LLT1-mediated activation of IFN-gamma production in human natural killer cells involves ERK signalling pathway.

Natural killer (NK) cell functions are regulated by a delicate balance of signals received through activating and inhibitory receptors expressed on the cell surface. Lectin-like transcript-1 (LLT1), expressed on a subpopulation of NK cells and other immune cells is a ligand for the NK cell inhibitory receptor, NKR-P1A (CD161). Previous studies showed that cross-linking surface LLT1 with a monoclonal antibody stimulated NK cell IFN-gamma secretion but had no effect on cytotoxicity. Here, we have examined the signalling pathways associated with LLT1-stimulated IFN-gamma secretion. We ligated LLT1 on NK92 cells with CD161 on target cells and analysed IFN-gamma production in the presence of pharmacological inhibitors specific for various signalling mechanisms. These results indicate that LLT1 employs Src-PTK, p38 and ERK signalling pathways, but not PKC, PI3K or calcineurin. Phosphorylation studies of the signalling adaptor molecules confirmed that the ERK signalling pathway is associated with LLT1-mediated IFN-gamma production. LLT1 ligation is not associated with any change in detectable IFN-gamma mRNA levels suggesting that LLT1-stimulated IFN-gamma production in NK cells may involve post-transcriptional or translational events.

Altered expression of signalling lymphocyte activation molecule (SLAM) family receptors CS1 (CD319) and 2B4 (CD244) in patients with systemic lupus erythematosus.

CS1 (CRACC, CD319) and 2B4 (CD244), members of the signalling lymphocyte activation molecule (SLAM) family receptors, regulate various immune functions. Genes encoding SLAM family receptors are located at 1q23, implicated in systemic lupus erythematosus (SLE). In this study, we have investigated the expression and alternative splicing of CS1 and 2B4 in immune cells from SLE patients. The surface expression of CS1 and 2B4 on total peripheral blood mononuclear cells (PBMCs), T, B, natural killer (NK) cells and monocytes in 45 patients with SLE and 30 healthy individuals was analysed by flow cytometry. CS1-positive B cell population was increased significantly in SLE patients. Because CS1 is a self-ligand and homophilic interaction of CS1 induces B cell proliferation and autocrine cytokine secretion, this could account for autoreactive B cell proliferation in SLE. The proportion of NK cells and monocytes expressing 2B4 on their surface was significantly lower in patients with SLE compared to healthy controls. Our study demonstrated altered expression of splice variants of CS1 and 2B4 that mediate differential signalling in PBMC from patients with SLE.

2B4 (CD244)-mediated activation of cytotoxicity and IFN-gamma release in human NK cells involves distinct pathways.

2B4 (CD244), a member of the CD2 subset of the Ig superfamily receptors, is expressed on all human NK cells, a subpopulation of T cells, basophils and monocytes. 2B4 activates NK cell mediated cytotoxicity, induces secretion of IFN-gamma and matrix metalloproteinases, and NK cell invasiveness. Although there have been several molecules shown to interact with 2B4, the signaling mechanism of 2B4-mediated activation of NK cells is still unknown. In this study, we found cross-linking of 2B4 on YT cells, a human NK cell line, results in the increased DNA binding activity of activator protein-1 (AP-1), an important regulator of nuclear gene expression in leukocytes. We investigated the possible role of various signaling molecules that may be involved in the activation of lytic function of YT cells via 2B4. Treatment of YT cells with various specific inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN-gamma release indicating distinct pathways are involved in cytotoxicity and cytokine release. In this study we also show that 2B4 constitutively associates with the linker for activation of T cells (LAT) and that 2B4 may mediate NK cell activation via a LAT-dependent signaling pathway. These results indicate that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways.

2B4 (CD244) and CS1: novel members of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer cells and other leukocytes.

2B4 is a member of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer (NK) cells and other leukocytes. It is the high affinity ligand for CD48. Engagement of 2B4 on NK-cell surfaces with specific antibodies or CD48 can trigger cell-mediated cytotoxicity, interferon-gamma secretion, phosphoinositol turnover and NK-cell invasiveness. The function of 2B4 in CD8+ T cells and myeloid cells remains unknown. The cytoplasmic domain of 2B4 contains unique tyrosine motifs (TxYxxV/I) that associate with src homology 2 domain-containing protein or signaling lymphocyte activation molecule (SLAM)-associated protein, whose mutation is the underlying genetic defect in the X-linked lymphoproliferative disease (XLPD). Impaired signaling via 2B4 and SLAM is implicated in the immunopathogenesis of XLPD. CS1 is a novel member of the CD2 subset that contains two of the unique tyrosine motifs present in 2B4 and SLAM. Signaling through 2B4, CS1 and other members of the CD2 subset may play a major role in the regulation of NK cells and other leukocyte functions.

