| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Cutting Edge |
*
Basel Institute for Immunology, Basel, Switzerland;
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110;
Omaha, NE 68164; and
Medical Virology Section, Laboratory of Clinical Investigation, National Institutes of Health, Bethesda, MD 20892
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
release. These effector responses are initiated by multiple NK cell
receptors, which activate signaling pathways involving protein tyrosine
kinases as well as mitogen-activated protein kinases
(MAPK)3
(1). One emerging group of activating NK cell receptors
encompasses cell surface molecules of the Ig superfamily homologous to
CD2 (2). The prototype of these receptors, known as
2B4/CD244 (3, 4, 5, 6, 7), stimulates cytotoxicity through a
signaling pathway, which is strictly dependent on the recruitment of an
adapter protein called SLAM-associated protein (SAP) or SH2D1A
(2, 7). Thus, NK cells derived from SAP-deficient
individuals are no longer activated through 2B4 (2, 8, 9, 10, 11, 12). SAP is also essential for the signal transduction of
other CD2 family receptors, such as SLAM/CD150, CD84, and Ly-9, which
are differentially expressed on cytotoxic lymphocytes, Th cells, B
cells, and myeloid cells (2, 13, 14). The lack of function
of all these receptors in SAP-deficient individuals results in a
complex deficit of NK, T, and B cell responses, which leads to
uncontrolled EBV infections and, ultimately, to the X-linked
lymphoproliferative disease (XLPD) (2, 13, 14). In an attempt to identify novel cell surface receptors potentially involved in controlling EBV infections, we searched the expressed sequence tag database for CD2-like molecules. By this approach we have identified a novel human receptor called CD2-like receptor activating cytotoxic cells (CRACC). Functional characterization revealed that CRACC triggers NK cell-mediated cytotoxicity through a unique SAP-independent extracellular signal-regulated kinase (ERK)-dependent signaling pathway.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
GenBank expressed sequence tagged database was searched with the amino acid sequences of CD244 (2B4), CD150 (SLAM), CD84, and Ly-9 using the tblastn algorithm. A contig assembled from five distinct cDNAs (accession nos. AA765813, AI422743, AA554342, H73135, and AA921765) contained an open reading frame encoding CRACC. CRACC cDNA was amplified from NK and CD8+ T cell RNA by RT-PCR, cloned into pCR2.1 (Invitrogen, Carlsbad, CA), and sequenced. PCR primers were: 5'-ATGGCTGGTTCCCAACAT and 3'-ATTAATAGGAATACTTCTAA.
Production of CRACC-HuIgG fusion protein and anti-CRACC mAb
To produce soluble CRACC, J558L mouse myeloma cells were
transfected with a chimeric gene encoding the CRACC extracellular
domain fused with human IgG1 constant regions (CRACC-HuIgG).
Anti-CRACC mAb 162 (mouse IgG2b, 
) was raised by immunizing BALB/c
mice against CRACC-HuIgG. F(ab')2 of mAb 162 were
prepared using the Fab'/F(ab')2 Kit (Pierce,
Rockford, IL).
Transient transfections
CRACC cDNA was subcloned into pCMV-1-FLAG (Kodak, Rochester, NY) and expressed as amino-terminal FLAG peptide fusion protein (CRACCFLAG) in 293 cells. Cell surface expression of CRACCFLAG was determined by flow cytometry with mAb M2 (anti-FLAG; Kodak).
Cells
PBMC and NK cell lines from normal controls and XLPD patients were obtained as previously described (8). NK92 is a human NK cell line which lacks the Fc receptor CD16. P815 is a murine mastocytoma cell line. Peripheral B cells and monocyte-derived dendritic cells (DC) were activated by incubation with CD40L-expressing cells and influenza virus strain PR8, respectively.
Flow cytometry
In four-color flow cytometric analysis, PBMCs were sequentially incubated with PBS-20% human serum, anti-CRACC mAbs 162, PE-conjugated human-adsorbed goat anti-mouse IgG2b (Southern Biotechnology Associates, Birmingham, AL) and PBS-20% normal mouse serum. One aliquot of cells was further incubated with anti-CD3-PC5, anti-CD19-FITC, and anti-CD56-APC mAbs (Immunotech, Marseille, France). A second aliquot was incubated with anti-CD3-PC5, anti-CD4-FITC, and anti-CD8-APC mAbs (Immunotech).
