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，鉴定自身反应性T细胞的T细胞受体（TCR）的配体是重要的。我们开发了一种新的系统，用于将抗原肽递送到MHC II类途径，使用不变链（Ii）。我们设计了CLIP-取代的人p33-形式，其中链球菌M12 p55 -68肽或人65 kDa谷氨酸脱羧酶（GAD 65）p116-129肽取代CLIP（II类相关不变链肽）。与野生型Ii相似，CLIP取代的Ii在细胞内与DR 4分子结合。我们研究了这些构建体的肽呈递功能。稳定表达M12/CLIP取代的Ii基因和HLA-DR 4基因的CHO细胞转染子可以加工并将M12 p55 -68呈递给M12肽特异性和DR 4限制性的CD 4 ^+ T细胞克隆，以诱导IFN-γ的产生。即使在96孔威尔斯培养板的一个孔中有100个CHO细胞转染子，其中容纳3 × 10^4个CHO细胞，T细胞克隆也产生大量的IFN-γ。此外，稳定表达HLA-DR 53基因和瞬时表达GAD 6S/CLIP取代的Ii基因的CHO细胞刺激了从日本胰岛素依赖型糖尿病患者建立的HLA-DR 53限制性和GAD 65自身反应性T细胞克隆中IFN-γ的产生。这些结果表明，取代Ii p323的CLIP的肽以与CLIP相同的方式与DR分子的沟结合，并且它们在不存在HLA-DM分子的情况下优先呈递给CD 4 ^+ T细胞克隆。该系统可用于DNA疫苗的免疫治疗，或用于构建表达不同HLA-II类加肽复合物的抗原呈递细胞库，用于鉴定TCR配体，后者的研究项目正在进行中。

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.