Contribution of CD3
to TCR regulation and signaling in human mature T lymphocytes
Pilar S. Torres1,
David A. Zapata1,
Alberto Pacheco-Castro1,
José L. Rodríguez-Fernández2,
Carlos Cabañas2 and
José R. Regueiro1
1 Inmunología and 2 Bioquímica y Biología Celular, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain
Correspondence to: J. R. Regueiro; E-mail: regueiro{at}med.ucm.es
Transmitting editor: F. Rosen
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Abstract
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CD3 proteins may have redundant as well as specific contributions to the intracellular propagation and final effector responses of TCR-mediated signals at different checkpoints during T cell differentiation. We report here on the participation of CD3
in the activation and effector function of human mature T lymphocytes at the antigen recognition checkpoint. Following TCRCD3 engagement of human CD3
-deficient T cell lines, and despite their lower TCRCD3 surface levels compared to normal controls, mature T cell responses such as protein tyrosine phosphorylation and the regulation of expression of several cell surface molecules, including the TCRCD3 itself, were either normal or only slightly affected. In contrast, other physiological responses like the specific adhesion and concomitant cell polarization on ICAM-1-coated dishes were selectively defective, and activation-induced cell death was increased. Our data indicate that CD3
contributes essential specialized signaling functions to certain mature T cell responses. Failure to generate appropriate interactions may abort cytoskeleton reorganization and initiate an apoptotic response.
Keywords: cell death, immunodeficiency disease, polarization, signal transduction, surface molecule
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Introduction
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Mature
ß T lymphocytes recognize pathogen-derived peptides on antigen-presenting cells by means of a multimeric membrane protein ensemble termed the TCRCD3 complex. The
ß TCRCD3 complex includes two clonally distributed variable chains that directly interact with antigen (TCR
and TCRß), and four invariant polypeptides that regulate assembly, expression and signal transduction (CD3
, CD3
, CD3
and CD3
) (1). TCRCD3 engagement initiates an intricate cascade of signal transduction events involving a growing family of downstream intracellular mediators (both adaptors and enzymes), which may result either in T cell activation, proliferation and effector function, or in anergy, or in apoptosis, depending on as yet unclear differences in the cytosolic integration of extracellular information (2,3). Signals relayed through the very same structure or through immature variants thereof (such as the pre-TCR sensor) are also responsible much earlier, during intrathymic development, for T cell selection (4). However, the relative contribution and intracellular connections of each invariant chain during TCRCD3-mediated signaling are still a matter of debate (5,6).
CD3 chains may have partially redundant functions, as all CD3 components share in their cytoplasmic tails at least one immunoreceptor tyrosine-based activation amino acid motif (ITAM), which accounts for the signaling properties of the individual chains (7). Indeed, isolated ITAM from CD3
or CD3
coupled to quimeric receptors, which lack all other CD3 subunits, transduce efficiently certain activation signals in T lymphocytes (8,9). Zap-70, one of the enzymes recruited earlier to TCRCD3 complexes, can bind to CD3 chains other than
(10) and certain ITAM (such as those of CD3
, CD3
or CD3
) are altogether dispensable for T cell activation or differentiation (1113).
However, CD3 molecules may also perform specialized functions, as ITAM sequences belonging to different CD3 chains show different affinities for downstream adaptors and enzymes, as well as different signaling functional outcomes [such as substrate tyrosine phosphorylation, apoptosis or calcium mobilization, reviewed in (11)]. Isolated CD3
or CD3
ITAM cannot induce mature T cell proliferation, suggesting that certain responses require a minimum complement of such ITAM (14,15). The specialized functions may not map to the ITAM, since a tail-less CD3
has been shown to rescue the selective defect observed in TCRCD3 signaling to ERK, but not other, MAP kinases when CD3
is lacking during thymocyte selection (12).
Lastly, ablation experiments in mice suggest that redundant and specific roles for the invariant chains of the TCRCD3 complex may co-exist, and can be exposed by carefully probing activation events at the different developmental checkpoints (6).
In contrast to their murine homologues, humans with natural CD3
and CD3
deficiencies have a substantial number of mature peripheral blood T lymphocytes, allowing the analysis of signal specification by incomplete receptors at the last TCRCD3-mediated checkpoint, i.e. antigen recognition (16). Our previous studies in human CD3
-deficient primary T cells, IL-2-dependent T cell lines and Herpesvirus saimiri (HVS)-transformed T lymphocytes indicated that, despite their decreased TCRCD3 expression levels, several mature T cell responses were normally induced via their mutant TCRCD3, including calcium flux, cytotoxicity, up-regulation of CD69 or CD154, proliferation and synthesis of specific cytokines, such as tumor necrosis factor (TNF)-
. In contrast, TCRCD3-induced synthesis of other cytokines like IL-2, and phorbol myristate acetate (PMA)-induced TCRCD3 down-regulation, were severely impaired (1720). These results suggested a specialized role for CD3
in coupling the remaining TCRCD3 chains to these particular signaling pathways. We have now extended these studies and explored the participation of CD3
in other relevant TCRCD3-induced T lymphocyte responses, such as regulation of the expression of several surface molecules including the TCR, adhesion, apoptosis and protein tyrosine phosphorylation in transformed CD3
-deficient T lymphocytes.
