IMMUNOLOGY
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`JOURNAL
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`THE
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`OF
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`CUTTING EDGE
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`Cutting Edge: CD96 (Tactile) Promotes NK Cell-Target
`Cell Adhesion by Interacting with the Poliovirus
`Receptor (CD155)
`Anja Fuchs, Marina Cella, Emanuele Giurisato, Andrey S. Shaw, and Marco Colonna1
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`activating receptor DNAM-1, also called CD226 (6, 7).
`DNAM-1 is a member of the Ig superfamily that stimulates NK
`cells by recruiting the protein tyrosine kinase Fyn (6, 7). In leu-
`lack ␤
`2 integrin,
`kocyte adhesion-deficient patients
`that
`DNAM-1 does not deliver a stimulatory signal, indicating that
`DNAM-1 is physically and functionally coupled with ␤
`2 inte-
`grin (8). It has been recently shown that DNAM-1 recognizes
`the poliovirus receptor (PVR,2 or CD155) and the poliovirus-
`related receptor 2 (PRR2 or CD112) on target cells (9). PVR
`and PRR2 belong to a large family of Ig-like molecules called
`nectins and nectin-like proteins, which mediate cell-cell adhe-
`sion, cell migration, and cell polarization by homotypic contact
`or heterotypic interaction with other nectins (10). In addition,
`nectins and nectin-like proteins serve as entry receptors for po-
`liovirus and HSVs (11). To date, it is unknown whether the
`multiplicity of nectins and nectin-like proteins is matched by a
`comparably diverse assortment of activating receptors and ad-
`hesion molecules on NK cells.
`In this study, we show that CD96 or T cell-activated in-
`creased late expression (Tactile) (12) is another NK cell receptor
`for PVR. CD96 promotes NK cell adhesion to target cells ex-
`pressing PVR, stimulates cytotoxicity of activated NK cells, and
`mediates acquisition of PVR from target cells. These results in-
`dicate that NK cells have evolved a dual receptor system to rec-
`ognize nectins and nectin-like molecules on target cells, which
`mediates NK cell adhesion and triggering of NK cell effector
`functions.
`
`Materials and Methods
`Cells and transfectants
`
`NK92 cells were kindly provided by M. L. Botet (University Pompeu-Fabre,
`Barcelona, Spain). Human NK cells were obtained from CD56⫹CD3⫺ PBMC
`as described (13). CD96, DNAM-1, and PVR full-length cDNAs were ampli-
`fied by RT-PCR, cloned into pEF6/V5-His A (Invitrogen, Carlsbad, CA), and
`transfected into P815 murine mastocytoma cells (P815-CD96 and P815-
`DNAM-1), human Jurkat T cells (Jurkat-PVR), or human Daudi B cells
`(Daudi-PVR).
`
`2 Abbreviations used in this paper: PVR, poliovirus receptor; PRR, poliovirus-related re-
`ceptor; Tactile, T cell-activated increased late expression; ED, ectodomain; GFP, green
`fluorescent protein; CEACAM, carcinoembryonic Ag-related cell adhesion molecule;
`ITIM, immunoreceptor tyrosine-based motif.
`
`The poliovirus receptor (PVR) belongs to a large family of
`Ig molecules called nectins and nectin-like proteins, which
`mediate cell-cell adhesion, cell migration, and serve as en-
`try receptors for viruses. It has been recently shown that
`human NK cells recognize PVR through the receptor
`DNAM-1, which triggers NK cell stimulation in associa-
`tion with ␤
`2 integrin. In this study, we show that NK cells
`recognize PVR through an additional receptor, CD96, or
`T cell-activated increased late expression (Tactile). CD96
`promotes NK cell adhesion to target cells expressing PVR,
`stimulates cytotoxicity of activated NK cells, and mediates
`acquisition of PVR from target cells. Thus, NK cells have
`evolved a dual receptor system that recognizes nectins and
`nectin-like molecules on target cells and mediates NK cell
`adhesion and triggering of effector functions. As PVR is
`highly expressed in certain tumors, this receptor system
`may be critical for NK cell recognition of tumors. The
`Journal of Immunology, 2004, 172: 3994 –3998.
