Potential pathways for regulation of NK and T cell responses: differential X-linked lymphoproliferative syndrome gene product SAP interactions with SLAM and 2B4
Joan Sayós,
Khuong B. Nguyen1,
Chengbin Wu,
Susan E. Stepp2,
Duncan Howie,
John D. Schatzle2,
Vinay Kumar2,
Christine A. Biron1 and
Cox Terhorst
Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
1 Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02192, USA
2 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Correspondence to:
C. Terhorst
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Abstract
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SAP, the gene that is altered or absent in the X-linked lymphoproliferative syndrome (XLP), encodes a small protein that comprises a single SH2 domain and binds to the cell-surface protein SLAM which is present on activated or memory T and B cells. Because defective NK cell activity also has been reported in XLP patients, we studied the SAP gene in NK cells. SAP was induced upon viral infection of SCID mice and shown to be expressed in NK cells by in vitro culturing in the presence of IL-2. Moreover, SAP was expressed in the NK cell lines YT and RNK 16. Because SLAM, the cell-surface protein with which SAP interacts, and 2B4, a membrane protein having sequence homologies with SLAM, also were found to be expressed on the surfaces of activated NK and T cell populations, they may access SAP functions in these populations. Whereas we found that 2B4 also binds SAP, 2B4SAP interactions occurred only upon tyrosine phosphorylation of 2B4. By contrast, SLAMSAP interactions were independent of phosphorylation of Y281 and Y327 on SLAM. As CD48, the ligand for 2B4, is expressed on the surface of EpsteinBarr virus (EBV)-infected B cells, it is likely that SAP regulates signal transduction through this pair of cell-surface molecules. These data support the hypothesis that XLP is a result of both defective NK and T lymphocyte responses to EBV. The altered responses may be due to aberrant control of the signaling cascades which are initiated by the SLAMSLAM and 2B4CD48 interactions.
Keywords: 2B4, NK, SAP, SLM, T cell, XLP
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Introduction
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X-linked lymphoproliferative disease (XLP) is a human immunodeficiency that was identified >25 years ago (1). Around 50% of patients with XLP develop fatal mononucleosis with an uncontrolled proliferation of B and T cell populations. Twenty-five percent of the cases develop malignant B cell lymphomas; these are often but not always EpsteinBarr virus (EBV) induced. While some immunodeficiency symptoms are detected in XLP patients prior to infection by EBV virus (hypogammaglobulinemia), it is after the patients become infected by the virus that the disease fully progresses (25). SAP, the gene that is altered or absent in XLP (68), encodes a small protein that comprises a single SH2 domain and binds to the cell-surface protein SLAM (6). SLAM is a glycosylated transmembrane protein that is present on the surface of activated T and B cells. In T cells, triggering of signals by anti-SLAM antibodies induces IFN-
production and redirects Th2 responses of antigen-specific T cell clones to a Th1 or Th0 phenotype (912). In B cells, SLAM triggering in conjunction with anti-CD40 antibodies leads to B cell proliferation, and production of IgM, IgG and IgA (13).
We have previously proposed that SAP is a natural inhibitor of the recruitment of SH2 domain-containing molecules to SLAM as it blocks binding of the enzyme SHP-2 to tyrosine phosphorylated SLAM (6). Because SLAM is a high-affinity self-ligand and as SAP is expressed in T cells, but not in B cells, SAP regulates signal transduction induced by engagement of SLAM in T lymphocytes. This is consistent with experiments showing that the XLP defect is evident in T lymphocytes and particularly in cytotoxic T lymphocytes (4). Since defects in NK cells and activity have, however, also been reported in XLP (14,15), the possibility of activation related expression of SAP and SLAM in human and mouse NK cells was explored.
Here we report that both SAP and SLAM are present in certain activated NK cell populations as well as in T cells. In addition to SLAM, we found that 2B4, a cell-surface molecule expressed on NK cells and sharing sequence homology with SLAM, both in its ectodomain as well as in its cytoplasmic tail, interacts with SAP and is also induced on a proportion of activated T cells.