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Methods
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T cell lines
HVS (strain 488C)-transformed T cell lines were derived from peripheral blood lymphocytes of a healthy congenital CD3
-deficient (
) individual or normal donors (
+), as previously described (17,18). The CD4+ and CD8+ T cell lines used in the experiments had been cultured for 6 years (DSF4, CD4+
), 5 years (CTO, CD8+
+ and DSF8, CD8+
), 4 years (AGU and RUT, both CD4+
+) or 3 years (APC, CD4+
+, and DPR, MAI, SLM and ANZ, CD8+
+). Cells have always been grown in parallel in 1:1 Panserin 401:RPMI [from PAN Biotech (Aidenbach, Germany) and Gibco/BRL (Paisley, UK) respectively] medium with 40 IU/ml human rIL-2 (Frederick Cancer Research and Development Center, NCI, Frederick, MD), 10% FCS (Flow, Rockville, MD, UK) and 1% glutamine (Biowhittaker, Verviers, Belgium) replaced every 34 days. All experiments were performed on resting cells, generated by starving cells overnight in Panserin/RPMI with 2% FCS, but no IL-2, the day before medium replacement was due. Cells were washed twice in PBS and resuspended in Panserin/RPMI 10% FCS without IL-2 before use.
Antibodies and flow cytometry
The expression of different surface markers was studied by flow cytometry according to a standard procedure (19). The following mAb were used (30 min, 4°C): CD2, CD5, CD8
, CD25 and CD71 from Caltag (Burlingame, CA); CD3 (UCHT1) and CD18 from Immunotech (Marseille, France); and CD3 (Leu4), CD4 (Leu3a), CD45R0 and CD80 (BB1/B7) from Becton Dickinson (Mountain View, CA). CD3 (F101.01) hybridoma supernatant was a generous gift of Dr Bent Rubin (CHU de Purpan, Toulouse, France). All commercial antibodies were FITC- or phycoerythrin-conjugated and, for F101.01, an FITC- or phycoerythrin-conjugated goat anti-mouse IgG (H + L) from Caltag was used. Background fluorescence was defined in all cases with an isotype-matched irrelevant mAb from Caltag. Cells were washed twice in PBS and analyzed in an Epics Elite Analyzer cytofluorometer (Coulter, Hialeah, FL).
For comparative stainings we used the MFI, defined as the average fluorescence value of the corresponding mAb referred to the logarithmic scale of fluorescence intensity along the x-axis of the histograms.
To follow the TCRCD3-induced regulation of several surface markers, 1 x 106 resting cells/ml were incubated at 37°C/5% CO2 in 96-well round-bottom plates (105 cells/well, in triplicate) in the presence of 1 µg/ml plastic-immobilized CD3-specific mAb (purified UCHT1) or (as a negative control) of an immobilized isotype-matched irrelevant mAb (Caltag), for several time periods (2048 h). Cells were then harvested, washed twice in PBS 1% FCS and analyzed for the expression of each surface molecule by flow cytometry. Expression changes were recorded as the percentage of mAb binding (estimated as MFI) in CD3-stimulated samples relative to the unstimulated control as a function of time.
TCRCD3 down-regulation and immunoprecipitation
CD3-induced TCRCD3 down-regulation was performed as described previously (21). Briefly, 1 x 106 resting cells/ml were incubated at 37°C/5% CO2 in 96-well round-bottom plates (105 cells/well) in the presence of different dilutions of soluble CD3-specific mAb (Leu4 hybridoma supernatant kindly donated by Dr B. Alarcón, Centro de Biología Molecular, Madrid, Spain) or (as a negative control) of soluble isotype-matched irrelevant mAb (Caltag) for several time periods (230 h). Cells were washed twice in PBS/1% FCS, incubated on ice with 5 µl of the same supernatant (Leu4) for 45 min, stained with phycoerythrin-conjugated goat anti-mouse IgG for 45 min and analyzed by flow cytometry. Expression changes were recorded as the percentage of Leu4 binding (estimated as MFI) in CD3-stimulated samples relative to the unstimulated control.
PMA-induced TCRCD3 down-regulation was done as described (22). Briefly, 5 x 105 resting cells/ml were incubated at 37°C (i) for 30 min in the presence or absence of increasing amounts of PMA (20160 ng/ml; doseresponse experiments) or (ii) in the presence or absence of 20 ng/ml PMA for several time periods (30120 min; kinetic analyses). After two washings with PBS/1% FCS, CD3 expression was monitored with phycoerythrin-conjugated Leu4 by flow cytometry. Results are given as the percentage of anti-CD3 binding in PMA-treated cells relative to unstimulated cells.
To analyze the surface CD3
and CD3
levels after PMA treatment, 40 x 106 resting cells were incubated with 20 ng/ml PMA for 30 min and monitored with Leu4 for TCR down-regulation relative to untreated cells, as explained above. The cells were then surface radioiodinated, lysed, immunoprecipitated with APA 1/1 (anti-CD3 ascitic fluid, donated by B. Alarcón) and digested with N-glycanase before electrophoresis, essentially as described (20). CD3
:CD3
ratios were estimated by electronic densitometry of the corresponding deglycosylated proteins on a Geldoc 2000 analyzer (Bio-Rad, Hercules, CA).
Cell attachment assays
Cell adhesion assays were performed as described (23). First, 96-well flat-bottom plates (Costar, Cambridge, MA) were precoated overnight at 4°C with 6 µg/ml ICAM-1Fc in adhesion buffer (20 mM TrisHCl and 150 mM NaCl, pH 8.2), blocked with 1% BSA in adhesion buffer for 1 h at room temperature and washed twice with PBS. Second, 1 x 106 resting cells/ml were incubated on ice for 30 min in the presence or absence (spontaneous adhesion) of (i) anti-CD3-specific mAb (1 µg/ml UCHT1 or 1/50 dilution F101.01), washed twice in PBS to remove mAb excess and cross-linked with 2 µg/ml of goat anti-mouse IgG for 10 min at 37°C), (ii) 10 µg/ml of the LFA-1-activating CD18-specific mAb KIM-127 (maximum adhesion control) or (iii) 100 nM phorbol ester PDBu (Sigma, St Louis, MO). Treated and untreated cells were incubated in the coated wells (3 x 105 cells/well in duplicates) for several time periods (1590 min) at 37°C. Plates were gently washed 4 times in prewarmed PBS, and bound cells were fixed with 1% formaldehyde in cold PBS and stained with 0.5% crystal violet in 2% methanol. Images were acquired with a SPOT-2 color digital camera (Diagnostic Copy, Sterling Heights, MI) coupled to an Axioplan-2 microscopy (Carl-Zeiss, Jena, Germany) at x20. Electronic cell counting of at least two representative fields was performed with MetaMorph software (MetaMorph Imagin System; Universal Imaging, Downingtown, PA). The percentage of specific adhesion was calculated for each time point as the ratio of 100 x [(experimental spontaneous cell binding)/(maximum spontaneous cell binding)]. Cell polarization, defined as the appearance of long, frequently unique, projections, was calculated from 10 randomly chosen fields of each condition by direct counting of total cells (500600) at x40. Only clearly polarized cells were considered.
