MIMICKING THE HUMORAL IMMUNE-RESPONSE IN-VITRO RESULTS IN ANTIGEN-SPECIFIC ISOTYPE SWITCHING SUPPORTED BY SPECIFIC AUTOLOGOUS T-HELPER CELLS - GENERATION OF HUMAN HIV-1-NEUTRALIZING IGG MONOCLONAL-ANTIBODIES FROM NAIVE DONORS
MIMICKING THE HUMORAL IMMUNE-RESPONSE IN-VITRO RESULTS IN ANTIGEN-SPECIFIC ISOTYPE SWITCHING SUPPORTED BY SPECIFIC AUTOLOGOUS T-HELPER CELLS - GENERATION OF HUMAN HIV-1-NEUTRALIZING IGG MONOCLONAL-ANTIBODIES FROM NAIVE DONORS
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DOI：
10.1002/eji.1830250305
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发表时间：
1995-03-01
影响因子：
5.4
通讯作者：
BORREBAECK, CAK
中科院分区：
文献类型：
--
作者：
CHIN, LT;MALMBORG, AC;BORREBAECK, CAK
通过模拟生发中心发生的信号传导，研究了人类 B 细胞抗原特异性同种型转换的分子和细胞要求。首先使用含有 T 和 B 细胞表位的合成免疫原对健康血清阴性献血者的外周血单核细胞进行体外初次免疫，从而产生特异性 IgM 分泌 B 细胞。我们使用 gp120 V3 环的顶点作为 B 细胞表位，与来自破伤风毒素的混杂 T 辅助表位连接。同时，建立了对免疫原的T表位具有特异性的CD4(+) T辅助细胞克隆。在二次体外刺激期间，我们在转染 CD32 的成纤维细胞上与抗 CD40 单克隆抗体共培养抗原特异性 T 细胞和 B 细胞。这导致同种型转换并检测到人类抗原特异性、分泌 IgG 的 B 细胞。这种反应严格依赖于自体 T 辅助细胞和免疫原的存在。随后对源自初次和二次体外免疫的抗原特异性人B细胞进行电场诱导的体细胞杂交，并分离分泌人抗V3 IgG单克隆抗体的杂交瘤。进一步表征了一种人抗体，并显示其对免疫抗原具有特异性，亲和常数为 24 nM。该抗体还可以有效中和 HTV-1 的不同分离株，在 0.46 μg/ml 浓度下实现 50% 中和。
Molecular and cellular requirements for antigen-specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM-secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4(+) T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD40 monoclonal antibody. This resulted in isotype switching and human antigen-specific, IgG-secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreting human anti-V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HTV-1, achieving a 50 % neutralization at 0.46 mu g/ml.