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The Journal of Immunology, 2000, 165: 3578-3583.
Copyright © 2000 by The American Association of Immunologists

Differential Requirement of ZAP-70 for CD2-Mediated Activation Pathways of Mature Human T Cells1

Edgar Meinl2,*,{dagger}, Doris Lengenfelder*, Norbert Blank{ddagger}, Rainer Pirzer*, Luis Barata§ and Claire Hivroz

* Institute for Clinical and Molecular Virology, University Erlangen-Nürnberg, Erlangen, Germany; {dagger} Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, Martinsried, Germany and Institute for Clinical Neuroimmunology, Ludwig-Maximilians-University, Munich, Germany; {ddagger} Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen-Nürnberg, Erlangen, Germany; § Primary Immunodeficiencies Unit, Department of Allergy and Clinical Immunology, Coimbra Paediatric Hospital, Coimbra, Portugal; and Institut National de la Santé et de la Recherche Médicale Unit 520, Institut Curie, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study addresses the role of the tyrosine kinase ZAP-70 in CD2-mediated T cell activation. Patients lacking ZAP-70 have few mature CD8+ T cells and high numbers of CD4+ T cells that are nonfunctional upon TCR triggering. Such a patient with a homozygous deletion in the zap-70 gene that resulted in the complete absence of ZAP-70 protein expression has been identified. Expression of the tyrosine kinases Lck, Fyn, and Syk was normal. The patient’s T cells were activated with two different pairs of mitogenic mAbs. CD2-induced phosphorylation of the {zeta}-chain and influx of Ca2+ was defective in the ZAP-70-deficient T cells, whereas CD2-induced phosphorylation of several other proteins, including Syk, was not affected. CD2-induced proliferation as well as production of TNF-{alpha} and IFN-{gamma} was abrogated in ZAP-70-deficient T cells, whereas PMA plus ionomycin induced normal activation of these cells. Together, this study shows that CD2-activation triggers ZAP-70-dependent and -independent pathways. Deletion of ZAP-70 affected CD2- and CD3-mediated proliferation and cytokine production in a similar way, suggesting that one of the different CD2 pathways converges with a CD3 pathway at or upstream of the activation of ZAP-70.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD2 is expressed on the surface of thymocytes, mature T cells, and NK cells. Its ligand in humans, CD58, is found on many different cell types. CD2-CD58 interactions strengthen the binding to APCs, increase the efficiency of Ag recognition (1), and induce cytoskeletal polarization of T cells in contact with APCs (2). Engagement of CD2 also augments the T cell response to cytokines (3). Simultaneous triggering of two distinct epitopes on CD2 by mAbs induces T cells to proliferate and secrete lymphokines in the absence of Ag and APCs. This has been referred to as the alternative pathway of T cell activation (4). The possibility to induce anergy (5), apoptosis (6), or even to reverse anergy (7) indicates potent effects of CD2.

T cell activation via CD2 or CD3 triggers a cascade of signaling events, which seem to be very similar (8, 9). In some studies, stimulation via CD2 or CD3 induced tyrosine phosphorylation of indistinguishable patterns of polypeptides (10). Despite these similarities, a number of differences between these two pathways have also been described (11, 12, 13).

Optimal CD2-mediated signaling in T cells or NK cells requires the presence of the {zeta}-chain, which is a component of both the CD3 complex and the CD16 complex on NK cells (14, 15). The precise molecular mechanism through which the {zeta}-chain mediates CD2 signaling is unknown. Upon TCR stimulation, the {zeta}-chain interacts with the Src-family tyrosine kinases Lck and Fyn, becomes phosphorylated on its immunoreceptor tyrosine-based activation motifs (ITAM),3 and recruits the Syk-family protein tyrosine kinase (PTK) ZAP-70 (16). CD2 stimulation also induces activation of Lck (17) and phosphorylation of {zeta} (8). However, several studies have led to conflicting data regarding the involvement of {zeta} and ZAP-70 in CD2-induced signaling: CD2-mediated signaling is also possible in TCR-positive T cells that lack {zeta}-chain-specific ITAMs (18). Evidence has been presented that stimulation via CD2 can bypass tyrosine phosphorylation and recruitment of ZAP-70 to the CD3-TCR complex for induction of phospholipase C-{gamma}1 activation (19) and activation of mitogen-activated protein kinases (20).

ZAP-70 is solely expressed in thymocytes, T cells, and NK cells and therefore shows a pattern of expression similar to CD2. ZAP-70 is essential for TCR-mediated activation of mature T cells, but it also plays a role in T cell maturation (16). Lack of ZAP-70 expression leads to severe T cell immunodeficiency (21, 22, 23). ZAP-70-deficient patients have very few CD8+ T cells in the periphery, whereas the number of peripheral CD4+ T cells is increased. However, these CD4+ T cells are nonfunctional, showing no proliferation in response to TCR triggering. Remarkably, lack of ZAP-70 affects T cell maturation differently in humans and in mice. In ZAP-70-/- mice, T cell maturation is arrested at the double-positive stage of differentiation, and, therefore, no mature CD4+ or CD8+ T cells are found in the periphery (24).

