1 Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan
2 Department of Molecular Genetics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
3 Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
4 PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
Correspondence to: T. Saito; E-mail: saito{at}rcai.riken.jp
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Abstract |
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Keywords: cell adhesion, expression cloning, Ig superfamily, subtraction
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Introduction |
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In addition to the NK activation receptors associated with ITAM-bearing adaptors, various other molecules are also involved in the regulation of NK cell activation. These surface molecules include 2B4 (CD244) and NTB-A that are associated with signalling lymphocyte activation molecule (SLAM)-associated protein (9), CD160-recognizing MHC class I, leukocyte function-associated antigen (LFA)-1 (10) and DNAM-1 (11, 12). Most of these surface receptors interact with the counter-receptor/ligand on the target cells. Stimulation through such adhesion molecules alone has been shown to activate NK cells. For example, an integrin LFA-1 mediates the adhesion with ICAM-1 and activates NK cells (13). Therefore, adhesion receptors also play a critical role in regulating the development and activation of NK cells. In addition, unlike T cell co-stimulation, there is no evidence for a dominant stimulatory receptor in NK cells, rather activation may instead be achieved by the summation or synergy of multiple different receptors.
In an attempt to identify new molecules regulating the activation of NK cells, we identified class I-restricted T cell-associated molecule (CRTAM) as one such molecule that is expressed on activated NK cells and CD8+ T cells. Furthermore, we cloned Nectin-like (Necl) molecule 2 as a ligand for CRTAM. As a result, we found that the heterotypic interaction between CRTAM and Necl2 induces strong cellcell adhesion, thus suggesting that CRTAMNecl2 interaction may play a role in tissue localization and/or the recruitment of activated NK cells and CTLs.
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Methods |
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cDNA subtraction
Mouse NK cells were purified as described previously (16). In brief, sIg, CD4 and CD8 splenocytes from C57BL/6 mice were stained with PE-conjugated DX5 mAb (eBioscience, San Diego, CA, USA), and anti-DX5+ cells were obtained using the MACS purification system (Miltenyi Biotech, Germany). DX5+ NK cells were cultured in the presence of 2000 U ml1 IL-2 (kindly provided by J. Hamuro, Ajinomoto Ltd, Japan) for 7 days. mRNA was extracted from purified NK cells using the mRNA Purification Kit (Amersham Bioscience, UK). cDNA was amplified with the SMART PCR cDNA Synthesis Kit (Clontech, Moutain View, CA, USA), and PCR-based cDNA subtraction was performed with the PCR-Select cDNA Subtraction Kit (Clontech).
Cell preparation
For PCR analysis freshly isolated NK cells were roughly purified as described above, stained with PE-conjugatedanti-NKR-P1C mAb (eBioscience) and then sorted by FACS Vantage (BD Bioscience, San Jose, CA, USA). NK cells were cultured as described above and used as LAK cells. sIg and CD4 splenocytes from C57BL/6 mice were stained with PE-conjugatedanti-CD8 (eBioscience) and sorted as CD8+ T cells. For CTL assay splenic CD8+ T cells were stained with MicroBeads-conjugated anti-CD8 mAb and then purified using a MACS system (Miltenyi Biotec).
Reverse transcriptionPCR analysis
Total RNA was isolated from purified fleshly isolated NK cells, LAK cells and CD8+ T cells, and semi-quantitative reverse transcription (RT)PCR was performed using the Super Script One-Step RTPCR Systems (Invitrogen, Carlsbad, CA, USA). The primers used for amplification were as follows: ß-actin, sense primer (5'-TCTACAATGAGCTGCGTGTG-3') and anti-sense primer (5'-GGTACGACCAGAGGCATACA-3'); CRTAM sense primer (5'-CGGGAGTTCTGTGAAGACGA-3') and anti-sense primer (5'-GAGGGCTTGGGAGGAGAG-3').
For real-time quantitative PCR, PCR was conducted using a SYBER green PCR kit (Qiagen) and analyzed by an iCycler (Bio-Rad Laboratories). The primers used for amplification were as follows: ß-actin, sense primer (5'-TGGAATCCTGTGGCATCCATGAAAC-3') and anti-sense primer (5'-TAAAACGCAGCTCAGTAACAGTCCG-3'); CRTAM, sense primer (5'-GTTTGCTGTTCTGGGTGCCGGTG-3') and anti-sense primer (5'-TTTCTCCCGTGCAAGCCCTCGTG-3'). The transcription level of CRTAM was normalized to the amount of ß-actin.
