII Ab have a biased representation of the VH1 gene family, and c) somatic mutations in the VH genes coding for FVIII specificity occur in the anti-FVIII Ab response, as evidenced by lack of expression of the VH-associated CRI.
接受人凝血因子VIII（FVIII）治疗的甲型血友病患者可能会产生抑制性抗体（抑制剂）。在克隆水平上对抑制剂进行表征可能有助于探索新的治疗策略。我们从抑制剂滴度高的甲型血友病患者外周血淋巴细胞中生成了产生抗FVIII抗体的淋巴母细胞系（LCLs）。我们将抗FVIII阳性的LCLs与异源骨髓瘤细胞融合，以产生FVIII特异性杂交瘤。我们通过酶联免疫吸附测定（ELISA）、免疫印迹和逆转录聚合酶链反应（RT-PCR）确定了分泌抗体（Ab）的特异性、同种型、独特型和免疫球蛋白（Ig）可变区重链（VH）基因家族谱。我们建立了8个产生高滴度抗FVIII Ab的杂交瘤。所有杂交瘤均分泌IgM Ab，与κ（5/8）或λ（3/8）轻链相关。通过RT-PCR对表达的VH基因进行分析表明，杂交瘤仅利用VH1（63%）或VH3（37%）基因家族。在我们测试的交叉反应独特型（CRIs）中，只有VH1和VK3b相关的CRIs由3个杂交瘤表达。对凝血酶消化的FVIII进行免疫印迹显示，杂交瘤分泌的单克隆抗体（MAb）具有不同的反应模式，这些单克隆抗体识别Fvm重链的A2结构域、轻链或两者。我们的研究结果表明：a）LCLs和杂交瘤克隆分泌的抗FVIII Ab的同种型（IgM）与体内抗FVIII Ab的同种型不同，体内抗FVIII Ab主要为IgG4：这表明在这些患者外周，同种型转换的FVIII特异性B细胞发生了阴性选择；b）抗FVIII Ab中VH1基因家族的代表性存在偏差，以及c）编码FVIII特异性的VH基因中的体细胞突变发生在抗FVIII Ab反应中这一点，可通过缺乏VH相关CRI的表达得到证明。
