An approach to generate a library of CHO cells expressing diverse HLA class II plus peptide complexes for identification of TCR-ligands by using CLIP-substituted invariant chains

为了了解自身免疫性疾病的发病机制，确定自身反应性T细胞的T细胞受体(TCR)的配体是非常重要的。我们开发了一种新的系统，利用不变链(II)将抗原肽输送到MHC II类途径。我们设计了以链球菌M12p55-68多肽或人65 kDa谷氨酸脱羧酶(GAD65)p116-129多肽取代Clip(II类相关不变链肽)的Clip-替换人p33形式。与野生型II类似，片段替换的II与DR4分子在细胞内结合。我们研究了这些结构的肽递呈功能。稳定表达M12/CLIP-替换II基因和人类白细胞抗原-DR4基因的仓鼠CHO细胞可将M12p55-68转化为M12-肽特异性和DR4限制性的CD4~+T细胞克隆，以诱导产生干扰素-γ。在一个可容纳3×10~(-4)个CHO细胞的96孔微孔培养板中，即使有100个CHO细胞转染体，T细胞克隆也能产生大量的干扰素-γ。此外，稳定表达HLADR53基因的CHO细胞和瞬时表达GAD6S/CLIP替换II基因的CHO细胞在从日本胰岛素依赖型糖尿病患者建立的HLADR53限制性和GAD65自身反应性T细胞克隆中刺激干扰素-γ的产生。这些结果表明，取代IIp323的多肽以与CLIP相同的方式结合到DR分子的沟槽上，并且在没有HLA-DM分子的情况下，它们优先呈现给CD4^+T细胞克隆。该系统可能用于DNA疫苗的免疫治疗，或用于构建表达不同的人类白细胞抗原II类加多肽复合体的抗原提呈细胞库，以鉴定TCR配体，后者的研究项目目前正在进行中。

In order to understand pathogenesis of autoimmune diseases, it is important to identify ligands for T cell receptor (TCR) of autoreactive T cells. We developed a new system for delivery of an antigenic peptide to the MHC class II pathway, using the invariant chain (Ii). We designed CLIP-substituted human p33-form us in which either streptococcal M12p55-68 peptide or human 65 kDa glutamic acid decarboxylase (GAD65) p116-129 peptide was substituted for CLIP (class II associated invariant chain peptide). Similar to the wild-type Ii, the CLIP-substituted Ii was associated intracellularly with DR4 molecules. We examined the peptide presenting function of these constructs. Hamster CHO cell transfectants stably expressing M12/CLIP-substituted Ii gene together with HLA-DR4 gene could process and present M12p55-68 to a M12-peptide specific and DR4-restricted CD4^+ T cell clone to induce production of IFN-gamma. Even in the presence of 100 CHO cell transfectants in a well of 96-wells micriculture plate in which 3*10^4 CHO cells were accomodated, the T cell cloneproduced a significant ammount of IFN-gamma. Furthermore, CHO cells expressing stably with HLA-DR53 gene and transiently with GAD6S/CLIP-substituted Ii gene stimulated production of IFN-gamma in a HLA-DR53-restricted and GAD65 autoreactive T cell clone established from a Japanese patient with insulin-dependent diabetes mellitus. These results indicate that the peptides substituted for CLIP of Ii p323 bound to the groove of DR molecules in the same manner as CLIP and they were preferentially presented to the CD4^+ T cell clone in the absence of HLA-DM molecules. This system may prove useful for immunotherapy with DNA vaccines or for construction of a library of antigen presenting cells expressing diverse HLA class-II plus peptide complexes for identification of TCR-ligands and the latter research project is now on going.