CS1, a novel member of the CD2 family, is homophilic and regulates NK cell function
Introduction
Natural killer cell functions are regulated by a delicate balance between signaling through activating and inhibitory receptors (Lanier, 2000). The killer cell receptors belong to two major groups, the immunoglobulin superfamily (IgSF) and the lectin superfamily. Most of the NK killer cell receptors recognize MHC class I molecules and inhibit NK cell functions (Long, 1999). However, members of the CD2 family do not recognize MHC molecules, but still play a major role in lymphocyte functions. For example, CD2 and CD58 interaction is important for T-cell mediated cytotoxicity, antigen and mitogen-induced T cell proliferation, and lymphokine production (Davis et al., 1998, Tangye et al., 2000a). CD150 is expressed on T cells and B cells and regulates T cell activation and production of Ig in B cells (Cocks et al., 1995). Another member of the CD2 subset, 2B4 (CD244), is expressed on NK cells, a subpopulation of T cells and monocytes and regulate NK-cell cytolytic activity, as well as cytokine production (Boles et al., 1999, Chuang et al., 2000, Kumaresan et al., 2000b, Mathew et al., 1993a). Recently, it has been shown that 2B4 on activated/memory T cells, by interacting with CD48 provides co-stimulatory like function for neighboring T cells (Kambayashi et al., 2001). Most of the CD2 family members have two or four signaling motifs (TxYxxI/V) called immunoreceptor tyrosine-based switch motif (ITSM) in their cytoplasmic tail. The ITSM present in the cytoplasmic domain of 2B4, CD150, CD84 and Ly-9 molecules is involved in the association of small SH2-containing adapter protein 1A (SH2D1A), also called Duncan’s disease SH2-protein (DSHP) or SLAM-associated protein (SAP) and regulates signaling through these receptors (Sayos et al., 1998, Sayos et al., 2001, Tangye et al., 1999a). Mutations in SAP gene lead to X-linked lymphoproliferative syndrome (XLP), an immunodeficiency associated with dysregulated proliferation of T and B lymphocytes in the setting of primary EBV infection (Coffey et al., 1998). This suggests that SAP is essential for the signal transduction of CD2 family receptors such as SLAM, 2B4,CD84 and LY9, which are differentially expressed on NK, T, B and monocytes. The lack of function of all these receptors in SAP-deficient individuals results in a complex of NK, T and B cell responses, which leads to uncontrolled EBV infections and ultimately, to the X-linked lymphoproliferative diseases (XLPD).
Recently, we have cloned and characterized CS1, also called CRACC (CD2-like receptor activating cytotoxic cells), a novel member of the CD2 family (Boles and Mathew, 2001). CS1 is expressed on CD8+ cytotoxic T lymphocytes, activated B cells, NK cells and mature dendritic cells (Boles and Mathew, 2001, Bouchon et al., 2001). CS1 shows highest homology to CD84, SLAM and 2B4 with 47, 44 and 40% similarity, respectively (Boles and Mathew, 2001). In addition, the cytoplasmic domain of CS1 contains unique tyrosine motifs (TxYxxI/V) similar to those present in the cytoplasmic domain of 2B4, CD150, CD84 and Ly-9 (Boles et al., 2001, Cocks et al., 1995, Mathew et al., 1993a, Sayos et al., 1998, Sayos et al., 2001). It has been shown that CS1 may activate NK mediated cytotoxicity through an ERK-mediated pathway in a SAP-independent manner (Bouchon et al., 2001). The CS1 gene is located on the long arm of the human chromosome 1 and localized at 1q 23–24, in between CD48 and Ly9 (Boles and Mathew, 2001).
Several receptor–ligand interactions between members of the CD2 subset have been described. It has been suggested that the members of the CD2 family diverged by gene duplication from a common ancestor originally encoding a homophilic cell adhesion molecule (Kingsmore et al., 1995). To support this, the ligands for the members of the CD2 family are from the CD2 family itself. It can be heterophilic as seen in 2B4–CD48 interaction (Brown et al., 1998, Latchman et al., 1998) or homophilic as seen in CD150 (Mavaddat et al., 2000) and CD84 (Martin et al., 2001). The first interaction reported was between human CD2 and CD58 (Selvaraj et al., 1987) and CD2 binds weakly with CD48. However, we have shown that 2B4 is the high affinity ligand for CD48 (Brown et al., 1998). In this report, we demonstrate that CS1 is a self-ligand and that CS1–CS1 interactions regulate NK cell cytolytic activity.
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Cells and reagents
Cell lines DB (human B cell line, Jurkat (human T cell line), K562 (human erythroleukemia cell line), YT (human NK cell line) were cultured in 4+ RPMI complete medium (RPMI 1640 supplemented with 10% FBS (Hyclone, Logan, UT), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, and 10 mM non-essential amino acids). All the cells were grown at 37 °C in a humidified 5% CO2/95% air incubator. All the cell culture reagents were obtained from Life technologies (Invitrogen, Carlsbad,
Generation of monoclonal (2C7) and polyclonal antibodies against CS1
CS1 peptide designed from CS1 extracellular region was used to generate the mouse anti-CS1 mAb (clone 2C7) and anti-CS1 polyclonal serum by intrasplenic immunization. The specificity of the anti-CS1 mAb (clone 2C7) and the anti-CS1 polyclonal sera was tested against the BW cells transfected with CS1 cDNA. As seen in Fig. 1A, mAb 2C7 showed binding to BW cells expressing CS1, whereas untransfected BW cells did not show any binding. Polyclonal antisera against CS1 also showed positive staining for
Discussion
In this study, we have identified CS1 as a self-ligand and show that homophilic interaction of CS1 regulate NK cell cytolytic activity. We found that soluble CS1-Ig fusion protein containing the extracellular domain of CS1 bound to CS1-transfected cells, but not cells transfected with cDNA for other members of the CD2 family. This type of homophilic interaction is also observed in other members of the CD2 subset. For example, CD150 and CD84 display homophilic interactions resulting in the
Acknowledgements
Research supported by NIH Grant CA85753.
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