The aim of this study was to analyze the role of ZAP-70 in CD2-mediated activation of mature T cells. We have identified a patient with a severe immunodeficiency due to a homozygous deletion in the zap-70 gene leading to a lack of expression of ZAP-70 protein. Analysis of mature T cells from this patient revealed a differential requirement of ZAP-70 for CD2-mediated signaling events: ZAP-70 was absolutely required for CD2-mediated Ca2+ influx, for persistent phosphorylation of {zeta}, for proliferation, and cytokine production. However, several proteins, including Syk, were phosphorylated after CD2 activation in the patient’s T cells, indicating that CD2 also induces ZAP-70-independent signaling events.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Case report

The patient described in this study, a second child of unrelated parents, developed pneumonia at 3 mo of age due to Pneumocystis carinii and, at 6 mo, developed an extensive varicella infection. Severe combined immunodeficiency was diagnosed at the age of 7 mo. The lymphocyte blood count was high (15,000–20,000 cells/µl). Less than 1% of the CD3+ cells, either TCR{alpha}ß or TCR{gamma}{delta}, expressed CD8. B cells and NK cells were found at a normal proportion.

zap-70 sequencing and segregation of the mutation

Total RNA and cDNA from the PBMC of the patient and from a normal donor and genomic DNA of the patient, her parents, and a control were prepared. Amplification of cDNAs was performed by PCR using zap-70-specific pairs of primers. The upper primer, 5'-TTTGCCTGGACATCCACCTGTACGTCC-3', is located upstream of the ATG, and the lower primer, 5'-CAGCTGTGTGTGGAGACAACCAA-3', binds 3' of the stop codon. PCR products were subcloned into the pGemT vector before DNA sequencing using the Thermosequenase kit (Amersham, Braunschweig, Germany). Four independent clones were sequenced. Analysis of the segregation of the mutation was performed on genomic DNA using one intronic primer upstream of the deletion, 5'-CCTGATCCAGCAGCATCTCCC-3', and one exonic primer downstream of the deletion, 5'-CTTGCCCTGCTCGATGAAGGC-3'. PCR were conducted in the presence of 2 µCi of [{alpha}-32P]dCTP per reaction. Samples were separated using a 6% denaturing PAGE gel and exposed to autoradiography. The experiments determining the segregation of the mutation were done three times.

T cell culture and activation

PBMC from the ZAP-70-deficient patient and two healthy donors were stimulated with 10 ng/ml PMA (Sigma, Deisenhofen, Germany) and 1 µg/ml ionomycin (Sigma). Culture medium consisted of 45% Panserin 401 (Pan, Aidenbach, Germany), 45% RPMI 1640, and 10% FCS (Boehringer Mannheim, Mannheim, Germany) supplemented with 2 mM glutamine and 50 µg/ml gentamicin (Life Technologies, Berlin, Germany). Recombinant human IL-2 (Chiron, Ratingen, Germany) was added at a concentration of 100 U/ml 2 days later. The growing cells were expanded in the IL-2-containing medium for at least 2 wk.

Flow cytometry

mAbs directed to CD3, CD4, CD8, TCR{alpha}ß, TCR{gamma}{delta}, HLA-DR, and labeled isotype controls were obtained from Becton Dickinson (Heidelberg, Germany). CD2 expression was detected with supernatant of the hybridoma TS2/18.1.1 (American Type Culture Collection, Manassas, VA) and a FITC-labeled goat-anti-mouse-IgG F(ab')2 fragment (Dianova, Hamburg, Germany). Each surface marker was analyzed two to three times.

Activation via CD2 and CD3, assessment of proliferation, and cytokine production

For CD2-mediated activation, two different pairs of mAbs were used: either a combination of the mAb 39C1.5, which recognizes the T11.1 epitope, and the mAb 6F10.3, which recognizes the T11.2 epitope (both from Coulter Immunotech, Hamburg, Germany), or a combination of the mAb X11, which recognizes the T11.1 epitope of CD2, and the mAb D66, which recognizes the CD2R cryptic epitope (kindly provided by Dr. L. Boumsel, INSERM U448, Créteil, France). For CD3-mediated activation, 96-well U-bottom plates were coated overnight with 1 µg/ml of purified mAb OKT3. Additionally PMA (10 ng/ml) plus ionomycin (1 µg/ml) were used to activate T cells. About 7 x 104 T cells were seeded per well in a volume of 200 µl medium without IL-2. All experiments were performed in triplicates. The supernatant was collected 24 h after activation. Production of IFN-{gamma} and TNF-{alpha} was determined by ELISA. Two days after activation, 0.2 µCi of [3H]thymidine (Amersham) was added for another 16 h. Cultures were harvested using a Matrix TM96 (Packard, Frankfurt, Germany). The filters were dried, exposed overnight on a [3H]thymidine-sensitive screen, and analyzed with a BAS2000 imaging system (Fuji Raytest, Straubenhardt, Germany). Comparative measurements of the same filters with the direct beta-counter (Packard) and the BAS2000 showed that these two evaluation systems had a correlation of >0.95 and a comparable sensitivity. Cpm values obtained with the direct beta-counter are about 20% of those obtained with a liquid scintillation counter. To analyze cytokine production and proliferation after CD2 and CD3 activation, three independent experiments were performed with two ZAP-70-expressing control cell lines included in each experiment.