Establishment of anti-CRTAM mAb
Anti-mouse CRTAM mAb was established as follows (17). Ten-week-old Wistar rats were immunized with CRTAM-transfected NRK cells followed by immunization with CRTAM-Ig with Titer Max Gold (CytRx, Los Angeles, CA, USA). After the immunizations, lymph node cells were fused with the SP2/0 cell line, and clones (115 and 11E1) that stained CRTAM transfectants specifically were selected.
Retrovirus cDNA library
A retrovirus cDNA library was constructed as reported previously (16). In brief, cDNA was generated from mRNA purified from the B16 cell line using the Superscript plasmid system (Invitrogen) and it was cloned into SalI and NotI sites of the pMxs retrovirus vector (18). Ligated cDNA was transformed into ElectroMAX competent cells (Invitrogen), and plasmids were purified using a MAXI prep plasmid purification kit (Qiagen).
Expression cloning of Necl2
A total of 2 x 107 2B4 cells were infected with a retrovirus cDNA library from B16 at an efficiency of 30%. Two days after infection, the cells were stained with CRTAM-Ig and PE-conjugated goat anti-human IgG, and the stained cells were purified using the FACS Vantage. After expansion of the purified cells, the cells were stained with CRTAM-Ig plus PE-conjugated anti-human IgG and FITC-conjugated anti-Fc receptor mAb (2.4G2; BD Pharmingen, San Diego, CA, USA), and cells stained with CRTAM-Ig, but not with 2.4G2 mAb, were obtained. Single-cell clones stained by CRTAM-Ig were isolated using a Clonocyte (BD Bioscience). Library-derived genes were amplified by PCR using sense primer (5'-GGTGGACCATCCTCTAGACT-3') and anti-sense primer (5'-TTTATTTTATCGTCGATCGACC-3'). Amplified cDNA was cloned into the pMxs retrovirus vector, and the nucleic acid sequences were determined and thereafter the vector was used for generating transfectants.
Preparation of Ig fusion proteins and a FACS analysis
DNA fragments corresponding to the extracellular domains lacking the signal sequence of mouse CRTAM or Necl2 were inserted into either the XhoI site of a modified pME18S expression vector containing a mouse CD150 leader segment at the 5' terminus, and the XhoI cloning site and the Fc segment of human IgG1 at the 3' terminus. Various Ig fusion proteins including mutants were prepared using the following primers: CRTAM-Ig, sense primer (5'-AAT CTC GAG TTT CTG AAA ATG GAG ACC GTC ACG-3') and anti-sense primer (5'-AAT CTC GAG ACC ACT CTT CCT CCG GGC-3'); CRTAM first Ig, sense primer (5'-AAT CTC GAG TTT CTG AAA ATG GAG ACC GTC ACG-3') and anti-sense primer (5'-AAT CTC GAG TTT CAG CAC AAC CGA TTT CTC-3'); CRTAM second Ig, sense primer (5'-AAT CTC GAG TTG CAC TAC GGG AGT TCT GTG-3') and anti-sense primer (5'-AAT CTC GAG ACC ACT CTT CCT CCG GGC-3'); Necl2-Ig, sense primer (5'-TTT TTT TTT TGT CGA CCA GAA TCT GTT TAC TAA AGA CG-3') and anti-sense primer (5'-TTT TTT TTT TGT CGA CGT GGT CCA CTG CCC C-3'); Necl2 first Ig, sense primer (5'-TTT TTT TTT TGT CGA CCA GAA TCT GTT TAC TAA AGA CG-3') and anti-sense primer (5'-AAT CTC GAG GTT GAC TTC AAT CTC CTC CC-3'); Necl2 second + third Ig, sense primer (5'-TTT TTT TTT TGT CGA CCA GCT CTA CAC GGA CCC CC-3') and anti-sense primer (5'-TTT TTT TTT TGT CGA CGT GGT CCA CTG CCC C-3'). Ig fusion constructs were transfected to Cos 7 cells and the fusion proteins in the culture supernatant were purified. The cells were incubated with saturating concentrations of various Ig fusion proteins or mAbs for 30 min on ice, followed by incubation with F(ab')2 fragments of PE-conjugated goat anti-human IgG Fc or anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 30 min. For staining with PE-labeled secondary antibody Ig fusion protein complex, 10 µg ml1 Ig fusion proteins were mixed with 4 µg ml1 PE-conjugated goat anti-human IgG Fc (Jackson ImmunoResearch) for 30 min on ice, followed by the addition of 30 µg ml1 of normal human IgG (Cappel, Irvine, CA, USA) for 30 min. PEanti-mCD25 mAb and PEanti-mCD62L were from eBioscience. The stained cells were analyzed using a FACS CaliburTM (BD Bioscience).
