Natural Killer Cell Protocols pp 73-79

Part of the Methods in Molecular Biology book series (MIMB, volume 121)

Hybrid Resistance by Mouse NK Cells In Vitro

  • P. V. Sivakumar
  • Thaddeus George
  • Igor J. Puzanov
  • Noelle S. Williams
  • Susan Stepp
  • Jingxuan Liu
  • John Schatzle
  • Wayne C. Lai
  • Michael Bennett
  • Vinay Kumar

Abstract

The study of hybrid resistance was greatly aided by the development of an in vitro assay by Chadwick and Miller (1). The assay allows one to test for the presence of negative or positive signaling in natural killer (NK) cells, and the ability of monoclonal antibodies (MAbs) or F(ab′)2 reagents to block those signals. There is already good evidence for negative signals between Ly49 NK cell receptors and their specific class I antigens, e.g., Ly49A and H2-Dd (2). Thus, the inability of H2b Ly49A purified NK cells to kill H2d target cells can be reversed by adding MAb reagents that bind to either Ly49A or H2-Dd. We and others have extended these early findings to the Ly49C and I subsets of NK cells, and have recently detected positive signaling from D8 (H2b, Dd transgene) to purified H2b Ly49D+ NK cells that can be blocked by antibodies or their F(ab′)2 fragments to Ly49D. The assay can also be used to study signaling properties of fetal and neonatal NK cells lacking Ly49 receptors, which suggest that inhibitory non-Ly49 NK receptors are expressed on such NK cells and that these receptors can receive negative signals from class I molecules (3,4). The stimulation of bone marrow NK precursor cells with various cytokines, including IL-15, leads to generation of lytic NK1.1+Ly49- cells whose function is similar to that of fetal NK cells, i.e., they can lyse YAC-1 tumor cells and class I deficient cells, but cannot lyse normal lymphoblasts and lack Ly49 receptors (5). These two types of in vitro systems allow the analysis of factors necessary for various stages in NK cell differentiation and positive and negative signaling to NK cells at different stages of maturation.

References

  1. 1.
    Chadwick B. S. and Miller R.G. (1992) Hybrid resistance in vitro. J. Immunol. 148, 2307–2313.PubMedGoogle Scholar
  2. 2.
    Karlhofer F. M., Ribaudo R. K., and Yokoyama W.M. (1992) MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 358, 66–70.PubMedCrossRefGoogle Scholar
  3. 3.
    Manoussaka M., Georgiou A., Rossiter B., Shrestha S., Toomey J. A., Sivakumar P. V., Bennett M., Kumar V., and Brooks C. G.(1997) Phenotypicand functional characterization of long-lived NK cell lines of different matura-tional status obtained from mouse fetal liver. J. Immunol. 158, 112–119.PubMedGoogle Scholar
  4. 4.
    Sivakumar P. V., Bennett M., and Kumar V. (1997) Fetal and neonatal NK1.1+Ly-49- cells can distinguish between major histocompatibilty class Ihi and classIlow target cells evidence for a Ly-49--independent negative signaling receptor.Eur. J. Immunol. 27, 3100–3104.PubMedCrossRefGoogle Scholar
  5. 5.
    Williams N.S., Moore T. A., Schatzle J. D., Puzanov I. J., Sivakumar P. V., Zlotnik A., Bennett M., and Kumar V. (1997) Generation of NK1.1+, Ly-49- cells from multipotential murine bone marrow progenitors in a stroma-free culture:definition of cytokine requirements and developmental intermediates. J. Exp. Med. 186, 1609–1614.PubMedCrossRefGoogle Scholar
  6. 6.
    Savary C. A., Phillips J. H., and Lotzová E (1979) Inhibition of murine naturalkiller cell-mediated cytotoxicity by pretreatment with ammonium chloride.J. Immunol. Methods 25, 189–192.PubMedCrossRefGoogle Scholar
  7. 7.
    V ujanovic N. L., Herberman R. B., Maghazachi A. A., and Hiserodt J. C. (1988) Lymphokine-activated killer cells in rats. III. A simple method for the purification of large granular lymphocytes and their rapid expansion and conversion intolymphokine-activated killer cells. J. Exp. Med. 167, 15–29.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • P. V. Sivakumar
    • 1
  • Thaddeus George
    • 1
  • Igor J. Puzanov
    • 1
  • Noelle S. Williams
    • 1
  • Susan Stepp
    • 1
  • Jingxuan Liu
    • 1
  • John Schatzle
    • 1
  • Wayne C. Lai
    • 1
  • Michael Bennett
    • 1
  • Vinay Kumar
    • 1
  1. 1.Department of PathologyUniversity of Texas Southwestern Medical SchoolDallas

Personalised recommendations