Elsevier

Molecular Immunology

Volume 42, Issue 4, February 2005, Pages 463-469
Molecular Immunology

Review
PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in tumor cell lysis

https://doi.org/10.1016/j.molimm.2004.07.028Get rights and content

Abstract

The capability of NK lymphocytes to kill tumor cells depends on different receptors/ligands interactions. In order to identify the cellular ligands recognized by “orphan” triggering receptors, mice were immunized with NK susceptible target cells. mAbs were selected that inhibited NK cytotoxicity and recognized two different molecules of 70 and 60–65 kDa. Tryptic digestion and mass spectra analysis of purified proteins identified these molecules as PVR and Nectin-2, respectively. PVR-Fc and Nectin-2-Fc chimeric molecules stained COS-7 cells expressing the DNAM-1 activating receptor and conversely, PVR and Nectin-2 CHO-K cell transfectants were stained by DNAM-1-Fc. Thus, both PVR and Nectin-2 represent specific ligands for DNAM-1. Importantly, the specific interaction between DNAM-1 (in NK cells) and PVR or Nectin-2 (in target cells) enhanced the NK-mediated lysis of tumor cells that was downregulated by mAb-mediated masking of the receptor or its ligands.

Introduction

In humans NKp46, NKp30 and NKp44 (collectively termed “Natural Cytotoxicity Receptors”, NCR) and NKG2D (Moretta et al., 2001, Cerwenka and Lanier, 2001) represent the main triggering receptors expressed by NK cells that are involved in target cell lysis. However, additional surface molecules have been implicated in this process; these include the 2B4 (CD244), NTB-A and NKp80 co-receptors and the leukocyte adhesion molecule DNAM-1 (CD226) (Shibuya et al., 1996). Although functional data would indicate that these molecules recognize ligands expressed at the target cell surface, so far only few of these have been identified. For example NKG2D recognizes the stress-inducible MICA/B and ULBPs molecules (Bauer et al., 1999, Cosman et al., 2001, Pende et al., 2002), 2B4 interacts with CD48 and NTB-A displays homophilic recognition (Falco et al., 2004). The aim of this study was to identify ligands for orphan activating receptors.

Section snippets

Monoclonal antibodies

M5A10 and M2C24 or L95 and L14 mAbs were obtained by immunizing a 5-week-old BALB/c mouse with M14 (human melanoma) or 293T (embryonic fibroblast) cell lines respectively. The following mAbs were selected in our lab: GN18 (IgG3, anti-DNAM-1), KL247 (IgM, anti-NKp46), F252 (IgM, anti-NKp30), BAT221 (IgG1, anti-NKG2D).

Polyclonal NK cells and NK cell lines

Polyclonal activated NK cell populations were obtained as previously described (Pende et al., 2002). NK92 and YT NK cell lines were kindly provided by E.O. Long (NIAID, NIH,

Isolation of mAbs against NK-susceptible target cells

In an attempt to identify novel cell surface ligands for orphan triggering NK receptors, mice were immunized with the NK-susceptible M14 (human melanoma) or 293T (human embryonic fibroblasts) cell lines. After cell fusion, mAbs were screened for their capability of inhibiting the NK cytotoxicity against the immunizing cells. Since the target cells expressed NKG2D ligands (ULBP2 on M14 while MICA, ULBP2 and ULBP3 on 293T), in order to select mAbs directed to ligands different from MICA and

Discussion

PVR (Mendelsohn et al., 1989) and Nectin-2 (Lopez et al., 1998) are closely related molecules encoded by genes located in the 19q13 chromosomal region. Alternative splicing of PVR (α and δ) and Nectin-2 (α and δ) yields to surface molecules characterized by identical extracellular portions, formed by three Ig-like domains, in the order V/C/C, and by different transmembrane and cytoplasmic regions. Importantly, both PVR and Nectin-2 are highly expressed in tumor cell lines of epithelial or

Conclusions

In this study, we have identified PVR and Nectin-2 as specific ligands for the human DNAM-1 activating receptor. Importantly, the functional interaction between DNAM-1 and its ligands participate to the process of NK-mediated recognition and killing of tumor cell targets.

Acknowledgments

This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Istituto Superiore di Sanità (I.S.S.), Ministero dell’Istruzione, dell’Università e della Ricerca (M.I.U.R), Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie, EU QLRT-2000-01495 Project “Engineering human antibody derivatives, which specifically recognize and ablate new blood vessels, for the therapy of angiogenesis-related pathologies”, INSERM, the Ligue Nationale

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