Apoptosis assays
Resting cells (5 x 105 /ml) were incubated in 96-well round-bottom plates (1 x 105 cells/well in triplicates) in the absence (negative control) or presence of 1 µg/ml plastic-immobilized CD3-specific mAb (UCHT1; Immunotech, Marseille, France), 10 ng/ml PMA (Sigma) plus 750 ng/ml ionomycin (Sigma) or 1 µg/ml phytohemagglutinin (Difco, Detroit, MI) for several time periods (1848 h) at 37°C/5% CO2. Apoptosis was analyzed using a commercial kit for such purpose (Bender Medsystems; Sigma). Briefly, after stimulations, 2 x 105 cells were washed once in PBS, resuspended in 200 µl binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2), in the presence of 5 µl FITC-conjugated Annexin V, transferred to cytometer tubes and incubated for at least 10 min in the dark at room temperature. Then cells were washed once in binding buffer and resuspended in 300 µl of the same solution. Finally, 15 µl of propidium iodide (PI, 20 µg/ml) was added to the tubes and cells were analyzed by flow cytometry. Untreated cells were used as negative apoptosis control. Necrotic (N) cells were distinguished from apoptotic (A) cells, among Annexin V+ lymphocytes, by their susceptibility or resistance to PI staining respectively. Relative cell death was calculated as the percentage of apoptotic or necrotic cells after stimulation relative to unstimulated cells (spontaneous cell death) as a function of time. Results are given as the mean ± SD of three independent experiments.
Phosphorylation assays
Tyrosine-phosphorylated proteins in response to anti-CD3 treatment were detected by Western immunoblotting of cell lysates with the anti-phosphotyrosine-specific mAb 4G10 (kindly donated by N. Taylor, Institut de Genetique Moleculaire, CNRS, Montpellier) as described elsewhere (24). Briefly, 6 x 106 resting cells/ml were stimulated with a CD3-specific mAb (50 µl of an 1/50 dilution of Leu4 supernatant) at 37°C for several time periods (110 min). Cells were then washed in cold PBS and lysed on ice with 500 µl of lysis buffer (1% NP-40, 140 mM NaCl, 10 mM Tris pH 7.5, 5 mM EDTA, 10 mM PMSF, 1 µg/ml aprotinin and 1 mM sodium orthovanadate) for 30 min. Lysates were then centrifuged at 12,000 g for 30 min and supernatants containing the NP-40-soluble fraction were subjected to SDSPAGE on 7.5% gels. Proteins were then transferred onto a nitrocellulose membrane using a semidry transfer system for 30 min at 15 V (Bio-Rad). Immunoblots were stained with Ponceau S to control the quality of the transfer and then blocked with 0.5% blocking reagent (Roche, Mannheim, Germany) in PBS with 1% Tween 20. After 3 washes in TBS, blots were incubated with 1/100 4G10 in 0.5% blocking reagent, 0.1% Tween 20 in TBS for 1 h at room temperature and washed 4 times with 0.1% Tween 20 in TBS. Bound anti-phosphotyrosine mAb was developed with a horseradish peroxidase-labeled sheep anti-mouse IgG (Amersham, Little Chalfont, UK), and detected by enhanced chemiluminescence (ECL; Amersham). The films were electronically scanned on a Bio-Rad Geldoc 2000 analyzer (Bio-Rad) to determine the intensity and molecular weight of each protein.
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Results
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Phenotype of CD3
-deficient HVS-transformed mature T lymphocytes
Normal (
+) and mutant (
) T cells expressed comparable levels of several relevant molecules (Fig. 1). However, CD45RO was strongly overexpressed in transformed CD8+
cells, perhaps as a compensatory mechanism to ensure transformation (19,25), as such difference was not observed in primary T cells [(26) and Zapata, unpublished]. CD3 expression levels were clearly positive but lower in
(particularly CD8+) than in
+ T cells with all tested monoclonals, as described (20).
TCRCD3-induced regulation of cell surface molecules
Signaling through the mutant
TCRCD3 complex was next studied by analyzing changes in the surface levels of several cell surface molecules after CD3 engagement (Fig. 2). The results indicated that
T cells were essentially normal by this functional assay, both for the up-regulation (CD25, CD71 and CD80) or down-regulation (CD4/8) of the analyzed molecules. However, a slight but consistent delay was observed in
cells for the up-regulation of CD25 in CD8+ cells, or for the down-regulation of CD4 or CD8 in the respective lineages. Larger differences in CD25 induction were observed when other normal controls were used for comparison (data not shown). Impaired CD3-induced regulation of CD25 has been reported previously in murine
thymocytes (27). Lower TCRCD3 expression by
T cells did not affect the induction of CD71, suggesting that the observed delay in the regulation of other cell surface molecules was CD3
specific. We conclude that
T cells can regulate the expression of several cell surface molecules after TCRCD3 triggering, although with delayed kinetics in some cases.
