This application aims at studying HLA-G as a factor in the regulation of feto-maternal immune interactions. Generation of antibody reagents specific for HLA-G (conventional polyclonal sera, monoclonal antibodies and single chain Fv fragments) suitable for biochemical and immunocytochemical analysis will be the first priority, as no such reagents are available at present. For comparative purposes, the expression of HLA-E will be analyzed under conditions where viral evasion eliminates expression of the classical Class I gene products HLA-A,-B, using reagents to be generated in parallel with those specific for HLA-G. Recombinantly produced HLA-G, in conjunction with HLA-G specific detection methods, should allow the evaluation of sera from women who have undergone recurrent spontaneous abortions for the presence of both soluble HLA-G and antibodies against HLAG. Preliminary data further suggest the possibility of a murine HLA-G homolog. Biochemical purification of this product, its analysis by tryptic digestion and LC/MS, MS/MS is proposed for obtaining sequence information to allow subsequent cloning of the gene that encodes it. Mice transgenic for HLA-G expressed under a placenta-specific or a more broadly specific promoter will be used not only for validation of the HLA-G specific reagents obtained but also to examine the possible contributions of HLA-G to the generation and maintenance of correspondingly restricted T-cell populations in the mouse. This analysis will benefit from the availability of newly generated mice that lack expression of all classical Class I molecules, yet are fully proficient for B2m and TAP, to be crossed with HLA-G transgenic animals. Such HLAG transgenic animals will further be used to explore the possibilities of HLA-G's contribution to fetal oral tolerance and maternal anergy. Independent of HLA-G's role in the immunology of feto-maternal interactions, the contribution of the Class I and Class II restricted presentation pathways to generation and maintenance of intra-epithelial T lymphocytes in the reproductive tract will be determined.