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Methods
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Cells and antibodies
YT and Jurkat cells were grown in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. COS-7 cells were grown in DMEM (Whittaker, Walkersville, MD) supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin and 100 µg/ml streptomycin. An anti-human 2B4 mAb (C1.7) was purchased from Immunotech and monoclonal anti-mouse 2B4 from PharMingen (San Diego, CA). Rabbit anti-SAP serum and monoclonal anti-human SLAM (2E7) have previously been described (6), and monoclonal anti-human SAP was obtained by standard procedures by immunizing BALB/c mice with the synthetic peptide CQGTTGIREDPDV coupled to keyhole limpet hemacyanin (Pierce, Rockford, IL). Rat anti-mouse SLAM is described elsewhere (D. Howie et al., manuscript in preparation). Phosphotyrosine mAb cocktail horseradish peroxidase-conjugated (PY-7E1, PY-1B2 and PY20) and horseradish peroxidase-conjugated streptavidin were from Zymed (San Francisco, CA). Antibody against SHP2 was obtained from Santa Cruz (Santa Cruz, CA).
Cell activation, immunoprecipitation and immunoblotting
COS-7 cells (107) were transfected by the DEAEdextran method and biotinylated with sulfo-NHS-LC-biotin (Pierce) as described before (6). YT cells (50x106/ml) were activated with pervanadate 1 mM for 20 min at 37°C. Lysis was carried out with 2% Triton X-100 (YT cells and A-LAK) or CHAPS 0.5% (COS cells) as described before (6). Cell lysates were clarified by centrifugation at 14,000 g for 15 min at 4°C and the crude lysate was precleared using 50 µl of Protein Gagarose beads (Gibco/BRL, Gaithersburg, MD) and 5 µl of normal mouse serum for 1 h. Immunoprecipitations were carried out using 1 µg of the indicated antibody and 30 µl of Protein Gagarose beads for 3 h at 4°C. Beads were then washed as described (6). Crude lysates and immunoprecipitates were subjected to SDSPAGE and transferred onto nitrocellulose filters (Millipore, Bedford, MA). Filters were blocked for 1 h with 5% skim milk (or 3% BSA) and then probed with the indicated antibodies. Bound antibody was revealed using horseradish peroxide-conjugated secondary antibodies using enhanced chemiluminescence (Supersignal; Pierce). For anti-phosphotyrosine blotting we used a directly horseradish peroxide-conjugated antibody cocktail (Zymed).
Yeast two-hybrid system and ß-galactosidase assay
Human SLAM and murine 2B4 cDNAs encoding the cytosolic domain of these proteins were subcloned in the vector pGAD424, while SAP cDNA was cloned in the vector pBRIDGE (Clontech, Palo Alto, CA). Competent yeast cells (strain Y197) were co-transformed using the standard LiAc/TE/PEG transformation protocol, and transformants were selected in media lacking tryptophan and leucine. Liquid culture assay using ONPG as substrate was used to measure ß-galactosidase activity (Yeast Protocols Handbook; Clontech).
Animal protocols
Pathogen-free C57BL/6 immunocompetent mice were purchased from Taconic Laboratory Animals and Services (Germantown, NY) and C57BL/6 SCID mice were purchased from Jackson Laboratories (Bar Harbor, ME) or obtained from Brian Gordon (Carolinas Medical Center, Charlotte, NC). Mice were infected i.p. with 2x104 p.f.u. of a lymphocytic choriomeningitis virus (LCMV) clone from Armstrong (16,17) or 5x104 p.f.u. of Smith strain murine cytomegalovirus (MCMV) as described (18).
In vitro IL-2-activated NK cells
A single-cell suspension of C57BL/6 SCID spleen cells was prepared and isolated with a one-step gradient of Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) as described (19). IL-2-activated NK cells were generated in vitro by culturing spleen cells at 10% CO2 in LAK culture media: DMEM (Gibco/BRL) supplemented with 10% FCS (Hyclone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2.25x105 M 2-mercaptoethanol (all from Gibco/BRL), 1 µg/ml indomethacin (Sigma) and 1000 U/ml human rIL-2 (Chiron, Emeryville, CA). On days 3 and 6 the cells were fed with one-half conditioned media and one-half LAK culture media containing 2xrIL-2. (19)
RNA analysis
For Northern blotting, total mRNA from SCID mouse spleen was used as described (6) with a radiolabeled mouse SAP-specific cDNA probe. SAP and SLAM mRNAs were quantified with RT-PCR ELISA according to manufacturer's instructions (Roche, Indianapolis, IN). Briefly, equal amounts of total RNA (0.11 µg) from different samples were used in RT-PCR. The RT-PCR product was labeled with digoxigenin (DIG) during the reaction and immobilized onto streptavidin-coated 96-well plates using a biotinylated oligonucleotide probe that is complementary to an internal sequence of the RT-PCR product. Horseradish peroxidase-conjugated anti-DIG was then added to the plate. After incubation and washing, the samples were developed with manufacturer's developing solution (Roche) and detected with a micro plate reader at 415 nm. A standard curve was constructed for each mRNA species using different RT-PCR and assay conditions to ensure that the sample readings were within the proper range. Each sample reading was also normalized with GAPDH. Samples were assayed in triplicate.