51Cr release assay
NK cell cytotoxicity was tested against [51Cr]-labeled P815 cells in the presence of 10 µg/ml of either mAb 162, mAb 1C7 (anti-2B4, IgG1; Refs. 6, 7), mAb 9E2 (anti-NKp46, IgG1 (8), mAb (anti-CD16, IgG1 (DAKO, Carpinteria, CA)) or a control mouse IgG (Immunotech). F(ab')2 of mAb 162 were used as indicated. In some experiments, NK92 cells were preincubated for 1 h with the mitogen-activated protein/ERK (MEK) inhibitor PD98059 (20 µM) (Calbiochem, San Diego, CA) before coincubation with 51Cr-labeled P815 target cells. Control NK92 cells were incubated with DMSO, which was used as a solvent for PD98059.
Surface biotinylation, pervanadate treatment, and immunoprecipitations
Immunoprecipitations with mAb 162 or control IgG from biotinylated NK92 cells were performed and analyzed as previously described (4). Lysates from pervanadate-treated cells were subjected to immunoprecipitation with mAbs 162, 1C7, Z199 (anti-NKG2A; Immunotech), or control IgG1. Western blot analyses of immunoprecipitates were performed with anti-phosphotyrosine PY20-HRP (BD Transduction Laboratories, Lexington, KY), anti-SAP (kindly provided by S. Tangye, University of Sidney, Sidney, Australia, and H. Nakajima, National Institute for Longevity Sciences, Aichi, Japan), anti-SHP-1 (BD Transduction Laboratories), anti-SHP-2 (BD Transduction Laboratories), anti-SHIP (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-EAT2 rabbit antisera. Anti-EAT2 antiserum was generated by immunizing rabbits with the keyhole limpet hemocyanin-conjugated peptide DLPYYHGRLTKQDCETL. Western blot analysis with anti-phospho-ERK1/2 and anti-ERK1/2 Abs (New England Biolabs, Beverly, MA) was performed on NK92 cells following stimulation with mAb 162 or a control IgG mAb in the presence of a cross-linking Ab (goat anti-mouse IgG, F(ab')2; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1, 5, and 10 min. In some experiments, NK92 cells were preincubated for 1 h with the MEK inhibitor PD98059 (20 µM).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CRACC cDNA encodes a protein of 335 amino acids with a predicted
molecular mass of 
37 kDa (Fig. 1
). A
putative hydrophobic signal peptide is followed by an extracellular
region composed of two Ig superfamily domains containing seven
potential N-glycosylation sites. The membrane-distal V-type
Ig fold lacks the inter-
sheets disulfide bridge. This feature is a
hallmark of the CD2 family members (15). The
membrane-proximal Ig fold is of the C2 type. The hydrophobic
transmembrane domain is followed by a cytoplasmic domain, which
contains four tyrosine-based motifs. Some of these motifs closely
resemble those recruiting the adapter protein SAP (2),
which is essential for 2B4-mediated activation (Fig. 1) (2, 7, 8, 9, 10, 11, 12, 13, 14). CRACC cDNA was amplified by RT-PCR from human NK cells
and CD8+ T cells (data not shown). Therefore, we
designated this molecule CD2-like receptor
activating cytotoxic cells, CRACC. An alignment
of the extracellular domains of CD2 family members showed that
CRACC is most closely related to Ly-9 and CD84 (28% identity) (Fig. 1 and data not shown). The gene encoding CRACC was identified within
the human chromosome 1 genomic sequence performed by the Sanger
Center (Cambridge, U.K.; accession no. AL121985, tentative gene
designation LOC57823, tentative protein designation 19A24). It maps on
human chromosome 1q23–24, telomeric of CD48, CD150, and CD84, and
centromeric of Ly-9 (CD229) and 2B4. A cDNA corresponding to CRACC was
also recently cloned from NK cells by Boles and Mathew
(16) (protein designation CS1, accession no.
AF291815).
|
66-kDa cell-surface glycoprotein selectively
expressed on NK cells, a subset of cytotoxic T cells, and activated B
cells and DCs
To investigate the cellular distribution of CRACC, we produced an
anti-CRACC mAb, which specifically stained CRACC-transfected 293
cells, as compared with control transfectants (Fig. 2A). In human peripheral
blood, CRACC was expressed on virtually all NK cells, a large subset of
CD8+ T cells, and a very small percentage of
peripheral CD4+ T cells (Fig. 2B).