Biochemical analysis of CRTAM
Transfectants and a cell line were surface biotinylated with EZ-link Sulfo-NHS-LC-Biotin (Pierce Biotechnology, Inc., Rockford, IL, USA) and lysed with 1% digitonin (SigmaAldrich, St Louis, MO, USA) lysis buffer containing 50 mM TrisHCl (pH 7.6), 150 mM NaCl, 10 µg ml1 aprotinin, 10 µg ml1 leupeptin, 1 mM phenylmethylsulfonylfluoride and 10 mM iodoacetamide (Sigma) at a concentration of 107 cells ml1. The lysate was precipitated with protein A-sepharose beads coated with Ig fusion protein. Next, the precipitated protein was loaded to 520% polyacrylamide gel (Atto Co., Japan) and subjected to SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Billerica, MA, USA). The biotinylated proteins were detected using streptavidin-peroxidase (VECSTAIN Elite ABC kit; Vector Laboratories, Burlingame, CA, USA), Super Signal West Fetmo (Pierce) and LAS1000 (Fuji film, Japan).
Chemical cross-linking was performed as previously described (19, 20). Briefly, 2B4-CRTAM (1 x 106 cells ml1) was incubated in PBS with 1 mM Bis(sulfosuccinimidyl) suberate (BS3) cross-linker (Pierce) at 14°C for 15 min. After the incubation, the reaction was stopped by the addition of 10 mM TrisHCl (pH 7.5). Thereafter, the cells were washed, suspended in SDS-PAGE sample buffer and the lysates were subjected to SDS-PAGE.
Cytotoxic assay for NK cells
NK cells were purified and cultured as described above. The cytotoxicity of various number of NK cells against 1 x 103 NIH3T3 transfectants were analyzed by 51Cr-release assays using standard techniques. A total of 1 x 105 NK cells were co-cultured with 1 x 104 YAC-1 transfectants in 96-well plates. After 24 or 48 h, culture supernatants were collected, and amounts of IFN- were measured by using mIFN-
ELISA kit (BD Pharmingen).
Cytotoxic assay for CTLs
Purified CD8+ T cells from RT-1 Tg mice were cultured with the peptide and irradiated whole spleen cells (35 Gy) from BALB/c mice for 2 or 3 days, and peptide-specific CTL was induced. The antigenic peptide P18IIIB (315329:RIQRGPGRAFVTIGK) derived from HIV-IIIB was used at 1 µM (synthesized by T. Akizawa, Setsunan University, Osaka, Japan). For the CTL assay, various numbers of effector cells were cultured for 5 h in triplicate in 96-well round-bottom plates with 5 x 103 NIH3T3 target cells pre-incubated with or without P18IIIB peptides. Cytotoxicity was analyzed by standard 51Cr-release assays and calculated as follows: (experimental release spontaneous release)/(maximum release spontaneous release) x 100.
Analysis of cell aggregation
A total of 1 x 105 2B4 transfectants labeled with 2 µg ml1 CFSE dye (Molecular Probes, Eugene, OR, USA), according to manufacturer's instructions, were mixed and cultured with an equal number of 2B4 transfectants labeled with 0.5 µM CMTR dye (Molecular Probes) in 96-well plastic plates for 3 h. After incubation, fluorescence-labeled cells were visualized with an epi-fluorescence microscope, IX8 (Olympus, Japan). In the blocking experiment with CRTAM-Ig, 1 x 105 non-labeled 2B4 transfectants were cultured in a 96-well plastic plate for 3 h in the presence or absence of purified CRTAM-Ig or Necl2-Ig (1.25 µg ml1). The adhesion assays were performed as follows: 1.5 x 105 NIH3T3 transfectants were plated on 24-well plates the day before the assay. A total of 2 x105 2B4 transfectants or CD8+ T cells stimulated with immobilized anti-TCRß mAb for 24 h were suspended and plated onto NIH3T3 transfectants. The plates were briefly centrifuged and cultured for 30 min at 37°C. Next, any floating cells were collected after gentle mixing, and counted.