`
`N atural killer cells recognize virally infected and tumor
`
`cells using multiple receptors with diverse structures,
`specificities, and signaling properties (1, 2). These re-
`ceptors activate cytoplasmic protein tyrosine kinases, phospho-
`inositol kinases, and mitogen-activated protein kinases, which
`trigger NK cell secretion of cytotoxic granules and IFN-␥ (3).
`NK cell functions are also critically dependent on cell surface
`molecules that mediate adhesion of NK cells to other cells (4).
`Typically, these adhesion molecules include ␤
`
`2 and ␤1 inte-
`grins, which interact with ICAM-1 and -2 and VCAM, respec-
`tively. By promoting NK cell-target cell adhesion, integrins al-
`low triggering of activating NK cell receptors by their cognate
`ligands. In turn, some activating receptors further strengthen
`the NK cell adhesion mediated by integrins (5).
`The close cooperation between activating receptors and ad-
`hesion molecules in stimulating NK cells is exemplified by the
`
`Department of Pathology and Immunology, Washington University School of Medicine,
`St. Louis, MO 63110
`
`Received for publication December 16, 2003. Accepted for publication February 3, 2004.
`
`The costs of publication of this article were defrayed in part by the payment of page charges.
`This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
`Section 1734 solely to indicate this fact.
`1 Address correspondence and reprint requests to Dr. Marco Colonna, Department of Pa-
`thology and Immunology, Washington University School of Medicine, 660 South Euclid,
`St. Louis, MO 63110. E-mail address: mcolonna@pathology.wustl.edu
`
`Copyright © 2004 by The American Association of Immunologists, Inc.
`
`0022-1767/04/$02.00
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`FIGURE 1. NK92 does not express DNAM-1 but binds PVR-ED-IgG. A,
`Staining of NK92 with the anti-DNAM-1 mAb 11A8 (gray profile) coincides
`with background staining (solid line). B, The soluble PVR ED fused with hu-
`man IgG Fc (PVR-ED-IgG) binds NK92 (gray profile). Binding is inhibited by
`the anti-PVR mAb SKII.4 (thick solid line) but not by the anti-DNAM-1 mAb
`11A8 (dashed line that overlaps with the gray profile). Background staining of
`NK92 is indicated (thin solid line).
`
`To identify the receptor for PVR expressed on NK92 we im-
`munized mice with NK92, produced anti-NK92 mAbs and,
`among these, selected the mAb NK92.39, which specifically
`blocked the interaction between PVR-ED-IgG and NK92 (Fig.
`2A). Flow cytometric analysis revealed that the NK92.39 Ag is
`
`FIGURE 2. mAb NK92.39 blocks binding of PVR-ED-IgG to NK92 and
`recognizes CD96. A, mAb NK92.39 blocks binding of PVR-ED-IgG to NK92
`cells as the anti-PVR mAb does. B, Cell surface expression of NK92.39 Ag on
`PBMC. We analyzed lymphocytes, monocytes, and granulocytes using separate
`forward scatter (FSC)/side scatter gates with the exception of natural IFN-pro-
`ducing cells. CD2⫹ lymphocytes, CD56⫹ NK cells, CD4⫹ and CD8⫹ T cells
`and a few CD20⫹ B cells express the NK92.39 Ag. CD14⫹ monocytes,
`CD16⫹ granulocytes, and BDCA2⫹ IFN-producing cells lack the NK92.39
`Ag. C, mAb NK92.39 stains P815-cell transiently transfected with CD96
`cDNA but not untransfected control cells. Transfected and untransfected cells
`stained with a control Ab fell into the lower right quadrant.
`
`The Journal of Immunology
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`PVR-ED-IgG and PVR-D1-IgG
`
`The PVR ectodomain (PVR-ED) and membrane distal Ig domain (PVR-D1)
`were amplified from PVR cDNA by PCR using a common forward primer
`(5⬘-ATGGCCCGAGCCATGGCCGCCGCGTGG-3⬘) and specific reverse
`primers (PVR-ED: 5⬘-GTTACGGGACATGCCTGAGTGC-3⬘, PVR-D1:
`5⬘-CTTGGCAAGCACTCGGAGCCA-3⬘). Amplified products were cloned
`into pHuIgG1 and expressed as human IgG fusion proteins as previously de-
`scribed (14). Binding of PVR-ED-IgG and PVR-D1-IgG (200 ␮g/ml) to cells
`was detected by flow cytometry using a biotinylated goat anti-human IgG-Fc
`followed by streptavidin-allophycocyanin (Molecular Probes, Eugene, OR).
