Assembly of MHC Class I molecules in vitro and in vivo

Whitehead Institute For Biomedical Res, Cambridge MA

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Abstract

A Class l-restricted I cell response deployed against avirus-infectedcell constitutes a formidable selective pressure on the pathogen. To escape immune detection, the Herpesviruses have evolved and perfected a number of post-translational strategies to avoid antigen presentation via Class I molecules. A fuller understanding of how viruses manage to escape from the immune system should help us understand better the mechanistic and functional iaspects of antigen presentation. We should be able to exoloit this information not only to design better strategies for anti viral therapies, but we could perhaps also learn from these viruses how to avoid and unwanted attack by T cells as a,nadjunct to organ transplantation or gene therapy. The present proposal will address, using biochemical, cell- biological and immunological methods,a number of key aspects of Class I restricted antigen presentation. First, we shall develop novel pharmacological tools with which to manipulate cytosolic proteolysis, and explore proteolytic pathways that manifest themselves when proteasomes are inactivated by active-site directed covalent inhibitors. Second, we shall exploit the occurrence of a natural inhibitor of the MHC-eicoded peptide transporter, TAP, to explore how the Herpes Simplex Virus manages to escape the detection of cytotoxic T cells. The protein responsible for this evasion, ICP47, has been produced through complete synthesis and offers a well-nigh-ideal target for detailed structure-function analysis through a peptide-synthetic approach. The stable membrane insertion or assembly of MHC class I molecules is targeted by HCMV through expression of the US2 and US11 gene products. These proteins catalyze a reaction in which newly synthesized Class I molecules are dislocated for the ER<discharged into the cytosol, and destroyed by the proteasome. Experiments are proposed to examine, in mammalian cells and in yeast, the mechanistic details of this reaction, which sheds altogether new lights on the phenomenon of protein of murine Class I Mice rendered Class I deficient through targeted deletions of the H-2K and -D loci, produced by selecting form intra-H2 recombinants produced in an F1 cross between the respective single knock-outs, should prove useful tools in addressing questions pertaining to T cell development. The production of recombinant Class I molecules, which can be loaded in vitro with peptide mixturesof any desired complexity, will allow their introduction into a tissue culture system known to support T cell development in vitro (fetal thymus organ culture), based on thymus from the Class l-deficient animals. By the addition of known quantities of Class I molecules, occupied with peptide mixtures of known complexity, to developing T cells exposed to all the necessary non-Class I signals as provided by thymic epithelial cells, it should be possible to dissect more systematically the requirements forpositive selection. /