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Results |
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Molecular cloning of Necl2 as a CRTAM ligand
We next intended to identify the ligand of CRTAM. For this purpose, CRTAM-Ig fusion protein (CRTAM-Ig) was generated to stain the CRTAM ligand on the cell surface. When various mouse tumor cell lines were stained with the CRTAM-Ig, B16 melanoma cells, both Hepa 16 hepatoma cells and the IC-21 macrophage cell line were positively stained with CRTAM-Ig (Fig. 3A). However, most of other tumor cell lines, including J774.1 or P388D1 macrophage cell lines, were not recognized by CRTAM-Ig (data not shown). These results indicate that some cell lines such as B16 melanoma express a putative CRTAM ligand.
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Interaction between CRTAM and Necl2
When the cloned Necl2 was transfected into NIH3T3 cells, Necl2 cells, but not mock-transfected NIH3T3 cells, were clearly stained with CRTAM-Ig (Fig. 3B, a and c). To compare the binding affinity of the CRTAMNecl2 heterotypic interaction with the Necl2 homotypic interaction, we also generated the Necl2-Ig fusion protein and stained CRTAM- and Necl2-transfected NIH3T3 cells. As predicted, CRTAM-transfectants were clearly stained with Necl2-Ig (Fig. 3B, b). In spite of the previous report that Necl2 mediates homotypic interaction (23), Necl2-transfectants were not stained with Necl2-Ig (Fig. 3B, c). However, when Necl2-Ig was pre-mixed with anti-human IgG Fc antibody to generate aggregated Ig fusion protein and then used for staining, Necl2-transfectants were significantly stained by the aggregated Necl2-Ig/anti-Ig (Fig. 3B, f). In contrast, CRTAM-transfectants were stained by neither CRTAM-Ig nor aggregated CRTAM-Ig/anti-Ig (Fig. 3B, b and e). Considering that the aggregated form of Ig fusion proteins may exhibit higher affinity to its ligand than simple dimeric Ig fusion proteins, these results suggest that the Necl2CRTAM interaction has a higher affinity than the Necl2Necl2 homotypic interaction, and that unlike Necl2, CRTAM may not exert a homotypic interaction.
We analyzed Necl2 on the cell surface of the transfectants by surface biotinylation, and pull-downed with CRTAM-Ig. Necl2-transfected 2B4 cells (2B4-Necl2) and B16 melanoma cells were lysed with 1% digitonin and pull-down assay was performed using CRTAM-Ig. As shown in Fig. 3(C), Necl2 was precipitated as a 90-kDa protein from the lysates of both 2B4-Necl2 and B16 cells but not parental 2B4.
Analysis of the domains responsible for the CRTAMNecl2 interaction
Necl2 has three Ig-like domains in the extracellular region whereas CRTAM has two as illustrated in Fig. 4(A). To determine the domains of CRTAM and Necl2 responsible for mutual binding, we constructed various Ig fusion proteins of CRTAM and Necl2 based on the structure of Ig domainsNecl2-Ig with first Ig-loop and second + third Ig-loop as well as CRTAM-Ig with a first Ig-loop and second Ig-loop. The CRTAM-transfectants were stained with Necl2-Ig with an N-terminal first Ig-loop while those with Necl2-Ig with a second + third Ig-loop of Necl2 were not stained (Fig. 4B), thus demonstrating that the first Ig domain is mainly responsible for binding. On the other hand, Necl2 transfectants were stained with CRTAM-Ig bearing first Ig-loop but not second Ig-loop (Fig. 4C). These data indicate that the N-terminal Ig domain (V-like) of both CRTAM and Necl2 are responsible for the heterotypic interaction.
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The heterotypic interaction between CRTAM and Necl2 may reflect the heterotypic interaction between CRTAM+T cells and Necl2 expressing other type of cells such as fibroblasts. Accordingly, we tested the binding of CRTAM-expressing T cells to Necl2-expressing NIH3T3 cells. First, NIH3T3 cells adhered to the plate and 2B4 T cells were added to NIH3T3 cells and the non-adherent fraction was counted (Fig. 7C). Indeed, 2B4-CRTAM specifically bound to NIH3T3-Necl2, but not to parental NIH3T3 cells. The addition of CRTAM-Ig significantly inhibited the binding of 2B4-CRTAM to 3T3-Necl2 (Fig. 7C). 2B4-Necl2 also bound to NIH3T3-Necl2, though less efficiently, in comparison to the binding of 2B4-CRTAM to NIH3T3-Necl2 (data not shown). Furthermore, we analyzed similar heterotypic cell binding using activated normal CD8+ splenic T cells. Indeed, similar to CRTAM-transfected 2B4, activated CD8+ T cells specifically bound to NIH3T3-Necl2 and the binding was significantly blocked by the addition of CRTAM-Ig (Fig. 7D), thus demonstrating that activated normal CD8+ T cells expressing endogenous CRTAM bind to Necl2-expressing cells.
