The human major histocompatibility complex (MHC) is defined by the highly polymorphic class I (HLA-A, -B and -C) and class 11 (HLA-DR, -DQ and -DP) immune response genes. These encode cell surface molecules, which bind peptides derived from antigen and mediate the activation of cytotoxic and helper T lymphocytes, respectively. Based on functional and genetic evidence, the MHC encodes additional genes involved in immune recognition, which are unrelated to the class I and class II gene families. The identification and characterization of these genes is the goal of the proposed research.
SPECIFIC AIM I : In several MHC deletion mutant cell lines, the intracellular assembly and subsequent expression at the cell surface of class I heavy chains and beta2-microglobulin (beta2m) is impaired. Presumably, this mutant phenotype is due to deletion of a chain assembly factor (CAF) gene from the class II or central MHC class III region. CAF will be identified by mapping homozygous deletions in mutants, localizing candidate genes in cloned cosmids and complementing the mutant phenotype by transfection of cosmids and cDNA expression constructs. CAF will be characterized structurally by sequence analysis and functionally at the protein level by immunochemistry. CAF might function in supplying the endoplasmic reticulum with peptides or in peptide loading of class I molecules. Experiments are designed to explore a physical interaction of CAF with class I heavy chains and/or beta2M. Moreover, the subcellular distribution of CAF and co-localization at a specific site with class I heavy chains and beta2m will be examined by immunoelectron microscopy. Altogether, these studies may provide new insights into the complexity of the class I antigen processing and presentation pathway.
SPECIFIC AIM II : The susceptibility of target cells to lysis by alloreactive NK cells is controlled by a gene, EC], which is encoded between C2 andHLA-B in the central MHC class Ill region. A number of candidate genes for EC1 have been mapped previously within this cloned 550 kilobases (kb) interval. Among these, EC] will be identified by segregation analysis of a series of allelic restriction fragment length polymorphisms (RFLPs) and hypervariable microsatellites in informative families with and without recombinant MHC haplotypes and in random individuals. The structural and functional characterization of EC1 may be of medical importance in bone marrow transplantation and cancer immunology.
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