Defective phorbol ester-induced and reduced TCRCD3-induced CD3 down-regulation
In a second set of functional experiments,
T cells were stimulated with anti-CD3 to analyze the down-regulation of the TCRCD3 itself. As a control, cells were stimulated with phorbol ester PMA, which is known to be strictly dependent on CD3
for TCRCD3 down-regulation (22,28). The results demonstrated that
T cells were resistant to PMA treatment (Figs 3B and 4A), whereas they clearly down-regulated their TCRCD3 upon CD3 (Leu4) engagement (Fig. 3). However, TCRCD3 down-regulation by
T cells was always lower than controls (Fig. 3A). Note that the clear differences in Leu4 staining between CD4+ and CD8+
cells (Fig. 1) did not affect their relative TCRCD3 down-regulation levels, suggesting that signaling for this particular function was independent of the number of engaged surface TCRCD3 complexes.
PMA stimulation consistently down-regulates
50% of normal surface TCRCD3 complexes, but hardly any when CD3
is absent (Fig. 4A) (22,2931). It has been proposed that the TCRCD3 complex is a mixture of at least two biochemically independent isoforms:
ß

and
ß

(32). It was therefore conceivable that only the former (i.e.
-containing) TCRCD3 isoforms were susceptible to PMA-induced internalization. To test this hypothesis, the surface
:
content ratio of
+ T cells (and, as a control, of
T cells) was evaluated by immunoprecipitation and N-glycanase digestion before and after PMA treatment (Fig. 4B). The results showed that the
:
ratio in
+ T cells was not modified by exposure to PMA, suggesting that the putative
-containing and
-containing TCRCD3 isoforms do not exist or are not functionally independent by this assay.
Increased TCRCD3- and lectin-induced apoptosis
TCR ligand-induced apoptosis kinetics using anti-CD3 or PHA was next analyzed. In contrast to anti-CD3, PHA binding to
+ and
T lymphocytes was similar (data not shown). As a TCRCD3-independent stimulus, cells were incubated with phorbol ester PMA plus calcium ionophore ionomycin. Figure 5A depicts the induction of necrosis (N) and apoptosis (A) relative to unstimulated cells. The results showed that both membrane stimuli (anti-CD3 and PHA), but not PMA + ionomycin, induced a stronger apoptotic response (A plots) throughout the analyzed timescale (see A plots in Fig. 5A, with representative profiles in Fig. 5B, A quadrants). The relative induction of necrosis, however, was similar in
and
+ T cells (N plots), although unstimulated transformed
T cell samples showed a high proportion of spontaneously necrotic, but not apoptotic, cells (Fig. 5B). We conclude that
T cells are specifically more prone to TCRCD3-induced apoptosis than
+ T cells.
Defective cytoskeleton control and TCRCD3-induced specific adhesion
Signals propagated by the TCRCD3 complex have been shown to functionally up-regulate the integrin LFA-1 (CD11a/CD18) and to induce adhesion on ICAM-1 (33). We thus next analyzed whether this physiological function was affected in the mutant
T cells. As a control, cells were stimulated with an activating mAb specific for CD18 (KIM-127, anti-LFA-1 in Fig. 6) or with phorbol ester PDBu, both of which are known to induce LFA-1-mediated adhesion to ICAM-1. Importantly, unstimulated
and
+ T cells originally expressed similar amounts of CD18 (Fig. 1) and CD11a (not shown), and their basal adhesion to ICAM-1 was also comparable (Fig. 6B). As shown for untransformed T cells (33,34), specific adhesion by transformed
+ T cells to ICAM-1-coated plates increased upon LFA-1 activation by any of the three stimuli (anti-CD3, anti-LFA-1 or PDBu), albeit with different kinetics and relative strength (Fig. 6A). In contrast, the number of ICAM-1-attached
T cells did not change after stimulation with two different anti-CD3 mAb (UCHT1, Fig. 6 and F101.01, not shown), whereas an increase comparable to that of
+ T cells was evident with anti-LFA-1 or PDBu. We conclude that TCRCD3-mediated signaling that led to enhanced adhesion onto ICAM-1 was selectively defective in
T cells.
The augmented adhesion was accompanied by profound morphological changes characterized by polarization (long, frequently unique, projections) in treated
+ T cells (Fig. 6A and B) (35). In contrast, almost no polarization was observed in
T cells with anti-CD3, whereas it was present (with delayed kinetics) when stimulated with anti-LFA-1. Anti-CD3 stimulation, however, did induce spreading in
T cells, confirming that the mutant TCRCD3 complex transduced certain signals involved in morphology control. Interestingly, PDBu stimulation did not result in any polarization of
T cells, despite their normal adhesion behavior, indicating that these two activation events are dissociated in
T cells. Normal adhesion induction with no cellular polarization has been reported previously in anti-LFA-1-stimulated T lymphoblasts treated with tyrosine kinase inhibitors (33). Thus, the results in
T cells suggested an abnormal cytoskeleton control via TCRCD3, which could not be corrected by direct protein kinase C (PKC) activation.
TCRCD3-induced tyrosine phosphorylation
Next, we analyzed protein tyrosine phosphorylation following TCRCD3 triggering (36,37). The results (Fig. 7) indicated that
T cells were essentially normal in terms of tyrosine phosphorylation specificity (arrows), although the response was slightly unsustained for certain proteins (40, 42, 70 and 81 kDa). We conclude that
T cells can undergo tyrosine phosphorylation of the appropriate target substrates after TCRCD3 triggering, although with slightly unsustained kinetics for some proteins.
