Non-classical MHC molecules such as CD ld or CD1b have evolved in parallel to classical class II molecules and I. They are however more specialized in the type of ligand they present to T cells. Typically, CD1 molecules present lipids or glycolipids to T cells. CD1 ligands are associated with T cells specific for M. tuberculosis. The studies in this project are based on a unique collaboration. The expertise of Dr. Grant Yeaman an assistant professor at Dartmouth Medical School (DMS) studies the role of carbohydrate antigens in endometriosis and HIV infection will be combined with that of Drs. William Wade and Bruce Reinhold. Dr. Wade is an associate professor whose research interests center on antigen presentation. Dr. Reinhold is an assistant professor of chemistry at the University of New Hampshire who studies CD1 ligands using mass spectrum analysis. This combination of expertise will be used to design synthetic peptide and carbohydrate/lipid antigens to investigate how BCR or Fc receptors internalizes and initiate the processing of these antigens to their effective form that induces T cell activation.
Aim 1 : To determine if BCR can internalize and target CDlb ligands for presentation to CDlb-restricted T cells. CDlb molecules can bind mycobacterial lipids. We will track internalization of CDlb ligands in the endocytic pathway by confocal microscopy, mass spectral analysis and activation T cells.
Aim 2 : To demonstrate that BCR can internalize and target CDld ligands for presentation to CDld-restricted T cells. The OVA hydrophobic peptide (IINFEKLTEWTSS) that binds CDld will be modified to contain class II, I-Ad OVA peptide and a B cell epitope for the V5 specific Ab. The construct will be linked to an anti-IgG2a (BCR) F(ab)2, and used to treat A20 B cells transfected with murine CDlda; galactosyl ceramide will also be examined in this system. The complexes will be followed functionally through the early endosome (EE), the MIIC and the lysosome by confocal microscopy, biochemical analysis and by T cell activation.
Aim 3 : Determine the trafficking of CDId/b ligands targeted to FcyRl on DCs and demonstrate that the distribution of CD1 differs with antigen internalization or maturation of the DC. DCs are likely the first cell to acquire mycobaterial antigens for priming T cells. Antigen is acquired through macropinocytosis or receptor-mediated endocytosis. We will use ex vivo DC and target immune complexes or particulate antigen such as bacteria to CD64 as a means of following the intracellular traffic of the complexes (antigen) biochemically and functionally.
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