`
`Antibodies
`
`We obtained mAbs against PVR (clone SKII.4, mouse IgG1), DNAM-1 (clone
`11A8, mouse IgG1), and CD96 (clone NK92.39, mouse IgG1) by immunizing
`mice with SK-N-S1 human neuroblastoma cells (American Type Culture Col-
`lection, Manassas, VA), human NK cell lines, and NK92 cells, respectively. We
`selected hybridomas that blocked NK cell-mediated lysis of various targets
`(SKII.4 and 11A8) or binding of PVR to NK92 (NK92.39). Abs against CD56,
`CD2, CD4, CD8, CD20, CD14, CD16, and BDCA-2 were mouse IgG2a
`(Beckman Coulter/Immunotech, Brea, CA and Miltenyi Biotec, Auburn, CA).
`Primary Abs were detected with FITC or PE-labeled goat anti-mouse IgG1 or
`IgG2a (Southern Biotechnology Associates, Birmingham, AL).
`
`Cell conjugations
`
`P815, P815-CD96, and P815-DNAM-1 were labeled with CFSE (Molecular
`Probes). Jurkat and Jurkat-PVR were stained with anti-CD45-allophycocyanin
`(Beckman Coulter/Immunotech). Labeled P815 cells (2 ⫻ 105) were mixed
`with 2 ⫻ 105 Jurkat cells, spun down, and incubated at 37°C for 1 h. Conju-
`gates were gently resuspended in a small volume of medium for flow cytometric
`analysis on FACSCalibur (BD Biosciences, San Jose, CA).
`
`Down-regulation of CD96 and acquisition of PVR by NK92
`NK92 cells (105) were mixed with Daudi-PVR or Daudi (5 ⫻ 104), spun down
`in a 96-well round-bottom plate and incubated for 2 h either at 37°C or at 0°C.
`Cell conjugates were dissociated by repeated pipetting, cells were stained on ice
`with anti-CD96 or anti-PVR mAbs followed by goat anti-mouse IgG1-PE
`(Southern Biotechnology Associates) and analyzed by flow cytometry. Addi-
`tional staining with anti-HLA-DR-FITC (mouse IgG2a; BD Biosciences) was
`performed to distinguish Daudi cells from NK92 in the conjugates.
`
`Confocal microscopy
`
`To visualize NK cells, we transfected NK92 with a cDNA encoding the adapter
`DAP12 (3) cloned into the retrovirus pMX-IRES-green fluorescent protein
`(GFP) (15). NK92 cells expressing the DAP12-GFP bicistronic transcript were
`conjugated to Daudi or Daudi-PVR cells at 1:1 to 1:10 ratios, briefly spun
`down, and incubated for 5–30 min at 37°C. After conjugation, cells were gently
`resuspended, placed onto poly-L-lysine-coated glass slides for 1 h atroom tem-
`perature, stained with the anti-PVR mAb SKII.4, followed by Cy3-conjugated
`goat anti-mouse IgG1 (Jackson ImmunoResearch Laboratories, West Grove,
`PA). Cell conjugates were visualized using a Zeiss LSM 510 laser-scanning con-
`focal microscope (Oberkochen, Germany). We examined ⱖ30 conjugated
`NK92 cells per slide.
`
`Results
`CD96 is a receptor for PVR
`It was recently shown that human NK cells recognize PVR on
`target cells through DNAM-1 (9). To further investigate NK
`cell recognition of PVR, we tested a variety of human NK cell
`lines for expression of DNAM-1 and their capacity to specifi-
`cally bind the ectodomain of PVR expressed as an IgG fusion
`protein (PVR-ED-IgG). Remarkably, the NK cell line NK92
`did not express DNAM-1 (Fig. 1A), yet strongly bound PVR-
`ED-IgG (Fig. 1B). This result suggested that NK92 expresses
`an as yet unknown receptor for PVR. Among potential PVRs,
`CD96 (also called Tactile) (12) and carcinoembryonic Ag-re-
`lated cell adhesion molecule (CEACAM) (16) were particularly
`attractive candidates in view of their similarity to DNAM-1.