Immunoprecipitation and Western blot analysis

For detection of ZAP-70, two different Abs were applied, a mAb (no. Z 24820; Transduction Laboratories, Lexington, KY) and a polyclonal rabbit Ab (kindly provided by Dr. B. Malissen, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique, Marseille, France). Syk was detected with a mAb (no. 05-434) from Upstate Biotechnology (Biozol, Eching, Germany) or a polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA), p56lck and p59fyn were detected with polyclonal Abs (Santa Cruz Biotechnology). Cleared cell lysates were separated under reducing conditions on an 8% SDS-PAGE and electroblotted on Immobilon P membrane (Millipore, Eschborn, Germany). Blots were blocked with PBS containing 5% low-fat milk and 0.05% Tween 20, incubated with 1 µg/ml of the primary mAbs, a peroxidase-conjugated secondary Ab, and developed by using the ECL detection system (Amersham). Immunoprecipitation for ZAP-70 and Syk was performed four times.

Purified primary T cells, obtained by rosetting with neuraminidase-treated sheep RBC or T cell blasts were left unactivated or activated for 3 min at 37°C in the presence of UCHT1 (ascitic fluid at 1/1000) or a combination of the mAbs X11 and D66, each used at 10 µg/ml. Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Nonidet P-40, 0.5% Na DOC, 0.1% SDS, 100 µM Na3VO4, protease inhibitors) for 20 min at 4°C. Nuclei and cell debris were removed by centrifugation. Protein concentrations were determined in the postnuclear lysates using a Bio-Rad kit (Bio-Rad, Richmond, U.K.). The same amount of lysates was precleared at 4°C by rocking with mouse or rabbit purified IgG for 1 h at 4°C. Then, protein G-Sepharose beads were added, and the nonspecific immunoprecipitates were recovered by centrifugation. After this preclearing, lysates were incubated overnight with anti-Syk Abs (Santa Cruz Biotechnology; sc-1077), anti-{zeta} mAb (Santa Cruz Biotechnology; sc-1239), or anti-ZAP-70 Abs (polyclonal rabbit anti-serum, kind gift from Dr. B. Malissen). Specific immunoprecipitates were recovered by addition of protein G-Sepharose beads for 1 h and washed three times in lysis buffer. Immunoprecipitates were then run on standard SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore). Nonspecific binding was blocked with 5% BSA in PBS/0.05% Tween, then the anti-phosphotyrosine mAb 4G10, an anti-Syk mAb (Upstate Biotechnology), an anti-{zeta} mAb, or anti-ZAP-70 Abs (Santa Cruz Biotechnology; sc-574) were applied. The Ab/Ag complexes were visualized by an enhanced chemiluminescence detection system according to the manufacturer’s instruction (ECL; Amersham) using anti-mouse or anti-rabbit Ig Abs coupled to HRP as secondary Abs. Experiments detecting activation induced tyrosine phosphorylation and specific phosphorylation of Syk and {zeta} were performed twice.

Measurement of Ca2+ influx

The procedure of measuring [Ca2+]i was adapted from previously described protocols (25, 26). In brief, 1 x 107 cells were incubated in 1 ml of their culture medium with Fluo3-AM (4 µM) on an agitator for 30 min at room temperature. To facilitate dye equilibration, 4 ml culture medium was added and cells were incubated for another 30 min (27). Rapid dye exclusion was prevented by the use of sulfinpyrazone (250 µM) in each solution. After the loading procedure was completed, cells were split in samples of 1 x 106 cells for each experiment, spun down, and resuspended in a simplified saline solution containing 1 mM Ca2+. The fluorescence of the cells was detected in a PTI (South Brunswick, NJ) spectrometer at 37°C under constant stirring. The excitation wavelength was 505 nm and emission wavelength was 530 nm. Experiments were started when the baseline was constant over 2–3 min. The Kd of Fluo3-AM for Ca2+ binding at 37°C was 864 nM as was described (26) and cytosolic Ca2+ was calculated as [Ca2+]i = Kd (F - Fmin)/(Fmax - F). The fluorescence maximum (Fmax) was calibrated using Ionomycin (5 µM). A control sample of 1 x 106 cells was not incubated with dye to determine autofluorescence (Fmin) (26). The experiments determining Ca influx were performed three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutation in the zap-70 gene and expression of tyrosine kinases in the patient’s T cells

Immunological findings in this child were reminiscent of a defect in ZAP-70 (23) and prompted us to study the coding region of zap-70. We amplified the complete coding sequence for ZAP-70 from cDNA and sequenced independent clones. A 13-bp deletion involving nucleotides 1719–1731 was found (Fig. 1Go, top). This mutation has already been detected in another, unrelated patient and has been shown to induce instability of the protein (28). Segregation analysis revealed that the patient was omozygous, but the parents were heterozygous for this mutation (Fig. 1Go, bottom).



