Biophysical parameters of self-reactive TCR engagement in T1D

摘要 主要组织相容性基因座(MHC)是自身免疫易感性的最大遗传贡献者，包括 1型糖尿病。我们的新观察表明，T细胞受体的生物物理参数与 多肽-MHC在易感MHC等位基因的背景下发生改变。T细胞间相互作用的解剖 受体和自身抗原需要敏感的技术来测量亲和力和结合寿命。T细胞 对TCR和pMHC抗原复合体之间的结合施加作用力，这最终反映在 蛋白质相互作用的时间长短。我们研究了T细胞的亲和力、键寿命和作用力 非自身免疫性I-Ab与模型抗原、感染性抗原和自身抗原的受体相互作用及比较 这些观察用I-Ag7自身免疫的MHC限制了T细胞。我们一直观察到两倍的差异 在非自身免疫性MHC限制性反应中，效应器和Foxp3+调节性T细胞之间有效， Tregs拉高了20pN的力。然而，I-Ag7限制性效应性T细胞能够拉高类似的水平 20pN的力量，而特雷格斯失去了2倍的力量优势。此外，人源性β细胞抗原特异性 自身免疫性HLADR4的TCR表现出类似的高20pN的作用力。我们的总体假设是Foxp3+ Treg的疗效取决于峰值TCR力水平，与传统T细胞相差2倍，这种差异 在自身免疫性MHC的背景下是缺失的。自身免疫性MHC、多肽和 TCR控制TCR/pMHC相互作用的稳定性没有完全解决，特别是在力的背景下 测量。我们最近发表和未发表的观察结果指出，TCR的CDR2环路为 在确定武力水平方面很重要。此外，目前还不清楚MHC对Treg的易感性。 抑制功能，以及为什么Tregs在T1D期间摇摇欲坠。因此，对β细胞的透彻了解- 效应器和调节性T细胞的特异性反应性需要充分理解和潜在地利用它们 自身免疫性糖尿病的治疗潜力。我们设计了两个目标来检验这一假设： 目的1.测定自身免疫MHC限制的TCR生物物理参数及其对自身免疫的影响 和调节性T细胞功能；以及目标2.确定TCR的结构成分，这些结构成分特异性地调节 力和键寿命，但不影响相互作用的特异性或亲和力。这个项目将是第一个 研究不同水平的力/键寿命作为T细胞功能和Treg功能丧失的指标 自身免疫，并将TCR亲和力与疾病的最终结果联系起来。此外，它还将提供 T1D中T细胞耐受功能障碍机制的新认识

ABSTRACT Major histocompatibility loci (MHC) are the largest genetic contributors to autoimmune susceptibility, including type 1 diabetes. Our novel observations show that biophysical parameters of T cell receptor interactions with peptide-MHC are altered in the context of susceptible MHC alleles. Dissecting the interaction between T cell receptor and self-antigens requires sensitive technologies to measure the affinity and bond lifetimes. T cells apply force to the bond between TCR and pMHC antigenic complex, which is ultimately reflected by changes in how long the proteins interact. We have surveyed affinities, bond lifetimes, and force that form during T cell receptor interaction with model, infectious, and self-antigens presented on non-autoimmune I-Ab and compared these observations with I-Ag7 autoimmune MHC restricted T cells. We consistently observed 2-fold difference in force between effector and Foxp3+ regulatory T cells in non-autoimmune MHC restricted responses, with Tregs pulling higher 20pN force. However, I-Ag7 restricted effector T cells are capable of pulling similar high 20pN force, and Tregs loose the 2-fold force advantage. Moreover, human-derived beta cell antigen specific TCR restricted to autoimmune HLA-DR4 exhibited similar high 20pN force. Our overall hypothesis is that Foxp3+ Treg efficacy is dependent on peak TCR force levels that differ 2-fold from T conventional cells, this difference is absent in the context of autoimmune MHCs. How structural components of autoimmune MHC, peptide, and TCR control the stability of the TCR/pMHC interaction is not fully resolved, especially in the context of force measurements. Our recently published and unpublished observations point to CDR2 loops of the TCR as important in establishing the level of force. Moreover, it is unknown how susceptible MHC effects Treg suppressive function, and why Tregs falter during T1D. Therefore, a thorough understanding of the beta cell- specific reactivity of effector and regulatory T cells is needed to fully understand and potentially exploit their therapeutic potential for treatment of autoimmune diabetes. We have devised two aims to test this hypothesis: Aim 1. Determine TCR biophysical parameters restricted by autoimmune MHC and their impact on autoimmune and regulatory T cell function; and Aim 2. Determine structural components of TCR that specifically regulate the force and bond-lifetime, but do not influence specificity or affinity of the interaction. This project will be the first to investigate various levels of force/bond lifetimes as indicators for T cell function and loss of Treg function in autoimmunity, and connect TCR affinity vs force to the ultimate outcome in disease. Furthermore, it will provide novel insight into the mechanisms governing dysfunction of T cell tolerance during T1D.