Molecular Genetic Analysis Of Lymphocyte Function

第一部分，这个项目的MHC-I方面，旨在了解CD8 T细胞反应的细节，它的时间过程和特异性。使用几种表达HIV-1包膜的载体，我们发现CD8细胞以不同的精细特异性和动员动力学被诱导。结果表明，该免疫优势肽的几种变体肽比亲本包膜衍生肽更能结合MHC-I限制元件H-2Dd，并通过不同载体诱导出具有不同特异性的T细胞克隆。高分辨率x射线晶体结构揭示了肽抗原表位残基在溶剂暴露中的主要差异，这与不同群体的T细胞一致。这些发现表明，不同的基于基因的载体在MHC-I中产生具有不同构象的肽，在接种疫苗后引起不同的T细胞反应。与较高水平的抗原敏感性和较慢的同源四聚体解离率一致，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细胞在体内以每个细胞为基础介导了更大的细胞溶解活性。这些不同的命运与转录因子eomesodermin的不同表达水平有关。因此，基于基因的疫苗中的单个表位可以诱导CD8+ T细胞募集的不同模式，这些模式随着传递给单个抗原特异性克隆型的信号强度而变化。

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.