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  		FIGURE 1. Determination of the zap-70 mutation and its segregation. Top, Sequence of cloned zap-70 cDNA obtained by RT-PCR from the patient’s mRNA or a control. A deletion of 13 bp between codons 1718 and 1730 was observed in two independent cDNA clones from the patient. Bottom, An informative region of the zap-70 gene of the patient, of the parents, and of an unrelated control was amplified in the presence of [{alpha}-32P]dCTP. Radioactive products were separated by PAGE and exposed to autoradiography. WT, Wild type.

 
This mutation leads to a total absence of ZAP-70 protein expression as shown on immunoprecipitates of total lysates (Fig. 2Go). The absence of ZAP-70 was seen with a commercially available mAb and also with a polyclonal anti-ZAP-70 Ab (a kind gift of Dr. B. Malissen). Levels of expression of Syk, p56lck, and p59fyn were compared in postnuclear lysates of CD4+ T cells from a control and from the patient. As shown in Fig. 2Go, these three tyrosine kinases were expressed at the same level in both T cell populations.



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  		FIGURE 2. Tyrosine kinase expression in patients’ T cells. A, ZAP-70 was precipitated from lysates obtained from control CD4+ T lymphocytes or patient T lymphocytes. The immunoprecipitated ZAP-70 was revealed by Western blot analysis using a ZAP-70 polyclonal Ab. B, Expression of ZAP-70 in total cell lysates was analyzed by Western blot with a commercially available mAb. Postnuclear lysates of T cells from a control or from the patient were also analyzed by Western blot with an anti-Syk mAb (C), anti-p56lck (D), or anti-p59fyn (E). Bands corresponding to Syk, p56lck, p59fyn, or the heavy chains of the ZAP-70 immunoprecipitating Ab (IgH) are indicated by arrows.

 
Culture and phenotype of ZAP-70-defective T cells

PBMC from the patient described above were stimulated with PMA plus ionomycin and cultured with IL-2. Under these conditions, the proliferative response of the patient’s T cells was similar to the one observed with control T cells. After at least 2 wk of culture, these cells had the phenotype of mature activated T cells. They were largely CD4+CD8- (71%), but contained a CD4-CD8- (15%) and a CD8+CD4- (13%) subpopulation. Activation markers such as CD69, CD25, and HLA-DR were expressed. The outgrowing cells were homogeneously (98%) positive for CD3, 81% expressed an {alpha}ß TCR, whereas a {gamma}{delta} TCR was found on 18%. CD2 was strongly expressed, as on T cells from healthy controls (Fig. 3Go).



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  		FIGURE 3. Phenotype of cultured ZAP-70-deficient T cells. T cell blasts from the ZAP-70-deficient patient were activated with PMA plus ionomycin and expanded in IL-2 for 2 wk. In the histograms, the open line represents the isotype control and the closed graph represents the specific staining for the indicated marker. The number below the marker gives the percentage of cells scored positive. In the dot blot graph, the lower left quadrant represents the negative control. The numbers in each quadrant represent the percentage of positive cells.

 
Lack of ZAP-70 affects CD2 signaling

To study the role of ZAP-70 in CD2 signaling, we activated T cell blasts obtained from the patient described herein with a combination of the CD2 mAbs X11 and D66. We first analyzed the protein tyrosine phosphorylation in total cell lysates induced by triggering of CD3/TCR or CD2. As shown in Fig. 4GoA, CD3 and CD2 activation led to tyrosine phosphorylation of several proteins in T cell blasts from both the control and the ZAP-70-deficient patient. The basal level and CD3- or CD2-induced tyrosine phosphorylations were reproducibly slightly reduced in the patient’s T cell blasts, but the pattern of tyrosine phosphorylated proteins was grossly similar in normal and ZAP-70-deficient blasts. However, we consistently noted that a phosphorylated band around 21 kDa induced by both CD3 and CD2 was absent in the ZAP-70-deficient blasts. Because this molecular mass was reminiscent of one of the phosphorylated forms of {zeta}, we immunoprecipitated {zeta} and studied its phosphorylation status in quiescent or activated T cells. As shown in Fig. 4GoC, we observed tyrosine phosphorylated {zeta} in the quiescent control cells; this phosphorylation was enhanced by CD3 or CD2 triggering. In marked contrast, we did not see any {zeta} phosphorylation, neither in quiescent nor in activated blasts from the patient.