CRACC was also detectable on a small subset of peripheral B cells (Fig. 2B) and became strongly expressed on all B cells upon
activation through CD40 (Fig. 2C). CRACC was not expressed
on monocytes and immature DC derived in vitro from monocytes, but was
up-regulated upon DC maturation induced by influenza virus (Fig. 2D), lipopolysaccharide, and CD40L (data not shown). To
determine CRACC biochemical characteristics, we immunoprecipitated
CRACC from the NK cell line NK92, detecting a broad band of 66 kDa
under reducing conditions (Fig. 3). After
deglycosylation, the immunoprecipitate appeared as a sharp band of
37 kDa, which corresponds to the predicted molecular mass of CRACC
polypeptide (Fig. 3). Together, these results identify CRACC as an
66-kDa glycoprotein preferentially expressed on cytotoxic
lymphocytes, activated B cells, and mature DCs.
|
|
Expression of CRACC on NK cells and CD8+ T lymphocytes
suggested it was involved in the activation of cell-mediated
cytotoxicity. This hypothesis was investigated by reverse Ab-dependent
cell-mediated cytotoxicity (rADCC). In these experiments, the Fc
receptor (FcR)+ murine mastocytoma cell line P815 was
incubated with NK cells in the presence of different mAbs which bind
the FcR on target cells and triggering receptors on NK cells, thereby
mimicking the stimulatory ligands. Anti-CRACC mAb activated lysis of
P815 cells by NK92, whereas the F(ab')2 of the same Ab had
no effect (Fig. 4A). The
lysis triggered by simultaneous engagement of CRACC and CD16 or CRACC
and NKp46 was approximately equivalent to the sum of the lyses induced
by each receptor separately (Fig. 4B). Thus,
CRACC-mediated pathway does not synergize with those initiated by CD16
or NKp46.
|
, C–F),
as did the anti-NKp46 Ab. In contrast, the anti-2B4 mAb
triggered lysis of P815 only by normal NK cells. Thus, CRACC-mediated
activation of NK cells is SAP-independent. CRACC recruits 19- and 39-kDa phosphoproteins upon tyrosine phosphorylation and does not associate with SAP, EAT2, or protein tyrosine phosphatases
To characterize the CRACC signaling pathway, CRACC was
immunoprecipitated from NK92, which was either unstimulated or
stimulated with sodium pervanadate. Anti-phosphotyrosine blot of CRACC
immunoprecipitates showed a substantial tyrosine
phosphorylation of CRACC in pervanadate-treated cells
together with the association of a 19-kDa tyrosine
phosphorylated protein (Fig. 5A). A weak 39-kDa
phosphoprotein was also observed which was reminiscent of the linker
for activation of T cells (LAT) previously shown to associate to 2B4
(17). However, LAT was not detectable by Western blot
analysis of CRACC immunoprecipitates (data not shown). Anti-SAP
immunoblotting demonstrated lack of SAP association, in agreement with
the results obtained in rADCC experiments (Fig. 5B). We also
investigated the potential recruitment of other proteins previously
found to be associated with CD2-like receptors, such as SHP-1
(11), SHP-2 (2, 7), SHIP (18),
or EAT-2, which is a SAP-homologous adapter protein encoded on human
chromosome 1 (19). However, we could not detect
association of CRACC with any of these proteins by specific immunoblot
analysis (Fig. 5B). Thus, in pervanadate-treated NK cells,
CRACC is tyrosine phosphorylated, is associated with 19-
and 39-kDa phosphorylated proteins, and does not recruit
LAT, SAP, EAT-2, SHP-1, SHP-2, or SHIP.
