Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2)
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)
Mason
L H
LH
Laboratory of Experimental Immunology, PRI/DynCorp, NCI-FCRDC, Maryland 21702-1201, USA.
Ortaldo
J R
JR
Young
H A
HA
Kumar
V
V
Bennett
M
M
Anderson
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000950:Antigens, Ly; D001483:Base Sequence; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D018076:DNA, Complementary / Q000235:genetics; D005796:Genes; D006183:H-2 Antigens / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology*; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D005810:Multigene Family; D051381:Rats; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology; D055630:Receptors, NK Cell Lectin-Like; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000911:Antibodies, Monoclonal; D000950:Antigens, Ly; D018076:DNA, Complementary; D006183:H-2 Antigens; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; D055630:Receptors, NK Cell Lectin-Like