`Nectin and nectin-like family members, which include PVR,
`were also plausible receptors for PVR as they mediate adhesion
`by heterotypic interactions with other nectins (10).
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`3996
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`CUTTING EDGE: ADHESION MOLECULE CD96 BINDS THE PVR
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`FIGURE 4. Conjugate formation between transfectants expressing CD96
`and PVR. P815-CD96 transfectants (top panels) and P815-DNAM-1 transfec-
`tants (middle panels) make conjugates with Jurkat-PVR but not with untrans-
`fected Jurkat. Percentage of conjugates are indicated in the upper right quad-
`rants. P815 did not make conjugates with either Jurkat-PVR or untransfected
`Jurkat (bottom panels).
`
`NK92 was selectively reduced after conjugation at 37°C but not
`at 0°C (Fig. 5A), indicating that CD96 is down-regulated upon
`interaction with PVR, possibly by an active process of internal-
`ization. Moreover, conjugation of NK92 with Daudi-PVR re-
`sulted in acquisition of PVR by NK92 (Fig. 5B). Interestingly,
`acquisition of PVR by NK92 was detected after 2 h ofconju-
`gation at 0°C and was markedly increased at 37°C (Fig. 5B).
`Thus, NK cells may acquire PVR in part through an active pro-
`cess of transport as well as by detouching it from transfectants
`when the cells are pulled apart, due to the high affinity of CD96
`for PVR. Confocal microscopy of conjugates between NK92
`and Daudi-PVR revealed PVR clusters not only at the site of
`cell-cell contact but also in NK92 cells at sites distal to the con-
`tact site, corroborating PVR acquisition by NK92 after CD96-
`PVR interaction (Fig. 5, C and D). We conclude that CD96-
`PVR-mediated adhesion results in exchange of membrane
`molecules between NK cells and cells expressing PVR, which
`may also involve internalization of CD96-PVR clusters.
`
`CD96 triggers cytotoxicity of activated NK cells
`Because DNAM-1 transduces activating signals upon engaging
`PVR (6, 9), CD96 may also function as an activating receptor
`for PVR. However, the CD96 cytoplasmic domain contains ty-
`rosine-based motifs that resemble immunoreceptor tyrosine-
`based motifs (ITIM), which may in fact trigger inhibitory sig-
`nals (3). To investigate CD96 signaling properties, we tested
`the cytotoxicity of human polyclonal NK cell lines and NK92
`against P815 cells in the presence of Abs that bind the FcR on
`P815 and engage CD96, DNAM-1, or the activating receptors
`NKp44 and NKp30 on NK cells. Engagement of CD96 in-
`creased the lysis of P815 by human polyclonal NK cell lines,
`although not as efficiently as did engagement of DNAM-1,
`NKp30, and NKp44 (Fig. 6A). In addition, coengagement of
`CD96 and NKp30 did not reduce the lysis of P815 triggered by
`NKp30 alone, demonstrating that CD96 does not transduce
`inhibitory signals (Fig. 6B). However, CD96 did not stimulate
`cytotoxicity of NK92 cells, suggesting that CD96-mediated
`
`expressed on all peripheral blood NK cells as well as all CD4⫹
`and CD8⫹ T cells and a few B cells. In contrast, mAb NK92.39
`did not stain monocytes, granulocytes, IFN-producing cells
`(Fig. 2B) or epithelial cell lines (data not shown). This cellular
`distribution was more consistent with that reported for CD96
`(12) than CEACAM or other nectins. Staining of P815 cells
`transiently expressing CD96 cDNA with mAb NK92.39 con-
`firmed that the NK92.39 Ag is CD96 (Fig. 2C).
