One of the major reasons for the high incidence of graft failure and GVHD in MHC matched unrelated marrow transplants is believed to be undetected microvariant mismatches of the MHC molecules. The overall goals of this proposal is to identify the undetected microvariants and to evaluate the impact of these mismatches on marrow transplants. From these studies, we plan to determine the acceptable limits of HLA incompatibility, and from there develop tissue typing strategies applicable to clinical HLA typing for patient-donor matching. To achieve these goals, this project is organized into two parts; studies focusing on structural polymorphism, with emphasis on the identification of microheterogeneity in class I molecules (specific aim 1) and studies focusing on the determination of the biological significance of those micro differences in inducing alloreactivity following marrow transplants (specific aim 2). A special emphasis is placed on the structural and functional characterization of the HLA-C molecules, whose allelic definition and functions are still unclear. The goal of specific aim 1 is to provide a fine definition of class I alleles and to develop a detection system to identify them. Direct sequencing and various PCR-based DNA typing methods (SSOP, SSCP, heteroduplex formation) will be used to provide the means for a retrospective evaluation of the phenotypically matched unrelated marrow transplanted pairs. The goal of specific aim 2 is to examine the biological significance of class I subtype mismatches in marrow transplants. Thus, the major focus of specific aim 2 is placed on the functional evaluation of the class I mismatches involving class I IEF variants or IEF undetectable microvariants on the induction of alloreactive cytolytic T cells and alloreactive NK cells. An in vitro system using transfectants that express a single class I allele will be employed to measure the CTL-p and NK-p frequencies in pairwise examination, and also to study the class I recognition motifs used by alloreactive CTLs and NK cells. Ultimately, the results will aid us in the development of strategies for the prospective typing of unrelated marrow donors and recipients.
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