Ishii Y, Nakano T, Ishizaka K
Division of Immunology, La Jolla Institute for Allergy and Immunology, CA 92037, USA.
J Immunol. 1996 Mar 1;156(5):1735-42.
We isolated a unique TCR alpha-chain derived from the OVA-specific Ts hybridoma, 231F1. The TR alpha-chain consists of V alpha 11.3, a unique J alpha and a complete sequence of C alpha region. Transfection of the TCR-alpha cDNA into a TCR-alpha-, TCR-beta+ T cell line, 175.2, resulted in the expression of TCR-alpha beta, and the transfectant contained a 35-kDa peptide having the TCR- alpha-specific antigenic determinant. However, the stable transfectant failed to release a peptide with the TCR-alpha determinant upon stimulation with anti-CD3. In contrast, overexpression of the cDNA in the 231 F1 cells markedly increased the formation of the 55-kDa peptide, which reacted with both anti-glycosylation-inhibiting factor (GIF) and the mAb H28-710. Definitive evidence for the relationship between the 55-kDa peptide and the TCR alpha-chain was obtained by transfection of the cDNA of the TCR alpha-chain with histidine tag into the 231F1 cells. The 55 kDa GIF peptide formed by stable transfectants of the TCR-alpha-tag cDNA bound to Ni+-nitrilotriacetic acid-agarose. Upon stimulation with anti-CD3, a stable transfectant of the TCR-alpha cDNA formed OVA-specific GIF which contained the 55-kDa GIF peptide, and bound not only to anti-TCR-alpha column but also to anti-TCR-beta column. The results indicate that the OVA-specific GIF consists of the TCR-alpha+ 55-kDa GIF and another peptide with TCR-beta determinant. It was found that the association of the TCR-beta+ peptide with the 55-kDa GIF is required for binding of the factor to OVA, but not essential for the formation and release of the latter peptide.
我们从OVA特异性Ts杂交瘤231F1中分离出一条独特的TCRα链。该TRα链由Vα11.3、一条独特的Jα和完整的Cα区域序列组成。将TCR-α cDNA转染到TCR-α-、TCR-β+ T细胞系175.2中，导致TCR-αβ表达，转染子含有一条具有TCR-α特异性抗原决定簇的35 kDa肽。然而，稳定转染子在用抗CD3刺激时未能释放出具有TCR-α决定簇的肽。相比之下，cDNA在231 F1细胞中的过表达显著增加了55 kDa肽的形成，该肽与抗糖基化抑制因子（GIF）和单克隆抗体H28-710均发生反应。通过将带有组氨酸标签的TCRα链cDNA转染到231F1细胞中，获得了55 kDa肽与TCRα链之间关系的确切证据。由TCR-α-标签cDNA的稳定转染子形成的55 kDa GIF肽与Ni+-次氮基三乙酸-琼脂糖结合。在用抗CD3刺激时，TCR-α cDNA的稳定转染子形成了包含55 kDa GIF肽的OVA特异性GIF，它不仅与抗TCR-α柱结合，还与抗TCR-β柱结合。结果表明，OVA特异性GIF由TCR-α+ 55 kDa GIF和另一条具有TCR-β决定簇的肽组成。发现TCR-β+肽与55 kDa GIF的结合是该因子与OVA结合所必需的，但对于后一种肽的形成和释放并非必不可少。