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Discussion
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Effects of HVS on T cell signaling
HVS-transformed T cells have proved to be a good model system to study T lymphocyte physiopathology (38). The fact that they are pure antigen- and mitogen-independent homogeneous cell lines, as well as their growth kinetics, makes them particularly suited for biochemical and functional studies of congenital human T cell disorders, where enough blood samples are a major ethical hurdle (20). However, the effect of HVS on T cell signal transduction has to be considered in relation to our observations. HVS infection transforms lymphocytes into autocrine antigen-independent growth by means of only two constitutive viral proteins, tyrosine kinase-interacting protein (Tip) and HVS transformation-associated protein of C strains (Stp-C) (39). Both viral proteins interact with, and probably activate, the cytosolic host signaling proteins Lck and Ras (38). Therefore, viral interference may change or even restore the original signaling defects. It has been reported that HVS restored certain CD2- and CD3-mediated signaling dysfunctions in Zap-70-deficient T cells, such as calcium flux, MAPK activation, cytokine production and proliferation (40). However, activation-induced tyrosine phosphorylation of
remained defective in the same study and others have shown that a subset of primary Zap-70-deficient T cells in fact already have partially restored CD3-induced responses (24). Therefore, rather than actually restore signaling as a side-effect of transformation on any given T cell, HVS may confer a selective growth advantage in vitro to those primary T cells which were originally different anyway. Indeed, restored signaling correlated with high content of the Zap-70-related Syk protein tyrosine kinase, both in transformed and in primary Zap-70-deficient T cells. CD45RO overexpression in transformed, but not primary, CD8+
cells (Fig. 1) could be due to a similar mechanism. Together, available evidence indicates that, if anything, HVS could restore, but not cause, T cell signaling defects, so that we may be under- rather than over-estimating the contribution of CD3
to TCRCD3 regulation and signaling. The present work identified TCRCD3-induced mature T cell responses which require the involvement of CD3
, and distinguished them from other responses where CD3
seems redundant. Alternatively, the affected responses may require a higher signaling strength, which the mutant TCRCD3 complex is unable to deliver.
Contribution of CD3
to surface TCRCD3 regulation
The results presented herein demonstrate that high numbers of
TCRCD3 complexes reach the cell surface and can be down-regulated by anti-CD3, but not by PMA (Fig. 3 and 4). Preserved ligand-induced TCRCD3 down-regulation has also been reported in fresh
PBL (41) and confirms a wealth of data showing that ligand-induced TCRCD3 internalization may be mediated by CD3 chains other than
(28). However, the extent of receptor down-regulation in CD3
-deficient T cells was consistently lower than in normal cells (Fig. 3), indicating that CD3
contributes to, but is not absolutely required for, TCRCD3 down-regulation, as shown in Jurkat
T cell mutants (42). These results contrast with those recently published in murine
peripheral blood lymphocytes showing no ligand-induced TCRCD3 down-regulation (11). Further studies will be required to settle this discrepancy, although inter-species differences in the regulation of TCRCD3-mediated signaling cannot be discarded. In this regard, it is known that the peripheral T cell compartment of humans is less affected than that of mice by the lack of CD3
or
(5).
Defective PMA-induced TCRCD3 down-regulation in the absence of CD3
is consistent with the crucial role demonstrated in this process for certain phosphoserine-dependent leucine-based endocytic signals mapping to the cytosolic tail of CD3
(22). Constitutive TCRCD3 internalization and recycling in unstimulated T cells is believed to be controlled also by serine phosphorylation of CD3
(43). Therefore, the lower basal TCRCD3 expression observed in unstimulated T cells (Fig. 1) could be due to an impaired assembly rate, as internalization and recycling should be absent or reduced (44,45). Our previous biochemical results which demonstrated an impaired association of
TCRCD3 to CD3
support this contention (18,20,46). Experiments are underway to test this hypothesis. Alternatively, or in addition, CD3
could improve the stability or accessibility of the TCRCD3 complex.
Contribution of CD3
to TCRCD3-mediated signaling
Following receptor engagement, we observed normal (CD71 and CD80) or slightly delayed (CD3, CD4, CD8 and CD25) regulation of different cell surface molecules (Figs 2 and 3). However, LFA-1-dependent adhesion was decreased (Fig. 6) and apoptosis was increased (Fig. 5), perhaps as a consequence of an unsustained phosphorylation of downstream substrates (Fig. 7). Our previous studies in human CD3
-deficient primary T cells, IL-2-dependent T cell lines and HVS-transformed T lymphocytes indicated that, despite its negative effect on TCRCD3 expression, CD3
is dispensable for several TCRCD3-induced mature T cell responses, including calcium flux, cytotoxicity, CD69 or CD154 up-regulation, proliferation and synthesis of certain cytokines (TNF-
). In contrast, PMA-induced TCRCD3 down-regulation and TCRCD3-induced synthesis of other cytokines (IL-2) were severely impaired (1720). Normal, reduced, delayed or abrogated T cell responses have also been reported in murine mature CD3
-deficient fresh T lymphocytes (11). For instance, relatively normal proliferation, cytokine production and cytolysis were observed in the murine model under certain conditions, whereas suboptimal kinetics or stimuli revealed defective responses. However, these assays were performed using fresh lymphocytes, which contain a variable mixture of heterogeneous CD4+ and CD8+ cells, the percentage of which may differ from normal controls, as reported in the human homologues (16). For instance, murine
lymphocyte samples contained relatively more CD8+ and less CD4+ cells compared to controls (11). Transformed T cells are clearly more homogeneous (Fig.1) and therefore allow a more precise molecular dissection of the signaling circuitry of T cells, albeit only for those responses that are preserved. This characteristic may explain, at least in part, the minor discrepancies between fresh human or murine
T cells and the results that we obtained using pure transformed T cells.