FACS staining
Single-cell suspensions of spleen cells from wild-type and SCID mice were prepared as described (1618). Three- and four-color staining of the cells was performed as previously described (16,17). Briefly, 1x106 cells were incubated with a blocking solution of 20% FBS containing 25 µg/ml of mouse IgG for 30 min. For SLAM staining, the cells were then incubated with a rat anti-mouse SLAM antibody (50 µl of 7D4 hybridoma culture medium) for 30 min. The cells were washed and incubated for 30 min with a biotinylated goat anti-rat IgG (PharMingen) diluted 1/50 in blocking buffer. The cells were washed again and incubated for 30 min with optimal concentrations of streptavidinallophycocyanin plus CyChrome-, FITC- and phycoerythrin-conjugated antibodies (0.5 µg each antibody/sample). Directly conjugated mAb for 2B4, NK1.1, CD3, CD8 and CD4 staining were purchased from PharMingen. After washing, cells were fixed for 30 min with 4% paraformaldehyde, until analyzed with a FACScalibur using CellQuest software (Becton Dickinson, San Jose, CA).
Immunofluorescence microscopy
YT cells were labeled in suspension with anti-human 2B4 (C1.7) or mouse IgG1 isotypic control (10 µg/ml) at 4°C for 30 min. After washing 2 times with ice-cold PBS, cells were incubated with 30 µg/ml of FITC-conjugated anti-mouse IgG1 for 15 min at 4°C (control) or at 37°C (capping). Cells were then washed twice with ice-cold PBS, immobilized in poly-lysine treated cover-slips at 4°C for 15 min and fixed in 20°C methanol for 15 min. After washing twice, cells were incubated for 30 min at room temperature with blocking buffer (PBS containing 0.2% skim milk, 2% FBS, 1% BSA and 0.1 mM Gly) and then with CY3-conjugated anti-SAP antibody (10C4.2) or with CY3-conjugated mouse IgG1 isotypic control (0.5 µg/ml) for 30 min at room temperature, washed twice with PBS and mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, Alabama). Fluorescence images were obtained with a Nikon Optiphot-2 microscope connected to a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI) and analyzed using SPOT software version 2.2.
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Results
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SAP is expressed in NK cells and its expression is up-regulated after NK cell activation
Although XLP is primarily thought to be a syndrome caused by abnormal functioning of T cells, NK cells are reported to be affected as well (14,15). In XLP patients NK cytotoxicity was found to be defective and this defect was not associated with a reduction in the number of NK cells. In this study two viruses that elicit a rapid NK cell response in mice were used. Because it is known that both MCMV and LCMV have the ability to activate NK cells (1618), SCID mice infected with either virus were examined using a semi-quantitative RT-PCR assay and Northern blotting. As shown in Fig. 1
(A and B), the amount of SAP-specific mRNA increases significantly in the day 3 activated splenocytes. Since no T or B cells are present in the spleen of SCID mice and the number of NK cells is known to remain constant in spite of their activation (1618), the increase in mRNA shown here represents up-regulation of the SAP gene in non-T and non-B cell populations. These data suggest that SAP expression may be induced in activated NK cells in response to viral infection.

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Fig. 1. SAP mRNA is up-regulated in cells from the spleen of SCID mice infected with LCMV and MCMV. (A) SAP mRNA from spleen of LCMV (open bars)- and MCMV (hatched bars)-infected SCID mice was quantified with RT-PCT ELISA as described in Methods. (B) Northern blotting of spleen cells from LCMV- and MCMV-infected mice was carried out with a mouse SAP cDNA probe as described in methods.