`To corroborate that CD96 is a receptor for PVR, we tested
`binding of P815 cells stably transfected with CD96 cDNA
`(P815-CD96) with PVR-ED-IgG and a PVR-IgG fusion pro-
`tein that included only the membrane-distal Ig domain of PVR
`(PVR-D1-IgG). PVR-ED-IgG and PVR-D1-IgG bound
`P815-CD96 equally well and the binding was inhibited by
`NK92.39 and anti-PVR Abs (Fig. 3). In addition, both PVR-
`ED-IgG and PVR-D1-IgG specifically bound P815 cells ex-
`pressing DNAM-1 (Fig. 3). We conclude that CD96 specifi-
`cally recognizes PVR and that the first Ig domain of PVR is
`sufficient to mediate interaction with CD96 and DNAM-1.
`
`CD96-PVR interaction mediates cell-cell adhesion
`To determine whether CD96-PVR interactions mediate cell-
`cell adhesion, we mixed P815-CD96 cells with Jurkat T cells
`stably transfected with PVR cDNA (Jurkat-PVR) or control Ju-
`rkat. After a 30-min incubation at 0°C or 37°C, we measured
`formation of conjugates by two-color flow cytometry. Under
`both conditions, P815-CD96 made abundant conjugates with
`PVR-Jurkat but not with Jurkat (Fig. 4). Conjugate formation
`was either entirely or partially blocked by anti-PVR and anti-
`CD96 Abs, confirming the specificity of the interaction (data
`not shown). The conjugation frequency of CD96-PVR was
`similar to that obtained with P815-DNAM-1 transfectants and
`Jurkat-PVR (Fig. 4). No significant conjugation of untrans-
`fected cells was observed (Fig. 4).
`We also analyzed adhesion of NK92 with PVR transfectants,
`particularly with those made in the human B cell Daudi, as
`Daudi can be easily distinguished from NK92 within conju-
`gates by its characteristic expression of MHC class II. As ex-
`pected, NK92 cells formed abundant conjugates with Daudi-
`PVR (data not shown). Interestingly, expression of CD96 on
`
`FIGURE 3. CD96 binds PVR and the membrane distal PVR Ig domain is
`sufficient for binding. Gray profiles represent binding of PVR-ED-IgG (left
`panels) or PVR-D1-IgG (right panels) to P815-CD96 (top panels), P815-
`DNAM-1 (middle panels) and P815 (lower panels). Binding was performed in
`the presence of mAbs against PVR (dashed lines), CD96 (dotted lines),
`DNAM-1 (thick solid lines). Thin solid lines indicate background staining of
`transfected and untransfected P815.
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`FIGURE 5. Down-regulation of CD96 and acquisition of PVR by NK92
`during conjugation with PVR transfectants. A, Expression of CD96 on NK92
`after 2 h ofconjugation with Daudi-PVR (thick solid line), Daudi (thin solid
`line), or no conjugation (gray profile overlapping with thin solid line) at 0°C
`and 37°C. Cells stained with a control Ab fell within the marker. B, PVR ex-
`pression on NK92 cells after conjugation with Daudi-PVR (thick solid line),
`Daudi (thin solid line), or no cells (gray profile overlapping with thin solid line).
`C, NK92 cells expressing the DAP12-GFP bicistronic transcript (green) form
`conjugates with Daudi-PVR (red). PVR (red) appears not only on the cell sur-
`face of Daudi-PVR but also on the surface of NK92, especially at the site of
`cell-cell contact. D, PVR (red) acquired by NK92 (green) from PVR-Daudi
`(red) is clearly detectable at sites distal to the NK92-Daudi-PVR contact site.
`
`stimulation may require expression and/or functional activa-
`tion of additional molecules that are present in freshly estab-
`lished NK cell lines but not in NK92 (Fig. 6C).
`
`Discussion
`Our study presents manifold evidence that CD96 is an NK cell
`receptor for PVR: 1) our anti-CD96 mAb blocks binding of
`soluble PVR to NK92; 2) PVR-IgG specifically binds CD96
`transfectants; 3) CD96 transfectants effectively conjugate with
`PVR transfectants; 4) NK cells down-regulate CD96 after con-
`jugation with PVR transfectants. CD96 is a member of the Ig
`superfamily, with an ectodomain that includes three Ig do-
`mains and a long serine/threonine/proline-rich region typical of
`an O-glycosylated domain (12). Our study shows that the
`membrane distal Ig domain of PVR is sufficient for binding to
`CD96, as well as to DNAM-1, the other Ig superfamily mem-
`
`FIGURE 6. CD96 stimulates lytic activity of freshly established human NK
`cell lines. A, Lysis of P815 by a human NK cell line is augmented in the presence
`of Abs against CD96, DNAM-1, NKp30, NKp44 as compared with a control
`Ab. B, When the anti-CD96 and anti-NKp30 mAbs are used together, lysis of
`P815 is not reduced as compared with that induced by the anti-NKp30 mAb
`alone. C, Anti-CD96 mAb does not activate lysis by NK92, as compared with
`anti-NKp44 mAb. As expected, anti-DNAM-1 Ab has no effect because NK92
`do not express DNAM-1.