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  		FIGURE 4. Tyrosine phosphorylation induced by CD2 activation. T cell blasts from the ZAP-70-deficient patient and one healthy control were left unstimulated (0) or activated by the combination of anti-CD2 mAbs X11 and D66 (CD2) or by the anti-CD3 mAb UCHT1 (CD3) for 3 min. at 37°C, before lysis and immunoprecipitation with either anti-Syk or anti-{zeta}. Postnuclear lysates (A) or immune complexes (B and C) were resolved by SDS-PAGE. Subsequently, a Western blot analysis (WBA) was performed with a mAb directed to phosphotyrosine (4G10), Syk, or {zeta}. Bands corresponding to Syk, {zeta}, phospho-{zeta} (P {zeta}), or the heavy chain of the immunoprecipitating Ab (IgH) are indicated by arrows.

 
We then analyzed whether the lack of ZAP-70 affects the activation-induced phosphorylation of Syk. To this end, we immunoprecipitated Syk in unstimulated, CD2- or CD3-activated T cell blasts. As shown in Fig. 4GoB, the level of Syk expression was the same in the patient’s and control’s cells. Analysis of the tyrosine phosphorylation status of Syk revealed that CD3 and CD2 activation induced Syk phosphorylation in both normal and ZAP-70-deficient T cells. Finally, we measured the activation-induced Ca2+ influx. These experiments showed that ZAP-70 was essential for CD2-mediated Ca2+ influx (Fig. 5Go).



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  		FIGURE 5. Activation-induced [Ca2+]i. T cell blasts from a control (A and C) and the ZAP-70-deficient patient (B and D) were labeled with Fluo3-AM and analyzed for [Ca2+]i after CD2 stimulation using 1–1.5 x 106 cells per cuvette in a volume of 1 ml. Two different pairs of mAbs were used. Arrows in each figure indicate the addition of Abs. A and B, The Abs X11 (dilution 1:50), D66 (dilution 1:50), and goat-anti-mouse Ig (10 µg/ml, dilution 1:150 in 6.6 µl) were used. C and D, The Abs 6F10.3 (4 µg/ml, dilution 1:50), 39C1.5 (4 µg/ml, dilution 1:50), and goat-anti-mouse-Ig (10 µg/ml, dilution 1:150) were added. Addition of 20 µl solution with Abs (dilution 1:50) induced a drop of the baseline due to dilution of the buffer solution.

 
Lack of ZAP-70 affects CD2-mediated proliferation and cytokine production

Activation via CD2 triggered normal T cells to produce TNF-{alpha} and IFN-{gamma}, whereas the ZAP-70-deficient T cells showed no response (Fig. 6Go). Cells were also stimulated with solid-phase bound anti-CD3 and with PMA plus ionomycin. Although ZAP-70-deficient T cells did not respond to activation via CD3, stimulation with PMA plus ionomycin induced IFN-{gamma} and TNF-{alpha} secretion. These results confirm that ZAP-70-deficient T cells can be activated by signals bypassing the TCR membrane signaling (Fig. 6Go). Measurement of the proliferative response induced by CD2 triggering showed that no proliferation of the ZAP-70-deficient T cells could be induced in contrast to what was observed with T cells obtained from two control donors (Fig. 7Go). The ZAP-70-deficient cells did not show any proliferative response at any concentration of anti-CD2 mAbs. By contrast, at 10 µg/ml of anti-CD2 mAbs, the control cells EM-P gave a stimulation index of 8, the control cells I-65, which were triggered to a lower proliferation and had a lower background of spontaneous proliferation, displayed a stimulation index of 9 (Fig. 7Go).



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  		FIGURE 6. Activation-induced secretion of IFN-{gamma} and TNF-{alpha}. Cultured short-term T cell lines from the ZAP-70-deficient patient and two healthy controls (EM-P and I65) were stimulated with a mitogenic pair of anti-CD2 mAbs (A and C), solid-phase bound anti-CD3, or PMA plus ionomycin (B and D). Supernatants were collected 24 h after activation, and the amount of secreted IFN-{gamma} or TNF-{alpha} was determined.

 


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  		FIGURE 7. Activation-induced proliferative response. T cells from the ZAP-70-deficient patient and two healthy controls were stimulated with a mitogenic pair of mAbs to CD2 or with solid-phase bound anti-CD3. Two days later the cells were labeled with [3H]thymidine for another 16 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that ZAP-70 has a differential effect on different signaling pathways activated upon CD2-engagement. We have identified a patient with a homozygous deletion in the gene coding for ZAP-70 resulting in complete lack of ZAP-70 protein expression. T cells from this patient were stimulated with two different mitogenic pairs of mAbs to CD2. In contrast to normal T cells, CD2 activation did not induce persistent phosphorylation of {zeta}, Ca2+ influx, proliferation, or production of TNF-{alpha} and IFN-{gamma}. However, even in the ZAP-70-deficient T cells, stimulation of the CD2 pathway induced tyrosine phosphorylation of several proteins, including Syk.