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As the percentages of NK cells in the spleen are low even after virus infection, expression of SAP was also determined in A-LAK cells, which had been treated for 1 week with recombinant IL-2. Western blot analysis demonstrated that SAP was expressed in these in vitro generated NK blasts, but not in freshly isolated NK cells (Fig. 2A
). Relative numbers of NK cells remained the same before and after IL-2 treatment (data not shown). In order to establish whether SAP was expressed in NK cell lines, human YT cells and rat RNK-16 cells were used for immunoprecipitation with rabbit anti-human or rabbit anti-mouse SAP polyclonal antibodies respectively. As shown in Fig. 2
(B and C), SAP is expressed as a 15 kDa protein in both cell lines.

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Fig. 2. SAP protein is expressed in activated NK cells, and in the established NK cell lines YT and RNK16. (A) Fresh NK cells extracted from SCID mice spleen or after 7 days culture in the presence of IL-2 were lysed as described in methods. Protein was transferred to a PVDF membrane and blotted with a rabbit serum anti-mouse SAP. (B and C) YT and RNK cells (50x106 cells/ml) were immunoprecipitated with 1 µg of an irrelevant antibody or with 1 µg of anti-human anti-SAP mAb (10C4.2) or 3 µl of anti-mouse SAP polyclonal rabbit serum. Proteins were transferred to PVDF and blotted with anti-SAP (10C4.2) or anti-mouse SAP.
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Together these studies clearly demonstrate that SAP is expressed in activated NK cells, but is either not expressed or expressed at low levels in resting NK cells.
SLAM and 2B4 are expressed on the surface of activated NK and T cells
The finding of SAP expression in NK cells prompted us to investigate with which cell-surface protein SAP could be associated. SLAM is expressed primarily on the surface of activated B and T lymphocytes, and no studies about the expression of SLAM in NK cells have been reported. We decided to check the possibility that SLAM could be expressed by activated NK cells. Again SCID mice were infected with LCMV and MCMV virus, and using a semi-quantitative PCR technique we measured the level of expression of SLAM at different time points after viral infection. As shown in Fig. 3
(A), infection with both viruses produced an increase in the levels of SLAM mRNA in the spleen of the infected SCID mice. The expression was maximal between days 5 and 7 after infection.

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Fig. 3. SLAM mRNA, and SLAM and 2B4 protein expression can be up-regulated during viral infections. (A) SLAM mRNA from spleen of MCMV (open bars) and LCMV (hatched bars) infected SCID mice was quantified with RT-PCT ELISA as described in Methods. (B) Cell-surface expression of SLAM and 2B4 on NK cells in C57BL/6 and C57BL/6-SCID mice that were uninfected or infected with MCMV for 1.5 days. Gates were set on populations of NK1.1+ and CD3 cells. Results shown are contour plots for SLAM or 2B4 expression on these gated NK cells. Numbers given are means ± SE for three to six mice per group. (C) Cell-surface expression of SLAM and 2B4 on NK cells in C57BL/6 and C57BL/6-SCID mice that were uninfected or infected with LCMV for 2 days. Data are shown as in (B). Numbers given are means ± SE for three to six mice per group. (D) SLAM and 2B4 expression on T cells. C57BL/6 mice were uninfected or infected with LCMV for 7 days. Gates were set on populations of CD8+ and CD4 T cells. Results shown are contour plots for SLAM or 2B4 expression on these gated T cells populations. Numbers given are means ± SE for three mice per group.
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In order to verify that the increase of the levels of SLAM mRNA in the spleens of the infected mice correlated with up-regulation of the protein in activated NK cells, we used FACS analysis to measure the expression of SLAM on NK cells in wild-type and SCID mice infected with MCMV or LCMV virus. As we show in Fig. 3
(B), while resting NK cells have a low basal level of SLAM expression, NK1.1+ and CD3 NK cells activated during MCMV infection show a significant increase in the degree of expression of SLAM 1.5 days after infection. The increase in both wild-type and SCID was at ~1015% of the cells. Interestingly, induction of SLAM was not observed on NK cells during LCMV infection (Fig. 3C
). At times of T cell activation during LCMV infections, i.e. day 7, similar or greater up-regulation of SLAM was observed on CD8+ T cells (Fig. 3D
). Greater than 90% of the NK cells under any of the conditions examined expressed 2B4 (Figs 3B and C
). 2B4 expression on T cells was induced during LCMV infection on proportions of cells similar to those expressing SLAM (Fig. 3D
).