Taken together, these results and our previously published data (19) suggest a specialized contribution by CD3
to certain T cell responses, such as adhesion or IL-2 induction, and a redundant role for other responses, such as proliferation, cytotoxicity or TNF-
induction. Alternatively, these differential functional outcomes could result from the impaired TCRCD3 expression (11), structural instability (20) or inefficient signaling. However, we believe that this is unlikely for several reasons. First, despite their differences in surface TCRCD3 (Fig. 1), CD4+ and CD8+
T cells showed a similar regulation of different cell surface molecules (CD3, CD71, CD4 and CD8, see Figs 2 and 3), and other functional responses (19). Second, pure
T cells stimulated in identical conditions can show normal or defective responses, such as TNF-
versus IL-2 cytokine induction (see above). Third, TCR-stimulated
murine thymocytes showed impaired activation of the ERK, but not JNK or p38, pathway when compared to normal control cells selected for comparable TCR expression levels (12), suggesting that this differential signaling is not related to the surface numbers or structure of TCR complexes, Obviously, a quantitative interpretation cannot be excluded. Lack of
may decrease signal strength by the mutant TCRCD3 and affect those responses requiring higher signaling thresholds. However, this seems unlikely in view that quantitative control of TCRCD3 levels in transgenic mice has shown that reduced TCR levels results in attenuated responses, but also in markedly reduced response thresholds in terms of TCR numbers (as low as 1/20th of normal) (47).
The early intracellular propagation of signals through the mutant TCRCD3 complex requires further investigation. Calcium flux was normal (19,20), but tyrosine phosphorylation kinetics was slightly altered (Fig. 7). Particularly relevant to our results is the recent report demonstrating that the TCR-proximal transmembrane adaptor termed TRIM can inhibit TCRCD3 internalization and apoptosis, and regulate the cytoskeleton through its interaction with CD3
and, to a lesser extent, with CD3
and CD3
(48,49). An impaired association of TRIM to the mutant
TCRCD3 complex (perhaps, again, as a consequence of its impaired association to CD3
, see above) may potentially result in low constitutive TCRCD3 expression (Fig. 1), high TCRCD3-induced apoptosis (Fig. 5), and defective cell adhesion and cytoskeleton control (Fig. 6). In this regard, murine
T cells have been proposed to be more prone to activation-induced cell death in vivo as a consequence of impaired signal extinction due to defective TCR down-regulation (11).
The complex interactions of the TCR with the cytoskeleton are an area of intense research (50,51).
T cells showed no TCRCD3- or PDBu-induced cell polarization, whereas LFA-1-induced morphological changes were present, although with delayed kinetics. As the actin cytoskeleton has an important role in LFA-1-dependent polarization (32), the results suggest that CD3
is required for, or participates in, the induction of actin polymerization not only through the TCRCD3, but probably indirectly through the LFA-1 integrin as well (33). Indeed, LFA-1-induced T cell migration has been shown to require Zap-70, which is crucial for TCRCD3 signaling (52). In addition, LFA-1-induced T cell polarization, but not adhesion, can be blocked with tyrosine kinase inhibitors (33). The virtual absence of TCRCD3 down-regulation using the PKC activator PMA (Fig. 4) correlates with this dissociated behavior, and suggests that PKC-induced cell polarization (but not adhesion, Fig. 6) requires rapid TCRCD3 internalization. In human T cells, mutations in downstream effectors such as WASP also result in impaired actin polymerization (53), suggesting that CD3
is required for the efficient coupling of the TCR to these cytoskeleton activation pathways, perhaps through stabilization of CD3
(see above).
Our results indicate that CD3
contributes specialized structural and signaling functions, which may improve certain TCRCD3-induced mature T cell responses. Such specialized features may be essential for survival under certain circumstances, as dramatically illustrated by a CD3
-deficient patient who died before his third birthday (17). This particular patient showed a severe lymphoid depletion at necropsy (54), which may be related to the observed defects in T cell adhesion control (Fig. 6). More often, however, signaling is sufficient for protection, as two additional
individuals are presently healthy and over their first decade (16).
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Acknowledgements
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We greatly appreciate the generous supply of Leu4 mAb and technical help by Dr B. Alarcón (Centro de Biologia Molecular, Madrid, Spain), of KIM-127 mAb by Dr Martyn Robinson (Celltech, Slough, UK), of recombinant ICAM-1-Fc by Dr Reyes Tejedor (Hospital de la Princesa, Madrid, Spain) and of rIL2 by Dr Craig W. Reynolds (Frederick Cancer Research and Development Center, NCI, Frederick, MD). Dr N. Taylor (Institut de Genetique Moleculaire, CNRS, Montpellier, France) was instrumental in setting up the phosphorylation assays and several techniques were performed at the Centro de Técnicas Inmunológicas (Universidad Complutense, Madrid, Spain) facilities. This work was supported in part by Comisión Interministerial de Ciencia y Tecnología grant PM98/91, Comunidad Autonoma de Madrid grant 8.3/21/01 and Ministerio de Educación y Cultura grant HF1999/0029 (to J. R. R). P. S. T was supported by the Universidad Complutense de Madrid, and D. A. Z and A. P. C were supported by the Comunidad Autónoma de Madrid
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Abbreviations
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ITAMimmunoreceptor tyrosine-based activation motif
HVSHerpesvirus saimiri
PHAphytohemagglutinin
PIpropidium iodide
PKCprotein kinase C
PMAphorbol myristate acetate
TNFtumor necrosis factor
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References
|
---|
- Weiss, A. and Littman, D. R. 1994. Signal transduction by lymphocyte antigen receptors. Cell 7:263.