Signal transmission from the TCR is mediated by the sequential activation of two families of PTKs (29). Members of the Src family, Lck and Fyn, initiate this process by phosphorylating ITAMs in the cytoplasmic domains of the CD3 and {zeta}-chains. The phosphorylated ITAMs recruit ZAP-70 to the activated receptor complex (29). The clustered Src and ZAP-70 PTKs phosphorylate a series of cytoplasmic substrates, allowing the activation of various signaling pathways (29). ZAP-70 plays a key role in these signaling events. Mature T cells that do not express ZAP-70 cannot be activated through the CD3/TCR complex (21, 23, 28, 30).

Our studies show that the CD2-dependent signaling only partially depends on ZAP-70. ZAP-70 is essential for Ca2+ influx, persistent phosphorylation of {zeta}, cytokine production, and proliferation. This is consistent with earlier observations showing that CD2-mediated activation of T cells requires expression of the {zeta}-chain (14, 15) and induces {zeta} phosphorylation (8) and ZAP-70 tyrosine phosphorylation in Jurkat T cells, although to a lesser extent than activation through the TCR (13). Our finding that ZAP-70 is essential for CD2-mediated persistent phosphorylation of {zeta} in human mature T cells is consistent with a previous report showing that CD3 activation does not induce {zeta} phosphorylation in another ZAP-70-deficient model, the P116 Jurkat T cell clone (31). The SH2 domains of ZAP-70 might protect the phosphorylated ITAMs of the {zeta}-chain from dephosphorylation (32). This strongly supports a model in which CD2 activation induces recruitment of ZAP-70 to the phosphorylated {zeta}-chain.

The exact molecular requirement for CD2-induced {zeta} phosphorylation is currently unknown. The two Src-family PTKs Lck and Fyn have been shown to bind to CD2 and to be activated in response to CD2 triggering (17). Thus, it is possible that these PTKs phosphorylate ITAMs in the TCR/CD3/{zeta} complex, resulting in recruitment of ZAP-70 to this complex and its further phosphorylation and activation by Lck. Proteins interacting with the cytoplasmic region of CD2 have been identified (2, 33). The precise role of these proteins in the different functions mediated by CD2 and their potential involvement as a link between CD2 and the {zeta}-chain remain to be determined.

The essential role of ZAP-70 in both CD3- and CD2-mediated T cell proliferation and cytokine production suggests that some of the anti-CD3- and CD2-induced activation pathways converge at or upstream of the activation of ZAP-70. Although similarities do exist between CD2 and TCR activation, there is evidence that some signaling events are specific to CD2 activation. It has been demonstrated that CD2-mediated signaling can occur independently of the ITAMs of the {zeta}-chain (18). CD2 activation of the JCAM1.6 clone of Jurkat leukemia cells, which lacks Lck expression, can activate the c-Jun N-terminal kinase involved in IL-2 regulation of transcription (13). CD3-mediated activation of these cells induces neither c-Jun N-terminal kinase activation nor IL-2 transcription (13). The authors suggested that CD2 can trigger a Lck- and ZAP-70-independent activation pathway. Another recent study showed that CD2 activation can trigger activation of mitogen-activated protein kinases in the absence of ZAP-70 (20). These reports together with the results reported herein show that CD2 can induce ZAP-70-dependent and -independent activation events.

ZAP-70 is essential for CD3-mediated activation in mature T cells, but not in thymocytes (34), which is consistent with a differential requirement for ZAP-70 in TCR signaling and T cell development (16). In ZAP-70-/- mice, thymocyte development can be restored by Syk (35), indicating that ZAP-70 and Syk have overlapping functions. Syk is the PTK most closely related to ZAP-70. Our study shows that Syk, which is expressed at normal level in the ZAP-70-deficient T cells, gets phosphorylated upon CD2 engagement. Although it has been shown that Syk can replace ZAP-70 during thymocyte maturation (35), this study shows that Syk cannot substitute for ZAP-70 to mediate CD2-dependent Ca2+ influx, proliferation, and cytokine production in mature human T cells.

Because CD2 modulates a variety of biological effects in T cells, such as cytokine production, proliferation, and polarization of T cells, it is possible that different activation pathways induce these different effects. Some of them may be dependent on ZAP-70, whereas others may be independent of this PTK.

In conclusion, the present study establishes the essential role of ZAP-70 in CD2-mediated persistent phosphorylation of {zeta}, in Ca2+ influx, proliferation as well as production of IFN-{gamma} and TNF-{alpha} in human mature T cells. It also shows that CD2 activation can induce Syk phosphorylation, which cannot replace ZAP-70 in CD2-mediated proliferation and cytokine production. CD3- and CD2-mediated activation pathways are similarly affected by lack of ZAP-70, suggesting that these two pathways converge at or upstream of the activation of ZAP-70.