Collectively, these results indicate that the heterotypic CRTAMNecl2 interaction induces cellcell adhesion/aggregation, particularly between CRTAM+T cells and Necl2+ epithelial/fibroblast cells.
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Discussion |
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As we intended to isolate the molecules specifically expressed in activated NK cells, CRTAM is one of such molecules that expressed highly restricted types of immune cellsactivated NK cells, activated CD8+ T cells and activated mast cells (our unpublished results). CRTAM is expressed very early after stimulation and transiently redundant on NK and CTLs. The transcript starts as early as 2 h, reaches the maximum at 612 and 1224 h for NK cells and CTLs, respectively, thereafter decreases and disappears at 48 h. We found that the kinetics of protein and transcript of CRTAM was parallel, thus indicating that the expression is strictly regulated on the transcriptional level in these cells. The kinetics of CRTAM expression are therefore quite different from other activation markers such as CD25, CD69 and CD62L. This unique feature of such an early and transient expression of CRTAM on NK cells and CD8+ T cells may suggest that such expression plays an important role in the early stage of cell activation. The reason why CRTAM expression is quickly down-regulated could be to prove specific cellcell adhesion through the CRTAMNecl2 interaction and to avoid any unnecessary non-specific adhesion, or alternatively, related to transient cell movement/recruitment. Although CRTAM mRNA was detected in mast cells, we failed to detect CRTAM protein in these cells, thus raising a possibility that there might be additional cell type-specific regulation of CRTAM expression.
As a CRTAM ligand, we cloned Necl molecule 2 (Necl2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1) by expression cloning using a retroviral cDNA library. Necl2 has a cell-surface molecule with three Ig domains and it belongs to the family of Nectin and Necl molecules that contribute to various types of cellcell adhesion through Ca2+-independent interaction. In contrast to the restricted tissue expression of CRTAM, Necl2 is widely expressed in mouse tissues including the brain, testis, gallbladder, liver and pancreas but not in fibroblasts or endothelial cells, [(23), and N. Arase, unpublished results]. Necl2 is involved in the homotypic and heterotypic interaction with Nectin and Necl family molecules thus inducing cellcell adhesion and the expression of Necl2 alone induces spontaneous aggregation (2325). We found that the counter-receptor, CRTAM, also functions as an adhesion molecule. Our critical findings on CRTAMNecl2 binding is 2-fold. One is that CRTAM mediates only the heterotypic interaction with Necl2 and not homotypically by itself. The other is that the heterotypic CRTAMNecl2 interaction has a higher affinity than the homotypic Necl2Necl2 interaction, thus suggesting that Necl2 preferentially binds to CRTAM and CRTAM can therefore interfere with homotypic Necl2 binding. NectinNecl family molecules bearing three Ig-like domains mediate homotypic adhesion as well as heterotypic interaction such as Necl2Nectin-3 (23). In addition, CD226/DNAM-1 and CD96 have been recently shown to bind to Necl5 and Nectin-2 (12, 26), heterotypic CRTAM-Necl2 adhesion represents heterophillic cellcell interaction between different cell types, e.g. lymphocytes and epithelial cells. Since some macrophage cell lines as well as thioglycolate-induced peritoneal macrophages (our unpublished results) express Necl2, such CRTAMNecl2 interaction might therefore be involved in T cellantigen-presenting cell (APC) interaction.
These characteristics and the expression patterns of CRTAM and Necl2 suggest that CRTAMNecl2 interaction seems to play an important role in the regulation of CTL and NK cell function. However, cytotoxicity and IFN- production by NK cells were not affected by the expression of Necl2 on target cells. Although it has been shown that engagement of an adhesion molecule such as LFA-1 is sufficient to trigger NK cell activation (10), CRTAMNecl2 interaction is not sufficient for both immediate cytokine secretion and cytotoxicity in our system. Similarly, no difference was seen in the cytotoxicity of antigen-specific CTLs against Necl2-transfected and untransfected targets. Furthermore, we found that the development of CTLs by stimulating naive CD8+ T cells with Necl2+APC was similarly induced to that with Necl2 APC (our unpublished results). Using transfected NK cell line, the transfection of mouse CRTAM into NKL cells also failed to affect the cytotoxicity against Necl2-expressing targets, although CRTAM+NKL showed intensive aggregation with Necl2 transfectants (our unpublished results). We also analyzed cytotoxicity by transiently activated NK cells on which the maximum CRTAM expression was induced upon stimulation with immobilized NKR-P1C mAb against Necl2-transfected 3T3 cell line. However, the activated CRTAM+NK cells showed no significant difference in cytotoxicity against mock- and Necl2-transfectants (our unpublished results). Collectively, CRTAM expressed on activated NK cells and CTLs do not appear to be directly involved in the cell activation and effector function in vitro. However, the condition of the in vitro culture may not reflect the in vivo situation. Cellcell contact is extensive in an in vitro culture without these adhesion molecules, which might overcome the requirement of specific cellcell binding. The requirement of CRTAMNecl2 interaction regarding the in vivo function remains to be determined using either Tg or gene-knockout mice.
