Ligation Of 2B4 Research Articles

Targeted Disruption of the2B4Gene in Mice Reveals an In Vivo Role of 2B4 (CD244) in the Rejection of B16 Melanoma Cells

Murine 2B4 (CD244) is a cell surface receptor expressed on all NK cells, gammadelta-T cells, a subset of CD8(+) T cells, and all CD14(+) monocytes. 2B4 binds to CD48 with high affinity, and cross-linking 2B4 with anti-2B4 Ab in vitro causes activation of NK cells. To study its physiological role, we have generated, by gene targeting, mice deficient in the expression of this cell surface molecule. The expression of lymphoid cell surface markers on PBMC and splenocytes of mice homozygous for the mutation in 2B4 (2B4(-/-)) is identical to that in wild-type mice. However, thymocytes from female 2B4(-/-) mice, but not male 2B4(-/-) mice, have an increase in the immature CD4(-)/CD8(-) population. To investigate the in vivo role of 2B4, wild-type and 2B4(-/-) mice were injected with CD48(+) and CD48(-) metastatic B16 melanoma cells. Wild-type mice rejected CD48(+) melanoma poorly compared with CD48(-) tumor cells, suggesting that ligation of 2B4 by CD48 on melanoma cells is inhibitory. In keeping with this, male 2B4(-/-) mice showed enhanced ability to reject CD48(+) melanoma cells. However, female 2B4(-/-) mice poorly rejected both CD48(+) and CD48(-) melanoma cells, revealing a gender-specific and CD48-independent defect in mice lacking 2B4. In vitro and in vivo experiments reveal a complex role of NK cells in gender specificity.

Cutting edge: the NK cell receptor 2B4 augments antigen-specific T cell cytotoxicity through CD48 ligation on neighboring T cells.

2B4 is expressed on all NK and a subset of memory/effector CD8(+) T cells. 2B4 binds to CD48 and activates NK cytotoxicity, but its function on CD8(+) T cells is not clear. Furthermore, two isoforms of 2B4 (2B4S and 2B4L) exist in mice but the role of individual isoforms is not known. To address these questions, we generated primary T cell cultures from L(d)-specific 2C/Rag2(-/-) TCR transgenic mice and transduced them with 2B4S or 2B4L. 2B4S- or 2B4L-transduced T cells showed greater cytotoxicity over control cells against CD48(+) and CD48(-) targets, suggesting that ligation of 2B4 by CD48 on target cells was not necessary for 2B4 function. Rather, 2B4/CD48 interaction on adjacent T cells appeared to be critical for cytotoxicity. Therefore, 2B4 functions as a costimulator of CD8(+) T cells in MHC-restricted cytotoxicity. We conclude that 2B4/CD48 interactions among T cells themselves can augment CTL lysis of their specific targets.

NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244).

2B4 is an NK cell activation receptor that can provide a co-stimulatory signal to other activation receptors and whose mode of signal transduction is still unknown. We show that cross-linking of 2B4 on NK cells results in its rapid tyrosine phosphorylation, implying that this initial step in 2B4 signaling does not require coligation of other receptors. Ligation of 2B4 in the context of an NK cell-target cell interaction leads to 2B4 tyrosine phosphorylation, target cell lysis, and IFN-gamma release. Coligation of 2B4 with the inhibitory receptors killer cell Ig-like receptor (KIR)2DL1 or CD94/NKG2 completely blocks NK cell activation. The rapid tyrosine phosphorylation of 2B4 observed upon contact of NK cells with sensitive target cells is abrogated when KIR2DL1 or CD94/NKG2 are engaged by their cognate MHC class I ligand on resistant target cells. These results demonstrate that NK inhibitory receptors can interfere with a step as proximal as phosphorylation of an activation receptor.

Functional requirement for SAP in 2B4-mediated activation of human natural killer cells as revealed by the X-linked lymphoproliferative syndrome.

X-linked lymphoproliferative syndrome (XLP) is an immunodeficiency characterized by life-threatening infectious mononucleosis and EBV-induced B cell lymphoma. The gene mutated in XLP encodes SLAM (signaling lymphocytic activation molecule-associated protein)-associated protein (SAP), a small SH2 domain-containing protein. SAP associates with 2B4 and SLAM, activating receptors expressed by NK and T cells, and prevents recruitment of SH2 domain-containing protein tyrosine phosphatase-2 SHP-2) to the cytoplasmic domains of these receptors. The phenotype of XLP may therefore result from perturbed signaling through SAP-associating receptors. We have addressed the functional consequence of SAP deficiency on 2B4-mediated NK cell activation. Ligating 2B4 on normal human NK cells with anti-2B4 mAb or interaction with transfectants bearing the 2B4 ligand CD48 induced NK cell cytotoxicity. In contrast, ligation of 2B4 on NK cells from a SAP-deficient XLP patient failed to initiate cytotoxicity. Despite this, CD2 or CD16-induced cytotoxicity of SAP-deficient NK cells was similar to that of normal NK cells. Thus, selective impairment of 2B4-mediated NK cell activation may contribute to the immunopathology of XLP.

2B4-mediated activation of human natural killer cells

2B4 is a member of the CD2 subset of the immunoglobulin superfamily of cell surface receptors. Other members of this family include CD2, CD48, CD58, CD84, signaling lymphocytic activation molecule and Ly-9. Some of these molecules are activating structures expressed by natural killer cells and T cells. We have recently cloned and characterised the human homologue of 2B4 and found that the cytoplasmic domain of 2B4 can interact with SAP, a signaling adaptor protein that is mutated in the immunodeficiency X-linked lymphoproliferative disease (XLP). Additionally, the natural ligand of 2B4 has been identified as CD48. These findings have facilitated the investigation of the functional role of this receptor–ligand pair, and associated signal transduction pathways, on immune cells. In this study, it was found that the interaction between 2B4 on effector cells and CD48 on target cells induced NK-cell activation, as evidenced by increased cytotoxicity and secretion of IFN-γ. The responses induced by ligation of 2B4 could be reduced by the co-ligation of inhibitory receptors expressed by NK cells, demonstrating that activation signals delivered via 2B4 can be regulated by the action of certain inhibitory receptors. Because the signalling pathway of 2B4 involves SAP, it is possible that 2B4-mediated NK-cell activation may be compromised in patients with XLP due to mutations in SAP. This may contribute to the phenotype and progression of this disease.

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-γ 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.

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.

Activating interactions in human NK cell recognition: the role of 2B4-CD48.

2B4 is a cell surface glycoprotein of the immunoglobulin superfamily structurally related to CD2-like molecules. It was originally identified in the mouse as a receptor that mediates non-MHC-restricted cytotoxicity by NK cells and CD8+ T cells. Recently, 2B4 was shown to bind CD48 by molecular binding assays and surface plasmon resonance. Here, we have investigated the cell surface expression, biochemical characteristics and function of human 2B4. Our results show that 2B4 is expressed not only on NK cells and CD8+ T cells, but also on monocytes and basophils, indicating a broader role for 2B4 in leukocyte activation. In NK cells, engagement of 2B4 with a specific monoclonal antibody or with CD48 can trigger NK cell-mediated cytotoxicity. The contribution of 2B4-CD48 interaction to target cell lysis by different NK cell clones varies, probably dependent on the relative contribution of other receptor-ligand interactions. In T cells and monocytes, ligation of 2B4 does not lead to T cell or monocyte activation. Thus, it appears that the primary function of 2B4 is to modulate other receptor-ligand interactions to enhance leukocyte activation.