   Acknowledgments
 
We thank Professor B. Fleckenstein (Institute for Clinical and Molecular Virology, Erlangen, Germany) for continuous support and Dr. F. LeDeist and E. Jouanguy (Institut National de la Santé et de La Recherche Médicale, Unité 429, Paris, France) for their help.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB 466) and by the Wilhelm Sander-Stiftung (97.081.1). The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation. Back

2 Address correspondence and reprint requests to Dr. Edgar Meinl, Department of Neuroimmunology, Max-Planck-Institute of Neurobiology, D-82152 Martinsried, Germany. Back

3 Abbreviations used in this paper: ITAM, immune receptor tyrosine-based activation motif; PTK, protein tyrosine kinase. Back

Received for publication February 25, 2000. Accepted for publication July 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Moingeon, P., H. C. Chang, B. P. Wallner, C. Stebbins, A. Z. Frey, E. L. Reinherz. 1989. CD2-mediated adhesion facilitates T lymphocyte antigen recognition function. Nature 339:312.[Medline]
  2. Dustin, M. L., M. W. Olszowy, A. D. Holdorf, J. Li, S. Bromley, N. Desai, P. Widder, F. Rosenberger, P. A. van der Merwe, P. M. Allen, A. S. Shaw. 1998. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell 94:667.[Medline]
  3. Gollob, J. A., J. Li, E. L. Reinherz, J. Ritz. 1995. CD2 regulates responsiveness of activated T cells to interleukin 12. J. Exp. Med. 182:721.[Abstract/Free Full Text]
  4. Meuer, S.C., R. E. Hussey, M. Fabbi, D. Fox, O. Acuto, K. A. Fitzgerald, J. C. Hodgdon, J. P. Protentis, S. F. Schlossman, E. L. Reinherz. 1984. An alternative pathway of T-cell activation: a functional role for the 50 kd T11 sheep erythrocyte receptor protein. Cell 36:897.[Medline]
  5. Miller, G. T., P. S. Hochman, W. Meier, R. Tizard, S. A. Bixler, M. D. Rosa, B. P. Wallner. 1993. Specific interaction of lymphocyte function-associated antigen 3 with CD2 can inhibit T cell responses. J. Exp. Med. 178:211.[Abstract/Free Full Text]
  6. Rouleau, M., A. Bernard, O. Lantz, J. P. Vernant, B. Charpentier, A. Senik. 1993. Apoptosis of activated CD8+/CD57+ T cells is induced by some combinations of anti-CD2 mAb. J.Immunol. 151:3547.[Abstract]
  7. Boussiotis, V. A., G. J. Freeman, J. D. Griffin, G. S. Gray, J. G. Gribben, L. M. Nadler. 1994. CD2 is involved in maintenance and reversal of human alloantigen-specific clonal anergy. J. Exp. Med. 180:1665.[Abstract/Free Full Text]
  8. Monostori, E., D. Desai, M. H. Brown, D. A. Cantrell, M. J. Crumpton. 1990. Activation of human T lymphocytes via the CD2 antigen results in tyrosine phosphorylation of T cell antigen receptor {zeta}-chains. J. Immunol. 144:1010.[Abstract]
  9. Kanner, S. B., N. K. Damle, J. Blake, A. Aruffo, J. A. Ledbetter. 1992. CD2/LFA-3 ligation induces phospholipase-C {gamma}1 tyrosine phosphorylation and regulates CD3 signaling. J. Immunol. 148:2023.[Abstract]
  10. Ley, S. C., A. A. Davies, B. Druker, M. J. Crumpton. 1991. The T cell receptor/CD3 complex and CD2 stimulate the tyrosine phosphorylation of indistinguishable patterns of polypeptides in the human T leukemic cell line Jurkat. Eur. J. Immunol. 21:2203.[Medline]
  11. Hubert, P., P. Debre, L. Boumsell, G. Bismuth. 1993. Tyrosine phosphorylation and association with phospholipase C{gamma}-1 of the GAP-associated 62-kD protein after CD2 stimulation of Jurkat T cell. J. Exp. Med. 178:1587.[Abstract/Free Full Text]
  12. Jin, Y. J., D. R. Kaplan, M. White, G. C. Spagnoli, T. M. Roberts, E. L. Reinherz. 1990. Stimulation via CD3-Ti but not CD2 induces rapid tyrosine phosphorylation of a 68-kDa protein in the human Jurkat T cell line. J. Immunol. 144:647.[Abstract]
  13. Sunder-Plassmann, R., E. L. Reinherz. 1998. A p56lck-independent pathway of CD2 signaling involves Jun kinase. J. Biol. Chem. 273:24249.[Abstract/Free Full Text]
  14. Spruyt, L. L., M. J. Glennie, A. D. Beyers, A. F. Williams. 1991. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16). J. Exp. Med. 174:1407.[Abstract/Free Full Text]
