Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1995 Aug 1;182(2):293–303. doi: 10.1084/jem.182.2.293

Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2)

PMCID: PMC2192132  PMID: 7629495

Abstract

Large granular lymphocyte (LGL) 1 is a cell surface glycoprotein expressed on a subset (50%) of C57BL/6 natural killer (NK) cells. Immunoprecipitation experiments reveal that the LGL-1 protein exists as a disulfide-linked 40-kD homodimer. Functional studies of LGL-1+ cells indicate that selected H-2d target cells are not lysed efficiently by these interleukin (IL)-2-cultured NK cells. These findings suggested that LGL-1 may be a member of the Ly-49 gene family. Here we report the molecular cloning of the LGL-1 cDNA from a severe combined immunodeficient-adherent lymphokine-activated killer cell library transfected into Cos-7 cells and find LGL-1 to be homologous to the Ly- 49 gene at both the nucleotide (85%) and amino acid levels (73%). Sequencing of our LGL-1 cDNA has revealed it to be nearly identical to the Ly-49G2 cDNA recently isolated by cross-hybridization with an Ly-49 probe. LGL-1 represents a type II transmembrane protein of 267 amino acids with its carboxyl end exposed extracellularly. The LGL-1 protein contains 11 highly conserved cysteine residues and a 25-amino acid transmembrane region. Southern blot analysis demonstrates that there are a number of homologous genes in mouse DNA that hybridize strongly to LGL-1. Northern analyses using poly A+ RNA from LGL-1+ NK cells indicate that LGL-1 is expressed as a 1.4 kb mRNA. Two-color flow cytometry analysis (FCA) of C57BL/6 splenic NK cells demonstrates that LGL-1 and Ly-49 label overlapping subsets of cells. FCA identifies four subsets of NK cells as defined by LGL-1 versus Ly-49 staining. We have sorted these individual subsets, expanded them in IL-2, and performed cytotoxicity experiments to determine their target cell profiles in relation to class I expression. Results of these studies are complex, but indicate that Ly-49 may not be the only molecule that recognizes class I as an inhibitory signal for cytotoxicity. LGL-1+ cells also fail to lyse several H-2d-expressing tumor targets and concanavalin A lymphoblasts from BALB/c but not C57BL/6 mice. This inhibition of lysis by LGL-1+ NK cells is negated by addition of monoclonal antibody (mAb) 4D11 that recognizes the LGL-1 protein. When mAbs to the class I molecules H-2Dd and H-2Ld (alpha 1 alpha 2 domains only) are added to cytotoxicity assays, LGL-1+ cells lyse H-2d targets very effectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson S. K., Gallinger S., Roder J., Frey J., Young H. A., Ortaldo J. R. A cyclophilin-related protein involved in the function of natural killer cells. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):542–546. doi: 10.1073/pnas.90.2.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chadwick B. S., Miller R. G. Hybrid resistance in vitro. Possible role of both class I MHC and self peptides in determining the level of target cell sensitivity. J Immunol. 1992 Apr 1;148(7):2307–2313. [PubMed] [Google Scholar]
  3. Chervenak R., Wolcott R. M. Target cell expression of MHC antigens is not (always) a turn-off signal to natural killer cells. J Immunol. 1988 Jun 1;140(11):3712–3716. [PubMed] [Google Scholar]
  4. Dennert G., Landon C., Lord E. M., Bahler D. W., Frelinger J. G. Lysis of a lung carcinoma by poly I:C-induced natural killer cells is independent of the expression of class I histocompatibility antigens. J Immunol. 1988 Apr 1;140(7):2472–2475. [PubMed] [Google Scholar]
  5. Evans G. A., Margulies D. H., Shykind B., Seidman J. G., Ozato K. Exon shuffling: mapping polymorphic determinants on hybrid mouse transplantation antigens. Nature. 1982 Dec 23;300(5894):755–757. doi: 10.1038/300755a0. [DOI] [PubMed] [Google Scholar]
  6. Giorda R., Trucco M. Mouse NKR-P1. A family of genes selectively coexpressed in adherent lymphokine-activated killer cells. J Immunol. 1991 Sep 1;147(5):1701–1708. [PubMed] [Google Scholar]
  7. Kane K. P. Ly-49 mediates EL4 lymphoma adhesion to isolated class I major histocompatibility complex molecules. J Exp Med. 1994 Mar 1;179(3):1011–1015. doi: 10.1084/jem.179.3.1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Karlhofer F. M., Ribaudo R. K., Yokoyama W. M. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature. 1992 Jul 2;358(6381):66–70. doi: 10.1038/358066a0. [DOI] [PubMed] [Google Scholar]
  9. Kaufman D. S., Schoon R. A., Leibson P. J. MHC class I expression on tumor targets inhibits natural killer cell-mediated cytotoxicity without interfering with target recognition. J Immunol. 1993 Feb 15;150(4):1429–1436. [PubMed] [Google Scholar]
  10. Leiden J. M., Karpinski B. A., Gottschalk L., Kornbluth J. Susceptibility to natural killer cell-mediated cytolysis is independent of the level of target cell class I HLA expression. J Immunol. 1989 Mar 15;142(6):2140–2147. [PubMed] [Google Scholar]
  11. Ljunggren H. G., Sturmhöfel K., Wolpert E., Hämmerling G. J., Kärre K. Transfection of beta 2-microglobulin restores IFN-mediated protection from natural killer cell lysis in YAC-1 lymphoma variants. J Immunol. 1990 Jul 1;145(1):380–386. [PubMed] [Google Scholar]