- Schraven, B., Cardine, A. M., Hübener, C., Bruyns, E. and Ding, I. 1999. Integration of receptor-mediated signals in T cells by transmembrane adaptor proteins. Immunol. Today 20:431.[ISI][Medline]
- Germain, N. R. and Stefanová, I. 1999. The dynamics of T cell receptor signaling: Complex orchestration and the key roles of tempo and cooperation. Annu. Rev. Immunol. 17:467.[ISI][Medline]
- Levett, C. N. and Eichmann, K. 1995. Receptor and signals in early thymic selection. Immunity 3:667.[ISI][Medline]
- Kappes, D. J., Alarcón, B. and Regueiro, J. R. 1995. T lymphocyte receptor deficiencies. Curr. Opin. Immunol. 7:441.[ISI][Medline]
- Malissen, B., Ardouin, L., Lin, S. Y. and Malissen, M. 1999. Function of the CD3 subunits of the pre-TCR and TCR complexes during T development. Adv. Immunol. 72:103.[ISI][Medline]
- Wange, R. L. and Samelson, L. E. 1996. Complex complexes: signaling at the TCR. Immunity 5:197.[ISI][Medline]
- Letourneur, F. and Klausner, R. D. 1992. Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of CD3
. Science 255:79.[ISI][Medline]
- Eshhar, Z., Waks, T. and Gross, G. 1993. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the
or
subunits of the immunoglobulin and T-cell receptors. Proc. Natl Acad. Sci. USA 90:720.[Abstract]
- Strauss, D. and Weiss, A. 1993. The CD3 chains of the T-cell receptor associate with ZAP-70 tyrosine kinase and are tyrosine-phosphorylated after receptor stimulation. J. Exp. Med. 178:1523.[Abstract]
- Haks, M. C., Cordaro, T. A., van sen Brakel, J. H. N., Haanen, J. B. A. G., de Vries, E. F. R., Borts, J., Krimperfort, P. and Kruisbeek, A. M. 2001. A redundant role of the CD3
-immunoreceptor tyrosine-based activation motif in mature T cell function. J. Immunol. 166:2576.[Abstract/Free Full Text]
- Delgado, P., Fernández, E., Dave, V., Kappes, D. and Alarcón, B. 2000. CD3
couples T-cell receptor signaling to ERK activation and thymocyte positive selection. Nature 406:426.[ISI][Medline]
- Ardouin, L. C., Boyer, A., Gillet, J., Trucy, A., M. Bernard, J., Nunes, J., Deton, A., Trautmann, H. T. H., Malissen, B. and Malissen, M. 1999. Crippling of CD3
ITAM does not impair T cell receptor signaling. Immunity 10:409.[ISI][Medline]
- Sinkai, Y., Ma, A., Cheng, H. L. and Alt, F. W. 1995. CD3
and CD3
cytoplasmic domains can independently generate signals for T cell development and function. Immunity 2:401.[ISI][Medline]
- Brocker, B. and Karjaleinen, K. 1995. Signals through T-cell receptor
-chain alone are insufficient to prime resting T lymphocytes. J. Exp. Med. 181:1653.[Abstract]
- Zapata, D. A., Pacheco-Castro, A., Torres, P. S., Millán, R. and Regueiro, J. R. 2000. CD3 immunodeficiencies. Immunol. Allergy Clin. N. Am. 20:1.[ISI]
- Arnaiz-Villena, A., Timon, M., Corell, A., Pérez-Aciego, P., Martín-Villa, J. M. and Regueiro, J. R. 1992. Primary immunodeficiency caused by mutations in the gene encoding the CD3-
subunit of the T-lymphocyte receptor. N. Engl. J. Med. 327:529.[ISI][Medline]
- Pérez-Aciego, P., Alarcón, B., Arnaiz-Villena, A., Terhorst, C., Timón, M., Segurado, O. G. and Regueiro, J. R. 1991. Expression and function of a variant T cell receptor complex lacking CD3-
. J. Exp. Med. 174:319.[Abstract]
- Pacheco-Castro, A., Zapata, D. A., Torres, P. S. and Regueiro, J. R. 1998. Signaling through a CD3
-deficient TCRCD3 complex in immortalized mature CD4+ and CD8+ T lymphocytes. J. Immunol. 161:3152.[Abstract/Free Full Text]
- Zapata, D. A., Pacheco-Castro, A., Torres, P. S., Ramiro, A. R., San José, E., Alarcón, B., Alibaud, L., Rubin, B., Toribio, M. L. and Regueiro, J. R. 1999. Conformational and biochemical differences in the TCRCD3 complex of CD8+ versus CD4+ mature lymphocytes revealed in the absence of CD3
. J. Biol. Chem. 274:35119.[Abstract/Free Full Text]
- Lauritsen, J. P. H., Christensen, M. D., Dietrich, J., Kastrup, J., Odum, N. and Geisler, C. 1998. Two distinct pathways exist for down-regulation of the TCR. J. Immunol. 161:260.[Abstract/Free Full Text]
- Dietrich, J., Hou, X., Wegener, A. M. K. and Geisler, C. 1994. CD3
contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor. EMBO J. 13:2156.[Abstract]
- Dransfield, I., Cabañas, C., Craig, A. and Hogg, N. 1992. Divalent cation regulation of the function of the leukocyte integrin LFA-1. J. Cell Biol. 116:219.[Abstract]
- Noraz, N., Schwarz, K., Steinberg, M., Dardalhon, V., Rebouisson, C., Hipskind, R., Friedrich, W., Yssel, H., Bacon, K. and Taylor, N. 2000. Alternative antigen receptor (TCR) signaling in T cells derived from ZAP-70-deficient patients expressing high levels of syk. J. Biol. Chem. 275:15832.[Abstract/Free Full Text]
- Penninger, J. M., Irie-Sasaki, J., Sasaki, T. and Oliveira-dos-Santos, A. J. 2001. CD45: new jobs for an old acquaintance. Nat. Immunol. 2:389.[ISI][Medline]
- Timon, M., Arnaiz-Villena, A., Rodríguez-Gallego, C., Pérez-Aciego, P., Pacheco-Castro, A. and Regueiro, J. R. 1993. Selective disbalances of peripheral blood T lymphocyte subsets in human CD3