  15. Howard, F. D., P. Moingeon, U. Moebius, D. J. McConkey, B. Yandava, T. E. Gennert, E. L. Reinherz. 1992. The CD3 {zeta} cytoplasmic domain mediates CD2-induced T cell activation. J. Exp. Med. 176:139.[Abstract/Free Full Text]
  16. Chu, D. H., C. T. Morita, A. Weiss. 1998. The Syk family of protein tyrosine kinases in T-cell activation and development. Immunol. Rev. 165:167.[Medline]
  17. Danielian, S., A. Alcover, L. Polissard, M. Stefanescu, O. Acuto, S. Fischer, R. Fagard. 1992. Both T cell receptor (TcR)-CD3 complex and CD2 increase the tyrosine kinase activity of p56lck: CD2 can mediate TcR-CD3-independent and CD45-dependent activation of p56lck. Eur. J. Immunol. 22:2915.[Medline]
  18. Steeg, C., A. von Bonin, H. W. Mittrucker, B. Malissen, B. Fleischer. 1997. CD2-mediated signaling in T cells lacking the {zeta}-chain-specific immune receptor tyrosine-based activation (ITAM) motif. Eur. J. Immunol. 27:2233.[Medline]
  19. Hubert, P., V. Lang, P. Debre, G. Bismuth. 1996. Tyrosine phosphorylation and recruitment of ZAP-70 to the CD3-TCR complex are defective after CD2 stimulation. J. Immunol. 157:4322.[Abstract]
  20. Ueno, H., S. Matsuda, K. Katamura, M. Mayumi, S. Koyasu. 2000. ZAP-70 is required for calcium mobilization but is dispensable for mitogen-activated protein kinase (MAPK) superfamily activation induced via CD2 in human T cells. Eur. J. Immunol. 30:78.[Medline]
  21. Chan, A. C., T. A. Kadlecek, M. E. Elder, A. H. Filipovich, W. L. Kuo, M. Iwashima, T. G. Parslow, A. Weiss. 1994. ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 264:1599.[Abstract/Free Full Text]
  22. Arpaia, E., M. Shahar, H. Dadi, A. Cohen, C. M. Roifman. 1994. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase. Cell 76:947.[Medline]
  23. Hivroz, C., A. Fischer. 1994. Immunodeficiency diseases: multiple roles for ZAP-70. Curr. Biol. 4:731.[Medline]
  24. Negishi, I., N. Motoyama, K. Nakayama, S. Senju, S. Hatakeyama, Q. Zhang, A. C. Chan, D. Y. Loh. 1995. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 376:435.[Medline]
  25. Kao, J. P., A. T. Harootunian, R. Y. Tsien. 1989. Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J. Biol. Chem. 264:8179.[Abstract/Free Full Text]
  26. Merritt, J. E., S. A. McCarthy, M. P. Davies, K. E. Moores. 1990. Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+. Biochem. J. 269:513.[Medline]
  27. Vandenberghe, P. A., J. L. Ceuppens. 1990. Flow cytometric measurement of cytoplasmic free calcium in human peripheral blood T lymphocytes with fluo-3, a new fluorescent calcium indicator. J. Immunol. Methods 127:197.[Medline]
  28. Elder, M. E., D. Lin, J. Clever, A. C. Chan, T. J. Hope, A. Weiss, T. G. Parslow. 1994. Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science 264:1596.[Abstract/Free Full Text]
  29. Weiss, A., D. R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263.[Medline]
  30. Arpaia, E., M. Shahar, H. Dadi, A. Cohen, C. M. Roifman. 1994. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase. Cell 76:947.
  31. Williams, B. L., K. L. Schreiber, W. Zhang, R. L. Wange, L. E. Samelson, P. J. Leibson, R. T. Abraham. 1998. Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line. Mol. Cell Biol. 18:1388.[Abstract/Free Full Text]
  32. Chan, A. C., D. M. Desai, A. Weiss. 1994. The role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signal transduction. Annu. Rev. Immunol. 12:555.[Medline]
  33. Li, J., K. Nishizawa, W. An, R. E. Hussey, F. E. Lialios, R. Salgia, R. Sunder-Plassmann, E. L. Reinherz. 1998. A cdc15-like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2-triggered adhesion. EMBO J. 17:7320.[Medline]
  34. Gelfand, E. W., K. Weinberg, B. D. Mazer, T. A. Kadlecek, A. Weiss. 1995. Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes. J. Exp. Med. 182:1057.[Abstract/Free Full Text]
  35. Gong, Q., L. White, R. Johnson, M. White, I. Negishi, M. Thomas, A. C. Chan. 1997. Restoration of thymocyte development and function in zap-70-/- mice by the Syk protein tyrosine kinase. Immunity 7:369.[Medline]



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