Thymic negative selection in human T1D immune systems

在健康个体中，中枢和外周免疫耐受机制可预防自身免疫性疾病。一 在1型糖尿病（T1 D）患者中，这些机制中的一种或多种被破坏，导致自身免疫性破坏 产生胰岛素的胰岛β细胞。在NOD小鼠模型中，胸腺阴性选择的缺陷使 与疾病发展密切相关。胰岛素反应性T细胞受体（TCR）的引入 来源于T1 D患者的人源化小鼠的外周T细胞可以引发糖尿病，这与 自反应性T细胞从负选择中逃逸可能促进疾病的观点。另一方面，beta 细胞抗原反应性T细胞也已从健康对照（HC）个体的血液中分离， 缺陷的外周耐受机制可能在促进T1 D中是最重要的。遗传 研究已经确定了超过60种T1 D的风险变体，包括与参与T1 D基因缺失的基因的关联。 在胸腺阴性选择过程中产生自身反应性T细胞。然而，迄今为止， 可用于模拟人类患者特异性胸腺选择，导致不确定性的作用， 这一途径促进人类自身免疫性疾病。我们假设有特定的 T1 D个体中的造血干细胞（HSC）-内在和胸腺-内在遗传变异导致 致糖尿病T细胞受体（TCR）在胸腺中的阴性选择失败。我们有 建立了一个个性化免疫（PI）小鼠模型，使我们能够产生一个成年人的免疫 在接受患者HSC的免疫缺陷NOD/LtSz-scid IL 2 R γ敲除小鼠（NSG）中的从头系统 和部分HLA匹配的胎儿胸腺移植物我们还证明了成年患者的HSC可以被 慢病毒转导以表达TCR，随后用于重建NSG小鼠或人胸腺 胸腺器官培养测定中的组织。我们已经证明，致糖尿病胰岛素B（9-23）/HLA-DQ 8- 特异性自身反应性TCR通常在用HLA-DQ 8+构建的人源化小鼠的胸腺内缺失 胸腺组织和HSC。有了这些数据，并从T1 D和HC供体获得HSC，从T1 D和HC供体获得胸腺， 糖尿病胰腺器官捐献者网络（nPOD），我们已经证明了我们的可行性， 建议的研究。我们将使用这些创新的体外和体内模型来评估是否有一种 在源自T1 D患者的免疫系统中胸腺阴性选择的缺陷，并确定这种缺陷是否 缺陷是患者HSC或T1 D胸腺组织固有的。我们将把这种缺陷与遗传 风险变量，并解决关于可能影响负选择的特定变量的影响的假设， HSC或胸腺组织依赖性方式。这些研究不仅将提供前所未有的信息， 缺陷性负选择在T1 D发病机制中的作用以及这种缺陷的遗传决定因素，但将 还建立了一个患者特异性模型，用于评估阴性选择并确定其在治疗中的作用。 多种自身免疫性疾病的发展。

In healthy individuals, central and peripheral immune tolerance mechanisms prevent autoimmune disease. One or more of these mechanisms is disrupted in Type 1 diabetes (T1D) patients, resulting in autoimmune destruction of insulin-producing pancreatic beta cells. In the NOD mouse model, defects in thymic negative selection have been strongly implicated in disease development. Introduction of an insulin-reactive T-cell receptor (TCR) derived from a T1D patient to peripheral T cells of humanized mice can initiate diabetes, consistent with the notion that escape of autoreactive T-cells from negative selection can promote disease. On the other hand, beta cell antigen-reactive T-cells have also been isolated from the blood of healthy control (HC) individuals, raising the possibility that defective peripheral tolerance mechanisms may be most important in promoting T1D. Genetic studies have identified over 60 risk variants for T1D, including associations with genes implicated in deletion of developing autoreactive T cells during thymic negative selection. To date, however, there has been no method available to model human patient-specific thymic selection, resulting in uncertainty about the role of defects in this pathway in promoting human autoimmune diseases. We hypothesize that there are specific hematopoietic stem cell (HSC)-intrinsic and thymus-intrinsic genetic variants in T1D individuals that lead to the failure of negative selection of diabetogenic T-cell receptors (TCRs) in the thymus. We have established a Personalized Immune (PI) mouse model that allows us to generate an adult human's immune system de novo in immunodeficient NOD/LtSz-scid IL2R gamma null mice (NSG) mice receiving patient HSCs and a partially HLA-matched fetal thymus graft. We have also demonstrated that adult patient HSCs can be lentivirally transduced to express a TCR and subsequently used to reconstitute NSG mice or human thymus tissue in thymic organ culture assays. We have demonstrated that a diabetogenic insulin B(9-23)/HLA-DQ8- specific autoreactive TCR is normally deleted within the thymus of humanized mice constructed with HLA-DQ8+ thymic tissue and HSCs. With these data and with access to HSCs from T1D and HC donors and to thymi from the network for Pancreatic Organ Donors with Diabetes (nPOD), we have demonstrated the feasibility of our proposed study. We will use these innovative in vitro and in vivo models to assess whether or not there is a defect in thymic negative selection in immune systems derived from T1D patients and determine whether such defects are intrinsic to the patient HSCs or to T1D thymic tissue. We will associate such defects with genetic risk variants and address hypotheses about the impact of specific variants that may impact negative selection in an HSC- or thymic tissue-dependent manner. These studies will not only provide unprecedented information on the role of defective negative selection in T1D pathogenesis and the genetic determinants of this defect, but will also establish a patient-specific model for assessing negative selection and determining its role in the development of multiple autoimmune diseases.