Analysis of the Soluble Isoforms of HLA-G

mRNAs and Proteins
  • Judith L. Pace
  • Pedro J. Morales
  • Joan S. Hunt
  • Teresa A. Phillips
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 122)

Abstract

The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms.

Key Words

Enzyme-linked immunosorbent assay (ELISA) flow cytometry HLA-G human immunoblotting immunohistochemistry placenta reverse transcriptase polymerase chain reaction (RT-PCR) recombinant HLA-G trophoblast cells 

References

  1. 1.
    Janeway, C. A., Travers, P., Walport, M., and Capra, J. D. (1999) Immunobiology. Garland, New York, NY, pp. 509–516.Google Scholar
  2. 2.
    Ishitani, A., Sageshima, N., Lee, N., et al. (2003) Protein expression and peptide binding suggest unique and interacting functional roles for HLA-E, F, and G in maternal-placental immune recognition. J. Immunol. 171, 1376–1384.PubMedGoogle Scholar
  3. 3.
    McMaster, M. T., Librach, C. L., Zhou, Y., et al. (1995) Human placental HLA-G expression is restricted to differentiated cytotrophoblasts. J. Immunol. 154, 3771–3778.PubMedGoogle Scholar
  4. 4.
    Hunt, J. S. and Hutter, H. (1996) Current theories on protection of the fetal semiallograft, in HLA and the Maternal-Fetal Relationship (Hunt, J. S., ed.). Landes, Austin, TX: pp. 27–50.Google Scholar
  5. 5.
    Carosella, E. D., Dausset, J., and Rouas-Freiss, N. (1999) Immunotolerant functions of HLA-G. Cell Mol. Life Sci. 55, 327–333.CrossRefPubMedGoogle Scholar
  6. 6.
    Hunt, J. S. (2002) Immunogenetics: genetic regulation of immunity in pregnancy, in Reproductive Medicine: Molecular, Cellular and Genetic Fundamentals (Fauser, B. C. J. M., ed.). Parthenon, NY: pp. 153–167.Google Scholar
  7. 7.
    Ishitani, A. and 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. USA 89, 3947–3951.CrossRefPubMedGoogle Scholar
  8. 8.
    Paul, P., Cabestre, F. A., Ibrahim, E. C., et al. (2000) Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLA-G5,-G6,-G7 transcripts in human transfected cells. Hum. Immunol. 61, 1138–1149.CrossRefPubMedGoogle Scholar
  9. 9.
    Ober, C, Aldrich, C., Rosinsky, B., et al. (1998) HLA-G1 protein expression is not essential for fetal survival. Placenta 19, 127–132.CrossRefPubMedGoogle Scholar
  10. 10.
    Lee, N., Llano, M., Carretero, M., et al. (1998) HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc. Natl. Acad. Sci. USA 95, 5199–5204.CrossRefPubMedGoogle Scholar
  11. 11.
    Morales, P., Pace, J. L., Platt, J. S., et al. (2003) Placental cell expression of HLAG2 isoforms is limited to the invasive trophoblast phenotype. J. Immunol. 171, 6215–6224.PubMedGoogle Scholar
  12. 12.
    McIntire, R. H., Morales, P. J., Petroff, M. G., Colonna, M., and Hunt, J. S. (2004) Recombinant HLA-G5 and-G6 drive U937 myelomonocytic cell production of TGF-β1. J Leukoc Biol. 76 (6), 1220–1228.CrossRefPubMedGoogle Scholar
  13. 13.
    Fournel, S., Aguerre-Girr, M., Huc, X., et al. (2000) Soluble HLA-G1 triggers CD95/CD95 ligand-mediates apoptosis in activated CD8+ cells by interacting with CD8. J. Immunol. 164, 6100–6104.PubMedGoogle Scholar
  14. 14.
    LeMaoult, J., Krawice-Radanne, I., Dausset, J., and Carosella, E. D. (2004) HLAG1-expressing antigen-presenting cells induce immunosuppressive CD4+ T cells. Proc. Natl. Acad. Sci. USA 101, 7064–7069.CrossRefPubMedGoogle Scholar
  15. 15.
    Chu, W., Yang, Y., Geraghty, D. E., and Hunt, J. S. (1999) Interferons enhance HLA-G mRNA and protein in transfected mouse fibroblasts. J. Reprod. Immunol. 42, 1–15.CrossRefPubMedGoogle Scholar
  16. 16.
    Jung, T., Schauer, U., Heusser, C., Neumann, C., and Rieger, S. (1993). Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159, 197–207.CrossRefPubMedGoogle Scholar
  17. 17.
    Hunt, J. S., Pace, J. L., Morales, P. J., and Ober, C. (2003). Immunogenicity of the soluble isoforms of HLA-G. Mol. Hum. Reprod. 9, 729–735.CrossRefPubMedGoogle Scholar
  18. 18.
    Hviid, T. V. F, Hylenius, S., Rørbye, C., and Nielsen, L. G. (2003). HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels. Immunogenetics 55, 63–79.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Judith L. Pace
    • 1
  • Pedro J. Morales
    • 2
  • Joan S. Hunt
    • 2
  • Teresa A. Phillips
    • 3
  1. 1.Department of Molecular and Integrative PhysiologyUniversity of Kansas Medical CenterKansas City
  2. 2.Department of Anatomy & Cell BiologyUniv ersity of Kansas Medical CenterKansas City
  3. 3.Department of Internal MedicineUniversity of Kansas Medical CenterKansas City

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