Biophysical parameters of self-reactive TCR engagement in T1D

摘要 主要组织相容性位点（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细胞受体与 在易感MHC等位基因的背景下，肽-MHC被改变。剖析T细胞之间的相互作用 受体和自身抗原需要灵敏的技术来测量亲和力和键寿命。T细胞 对TCR和pMHC抗原复合物之间的键施加力，这最终通过TCR和pMHC抗原复合物的变化来反映。 蛋白质相互作用的时间我们已经调查了亲和力，键的寿命，以及在T细胞过程中形成的力， 受体与非自身免疫性I-Ab上呈递的模型、感染性和自身抗原的相互作用， 用I-Ag 7自身免疫性MHC限制性T细胞进行这些观察。我们一直观察到2倍的差异 在非自身免疫性MHC限制性应答中效应细胞和Foxp 3+调节性T细胞之间的作用， 张力拉高20 pN。然而，I-Ag 7限制性效应T细胞能够拉动类似的高水平， 20 pN的力，并且Tynth失去了2倍的力优势。此外，人源性β细胞抗原特异性 限制于自身免疫HLA-DR 4的TCR表现出类似的高20 pN力。我们的总体假设是Foxp 3 + Treg功效取决于与T常规细胞相差2倍的峰值TCR力水平，这种差异 在自身免疫性MHC中不存在。自身免疫性MHC的结构成分，肽， TCR控制TCR/pMHC相互作用的稳定性尚未完全解决，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CDR 2环， 这对建立武力水平很重要。此外，尚不清楚MHC如何影响Treg 抑制功能，以及为什么TlD期间TlD会发生变化。因此，彻底了解β细胞- 需要效应T细胞和调节T细胞的特异性反应性来充分理解和潜在地利用它们的功能。 治疗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： 目标1.确定受自身免疫性MHC限制的TCR生物物理参数及其对自身免疫性疾病的影响 和调节性T细胞功能;和Aim 2.确定TCR的结构组分，其特异性调节 力和键寿命，但不影响相互作用的特异性或亲和力。该项目将是第一个 研究各种水平的力/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亲和力与力与疾病的最终结果联系起来。此外，它将提供 T1 D期间T细胞耐受性功能障碍的机制的新见解。

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.