The long term goal of this project is to understand the molecular mechanism by which the peptide/MHC ligand on the surface of antigen presenting cells (APC) is recognized by, and stimulates, T-cells in health and disease. The T-cell activation process depends upon limited proteolysis of the antigen within APC to yield the peptide/MHC ligand, requires specific recognition of the ligand by the alpha beta T-cell receptor (TCR), and results in intracellular signaling events initiated by ligand occupancy of TCR, as well as, of CD4 or CD8 co-receptors. With well-characterized model systems and novel assays established in this laboratory, these events will be analyzed by functional and biochemical analysis of transfectants generated by gene transfer of key components. (1) Structure/function relationship of alpha beta TCRs specific for lysozyme peptide (HEL74-88)/Ab or Abm-12 MHC complex will be determined in the alpha beta- recipient cell lines by functional analysis of transfected alpha/beta TCR genes. This analysis will use the panel of APC expressing mutant Ab/bm-12 molecules and substituted HEL74-88 analogs. (2) Intracellular events, initiated by ligand occupancy of TCR/CD4 or TCR/CD8 receptors, and mediated by the cytoplasmic tyrosine kinase, p56ick will be biochemically analyzed in our TCR transfectants expressing mutant CD4 and CD8 co-receptors and in normal T-cells from TCR transgenic mice. Western blots with anti-phosphotyrosine antibodies will identify unique phosphorylated proteins induced by CD4 or CD8 bound p56ick in fractionated lysates from activated T-cells. (3) For the first time, a general strategy for expression cloning of TCR defined antigens will be developed using the unique single ell """"""""lacZ"""""""" assay for TCR occupancy, and transiently transfected COS cells expressing appropriate class II MHC molecules. (4) This unique strategy, pathology, will be tested by identifying the enigmatic H-Y gene product in male mice. The H-Y antigen is known only as a peptide presented by Db MHC expressing male cells, causes thymic deletion of self H-Y/Db specific T-cells, and may also play a role in sex-determination. (5) Finally, specificity of intracellular mechanisms, which yield naturally processed peptide/MHC complex within APC will be deduced by expression of endogenously expressed variants within and flanking the ovalbumin octapeptide SIINFEKL in Kb- expressing APC. The knowledge of which residues affect the efficiency of endogenous presentation are critical to developing algorithms for predicting T-cell epitopes within primary sequences and to design of vaccines using these epitopes.
| Blanchard, Nicolas; Shastri, Nilabh (2008) Coping with loss of perfection in the MHC class I peptide repertoire. Curr Opin Immunol 20:82-8 |
