The broad, long term objectives of this application are to open new areas of investigation related to genetic polymorphism in marrow transplantation and to further elucidate the function of the characterized nonclassical HLA genes. One of the goals of the previous application was to investigate the functions of the nonclassical antigens and to further the understanding of their contribution to the problems encountered in transplantation. While no polymorphic variants of HLA-G were discovered, two alleles of HLA-E were described and their distribution in the population was examined. The most remarkable aspect of HLA-E polymorphism was that both alleles were randomly distributed in the population and no linkage disequilibrium was found between the HLA-E alleles and most HLA-A or -B alleles. Thus, in an HLA-A, -B matched unrelated transplant, the likelihood of an HLA-E mismatched is high. As part of our efforts to open up new areas of investigation and to further our search for polymorphisms which might contribute to the outcome of a marrow transplant, we used probes from the HLA genes and pseudogenes to screen a yeast artificial chromosome (YAC) library and isolated clones spanning 2.5 million base pairs. From this we hope to obtain a better understanding of the large scale differences between HLA haplotypes and to identify regions possibly containing polymorphic genes. This work will provide a stimulus to bring new ideas and discoveries to the field of transplantation immunology. In summary, we now have new opportunities to further investigate the role of a nonclassical class I molecule and its involvement in marrow transplants well as to uncover previously hidden genes linked to the HLA class I region. The experimental design and methods are as follows.
Specific aim 1 will determine to what extent the class I region is polymorphic. This will be done by first constructing large scale maps of the class I region in diverse HLA haplotypes and measuring the differences between then.
Specific aim 2 will attempt to investigate the potential involvement of polymorphic genes in a marrow transplant. This will be done by characterizing the structural and functional features of polymorphic genes located within the class I region and defining and measuring the extent of polymorphism. If warranted, polymorphisms will be correlated with transplant outcome in unrelated HLA identical transplants. The third specific aim will generate monoclonal antibodies specifically reactive with HLA-E. This will be accomplished using methods successfully applied to the generation of HLA-G specific antibodies. Finally, specific aim 4 includes experimentation into the role of HLA-E as a histocompatibility determinant in a clinical marrow transplant. We will do this by first investigating the function of HLA-E in evoking a T cell response, second by examining an HLA-E alloreactive response in vitro and thirdly by correlating the mismatch of HLA-E in HLA-A, -B identical unrelated transplants with transplant outcome.
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