`
`ber that has been shown to recognize PVR (9). Interestingly,
`CD96, DNAM-1, and PVR share significant sequence identity
`among themselves and with other members of the nectin and
`nectin-like families. Thus, this family of receptors is reminis-
`cent of the CD2 family, which includes multiple receptors that
`mediate NK cell adhesion and activation by homotypic inter-
`actions and/or by heterotypic interactions with other CD2 fam-
`ily members (17).
`Our results indicate that the predominant function of CD96
`is to mediate adhesion of NK cells to other cells expressing PVR.
`The strong adhesion between CD96 and PVR promoted the
`exchange of cell surface molecules between NK cells and target
`cells, in particular, the acquisition of PVR by NK cells as well as
`possible internalization of CD96 bound to PVR. A similar phe-
`nomenon has been observed after interaction of MHC on APCs
`with the TCR on T cells (18) and the inhibitory receptors on
`NK cells (19). It has been suggested that transfer of MHC to
`Ag-specific T cells may stimulate neighboring T cells to kill
`those expressing captured MHC/Ag peptide, exhausting T cell
`responses (18). Similarly, the transfer of PVR from target cells
`to NK cells may make them susceptible to “fratricide”. Because
`some tumors express high levels of PVR (20), transfer of PVR
`from tumors to NK cells via CD96 may elicit NK cell fratricide,
`providing tumors with a mechanism of immunoevasion.
`We observed that CD96 can also promote NK cell activation,
`although less efficiently than DNAM-1 and other activating
`NK cell receptors. As NK cell surface expression of CD96 and
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`3998
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`CUTTING EDGE: ADHESION MOLECULE CD96 BINDS THE PVR
`
`DNAM-1 is similar (data not shown), they may trigger path-
`ways with different activating capability. Notably, CD96 stim-
`ulated freshly activated NK cells, but not NK92, suggesting that
`the stimulatory function of CD96 may require expression and
`functional cooperation of other molecules that are absent in
`NK92, just as DNAM-1 requires ␤
`2 integrin to trigger NK cells
`(8). Despite the presence of cytoplasmic ITIM-like motifs,
`CD96 did not initiate inhibitory signals. In fact, these motifs
`may mediate CD96 down-regulation by promoting receptor
`internalization. Alternatively, CD96 may be down-regulated by
`cell surface shedding or other mechanisms.
`In conclusion, our study reveals that NK cells express a dual
`receptor system that recognizes nectins and nectin-like mole-
`cules on target cells. During cell-cell contact, CD96, DNAM-1,
`and their ligands may accumulate at the cell-cell contact site,
`leading to the formation of a mature immunological synapse
`between NK cells and target cells. This may not only trigger
`adhesion and secretion of lytic granules and IFN-␥, but may
`also promote NK cell-target cell molecular exchanges, which
`could subsequently lead to resolution of NK cell responses.
`
`Acknowledgments
`We thank Susan Gilfillan for helpful comments and Bill Eades and
`Jackie Hughes (Alvin J. Siteman Cancer Center, Washington University School
`of Medicine) for expert cell sorting.
`
`References
`1. Cerwenka, A., and L. L. Lanier. 2001. Ligands for natural killer cell receptors: redun-
`dancy or specificity. Immunol. Rev. 181:158.
`2. Diefenbach, A., and D. H. Raulet. 2001. Strategies for target cell recognition by nat-
`ural killer cells. Immunol. Rev. 181:170.
`3. McVicar, D. W., and D. N. Burshtyn. 2001. Intracellular signaling by the killer im-
`munoglobulin-like receptors and Ly49. Sci. STKE 2001:RE1.