|
Recent evidence indicates that spontaneous cytotoxicity of NK
cells against target cells requires activation of ERK (1, 20). Thus, we asked whether CRACC activates ERK and, if this is
the case, whether ERK activation is essential for CRACC-mediated
cytotoxicity. Ab-mediated cross-linking of CRACC in NK92 induced
tyrosine phosphorylation of ERK1/2, as demonstrated by
anti-phospho-ERK1/2 immunoblotting (Fig. 6A). In addition,
CRACC-mediated rADCC was partially inhibited by pretreatment of NK92
with PD98059, a specific inhibitor of ERK
phosphorylation (Fig. 6B). Thus,
CRACC-mediated cytotoxicity occurs through an ERK-mediated pathway.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The characterization of CRACC is a further demonstration that NK cell-target cell recognition is highly complex and involves multiple interactions at the NK cell/target cell interface. Like other receptors of the CD2 family, CRACC may mediate homotypic interaction or bind to other members of the same receptor family on target cells (3). We detected no interaction of soluble CRACC-HuIgG fusion protein with 293 cells expressing each of the known CD2 family members by flow cytometry (data not shown). The binding affinity of CRACC for its ligand may be too low to be detected by this assay. Alternatively, CRACC may bind to new members of the CD2 family yet to be characterized, such as BLAME, which was very recently discovered (21). The expression of CRACC on CTLs is noteworthy. CRACC induced no CTL-mediated cytotoxicity in rADCC (data not shown). Thus, CRACC may instead costimulate CD8+ T cells by interacting with ligands expressed on target cells. The expression of CRACC in activated B cells and mature DC further supports the idea that CRACC may be involved in modulating not only innate responses but also Ag-specific responses to pathogens. This dual role may be important in controlling infections by pathogens other than EBV.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Marco Colonna at the current address: Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: mcolonna{at}pathology.wustl.edu
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; SAP, SLAM-associated protein; DC, dendritic cell; ERK, extracellular signal-regulated kinase; CRACC, CD2-like receptor activating cytotoxic cells; MEK, mitogen-activated protein/ERK kinase; rADCC, reverse Ab-dependent cell-mediated cytotoxicity; XLPD, X-linked lymphoproliferative disease; LAT, linker for activation of T cells.
Received for publication June 26, 2001. Accepted for publication September 10, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  V. Brune, E. Tiacci, I. Pfeil, C. Doring, S. Eckerle, C. J.M. van Noesel, W. Klapper, B. Falini, A. von Heydebreck, D. Metzler, et al. Origin and pathogenesis of nodular lymphocyte-predominant Hodgkin lymphoma as revealed by global gene expression analysis J. Exp. Med., September 29, 2008; 205(10): 2251 - 2268. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Kamperschroer, D. M. Roberts, Y. Zhang, N.-p. Weng, and S. L. Swain SAP Enables T Cells to Help B Cells by a Mechanism Distinct from Th Cell Programming or CD40 Ligand Regulation J. Immunol., September 15, 2008; 181(6): 3994 - 4003. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. D. Hsi, R. Steinle, B. Balasa, S. Szmania, A. Draksharapu, B. P. Shum, M. Huseni, D. Powers, A. Nanisetti, Y. Zhang, et al. CS1, a Potential New Therapeutic Antibody Target for the Treatment of Multiple Myeloma Clin. Cancer Res., May 1, 2008; 14(9): 2775 - 2784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Lee, S. O. Mathew, S. V. Vaidya, P. R. Kumaresan, and P. A. Mathew CS1 (CRACC, CD319) Induces Proliferation and Autocrine Cytokine Expression on Human B Lymphocytes J. Immunol., October 1, 2007; 179(7): 4672 - 4678. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Eissmann and C. Watzl Molecular Analysis of NTB-A Signaling: A Role for EAT-2 in NTB-A-Mediated Activation of Human NK Cells. J. Immunol., September 1, 2006; 177(5): 3170 - 3177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. van Erp, K. Dach, I. Koch, J. Heesemann, and R. Hoffmann Role of strain differences on host resistance and the transcriptional response of macrophages to infection with Yersinia enterocolitica Physiol Genomics, March 13, 2006; 25(1): 75 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Bhat, P. Eissmann, J. Endt, S. Hoffmann, and C. Watzl Fine-tuning of immune responses by SLAM-related receptors J. Leukoc. Biol., March 1, 2006; 79(3): 417 - 424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Stark and C. Watzl 2B4 (CD244), NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int. Immunol., February 1, 2006; 18(2): 241 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Tassi and M. Colonna The Cytotoxicity Receptor CRACC (CS-1) Recruits EAT-2 and Activates the PI3K and Phospholipase C{gamma} Signaling Pathways in Human NK Cells J. Immunol., December 15, 2005; 175(12): 7996 - 8002. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Saborit-Villarroya, J. M. Del Valle, X. Romero, E. Esplugues, P. Lauzurica, P. Engel, and M. Martin The Adaptor Protein 3BP2 Binds Human CD244 and Links this Receptor to Vav Signaling, ERK Activation, and NK Cell Killing J. Immunol., October 1, 2005; 175(7): 4226 - 4235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bloch-Queyrat, M.-C. Fondaneche, R. Chen, L. Yin, F. Relouzat, A. Veillette, A. Fischer, and S. Latour Regulation of natural cytotoxicity by the adaptor SAP and the Src-related kinase Fyn J. Exp. Med., July 5, 2005; 202(1): 181 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Martin, J. M. Del Valle, I. Saborit, and P. Engel Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229 J. Immunol., May 15, 2005; 174(10): 5977 - 5986. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. V. Mikhalap, L. M. Shlapatska, O. V. Yurchenko, M. Y. Yurchenko, G. G. Berdova, K. E. Nichols, E. A. Clark, and S. P. Sidorenko The adaptor protein SH2D1A regulates signaling through CD150 (SLAM) in B cells Blood, December 15, 2004; 104(13): 4063 - 4070. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Chtanova, S. G. Tangye, R. Newton, N. Frank, M. R. Hodge, M. S. Rolph, and C. R. Mackay T Follicular Helper Cells Express a Distinctive Transcriptional Profile, Reflecting Their Role as Non-Th1/Th2 Effector Cells That Provide Help for B Cells J. Immunol., July 1, 2004; 173(1): 68 - 78. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-i. Yusa, T. L. Catina, and K. S. Campbell KIR2DL5 Can Inhibit Human NK Cell Activation Via Recruitment of Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP-2) J. Immunol., June 15, 2004; 172(12): 7385 - 7392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-M. Lee, M. E. McNerney, S. E. Stepp, P. A. Mathew, J. D. Schatzle, M. Bennett, and V. Kumar 2B4 Acts As a Non-Major Histocompatibility Complex Binding Inhibitory Receptor on Mouse Natural Killer Cells J. Exp. Med., May 3, 2004; 199(9): 1245 - 1254. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Simarro, A. Lanyi, D. Howie, F. Poy, J. Bruggeman, M. Choi, J. Sumegi, M. J. Eck, and C. Terhorst SAP increases FynT kinase activity and is required for phosphorylation of SLAM and Ly9 Int. Immunol., May 1, 2004; 16(5): 727 - 736. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Begum, K. Ishii, M. Kurita-Taniguchi, M. Tanabe, M. Kobayashi, Y. Moriwaki, M. Matsumoto, Y. Fukumori, I. Azuma, K. Toyoshima, et al. Mycobacterium bovis BCG Cell Wall-Specific Differentially Expressed Genes Identified by Differential Display and cDNA Subtraction in Human Macrophages Infect. Immun., February 1, 2004; 72(2): 937 - 948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. Tangye, K. E. Nichols, N. J. Hare, and B. C. M. van de Weerdt Functional Requirements for Interactions Between CD84 and Src Homology 2 Domain-Containing Proteins and Their Contribution to Human T Cell Activation J. Immunol., September 1, 2003; 171(5): 2485 - 2495. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), and so does the
anti-2B4 mAb (
), as compared with a control mouse IgG (
). On
the contrary, an F(ab')2 of the anti-CRACC (
)
induces no activation. B, Cytotoxicity of an NK cell
clonal population was tested against 51Cr-labeled P815
cells at 5:1 ratio in the presence of 2-fold dilutions of anti-CD16
(circles) or anti-NKp46 mAbs (squares) in combination with 10
µg/ml of anti-CRACC mAb (filled symbols) or control mouse IgG
(open symbols). The maximum concentration of anti-CD16 and
anti-NKp46 was 50 µg/ml. In the first point of the curves,
anti-CD16 and anti-NKp46 are absent, showing the effect of
anti-CRACC alone. The spontaneous lysis of P815 by the NK cell line
was 1.6% (data not shown). C–F, NK cell
polyclonal populations derived from normal controls (C
and D) and XLPD patients (E and
F) were tested in rADCC against 51Cr-labeled
P815 cells in the presence of anti-CRACC (
). Cytotoxicity was tested against
51Cr-labeled P815 cells at the indicated E:T ratios in the
presence of 10 µg/ml mAb. It is of note that the spontaneous lysis of
P815 by different NK cell lines is variable and so is the increase of
lysis mediated by anti-CRACC. Such variability is most likely due
to the presence within different NK cell polyclonal populations of NK
cell clones with different activating and inhibitor receptor
repertoires, each contributing to the total P815 lysis.
) or control IgG1 (