  12. Mason L. H., Mathieson B. J., Ortaldo J. R. Natural killer (NK) cell subsets in the mouse. NK-1.1+/LGL-1+ cells restricted to lysing NK targets, whereas NK-1.1+/LGL-1- cells generate lymphokine-activated killer cells. J Immunol. 1990 Jul 15;145(2):751–759. [PubMed] [Google Scholar]
  13. Mason L. H., Yagita H., Ortaldo J. R. LGL-1: a potential triggering molecule on murine NK cells. J Leukoc Biol. 1994 Mar;55(3):362–370. doi: 10.1002/jlb.55.3.362. [DOI] [PubMed] [Google Scholar]
  14. Mason L., Giardina S. L., Hecht T., Ortaldo J., Mathieson B. J. LGL-1: a non-polymorphic antigen expressed on a major population of mouse natural killer cells. J Immunol. 1988 Jun 15;140(12):4403–4412. [PubMed] [Google Scholar]
  15. Nishimura M. I., Stroynowski I., Hood L., Ostrand-Rosenberg S. H-2Kb antigen expression has no effect on natural killer susceptibility and tumorigenicity of a murine hepatoma. J Immunol. 1988 Dec 15;141(12):4403–4409. [PubMed] [Google Scholar]
  16. Ozato K., Hansen T. H., Sachs D. H. Monoclonal antibodies to mouse MHC antigens. II. Antibodies to the H-2Ld antigen, the products of a third polymorphic locus of the mouse major histocompatibility complex. J Immunol. 1980 Dec;125(6):2473–2477. [PubMed] [Google Scholar]
  17. Ozato K., Mayer N. M., Sachs D. H. Monoclonal antibodies to mouse major histocompatibility complex antigens. Transplantation. 1982 Sep;34(3):113–120. doi: 10.1097/00007890-198209000-00001. [DOI] [PubMed] [Google Scholar]
  18. Piontek G. E., Taniguchi K., Ljunggren H. G., Grönberg A., Kiessling R., Klein G., Kärre K. YAC-1 MHC class I variants reveal an association between decreased NK sensitivity and increased H-2 expression after interferon treatment or in vivo passage. J Immunol. 1985 Dec;135(6):4281–4288. [PubMed] [Google Scholar]
  19. Ryan J. C., Turck J., Niemi E. C., Yokoyama W. M., Seaman W. E. Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation molecules. J Immunol. 1992 Sep 1;149(5):1631–1635. [PubMed] [Google Scholar]
  20. Seed B., Aruffo A. Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. Proc Natl Acad Sci U S A. 1987 May;84(10):3365–3369. doi: 10.1073/pnas.84.10.3365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith H. R., Karlhofer F. M., Yokoyama W. M. Ly-49 multigene family expressed by IL-2-activated NK cells. J Immunol. 1994 Aug 1;153(3):1068–1079. [PubMed] [Google Scholar]
  22. Stoneman E. R., Bennett M., An J., Chesnut K. A., Wakeland E. K., Scheerer J. B., Siciliano M. J., Kumar V., Mathew P. A. Cloning and characterization of 5E6(Ly-49C), a receptor molecule expressed on a subset of murine natural killer cells. J Exp Med. 1995 Aug 1;182(2):305–313. doi: 10.1084/jem.182.2.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Storkus W. J., Alexander J., Payne J. A., Cresswell P., Dawson J. R. The alpha 1/alpha 2 domains of class I HLA molecules confer resistance to natural killing. J Immunol. 1989 Dec 1;143(11):3853–3857. [PubMed] [Google Scholar]
  24. Storkus W. J., Alexander J., Payne J. A., Dawson J. R., Cresswell P. Reversal of natural killing susceptibility in target cells expressing transfected class I HLA genes. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2361–2364. doi: 10.1073/pnas.86.7.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Storkus W. J., Howell D. N., Salter R. D., Dawson J. R., Cresswell P. NK susceptibility varies inversely with target cell class I HLA antigen expression. J Immunol. 1987 Mar 15;138(6):1657–1659. [PubMed] [Google Scholar]
  26. Storkus W. J., Salter R. D., Alexander J., Ward F. E., Ruiz R. E., Cresswell P., Dawson J. R. Class I-induced resistance to natural killing: identification of nonpermissive residues in HLA-A2. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):5989–5992. doi: 10.1073/pnas.88.14.5989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Storkus W. J., Salter R. D., Cresswell P., Dawson J. R. Peptide-induced modulation of target cell sensitivity to natural killing. J Immunol. 1992 Aug 15;149(4):1185–1190. [PubMed] [Google Scholar]
  28. Yokoyama W. M., Jacobs L. B., Kanagawa O., Shevach E. M., Cohen D. I. A murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins. J Immunol. 1989 Aug 15;143(4):1379–1386. [PubMed] [Google Scholar]
  29. Yokoyama W. M., Kehn P. J., Cohen D. I., Shevach E. M. Chromosomal location of the Ly-49 (A1, YE1/48) multigene family. Genetic association with the NK 1.1 antigen. J Immunol. 1990 Oct 1;145(7):2353–2358. [PubMed] [Google Scholar]
  30. Yokoyama W. M., Ryan J. C., Hunter J. J., Smith H. R., Stark M., Seaman W. E. cDNA cloning of mouse NKR-P1 and genetic linkage with LY-49. Identification of a natural killer cell gene complex on mouse chromosome 6. J Immunol. 1991 Nov 1;147(9):3229–3236. [PubMed] [Google Scholar]
  31. Ziegler S. F., Levin S. D., Johnson L., Copeland N. G., Gilbert D. J., Jenkins N. A., Baker E., Sutherland G. R., Feldhaus A. L., Ramsdell F. The mouse CD69 gene. Structure, expression, and mapping to the NK gene complex. J Immunol. 1994 Feb 1;152(3):1228–1236. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES