Mori A, Thomas P, Tagaya Y, Iijima H, Grey H, Ishizaka K
Division of Immunobiology, La Jolla Institute for Allergy and Immunology, CA 92037.
Int Immunol. 1993 Aug;5(8):833-42. doi: 10.1093/intimm/5.8.833.
From the spleen cells of BALB/c mice primed with bee venom phospholipase A2 (PLA2), we established seven T cell hybridomas which constitutively secreted glycosylation inhibiting factor (GIF), expressed both CD3 and TCR alpha beta, and responded to antigen-pulsed antigen presenting cells (APC) for the formation of IgE-binding factor. Upon stimulation with antigen-pulsed APC, four of the seven hybridomas produced GIF having affinity for native PLA2. The antigen-binding GIF could suppress the anti-hapten antibody response of BALB/c mice to dinitrophenyl (DNP)-PLA2 conjugates in a carrier-specific manner and bound to immunosorbents coupled with either the mAb 14-12 or anti-TCR alpha chain, H28-710. Analysis of the epitope specificity of the TCR on the GIF-producing T hybridomas indicated that all of the hybridomas which could produce antigen-binding GIF upon antigenic stimulation recognized the synthetic peptide representing amino acid residues 19-34 in PLA2 molecules in the context of the product of the I-Ad subregion and the antigen-binding GIF formed by the cells had affinity for the peptide. The 3-D structure of bee venom PLA2 indicates that the sequence of amino acid 14-24 forms a loop in the PLA2 molecule and represents an external structure of the antigen, while peptide 25-37 forms an alpha helix. Evidence was obtained which suggests that the sequence of 25-34 contains amino acid residues interacting with Ia molecules, while peptide 19-24 contains residues involved in the interaction of p19-34-Ia complexes with TCR on the hybridomas. It was also found that not only the synthetic peptide 19-34, but also the peptides 13-28 and 19-30 inhibited the binding of antigen-binding GIF to PLA2-coupled Sepharose, while peptide 25-40 failed to do so. The results collectively indicate that the antigen-binding GIF and TCR on the cell source of the factor interact with a common epitope which is exposed on the surface of a nominal antigen.
从用蜂毒磷脂酶A2（PLA2）致敏的BALB/c小鼠的脾细胞中，我们建立了7个T细胞杂交瘤，它们组成性分泌糖基化抑制因子（GIF），表达CD3和TCRαβ，并对抗原脉冲化的抗原呈递细胞（APC）作出反应以形成IgE结合因子。在用抗原脉冲化的APC刺激后，7个杂交瘤中的4个产生了对天然PLA2具有亲和力的GIF。抗原结合性GIF可以以载体特异性方式抑制BALB/c小鼠对二硝基苯基（DNP）-PLA2偶联物的抗半抗原抗体反应，并与偶联有单克隆抗体14-12或抗TCRα链H28-710的免疫吸附剂结合。对产生GIF的T杂交瘤上TCR的表位特异性分析表明，所有在抗原刺激后能产生抗原结合性GIF的杂交瘤，在I-Ad亚区产物的背景下，都识别代表PLA2分子中氨基酸残基19-34的合成肽，并且细胞形成的抗原结合性GIF对该肽具有亲和力。蜂毒PLA2的三维结构表明，氨基酸14-24的序列在PLA2分子中形成一个环，代表抗原的外部结构，而肽25-37形成一个α螺旋。有证据表明，25-34序列包含与Ia分子相互作用的氨基酸残基，而肽19-24包含参与p19-34-Ia复合物与杂交瘤上TCR相互作用的残基。还发现，不仅合成肽19-34，而且肽13-28和19-30都能抑制抗原结合性GIF与PLA2偶联的琼脂糖的结合，而肽25-40则不能。这些结果共同表明，该因子细胞来源上的抗原结合性GIF和TCR与暴露在名义抗原表面的一个共同表位相互作用。