`4. Helander, T. S., and T. Timonen. 1998. Adhesion in NK cell function. Curr. Top.
`Microbiol. Immunol. 230:89.
`
`5. Barber, D. F., and E. O. Long. 2003. Coexpression of CD58 or CD48 with intercel-
`lular adhesion molecule 1 on target cells enhances adhesion of resting NK cells. J. Im-
`munol. 170:294.
`6. Shibuya, A., D. Campbell, C. Hannum, H. Yssel, K. Franz-Bacon, T. McClanahan,
`T. Kitamura, J. Nicholl, G. R. Sutherland, L. L. Lanier, and J. H. Phillips. 1996.
`DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lympho-
`cytes. Immunity 4:573.
`7. Shibuya, A., L. L. Lanier, and J. H. Phillips. 1998. Protein kinase C is involved in the
`regulation of both signaling and adhesion mediated by DNAX accessory molecule-1
`receptor. J. Immunol. 161:1671.
`8. Shibuya, K., L. L. Lanier, J. H. Phillips, H. D. Ochs, K. Shimizu, E. Nakayama,
`H. Nakauchi, and A. Shibuya. 1999. Physical and functional association of LFA-1
`with DNAM-1 adhesion molecule. Immunity 11:615.
`9. Bottino, C., R. Castriconi, D. Pende, P. Rivera, M. Nanni, B. Carnemolla,
`C. Cantoni, J. Grassi, S. Marcenaro, N. Reymond, et al. 2003. Identification of PVR
`(CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1
`(CD226) activating molecule. J. Exp. Med. 198:557.
`10. Takai, Y., K. Irie, K. Shimizu, T. Sakisaka, and W. Ikeda. 2003. Nectins and nectin-
`like molecules: roles in cell adhesion, migration, and polarization. Cancer Sci. 94:655.
`11. Campadelli-Fiume, G., F. Cocchi, L. Menotti, and M. Lopez. 2000. The novel re-
`ceptors that mediate the entry of herpes simplex viruses and animal alphaherpesviruses
`into cells. Rev. Med. Virol. 10:305.
`12. Wang, P. L., S. O’Farrell, C. Clayberger, and A. M. Krensky. 1992. Identification and
`molecular cloning of tactile: a novel human T cell activation antigen that is a member
`of the Ig gene superfamily. J. Immunol. 148:2600.
`13. Cella, M., and M. Colonna. 2000. Cloning human natural killer cells. Methods Mol.
`Biol. 121:1.
`14. Traunecker, A., F. Oliveri, and K. Karjalainen. 1991. Myeloma based expression sys-
`tem for production of large mammalian proteins. Trends Biotechnol. 9:109.
`15. Nosaka, T., T. Kawashima, K. Misawa, K. Ikuta, A. L. Mui, and T. Kitamura. 1999.
`STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in he-
`matopoietic cells. EMBO J. 18:4754.
`16. Thompson, J. A. 1995. Molecular cloning and expression of carcinoembryonic anti-
`gen gene family members. Tumour Biol. 16:10.
`17. Boles, K. S., S. E. Stepp, M. Bennett, V. Kumar, and P. A. Mathew. 2001. 2B4
`(CD244) and CS1: novel members of the CD2 subset of the immunoglobulin super-
`family molecules expressed on natural killer cells and other leukocytes. Immunol. Rev.
`181:234.
`18. Huang, J. F., Y. Yang, H. Sepulveda, W. Shi, I. Hwang, P. A. Peterson, M. R. Jackson,
`J. Sprent, and Z. Cai. 1999. TCR-mediated internalization of peptide-MHC com-
`plexes acquired by T cells. Science 286:952.
`19. Carlin, L. M., K. Eleme, F. E. McCann, and D. M. Davis. 2001. Intercellular transfer
`and supramolecular organization of human leukocyte antigen C at inhibitory natural
`killer cell immune synapses. J. Exp. Med. 194:1507.
`20. Masson, D., A. Jarry, B. Baury, P. Blanchardie, C. Laboisse, P. Lustenberger, and
`M. G. Denis. 2001. Overexpression of the CD155 gene in human colorectal carci-
`noma. Gut 49:236.
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