Bikoff E K
Department of Obstetrics, Gynecology, and Reproductive Sciences, Mount Sinai School of Medicine, New York, NY 10029.
Eur J Immunol. 1991 Jun;21(6):1411-7. doi: 10.1002/eji.1830210613.
To study the question when and where self peptides become associated with major histocompatibility complex class II molecules for tolerance induction, we recently developed a system in which the intracellular site(s) of antigen expression could be manipulated using gene cloning techniques. We previously constructed a truncated IgGa gene comprising a variable (V) domain and the CH3 domain (not including the membrane exons) from the IgG2ab heavy (H) chain. The secreted form of the V-CH3b protein was expressed at high levels under control of the Ig H chain enhancer in Ia+ B lymphoma cells and was efficiently recognized by class II-restricted IgG2ab-specific T cell hybrids. Here we describe a modified V-CH3b gene construct in which the sequences encoding the signal peptide were deleted. A strong argument can be made that the signal-less V-CH3b protein is predominantly expressed in the cytosol. We show that transfected L cell lines expressing the signal-less form of the V-CH3b protein can stimulate class II-restricted IgG2ab-specific T cells. Cell mixing experiments indicate that this response cannot be due to passive uptake of soluble antigenic peptides released into culture supernatants. These experiments demonstrate that a cytoplasmic protein having no obvious means of reaching the cell surface can be presented to class II-restricted T cells.
为了研究自身肽在何时何地与主要组织相容性复合体II类分子结合以诱导耐受性这一问题，我们最近开发了一种系统，利用基因克隆技术可以操纵抗原表达的细胞内位点。我们之前构建了一个截短的IgGa基因，它包含来自IgG2ab重链的可变（V）结构域和CH3结构域（不包括膜外显子）。V-CH3b蛋白的分泌形式在Ia+B淋巴瘤细胞中在Ig重链增强子的控制下高水平表达，并被II类限制性IgG2ab特异性T细胞杂交体有效识别。在此我们描述一种修饰的V-CH3b基因构建体，其中编码信号肽的序列被删除。有充分理由认为无信号的V-CH3b蛋白主要在细胞质中表达。我们表明，表达无信号形式V-CH3b蛋白的转染L细胞系可以刺激II类限制性IgG2ab特异性T细胞。细胞混合实验表明，这种反应不是由于被动摄取释放到培养上清液中的可溶性抗原肽所致。这些实验证明，一种没有明显途径到达细胞表面的细胞质蛋白可以呈递给II类限制性T细胞。