Although we did not observe a functional contribution of the CRTAMNecl2 interaction on in vitro cytotoxicity by NK cells and CD8+ T cells, it might have such a function in vivo. Since Necl2 is involved in cellcell interaction and is also expressed ubiquitously (23, 27, 28), while it is also localized in basolateral plasma membrane in the case of epithelial cells, NK cells or CD8+ T cells hardly interact with such Necl2-expressing epithelial cells, even after expressing CRTAM on the cell surface upon activation. However, under invasive situations such as tumorgenesis or severe infection, where the tissue structure is disrupted and normal cellcell junction with adherent and tight junctions cannot be orderly maintained, Necl2-expressing cells, such as epithelial cells, may emerge at such sites where lymphocytes could have contact. NK cells or CD8+ T cells may migrate into such tumor or inflammatory sites and thus be activated to express CRTAM. As a result, CRTAM-expressing NK or CTLs might stay transiently within the tumor/inflammatory site and exhibit an efficient cytotoxicity against tumor/infected target cells through specific cellcell adhesion.
Necl2 is also known to be a tumor suppressor gene (TSLC1), which inactivates tumors in nude mice (22) and is frequently inactivated in many non-small cell lung cancers (22, 29). Although the mechanism of anti-tumor activity is still largely unknown, a lack of Necl2 may induce a disruption of cell polarity and adhesion, thus resulting in neoplastic growth. The cytoplasmic domain of Necl2 contains two important motifs, a protein 4.1-binding motif near the transmembrane region and a PDZ-binding domain at the C-terminus, and recruits intracellular adaptors (28, 30, 31). One of the protein 4.1 family molecule DAL-1 acts to anchor Necl2 to the actin cytoskeleton, and plays a crucial role in the tumor suppression mediated by Necl2 (32). The PDZ-domain of Necl2 binds to MPP3 and Pals2; the former functions to organize cell junction and mediates tumor suppression (33) while the latter is involved in the localization of other transmembrane protein(s) (23).
On the other hand, CRTAM has a candidate sequence of a PDZ-binding site at the C-terminus (30), and a possible serin phosphorylation site at the same position as that of DNAM-1 that is essential for LFA-1-mediated co-stimulatory signals for T cells and NK cells (34). This serine residue has also been reported to play a role in the recruitment of DNAM-1 into lipid raft in T cells. Accordingly, CRTAM might be involved in the regulation of NK cell and CTL activation.
In addition, the CRTAMNecl2 interaction could be in part related to immune surveillance. An abnormal appearance of Necl2 in disrupted tissues and its recognition by CRTAM-expressing NK cells or CTLs in tumor sites may suggest that Necl2 is a possible target of immune surveillance which thus acts as a type of danger signal. In addition to such local emergencies, considering the distribution of Necl2, especially on epithelial cells but not on endothelial cells, the CRTAMNecl2 interaction might therefore be involved in the recruitment of activated NK and CD8+ T cells to peripheral tissues under physiological conditions, which is now under investigation using CRTAM-deficient mice.
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Acknowledgements |
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Abbreviations |
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APC | antigen-presenting cell |
BS3 | Bis(sulfosuccinimidyl) suberate |
CRTAM | class I-restricted T cell-associated molecule |
ITAM | immunoreceptor tyrosine-based activation motif |
ITIM | immunoreceptor tyrosine-based inhibitory motif |
SHIP | SH2-domein-containing inositol polyphosphate 5-phosphatase |
SHP | SH2-domein-containing protein tyrosine phosphatase |
LFA | leukocyte function-associated antigen |
Necl | nectin-like |
RT | reverse transcription |
Tg | transgenic |
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Notes |
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Received 21 May 2005, accepted 20 June 2005.
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References |
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