Diabetogenic CD4 T Cell Recognition of Hybrid Peptide Ligands

项目总结/摘要 这项提案的总体目标是了解致糖尿病T细胞受体（TCR）如何识别糖尿病T细胞。 MHCII和杂合肽复合物。CD 4和CD 8 T细胞的肽配体通常来源于 通过内吞途径和蛋白酶体将亲本蛋白质的连续片段转化为蛋白质。我们首先建议 胰岛素（Ins）B：9-23和WE 14，嗜铬粒蛋白A（ChgA）的天然切割产物，肽可以被剪接 与其他β细胞颗粒蛋白结合形成新抗原。我们还证明了融合肽是 许多致糖尿病T细胞的真正配体。Ins B：9-23肽的C-末端修饰和N-末端修饰都是通过修饰Ins B：9-23肽来实现的。 ChgA的WE 14肽的末端添加可以产生T细胞的超激动剂。我们提出肽 通过转肽融合作为对肽产生所需修饰的手段，并解释如何 驱动自身免疫的T细胞逃避胸腺中的阴性选择，但在靶中找到它们的抗原 组织.我们的结构和生物物理数据表明，Ins B：9-23和WXRM（D/E）的EclampLV部分 WE 14的一部分可能是这些杂合肽的共同受体。我们确定了两个 不同类型的小鼠TCR和人TCR，与它们的最佳形式的MHCII-Ins肽结合 配体。所有Ins反应性TCR均接合Ins B：9-23的N-末端部分（ESTIL），这表明Ins B：9-23的N-末端部分（ESTIL）具有相似性。 这些配体是如何在人类和小鼠中形成的。结构表明，特异性差异 小鼠A型和B型T细胞之间的差异在于它们如何与Ins的p8（B：21）处的氨基酸相互作用 肽。这些结构还显示了肽修饰如何对肽的形成至关重要。 复合物，支持肽在体内修饰以启动T1 D中的CD 4 T细胞应答的作用。 我们假设EALYLV和WXRM（D/E）是融合肽的受体，并形成共同的 除了作为T1 D免疫治疗的新靶点之外，糖尿病原性TCR对接点也是重要的靶点。此前我们 已经显示芳香族氨基酸在TCR交叉反应性中起重要作用。芳香族氨基酸（Y 和W）可能是致糖尿病TCR的共同表位，并且对形成杂交的广泛性至关重要。 肽反应性T细胞库。而杂合肽的供体肽有助于T Ins和ChgA自身抗原的细胞库。我们将研究以下具体目标： 致糖尿病性CD 4 TCR识别IAg 7和ChgA WE 14杂合肽;定义人Ins的性质 B：呈递给致糖尿病性CD 4 TCR的9-23杂合肽;检测特异性单克隆抗体（mAb） 对于T1 D发展中与MHC结合的杂合肽的受体部分。这些数据可以 指导针对TCR活化的特异性调节的治疗策略，这可能是治疗的基础。 治疗T1 D的新治疗方法。 研究策略第2页

PROJECT SUMMARY/ABSTRACT The overall goal of this proposal is to understand how diabetogenic T cell receptors (TCRs) recognize the MHCII and hybrid peptide complexes. Peptide ligands of CD4 and CD8 T cells are conventionally derived from contiguous fragments of the parental proteins by the endocytic pathway and proteasome. We first suggested insulin (Ins) B:9-23 and WE14, a natural cleavage product of chromogranin A (ChgA), peptides may be spliced with other beta cell granule proteins and form neo-antigens. We also demonstrated that fusion peptides are the true ligands for many diabetogenic T cells. Both C-terminal modification of the Ins B:9-23 peptide and N- terminal additions to the WE14 peptide of ChgA can create superagonists for T cells. We proposed peptide fusion by transpeptidation as a means of creating the required modifications to the peptides and explain how the T cells driving autoimmunity escape negative selection in the thymus but find their antigen in the target tissue. Our structural and biophysical data showed that the EALYLV portion of Ins B:9-23 and WXRM(D/E) portion of WE14 may be common acceptors of these hybrid peptides. We determined the structures of two different types of mouse TCRs and a human TCR, bound to their optimal versions of the MHCII-Ins peptide ligands. All the Ins reactive TCRs engage the N-terminal portion (EALYL) of Ins B:9-23, indicating similarities in how these ligands are formed in both human and mouse. The structures showed that the specificity differences among mouse Type A and Type B T cells lies in how they interact with the amino acid at p8 (B:21) of the Ins peptide. The structures also show how the peptide modifications were essential to the formation of the complexes, bolstering a role for modification of the peptide in vivo to initiate the CD4 T cell response in T1D. We hypothesize that EALYLV and WXRM(D/E) are the acceptors of the fusion peptides and form the common diabetogenic TCR docking spots, in addition to being novel targets for T1D immunotherapy. Previously, we have shown aromatic amino acids play important role in TCR cross-reactivity. The aromatic amino acids (Y and W) may be the common epitopes for diabetogenic TCRs and critical to shape the broadness of hybrid peptide reactive T cell repertories. Whereas, the donor peptides of hybrid peptides contribute to diversity the T cell repertoire to both Ins and ChgA autoantigens. We will study the following specific aims: Determine how diabetogenic CD4 TCRs recognize IAg7 and ChgA WE14 hybrid peptides; Define the nature of the human Ins B:9-23 hybrid peptides presented to the diabetogenic CD4 TCRs; Test monoclonal antibodies (mAbs) specific for the acceptor portions of the hybrid peptides bound to MHC in the development of T1D. These data may guide therapeutic strategies towards specific modulation of TCR activation, which could be the basis for a novel therapeutic approach in treatment of T1D. Research Strategy Page 2