A prominent role for activator protein-1 in the transcription of the human 2B4 (CD244) gene in NK cells.

The cell surface glycoprotein 2B4 (CD244) of the Ig superfamily is involved in the regulation of NK and T lymphocyte functions. We have recently identified CD48 as the high affinity counterreceptor for 2B4 in both mice and humans. The cytoplasmic domain of 2B4 associates with src homology 2 domain-containing protein or signaling lymphocyte activation molecule-associated protein, whose mutation is the underlying genetic defect in the X-linked lymphoproliferative syndrome. In this study, we report the molecular cloning and characterization of the human 2B4 (h2B4) promoter. Through primer extension analysis, we found that the transcription of the h2B4 gene initiates at multiple start sites. We isolated h2B4 genomic clones and PCR amplified the 5' untranslated region containing the promoter elements. We have identified a functional AP-1 site that lies between (-106 to -100) through transient transfection analysis in YT cells, a human NK cell line. EMSAs with Abs specific for various protein factors of the AP-1 family revealed that multiple members of the Jun family are involved in the regulation of the h2B4 gene. Mutation of the AP-1 site not only abolishes protein/DNA interactions but also promoter activity. These results demonstrate a significant role for AP-1 in the transcriptional regulation of the h2B4 gene.

Regulation of IFN-gamma production following 2B4 activation in human NK cells.

IFN-gamma is a cytokine that regulates various functions of the immune system. The major producers of IFN-gamma are T cells and NK cells. 2B4 is a novel activating receptor expressed on all human NK cells, a subset of CD8+ T cells, monocytes and basophils. Activation of human NK cells through surface 2B4 enhances NK cell cytolytic function and secretion of IFN-gamma. We have examined the regulation of IFN-gamma production by the human NK cell line YT upon activation through surface 2B4. Our data indicate that ligation of surface 2B4 by mAb C1.7, that specifically recognizes 2B4, induces transcriptional activation of IFN-gamma. Partial inhibition of transcription did not prevent the transcriptional upregulation of IFN-gamma. S1 nuclease protection analysis indicated that transcriptional activation as well as mRNA stability may account for the increased production of IFN-gamma by human NK cells following 2B4 stimulation.

2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness.

2B4 is a surface molecule found on all human natural killer (NK) cells, a subset of CD8+ T cells, monocytes and basophils. It was originally identified on mouse NK cells and the subset of T cells that mediate non-major histocompatibility complex (MHC)-restricted killing. Recently,9 we have cloned the human homologue of 2B4 (h2B4) and found h2B4 to also mediate non-MHC-restricted cytotoxicity. In this study, we examine h2B4 in regulating various functions of NK cells using a human NK cell line YT, with monoclonal antibody (mAb) C1.7, an antibody that specifically recognizes h2B4. Ligation of surface 2B4 with mAb C1.7 increases YT's ability to destroy tumour cells. In the presence of mAb C1.7, the production of interferon-gamma (IFN-gamma) by YT cells is greatly enhanced. Engagement of surface 2B4 by mAb C1.7 downregulates the expression of h2B4 at the cell surface as well as the expression of h2B4 mRNA. Also, signalling through h2B4 causes the increased expression of matrix metalloproteinase-2, a member of the matrix degrading proteinase family. Thus, in addition to modulating cytolytic function and cytokine production of NK cells, activation through surface 2B4 may play a role in upregulating the machinery for degradation of extracellular matrices to promote invasion of the tumour by NK cells.

Molecular cloning of transmembrane and soluble forms of a novel rat natural killer cell receptor related to 2B4.

Natural killer (NK)-cell recognition of target cells and cytolytic function are controlled by multiple receptor-ligand interactions. These receptors can transmit either positive or negative signals and belong to the lectin superfamily or immunoglobulin superfamily (IgSF). One member of the IgSF, 2B4, is expressed on the surface of all mouse and human NK cells and the subset of T cells that mediate NK-like killing. In both mouse and human, 2B4 is a transmembrane protein and is the counter-receptor for CD48. Northern blot analysis had indicated the existence of 2B4-related genes. Here we report the cloning of novel cDNAs (r2B4R) closely related to the rat 2B4. Unlike 2B4, rat NK cells express mRNA corresponding to both transmembrane (r2B4R-tm) and soluble (r2B4R-se) forms of r2B4R. r2B4R-tm contains an open reading frame encoding a polypeptide of 311 amino acid residues. The encoded protein has characteristics of type I transmembrane proteins with a 20-amino acid leader sequence, a 203-amino acid extracellular domain, a 23-amino acid transmembrane domain, and a 65-amino acid cytoplasmic domain. r2B4R-se encodes a protein of 205 amino acid residues without a putative transmembrane domain. Northern blot analysis and reverse transcriptase-PCR analysis revealed that both transmembrane and soluble forms of r2B4R are expressed in interleukin-2-activated NK cells.