SAP also associates with the tyrosine phosphorylated form of 2B4 only
The data presented above demonstrated that NK cells start off with and retain 2B4 expression, but subsets become SLAM+ during MCMV infection. In contrast, T cell populations start off negative for both cell-surface proteins, but proportions become SLAM+ and 2B4+ during LCMV infection. Recently it has been reported that SAP interacts with 2B4 (20). The fact that the cytoplasmic tail of 2B4 contains the SAP binding motif found in SLAM (T I/V Y x x V/I) (21) explains the interaction of SAP with both molecules.
In order to compare the SAP2B4 and SAPSLAM interactions, COS-7 cells were co-transfected with constructs encoding for mouse 2B4 or human SLAM in combination with SAP and the src kinase Fyn (Fig. 4A
). Interestingly, co-transfection of 2B4 with SAP, in the absence of Fyn results in tyrosine phosphorylation of 2B4 and binding of SAP. However, we did not detect association of this phosphorylated form of 2B4 with SHP2. In the presence of Fyn, a strong tyrosine phosphorylation of 2B4 was observed, and both SHP2 and SAP were associated with 2B4. The association of SHP2 with the phosphorylated form of 2B4 was decreased in the presence of SAP. In contrast, SAP blocked totally the interaction between SLAM and SHP2 (Fig. 4B
).

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Fig. 4. 2B4SAP and SLAMSAP interaction in COS cells. COS-7 cells were transfected as described in Methods with a combination of 2B4, SAP and Fyn (A) or SLAM, SAP and Fyn constructs (B). Cell-surface-expressed proteins were biotinylated 48 h after transfection and 2B4 constructs were specifically immunoprecipitated with 1 µg of antibody anti-mouse 2B4 (A) or anti-human SLAM (2E7) (B). Proteins were transferred to PDVF, and blotted with streptavidin and anti-phosphotyrosine, anti-SHP2 and anti-SAP antibodies.
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These results suggest that SAP can bind 2B4 only if 2B4 is phosphorylated on tyrosine residues. To further study the nature of this interaction, YT cells, a human NK cell line that naturally expresses both molecules, were used. Immunoprecipitation of 2B4 from YT cells did not result in co-precipitation of SAP (Fig. 5A
), but after pervanadate treatment of YT cells, 2B4 becomes tyrosine phosphorylated and co-precipitates SAP (Fig. 5A
). The importance of 2B4 tyrosine phosphorylation was confirmed using the yeast two-hybrid system. While we found interaction between SLAM and SAP using this technique (Fig. 5B
), no binding was detected between 2B4 and SAP (Fig. 5B
).

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Fig. 5. 2B4-SAP binding is dependent on 2B4 tyrosine phosphorylation. (A) YT cells (50x106 cells/ml) were incubated in the presence or absence of 1 mM pervanadate. Cells were lysed, precleared and immunoprecipitated with 1 µg of an irrelevant antibody or with 1 µg of anti-human anti-2B4 mAb (C1.7). Proteins were transferred to PVDF, and blotted with streptavidin, anti-phosphotyrosine and anti-human SAP (10C4.2). (B) No interaction was detected between SAP and 2B4 using the two-hybrid system, while a strong association of SAP to SLAM is shown (see Methods). Liquid culture assay using ONPG as substrate to measure ß-galactosidase activity was used to score the interaction of SAP with 2B4 and SLAM. For each construct at least three independent colonies were tested in the galactosidase assay. Open bars correspond to empty pBRIDGE vector and filled bars to SAP cloned on pBRIDGE.
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Taken together, these studies clearly demonstrate that the interaction of 2B4 and SAP is dependent upon phosphorylation of critical tyrosine residues in the cytoplasmic tail of 2B4. This is in strong contrast with the SLAMSAP interactions. Also in contrast to the SLAMSAP interactions is the observation that SAP does not completely block the recruitment of SHP-2 to the phosphorylated tyrosine motifs in the cytoplasmic domain of 2B4.
