Maternal acceptance of genetically disparate tissue during semiallogeneic pregnancy is believed to require strict regulation of HLA class I gene expression in trophoblast cells at the maternal-fetal interface. Because inappropriate expression might lead to pregnancy failure, it is of the utmost importance to elucidate expression, to establish the mechanisms regulating expression, and to identify pregnancy-associated functions of antigens derived from this multigene family. Significant progress toward these long-range goals has been made in the current funding period, where molecular approaches established differential expression of the HLA class I genes in subpopulations of trophoblast cells, showed that a novel member of the HLA class I gene family, HLA-G, is expressed in placentas, and identified a placenta-targeting 5' regulatory sequence in the HLA-G gene. Further studies suggest that availability of light chains, peptide transporters (TAP-1, TAP-2), proteasome components (LMP2, LMP7), cytokines and cytokine receptors may uniquely govern HLA class I expression in trophoblast cells. Experiments done on HLA-G transgenic mouse placentas have established the usefulness of this model for further studies on HLA class I gene expression and regulation, and other advances that include the technology for generating mouse trophoblast cell lines have been made. Molecular, biochemical, immunological and cell culture techniques will be used to achieve the four Specific Aims of the proposed research. The goal of Specific Aim 1 is to determine whether or not class Ia (HLA-A, -B, -C) and class Ib (HLA-G) genes are co-expressed in human placental cells and whether the proportions of Ia:Ib or their cellular locations change during the course of gestation.
In Specific Aim 2, transgenic mice are used for fine analysis of developmental regulation and cellular localization of HLA class Ia (represented by HLA-B27 transgenic mice) and Ib (HLA-G transgenic) through the course of pregnancy.
Specific Aim 3 addresses the hypothesis that limited availability of intracellular and/or extracellular promoters of HLA class I expression on cell membranes (light chains, TAP- 1, TAP-2, LMP2, LMP7, cytokines and cytokine receptors) controls expression in some trophoblast cell subpopulations.
In Specific Aim 4, trophoblast cell lines generated from the placentas of outbred mice, founder strain mice and mice transgenic for class Ia and Ib genes will be used to establish the role of cytokines in determining class Ia:Ib ratios, steady state levels of class I mRNA and the subcellular location(s) of HLA class I antigens. We expect the results of this research to shed considerable light on crucial issues of HLA-related fertility and reproduction.
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