Vital involvement of a natural killer cell activation receptor in resistance to viral infection.

Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.

Nonstochastic coexpression of activation receptors on murine natural killer cells.

Murine natural killer cells (NK) express lectin-like activation and inhibitory receptors, including the CD94/NKG2 family of receptors that bind Qa-1, and the Ly-49 family that recognizes major histocompatibility complex class I molecules. Here, we demonstrate that cross-linking of NK cells with a new specific anti-Ly-49H mAb induced NK cell cytotoxicity and cytokine production. Ly-49H is expressed on a subset of NK cells and can be coexpressed with Ly-49 inhibitory receptors. However, unlike Ly-49 inhibitory receptors, Ly-49H is not detectable on naive splenic CD3(+) T cells, indicating that Ly-49H may be an NK cell-specific activation receptor. In further contrast to the stochastically expressed Ly-49 inhibitory receptors, Ly-49H is preferentially expressed with the Ly-49D activation receptor, and expression of both Ly-49H and Ly-49D is augmented on NK cells that lack receptors for Qa-1 tetramers. On developing splenic NK1.1(+) cells, Ly-49D and Ly-49H are expressed later than the inhibitory receptors. These results directly demonstrate that Ly-49H activates primary NK cells, and suggest that expression of Ly-49 activation receptors by NK cells may be specifically regulated on NK cell subsets. The simultaneous expression of multiple activation receptors by individual NK cells contrasts with that of T cell antigen receptors and is relevant to the role of NK cells in innate immunity.

Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells.

Natural killer (NK) cells express C-type lectin-like receptors, encoded in the NK gene complex, that interact with major histocompatibility complex class I and either inhibit or activate functional activity. Human NK cells express heterodimers consisting of CD94 and NKG2 family molecules, whereas murine NK cells express homodimers belonging to the Ly-49 family. The corresponding orthologues for other species, however, have not been described. In this report, we used probes derived from the expressed sequence tag database to clone C57BL/6-derived cDNAs homologous to human NKG2-D and CD94. Among normal tissues, murine NKG2-D and CD94 transcripts are highly expressed only in activated NK cells, including both Ly-49A+ and Ly-49A- subpopulations. Additionally, mNKG2-D is expressed in murine NK cell clones KY-1 and KY-2, whereas mCD94 expression is observed only in KY-1 cells but not KY-2. Last, we have finely mapped the physical location of the Cd94 (centromeric) and Nkg2d (telomeric) genes between Cd69 and the Ly49 cluster in the NK complex. Thus, these data indicate the expanding complexity of the NK complex and the corresponding repertoire of C-type lectin-like receptors on murine NK cells.

Long-range disruption of gene expression by a selectable marker cassette.

Recent studies have suggested that the retention of selectable marker cassettes (like PGK-Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in "knockout" mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the beta-like globin gene cluster. The insertion of PGK-Neo into the granzyme B gene, the most 5' gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK-Neo cassette into the beta-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3' to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these-long distance effects are unknown, the expression of PGK-Neo can be "captured" by the regulatory domain into which it is inserted. These results suggest that the PGK-Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells.

Granzyme (Gzm) B-deficient mice obtained by gene targeting were used to assess the role of Gzm B in the mechanisms used by natural killer (NK) and lymphokine-activated killer (LAK) cells to destroy target cells. Gzm B-/- NK cells, LAK cells, and cytotoxic T lymphocytes (CTL) all are defective in their ability to rapidly induce DNA fragmentation/apoptosis in susceptible target cells. This defect can be partially corrected with long incubation times of effector and target cells. Moreover, Gzm B-/- NK cells (but not CTL or LAK cells) exhibit a defect in 51Cr release from susceptible target cells. This 51Cr release defect in Gzm B-deficient NK cells is also not overcome by prolonged incubation times or high effector-to-target cell ratios. We conclude that Gzm B plays a critical and nonredundant role in the rapid induction of DNA fragmentation/apoptosis by NK cells, LAK cells, and CTL. Gzm B may have an additional role in NK cells (but not in CTL or LAK cells) for mediating 51Cr release.

Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells.

We have generated H-2b mice with a homozygous null mutation in the granzyme (gzm) B gene. Gzm B is a neutral serine protease with Aspase activity that is found only in the granules of activated cytolytic T cells, natural killer cells, and lymphokine-activated killer cells. Gzm B-/- mice develop normally and have normal hematopoiesis and lymphopoiesis. In vitro, cytotoxic T lymphocytes (CTL) derived from gzm B-/- animals are able to induce 51Cr release from allotarget cells, but with reduced efficiency. However, gzm B-/- CTL have a profound defect in their ability to induce rapid DNA fragmentation and apoptosis in allogeneic target cells. This defect is kinetic since DNA fragmentation is partially compensated and 51Cr release is completely rescued with long incubation times. We conclude that gzm B serves a critical and nonredundant role for the rapid induction of target cell DNA fragmentation and apoptosis by alloreactive cytotoxic T lymphocytes.

Molecular cloning, chromosomal location, and tissue-specific expression of the murine cathepsin G gene.

We previously have characterized a cluster of genes encoding cathepsin G (CG) and two other CG-like hematopoietic serine proteases, CGL-1 and CGL-2, on human chromosome 14. In this report, we clone and characterize a novel, related murine hematopoietic serine protease gene using human CG (hCG) cDNA as the probe. This murine gene spans approximately 2.5 kb of genomic DNA, is organized into five exons and four introns, and bears a high degree of homology to hCG at both nucleic acid (73%) and deduced amino acid (66%) levels. The predicted cDNA contains an open reading frame of 783 nucleotides that encodes a nascent protein of 261 amino acids. Processing of a putative signal (pre) peptide of 18 residues and an activation (pro) dipeptide would generate a mature enzyme of approximately 27 Kd that has an estimated pI of 12.0. Conserved residues at His44, Asp88, and Ser181 form the characteristic catalytic triad of the serine protease superfamily. The gene is tightly linked to the CTLA-1 locus on murine chromosome 14, where the serine protease genes mCCP1-4 are clustered. Expression of this gene is detected only in the bone marrow and is restricted to a small population of early myeloid cells. These findings are consistent with the identification of the gene encoding murine CG.

Structure and expression of a cluster of human hematopoietic serine protease genes found on chromosome 14q11.2.

We previously identified a cluster of hematopoietic serine protease genes on chromosome 14 at band q11.2. This cluster contains the cathepsin G gene and the two related cathepsin G-like genes CGL-1 and CGL-2. The CGL-1 gene is identical with the cytotoxic T cell serine protease CSP-B (also called SECT, and in mice, CCP1, granzyme B, or CTLA-1). In this report, we determined that CGL-2 is identical with a recently described gene called h-CCPX. The coding sequences of CG, CGL-1, and CGL-2 are 65-75% identical at the DNA level. The intervening sequences are much less conserved, except for introns 3 of the CGL-1 and CGL-2 genes, which are 93% identical. Each of the genes has the same overall organization, with 5 exons and 4 introns, very short 5' untranslated regions, and identical splice phases for all of the introns. Cathepsin G is expressed at high levels in promyelocytes/promonocytes, and CGL-1/CSP-B is expressed at high levels in activated cytolytic T cells, lymphokine-activated killer (LAK), and natural killer (NK) cells. CGL-2/h-CCPX is expressed at much lower levels in activated peripheral blood lymphocytes, LAK and NK cells. To begin to define the regulatory elements that target expression of each of these genes to their specific lineages at specific times, the 5' flanking region of each gene was sequenced. The 5' flanking regions are minimally related and have few conserved consensus elements. Further experiments will be required to determine the critical cis-acting regulatory sequences required for tissue- and development-specific expression of each of these genes.