Activation of YT cells by anti-2B4 induces co-capping of 2B4 and SAP
Immunofluorescence microscopy was used to examine whether the SAP2B4 interaction functions in the NK cell. 2B4 was homogeneously distributed on the membrane of non-activated YT cells, whilst SAP was mostly expressed in the cytosolic compartment and to a lesser degree associated with the plasma membrane (Fig. 6A and B
). When YT cells were treated with anti-2B4 antibody (C1.7) followed by a goat anti-mouse antibody for 15 min at 37°C the 2B4 molecules were capped (Fig. 6C
). No capping was detected when cells were incubated in the same conditions with an isotypic control (IgG1) instead of anti-2B4 antibody (data not shown) or for 15 min at 4°C with FITC-conjugated goat-anti mouse IgG1 (Fig. 6A
). When 2B4 capped cells were stained with CY3-conjugated anti-SAP antibody (10C4.2) we found overlap of the SAP and the 2B4 distribution on the membrane of ~30% of the capped YT cells (Fig. 6D
). No co-capping was detected in YT cells stained with CY3-conjugated mouse IgG1 isotypic control (data not shown). This data together with the fact that SAP binds 2B4 after the phosphorylation of the tyrosine residues contained in its cytoplasmic domain strongly suggest that in physiological conditions, SAP could be recruited after 2B4 triggering by its ligand CD48.

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Fig. 6. Activation of YT cells by anti-2B4 induces co-capping of 2B4 and SAP. YT cells were incubated with 10 µg/ml of anti-2B4 for 30 min as indicated. The cells were then incubated with 30 µg/ml of FITC-conjugated goat anti-IgG1 antibody for 15 min at 4°C (A and B) or 37 °C (C and D). Cells were then stained with CY3-conjugated anti-SAP antibody (10C4.2) as described in Methods and subjected to immunofluorescence microscopy as indicated. Green fluorescence indicates 2B4, red indicates SAP. White arrows show co-capping of 2B4 and SAP.
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Discussion
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The failure of the immune system of XLP patients to eliminate EBV-infected B cells seems to be related to a defective EBV-specific T and NK cell response, rather than a B cell-specific defect (2,4). In non-XLP patients, EBV transformation and proliferation of B cells are controlled by T and NK cells, both by cellular interactions and by the release of lymphokines (22). We have originally proposed that mutation or deletion of SAP in XLP patients affects the signal transduction pathways that are initiated by homotypic interactions between SLAM molecules on the interface between T and B cells. The fact that both SLAM and SAP can be expressed in activated NK cells seems to indicate a more complex scenario, in which both T and NK cells interact via SLAM with EBV-infected B cells. SLAM engagement on T cells enhances IFN-
production and redirects Th2 responses of antigen-specific T cell clones to a Th1 or Th0 phenotype (11). While no data are available on the outcome of SLAM triggering on NK cells, it is likely that in both, T and NK cells, the lack of a functional SAP protein results in a defective SLAM signal transduction pathway with the subsequent lack of lymphokine production (IFN-
and others) necessary for the control of EBV-infected B cells.
As we show in Fig. 3
(A), both LCMV and MCMV viruses produced an increase in the levels of SLAM mRNA in the spleen of the infected SCID mice, and the expression was maximal between days 5 and 7 after infection. These results contrast with the FACS staining data showing that only NK cells from mice infected with MCMV virus expressed SLAM (Fig. 3B and C
). The most likely explanation for these data could be the fact that cells other than B, T and NK cells are able to express SLAM, and the levels of mRNA detected in the spleen of the infected animals correspond to more than one population of cells. The observation that SLAM is not up-regulated on NK cells after LCMV infection in contrast with the results obtained with the MCMV infection model could be related to the fact that activation conditions are very different during the two infections. In particular, biologically active IL-12 is induced at early times after MCMV but not LCMV infection (16,18). The kinetics of this IL-12 response coincides with induction of detectable NK cell IFN-
only in MCMV infection, but not after infection with LCMV. There appears to be a correlation between the lack of IL-12 production in response to LCMV infection and subsequent IFN-
production by NK cells with the absence of up-regulation of SLAM on NK cells. Human IL-12 was originally cloned and identified as a product of EBV-transformed B cell lines and recently it has been shown that transformation of primary B cells with EBV induces IL-12 expression (23,24). These data suggest that in human EBV infection IL-12 may be an important mediator of NK cell activation and that a complete NK cell activation possibly requires a functional SLAM pathway, a pathway that is non-functional in XLP patients.
In addition to SLAM, it is likely that 2B4 is involved in the defective EBV T and NK cell response described in XLP patients. 2B4 is a membrane protein that has sequence homologies with SLAM in both its ectodomain and cytoplasmic tail, and which is expressed constitutively on the surface of NK cells (25). More interestingly, 2B4 expression is up-regulated on CD8+ T cells after activation (Fig. 3D
). It has been reported that in NK cells 2B4 engagement induces cytokine secretion (IFN-
) and enhances non-MHC-restricted killing (2628). Another interesting point is the fact that the natural ligand of 2B4, CD48 (27,29), is strongly up-regulated on EBV-transformed B cells (30). All these data and the fact that SAP interacts with the cytosolic tail of 2B4 (20) strongly suggest the involvement of 2B4 in the control of EBV-transformed immunoblasts by NK and CD8+ T cells. Again, similar to SLAM, the absence of SAP could alter the signal pathway generated by the interaction between 2B4 and its ligand CD48 expressed on the surface of EBV-transformed lymphocytes.
Tangye et al. have previously reported the binding of SAP to 2B4 (20). In that study the interaction was shown using BAF3 cells transfected with Flag-2B4 and SAP-Myc. Our data confirms this result and shows that the interaction can occur in YT cells, which naturally express both 2B4 and SAP. We have also shown that SAP binds with dual modalities, in a phosphotyrosine-dependent manner when binding 2B4 and in a novel phosphotyrosine-independent manner with SLAM.
In contrast with the known SH2-containing adapter molecules, SAP consists of a single SH2 domain with a short C-terminal extension with no homology with any known protein domain. While adapter molecules act as a bridge between two other molecules, the existence of a single binding domain in SAP indicates that this molecule has another function. We have proposed that SAP is a natural inhibitor of proximal signaling molecule recruitment via SH2 domains for it blocks binding of the enzyme SHP-2 to phosphorylated SLAM. The crystal structure of the SAP SH2 domain in complex with SLAM peptides shows that in addition to the classical interaction between the phosphotyrosine residue and the phosphotyrosine pocket in the SH2 domain, specific interactions with amino acid residues N-terminal to the tyrosine residue in SLAM peptides take place (21). These additional interactions stabilize the SAPSLAM complex, and explain the interaction between SAP and SLAM in the absence of SLAM tyrosine phosphorylation. Two functions have been suggested for the extended interaction between SAP and SLAM. First, binding with non-phosphorylated sequences could serve to block the phosphorylation of these sites and block different types of interactions that do not involve SH2 domains. Second, tight and highly specific binding to phosphorylated sequences could prevent other molecules from binding to SH2 docking sites (21).
As we show in Fig. 5
(A and B), the interaction between 2B4 and SAP seems to be the classical interaction between an SH2 domain and a phosphotyrosine residue, because no interaction is detected in the absence of 2B4 tyrosine phosphorylation. In these conditions, we could expect that SAP will not be able to block the SHP-2 recruitment by 2B4. As we show in Fig. 4
(A), both SAP and SHP2 bind 2B4 after tyrosine phosphorylation by Fyn. No total blocking of SHP2 binding is detected, but rather a situation of competition that would be expected for two molecules that bind the same sites in 2B4 with similar affinity. These data suggest a different function for the SAP2B4 interaction. It is not unlikely that the SAP tail could be able to recruit downstream signaling molecules by an unknown mechanism, acting as an adapter.
Taken together, our observations are consistent with a model in which, in normal conditions, EBV-transformed B cell proliferation is controlled by T and NK cells through the SLAMSLAM and 2B4CD48 interactions. The absence of SAP in XLP patients produces a functional impairment of the SLAM and 2B4 pathways. This could result in a deficient or unbalanced production of cytokines (IFN-
), and in the ineffective NK and CD8+ T cell response in sustaining elimination of EBV-infected B cells.
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Acknowledgments
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This work is supported by grants AI-35714, CA-41268 and ES-07272 from the NIH. C. W. is supported by a grant from the Cancer Research Institute. D. H. is supported by a grant from the Leukemia and Lymphoma Society.
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Abbreviations
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DIG digoxigenin |
EBV EpsteinBarr virus |
LCMV lymphocytic choriomeningitis virus |
MCMV murine cytomegalovirus |
XLP X-linked lymphoproliferative syndrome |
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Notes
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Transmitting editor: J. F. Kearney
Received 1 June 2000,
accepted 11 September 2000.
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