The successful initiation of an immune response hinges on the ability of the products of the Ma Histocompatibility Complex to present peptides to T cell receptors. The induction of antibody products requires an antigen that includes both B cell epitopes and epitopes that will result in T cell help, provided CD4+ T cells upon encounter of the appropriate mHC Class II - peptide complex. The formation of peptide loaded Class II molecules requires delivery of Class II molecules to a specialized endocytic compartment, the signals for which are borne by the Class II associated invariant chain (Ii). It is by no means clear whether Ii is sufficient to achieve this targeting: mutant mice harboring a Class II beta chain with a truncated cytoplasmic tail appear to be defective in antigen presentation, though they express seemingly normal class II molecules at the cell surface. These mice will be used to explore trafficking of Class II molecules as dictated by signals other than those carried by Ii, with special reference to the effects of IL-4 and TNFalpha. While much is known about the structure of MHC products and their bound peptides, less clear is the biochemistry of formation of higher order structures comprised of MHC-peptide complexes. T cell receptor (TCR), the coreceptor CD4, and the attendant kinases. In particular, changes in their interrelationships in the course of a productive encounter - such as the possible dimerization of MHC Class II molecules - are likely to be essential in the successful triggering of a T lymphocyte. To study the requirements for such complex formation, properly assembled MHC Class II peptide complexes. TCR-CD3 complexes and coreceptor (CD4) will be produced by simultaneous in vitro translation of the appropriate mRNAs in membrane- supplemented systems. Interactions amongst these proteins will be investigated immunochemically and by chemical crosslinking. Post- translational modifications of complexes thus generated by tyrosine kinases such as p56lck fyn and AZAP70 will likewise be explored in these systems. The stoichiometry of the complexes formed will be examined; even for the TCR-CD3 complex this parameter has not been established unequivocally. The experiments would provide deeper insight into the requirements for stable T cell-target interactions by studying their key components in vitro, in a manner that is functionally relevant, yet not easily achieved using soluble recombinant proteins or intact cells. The information gathered should aid in the design of strategies aimed at improved antigen presentation and hence should prove valuable for vaccine design.
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