Molecular Genetic Analysis Of Lymphocyte Function

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

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.