The broad, long-term objectives of the proposal are aimed at testing the hypothesis that the nonclassical HLA class I antigens play unique and fundamental roles in the immune response. The fundamental role played by the classical class I genes have been identified in humans. These genes, named HLA-E,-F, and -G, each have patterns of expression distinguishing them from one another and from the HLA-A, -B, and -C antigens. Most recently, it has been demonstrated that HLA-G is expressed in the human placenta by trophoblast cells and may be expressed in two forms, one membrane bound and one water soluble. In this proposal, we present evidence that HLA-G may be expressed in those tissues so called immune priveledged including the placenta, the anterior eye, and sperm. We present evidence that alternate forms of the HLA-G protein may exist, resulting from alternate splicing of the primary transcript. Based on our having demonstrated a specific CTL response to the HLA-E protein and having examined the pattern of protein expression in transfected cell lines, we propose HLA-E does function in vivo as a classical restricting element and may have another unknown function in vivo. The experiments proposed in this application are designed to test the above hypothesis by further describing and explaining the expression and testing the function of the nonclassical antigens. The experimental design and methods are as follows.
Specific aim 1 is focused on expanding the knowledge of tissues and cells expressing the HLA-E, -F, and -G antigens. We will accomplish this through three successive levels of analysis: a) identification of tissues expressing HLA-E, -F, and -G mRNA. b) identification of antigen expression on specific cell subsets using monoclonal antibodies. c) characterization of the different antigen forms expressed by each cell type.
In specific aim 2 we undertake to more fully characterize the different protein forms expressed by the HLA-G gene, allowing for further testing of their function. this will be accomplished by achieving the following goals: a) construction of cell lines expressing the alternate forms of HLA-G. b) generation of sera and monoclonal antibodies reactive with these alternate forms. c) Purification of the soluble form of HLA-G. The studies outlined in specific aim 3 propose to study the mechanisms affecting the surface transport of HLA-E. We will do this by: a) a comparison of the HLA-E protein concentrating on those features which distinguish it from a classical antigen. b) an examination of the amino acid residues in the HLA_E protein affecting cell surface expression. Finally, specific aim 4 proposes to analyze the T cell populations reacting with HLA-E and HLA-G in two ways: a) We will analyze the HLA-E reactive cells by the characterization of their T cell surface markers including a molecular characterization of their T cell receptor. b) We will concentrate on the T cell reactivity of the soluble HLA-G protein by attempting to measure it's effect on normal T cell function in vitro.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Experimental Immunology Study Section (EI)
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Fred Hutchinson Cancer Research Center
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Bahram, S; Bresnahan, M; Geraghty, D E et al. (1994) A second lineage of mammalian major histocompatibility complex class I genes. Proc Natl Acad Sci U S A 91:6259-63
Fujii, T; Ishitani, A; Geraghty, D E (1994) A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4. J Immunol 153:5516-24
Geraghty, D E (1993) Structure of the HLA class I region and expression of its resident genes. Curr Opin Immunol 5:3-7
Ishitani, A; Geraghty, D E (1992) Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens. Proc Natl Acad Sci U S A 89:3947-51
Geraghty, D E; Pei, J; Lipsky, B et al. (1992) Cloning and physical mapping of the HLA class I region spanning the HLA-E-to-HLA-F interval by using yeast artificial chromosomes. Proc Natl Acad Sci U S A 89:2669-73