Molecular Genetic Analysis Of Lymphocyte Function

第一部分，本项目的MHC-I方面，旨在了解CD 8 T细胞应答的细节，其时间过程和特异性。 使用几种载体表达HIV-1包膜，我们表明，CD 8细胞引起不同的精细的特异性和动力学的动员。 免疫显性肽的几种变体肽显示出结合MHC-I限制性元件，H-2Dd比亲本肽衍生肽更好，并且通过不同载体引起具有不同特异性的T细胞克隆。 高分辨率的X射线晶体结构揭示了肽抗原的表位残基的溶剂暴露的主要差异，与不同的T细胞群体一致。 这些发现表明，不同的基因为基础的载体产生的肽与交替构象内的MHC-I，引起不同的T细胞接种疫苗后的反应。与较高水平的抗原敏感性和较慢的同源四聚体解离速率一致，P9细胞在细胞因子产生方面相对更多功能。然而，值得注意的是，P10细胞在每个细胞的基础上在体内介导了显著更大的溶细胞活性。这些不同的命运与转录因子eomesodermin的不同表达水平相关。因此，基于基因的疫苗内的单个表位可以诱导不同的CD 8 + T细胞募集模式，其随递送至个体抗原特异性克隆型的信号强度而变化。 我们的MHC-I研究的另一个方面包括努力探索内质网中肽装载的基本方面。 为此，我们正在确定MHC-I分子的一部分的结构，该部分经历伴随肽结合的构象转变。 该项目的第二部分集中在T细胞受体识别自身抗原的功能研究以及这如何导致自身免疫性疾病。我们目前的方法包括：1）在自身免疫性胃炎的转基因小鼠模型中由自身免疫性T细胞识别的抗原肽的表征; 2）与其抗原肽共价连接的MHC-II分子的结构测定;和3）负载有自身免疫原性肽的MHC-II分子的分子表征。我们已经从T细胞克隆中克隆并表达了两种不同的TCR，其对来自胃H/K ATP酶的两种肽具有特异性。在转移到免疫缺陷动物时，这些克隆中的一个引起Th 1型疾病，另一个引起Th 2样疾病。Th 2样疾病的特征在于产生IL 4、IL 5和IL 13的T细胞，并且在胃粘膜中显示白细胞浸润。已经产生并分析了表达来自这些克隆中的每一个的TCR的转基因动物。来自Th 1克隆的转基因TXA 23在出生后10天内发展成暴发性自身免疫性胃炎。来自Th 2克隆的转基因TXA 51具有较低的侵袭性疾病。这第二个模型提供了提供洞察如何炎性（Th 1）细胞因子影响自身免疫性疾病的方式不同于Th 2细胞因子。取自Th 2患病动物的细胞可以在体外维持为Th 2细胞，或者如果用由树突细胞呈递的渐进剂量的抗原肽刺激，则可以分化成Th 1细胞。已经研究了关于两种转基因株的差异细胞因子和疾病谱的几种假设：1）TXA 23 TCR对其MHC/肽复合物的固有亲和力是否大于TXA 51 TCR的固有亲和力;和2）TXA 23所见的抗原肽的加工和呈递效率是否优于TXA 51所见的肽的加工和呈递效率。仔细的功能剂量反应实验和IAd/肽四聚体染色实验与TXA 51 TCR对肽/MHC的亲和力大于TXA 23 TCR的亲和力的观点一致。细胞转移实验表明，转移到正常小鼠的TXA 23 CD 4 T细胞在胃淋巴结中广泛增殖，而TXA 51细胞不能增殖。 我们已经确定了高分辨率的X-射线结构的MHC-II分子，IAd，在复合物的抗原肽和两个合成的变体，所识别的TXA 51小鼠品系的三个相关的复合物。 肽与MHC分子结合的组织细节反映了分子相互作用强度的生化参数以及TXA 51转基因动物的自身免疫表型。

Part one, the MHC-I aspect of this project, is directed to understand the fine details of the CD8 T cell response, its time course and specificity. Using several vectors expressing an HIV-1 envelope we showed that CD8 cells were elicited with different fine specificities and kinetics of mobilization. Several variant peptides of the immunodominant peptide were shown to bind the MHC-I restricting element, H-2Dd better than the parent envelope-derived peptide, and T cell clones with distinct specificities were elicited by the different vectors. High resolution X-ray crystal structures revealed major differences in solvent exposure of epitopic residues of the peptide antigen, consistent with the different populations of T cells. These findings suggest that different gene-based vectors generate peptides with alternate conformations within MHC-I that elicit distinct T cell responses after vaccination. Consistent with higher levels of antigen sensitivity and slower cognate tetramer dissociation rates, P9 cells were relatively more polyfunctional with respect to cytokine production. Notably, however, P10 cells mediated substantially greater cytolytic activity in vivo on a per cell basis. These divergent fates were correlated with different expression levels of the transcription factor eomesodermin. Thus, a single epitope within a gene-based vaccine can induce distinct patterns of CD8+ T cell recruitment that vary with the signal strength delivered to individual antigen-specific clonotypes. Another aspect of our MHC-I studies includes