Molecular characterization of the rat NK cell receptor 2B4.

2B4 (CD244) is a cell surface glycoprotein of the immunoglobulin superfamily involved in the regulation of natural killer and T lymphocyte function. It is the high affinity counter-receptor for CD48. In mouse and human NK cells, crosslinking of 2B4 with a specific monoclonal antibody or with CD48 can trigger cell-mediated cytotoxicity, IFN-gamma secretion, phosphoinositol turnover and NK cell invasiveness. Recent reports of defective 2B4 signaling and NK cell function in X-linked lymphoproliferative syndrome suggest that this may contribute to the progression of this human disease. Here we describe the molecular characterization of the rat 2B4 gene. The cDNA encodes a protein of 395 amino acid residues that contain two Ig domains in the extracellular region and three unique tyrosine motifs (TxYxxV/I/A) in the cytoplasmic region. The predicted protein has 81 and 68% similarity with mouse 2B4 and human 2B4, respectively. Additionally, it has 94 and 89% similarity at the protein level with the recently reported rat 2B4 related genes, r2B4R-tm and r2B4R-se respectively. Northern blot analysis indicated the presence of multiple transcripts in rat LAK cells and RNK-16 cells. Immunoprecipitation and deglycosylation studies showed that rat 2B4 is glycosylated to similar extent as that of mouse and human 2B4. The cloning of r2B4 in the light of the availability of rat NK cell lines should facilitate in vitro and in vivo experiments to decipher the functional role of 2B4 in NK cell biology.

Perforin gene defects in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, rapidly fatal, autosomal recessive immune disorder characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines. Linkage analyses indicate that FHL is genetically heterogeneous and linked to 9q21.3-22, 10q21-22, or another as yet undefined locus. Sequencing of the coding regions of the perforin gene of eight unrelated 10q21-22-linked FHL patients revealed homozygous nonsense mutations in four patients and missense mutations in the other four patients. Cultured lymphocytes from patients had defective cytotoxic activity, and immunostaining revealed little or no perforin in the granules. Thus, defects in perforin are responsible for 10q21-22-linked FHL. Perforin-based effector systems are, therefore, involved not only in the lysis of abnormal cells but also in the down-regulation of cellular immune activation.

Molecular cloning and characterization of the promoter region of murine natural killer cell receptor 2B4.

Natural killer (NK) cells are bone marrow-derived lymphocytes that have the ability to kill certain tumor cells and virally infected cells. The activation of NK cells is mediated by a balance of negative and positive signals from cell-cell interactions and from responses to cytokines. However, the molecular basis of NK cell activation and recognition of target cells is poorly understood. We have previously identified, cloned and characterized a receptor, 2B4, expressed on murine NK cells. 2B4 is not only expressed on all NK cells, but also on a subset of T-cells which have NK-like killing properties. Structural analysis indicated that 2B4 belongs to the CD2 subset of immunoglobulin superfamily. We have also shown 2B4 to interact with CD48 with nine times more affinity than that of CD2-CD48 interaction. In order to understand the transcriptional regulation as well as the mechanisms controlling the restricted expression of the 2B4 gene, we obtained a genomic 2B4 clone including the sequence of the 5'-flanking region. To define the start site of transcription, we performed primer extension and 5'-RACE assays and found that the 2B4 gene may be initiated at multiple start sites and driven by a TATA-less promoter. Transient transfections of nested 5'-fragments of the 2B4 promoter to drive CAT expression revealed tissue specific expression in CTLL-2 cells, a mouse T-cell line. A promoter fragment of 348 bases upstream from the first base of the mouse 2B4 cDNA clone p2B4.8 produced maximal CAT activity in CTLL-2 cells. The presence of the region -653 to -540 on the other hand, drastically reduced transcription. Sequence analysis of this promoter region has identified potential recognition motifs for a number of lymphocyte-restricted in addition to ubiquitous transcription factors, which may play a role in the transcriptional regulation of the mouse 2B4 gene.

Cloning of a new lectin-like receptor expressed on human NK cells.

