Diabetogenic CD4 T Cell Recognition of Hybrid Peptide Ligands

项目摘要/摘要 这项提议的总体目标是了解糖尿病T细胞受体(TCR)如何识别 MHCII和杂交肽复合体。CD4和CD8 T细胞的多肽配体通常来源于 亲本蛋白的相邻片段通过内吞途径和蛋白酶体。我们首先建议 胰岛素(INS)B：9-23和嗜铬粒素A(ChgA)的天然切割产物WE14可以剪接 与其他β细胞颗粒蛋白形成新的抗原。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肽和N-肽的C-末端修饰 ChgA的WE14肽的末端添加可以为T细胞产生超级激动剂。我们提出了多肽 通过转肽法进行融合，作为一种对多肽进行所需修饰的方法，并解释如何 驱动自身免疫的T细胞逃脱了胸腺的负选择，但在靶标中找到了它们的抗原 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EALYLV部分 WE14的一部分可能是这些杂交肽的共同受体。我们确定了两个化合物的结构 不同类型的小鼠TCR和人TCR，与其MHCII-INS多肽的最佳版本结合 配基。所有的INS反应性TCR都与INS B：9-23的N末端部分(EALYL)结合，表明在 这些配体是如何在人类和小鼠体内形成的。结构分析表明，其特异性差异较大 小鼠A型和B型T细胞之间的区别在于它们如何与INS的p8(B：21)氨基酸相互作用 多肽。这些结构还显示了多肽修饰是如何对形成 复合体，支持在体内修饰多肽以启动T1D中的CD4T细胞反应的作用。 我们推测EALYLV和WXRM(D/E)是融合肽的受体，形成共同的 糖尿病TCR对接点，除了是T1D免疫治疗的新靶点外。此前，我们 表明芳香族氨基酸在TCR交叉反应中起着重要作用。芳香族氨基酸(Y 和W)可能是非生理性TCR的共同表位，对形成杂交种的广泛性至关重要 多肽反应性T细胞库。而杂交肽的供体肽对T细胞的多样性有贡献 INS和ChgA自身抗原的细胞谱。我们将研究以下具体目标：确定如何 致糖尿病的CD4TCR识别IAg7和ChgA WE14杂交肽；定义人类INS的性质 B：9-23个杂交肽呈递给致糖尿病的CD4TCR；检测特异性的单抗 对于T1D发育过程中与MHC结合的杂交肽的受体部分。这些数据可能 指导治疗策略，对TCR激活进行特定的调节，这可能是 治疗T1D的新途径。 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