efforts to explore fundamental aspects of peptide loading in the endoplasmic reticulum. To this end, we are determining the structure of a portion of the MHC-I molecule that undergoes a conformational shift concomitant with peptide binding. The second part of this project is focused on functional studies of T cell receptor recognition of autoantigens and how this leads to autoimmune disease. Our current approaches include: 1) the characterization of antigenic peptides recognized by the autoimmune T cells in transgenic mouse models of autoimmune gastritis; 2) the structural determination of MHC-II molecules covalently linked to their antigenic peptides; and 3) the molecular characterization of MHC-II molecules loaded with autoimmunogenic peptides. We have cloned and expressed two different TCR from T cell clones that show specificity for two peptides from the gastric H/K ATPase. On transfer to immunodeficient animals, one of these clones causes a Th1 type disease, and the other a Th2-like disease. The Th2-like disease is characterized by T cells producing IL4, IL5, and IL13 and shows leukocyte infiltrates in the gastric mucosa. Transgenic animals expressing the TCR from each of these clones have been produced and have been analyzed. The transgenic derived from the Th1 clone, TXA23, develops a fulminant autoimmune gastritis within 10 days of birth. The transgenic derived from the Th2 clone, TXA51, has a less agressive disease. This second model offers to provide insight into how inflammatory (Th1) cytokines influence autoimmune disease in a manner distinct from Th2 cytokines. Cells taken from the Th2 diseased animals can be maintained in vitro as Th2 cells, or if stimulated with progressive doses of antigenic peptide presented by dendritic cells, can differentiate into Th1 cells. Several hypotheses concerning the differential cytokine and disease profiles of the two transgenic strains have been investigated: 1) is the intrinsic affinity of the TXA23 TCR greater for its MHC/peptide complex than that of the TXA51 TCR; and 2) is the efficiency of the processing and presentation of the antigenic peptide seen by TXA23 better than that of the peptide seen by TXA51. Careful functional dose-response experiments and IAd/peptide tetramer staining experiments are consistent with the view that the TXA51 TCR affinity for peptide/MHC is greater than that of TXA23 TCR. Cell transfer experiments indicate that TXA23 CD4 T cells transferred to normal mice proliferate extensively in the gastric lymph node while TXA51 cells fail to proliferate. We have determined the high resolution X-ray structures of three related complexes of the MHC-II molecule, IAd, in complex with the antigenic peptide and two synthetic variants that are recognized by the TXA51 mouse strain. Details of the organization of the peptide as bound to the MHC molecule reflect biochemical parameters of the strength of the molecular interaction as well as the autoimmune phenotype of the TXA51 transgenic animals.