Natural killer (NK) cells constitute the third major population of lymphocytes. They possess the inherent capacity to kill various tumor and virally infected cells and mediate the rejection of bone-marrow grafts in lethally irradiated animals. A large family of NK cell receptors belong to the C-type lectin superfamily and are localized to the NK gene complex on Chromosome (Chr) 6 in the mouse and Chr 12 in the human. Genes in the NK gene complex encode type II receptors and examples include the families of NKR-P1, Ly-49, and NKG2 receptors. Examples of other C-type lectin-like NK cell receptors that occur as individual genes are CD94, CD69, and AICL. Here we report the molecular characterization and chromosomal mapping of a human lectin-like transcript (LLT1) expressed on NK, T, and B cells and localized to the NK gene complex within 100 kilobases of CD69. The cDNA encodes a predicted protein of 191 amino acid residues with a transmembrane domain near the N-terminus and an extracellular domain of 132 amino acid residues with similarity to the carbohydrate recognition domain of C-type lectins. The predicted protein of LLT1 shows 59 and 56% similarity to AICL and CD69, respectively. The predicted protein does not contain any intracellular ITIM motifs, suggesting that LLT1 may be involved in mediating activation signals.

Gene structure of the murine NK cell receptor 2B4: presence of two alternatively spliced isoforms with distinct cytoplasmic domains.

The NK cell receptor 2B4 is expressed on the surface of all murine NK cells and a subset of T cells. Ligation of 2B4 with monoclonal antibodies increases target cell lysis and IFN-gamma production. 2B4 is the high-affinity counter-receptor for CD48 in mice and humans. 2B4-L is a member of the CD2 subgroup of the immunoglobulin supergene family, which includes CD48, LFA-3, CD84, Ly9 and SLAM. Here we describe 2B4-S, a second 2B4 isoform, and the genomic structure of the 2B4 gene. 2B4-S is identical to the 5' end of 2B4-L, differing only at the 3' end, corresponding to a portion of the cytoplasmic domain and the 3' untranslated sequence. Both 2B4-L and 2B4-S are expressed on IL-2-activated NK cells. The genomic clone of 2B4 reveals that the two cDNA clones are products of alternative splicing. Since they differ only in a portion of the cytoplasmic domain, it is likely that they transduce different signals.

Molecular characterization of a novel human natural killer cell receptor homologous to mouse 2B4.

Natural killer (NK) cells spontaneously detect and kill cancerous and virally infected cells through receptors that transduce either activating or inhibiting signals. The majority of well studied NK receptors are involved in inhibitory signaling. However, we have previously described an activating receptor, 2B4, expressed on all murine NK cells and a subset of T cells that mediate non-major histocompatibility complex (MHC) restricted killing. Anti-2B4 monoclonal antibodies directed against IL-2-activated NK cells enhanced their destruction of tumor cells. Recently, we determined binding of 2B4 to CD48 with a much higher affinity than CD2 to CD48. Here we describe the molecular characterization of a cDNA clone homologous to mouse 2B4, isolated from a human NK cell library. The cDNA clone contained an open reading frame encoding a polypeptide chain of 365 amino acid residues. The predicted protein sequence showed 70% similarity to murine 2B4. Additionally, it has 48, 45, and 43% similarity to human CD84, CDw150 (SLAM), and CD48, respectively. RNA blot analysis indicates the presence of 3 kb and 5 kb transcripts in T- and NK-cell lines. A single transcript of 3 kb is identified in poly(A)+ RNA from human spleen, peripheral blood leukocytes, and lymph node, whereas, the level of expression in bone marrow and fetal liver was indeterminate. Preliminary functional data suggests that NK-cell interaction with target cells via 2B4 modulates human NK-cell cytolytic activity.

Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4.

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and gamma-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.

2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48.

2B4 is a cell surface glycoprotein related to CD2 and implicated in the regulation of natural killer and T lymphocyte function. A recombinant protein containing the extracellular region of mouse (m)2B4 attached to avidin-coated fluorescent beads bound to rodent cells, and binding was completely blocked by CD48 monoclonal antibodies (mAbs). Using surface plasmon resonance, we showed that purified soluble mCD48 bound m2B4 with a six- to ninefold higher affinity (Kd approximately 16 microM at 37 degreesC) than its other ligand, CD2. Human CD48 bound human 2B4 with a similar affinity (Kd approximately 8 microM). The finding of an additional ligand for CD48 provides an explanation for distinct functional effects observed on perturbing CD2 and CD48 with mAbs or by genetic manipulation.