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-2(d) 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 how 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-2(d)-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-2D(d) and H-2L(d) (alpha(1) alpha(2) domains only) are added to cytotoxicity assays, LGL-1(+) cells lyse H-2(d) targets very effectively. Therefore, LGL-1 recognizes regions of the class I-specific molecules H-2D(d) and H-2L(d). This specificity distinguishes LGL-1 from Ly-49, whose killing was only reversed by antibodies to H-2D(d). The differential specificities recognized by LGL-1 versus Ly-49 support the hypothesis that this family of genes recognizes diverse class I molecules and regulates the lytic activity of NK cells.