The basic goals of the studies outlined in this grant are to gain information about the role played by the HLA-G class I MHC molecules in the maternal-placental immune interaction. We have been characterizing the HLA-G gene products and have made progress in understanding their expression and in developing reagents necessary for further investigation of their functions. We found that HLA-G mRNA is expressed in five forms due to alternative splicing of the primary transcript. Further, our analysis has shown that two of these forms, a membrane bound and a soluble counterpart, bind a diverse array of peptides typical of a class I molecule. We were successful in generating HLA-G specific antibodies, and were able to use them confirm that both proteins are expressed in placental tissue and to obtain evidence for the presence of the soluble form in both mothers and offsprings blood. The experiments proposed in this application are designed to further investigate the expression of the alternative HLA-G proteins and to use the information gained to test hypotheses about the unique roles these proteins may play in the maternal- placental immune interaction. We propose that two forces are acting on the HLA-G locus, the need to present viral peptide to the maternal immune system for protection against infection, a force thought to select for polymorphism, and the opposing need for low polymorphism in order to avoid an alloreactive maternal immune response. We further propose that one of the mechanisms that has evolved to help accommodate additional allelic polymorphism is the expression of soluble G, which may help to abrogate an alloresponse by acting to anergize alloreactive T cells. Our experimental plan utilizes a combination of biochemical, molecular, and cellular approaches, carried out by a collaborative effort among experts in these fields. We propose to study the expression of the unique HLA-G2 and -G3 MHC molecules in order to understand their role in maternal-placental immune accommodation. HLA-G protein expression in placental and maternal tissues and soluble G protein in maternal blood will be examined, emphasizing potential differences in abnormal pregnancies. In order to gain functional data and to elucidate post-translational mechanisms controlling soluble and membrane HLA-G expression in fetal tissue we propose to examine peptide binding by soluble and membrane HLA-G proteins. This will include analysis of peptides bound to HLA-G expressed in a tap- deficient cell line, from placentas, and a measurement of binding affinity of peptides derived from-viral antigens. In order to study the interaction of HLA-G with maternal immune cells, we will ask if membrane HLA-G functions as a restricting element for antigen presentation to maternal T cells, a question aimed at provided basic information about the control of transmission of infectious maternal disease to the fetus. We will further examine HLA-G function by comparing the ability of soluble and membrane HLA-G to suppress maternal NK activity.