deficiency. Eur. J. Immunol. 23:1440.[ISI][Medline]
- Haks, M. C., Krimpenfort, P., Borst, J. and Kruisbeek, A. M. 1998. The CD3
chain is essential for development of both the TCR
ß and TCR
lineages. EMBO J. 17:1871.[Free Full Text]
- Alcover, A. and Alarcón, B. 2000. Internalization and intracellular fate of TCRCD3 complexes. Crit. Rev. Immunol. 20:325.[ISI][Medline]
- Dietrich, J., Kastrup, J., Nielsen, B. L., Odum, N. and Geisler, C. 1997. Regulation and function of the CD3
DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro. J. Cell Biol. 138:271.[Abstract/Free Full Text]
- Dietrich, J., Bäckström, T., Lauritsen, J. P. H., Kastrup, J., Christensen, M., von Bülow, F., Palmer, E. and Geisler, C. 1998. The phosphorylation state of CD3
influences T cell responsiveness and controls T cell receptor cycling. J. Biol. Chem. 273:24232.[Abstract/Free Full Text]
- Geisler, C., Dietrich, J., Nielsen, B. L., Kastrup, J., Lauritsen, J. P., Odum, N. and Christensen, M. D. 1998. Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane. J. Biol. Chem. 273:21316.[Abstract/Free Full Text]
- San José, E., Sahuquillo, A. G., Bragado, R. and Alarcón, B. 1998. Assembly of the TCRCD3 complex: CD3
/
and CD3
/
dimers associate indistinctly with both TCR
and TCRß chains. Evidence for a double TCR heterodimer model. Eur. J. Immunol. 28:12.[ISI][Medline]
- Rodríguez-Fernández, J. L., Gómez, M., Luque, A., Hogg, N., Sánchez-Madrid, F. and Cabañas, C. 1999. The interaction of activated integrin lymphocyte function-associated antigen 1 with ligand intercellular adhesion molecule 1 induces activation and redistribution of focal adhesion kinase and proline-rich tyrosine kinase 2 in T lymphocytes. Mol. Biol. Cell. 10:1891.[Abstract/Free Full Text]
- Grakoui, A., Bromley S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. and Dustin, M. L. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.[Abstract/Free Full Text]
- Serrador, J. M., Nieto, M. and Sánchez-Madrid, F. 1999. Cytoskeletal rearrangement during migration and activation of T lymphocytes. Trends Cell Biol. 9:228.[ISI][Medline]
- Mittrücker, H.-W., Müller-Fleckenstein, I., Fleckenstein, B. and Fleischer, B. 1993. Herpesvirus saimiri-transformed human T lymphocytes: normal functional phenotype and preserved T cell receptor signaling. Int. Immunol. 5:985.[Abstract]
- Bröker, B. M., Tsygankov, A. Y., Müller-Fleckenstein, I., Guse, A. H., Chitaev, N. A., Biesinger, B., Fleckenstein, B. and Emmrich, F. 1993. Immortalization of human T cell clones by Herpesvirus saimiri. J. Immunol. 151:1184.[Abstract/Free Full Text]
- Meinl, E. and Hohfeld, R. 2000. T cell transformation with Herpesvirus saimiri: a tool for neuroimmunological research. J. Neuroimmunol. 103:1.[ISI][Medline]
- Duboise, S. M., Guo, J., Czajak, S., Desrosiers, R. C. and Jung, J. U. 1998. STP and Tip are essential for Herpesvirus saimiri oncogenicity. J. Virol. 72:1308[Abstract/Free Full Text]
- Meinl, E., Derfuss, T., Pirzer, R., Blank, N., Lengenfelder, D., Blancher, A., Le Deist, F., Fleckenstein, B. and Hivroz, C. 2001. Herpesvirus saimiri replaces ZAP-70 for CD3- and CD2-mediated T cell activation. J. Biol. Chem. 276:36902.[Abstract/Free Full Text]
- Rodríguez-Gallego, C., Corell, A., Pacheco, A., Timón, M., Regueiro, J. R., Allende, L. M., Madroño. A. and Arnaiz-Villena, A. 1996. Herpesvirus saimiri transformation of T cells in CD3
immunodeficiency: phenotypic and functional characterization. J. Immunol. Methods 198:177.[ISI][Medline]
- von Essen, M., Menne, C., Nielsen, B. L., Lauritsen, J. P., Dietrich, J., Andersen, P. S., Karjalainen, K., Odum, N. and Geisler, C. 2002. The CD3 gamma leucine-based receptor-sorting motif is required for efficient ligand-mediated TCR down-regulation. J. Immunol. 168:4519.[Abstract/Free Full Text]
- Krangel, M. S., Al-Haideri, M., Pernis, B., Cantor, C. R. and Wang, C. Y. 1987. Endocytosis and recycling of the T3T cell receptor complex. J. Exp. Med. 165:1141.[Abstract]
- Dietrich, J., Hou, X., Wegener, A. M. K., Ostegaard Pedersen, L., Odum, N. and Geisler, C. 1996. Molecular characterization of the di-leucine-based internalization motif of the T cell receptor. J. Biol. Chem. 271:11441.[Abstract/Free Full Text]
- Liu, H., Rhodes. M., Wiest., D. L. and Vignali, D. A. 2000. On the dynamics of TCR:CD3 complex cell surface expression and downmodulation. Immunity 13: 665.
- Alarcón, B., Regueiro, J. R. and Arnaiz-Villena, A. 1993. Familial defect in the surface expression of the T-cell receptorCD3 complex. N. Engl. J. Med. 319:1203.[ISI][Medline]
- Labrecque, A., Simon Whitfield, L., Obst, R., Waltzinger, C., Benoist, C. and Mathis, D. 2001. How much TCR does a T cell need? Immunity 15:71.[ISI][Medline]
- Bruyns, E., Marie-Cardine, A., Kirchgessner, H., Sagolla, K., Shevchenko, A., Mann, M., Autschbach, F., Bensussan, A., Meuer, S. and Schraven, B. 1998. T cell receptor (TCR) interacting molecule (TRIM), a novel disulfide-linked dimer associated with the TCRCD3-
complex, recruits intracellular signaling proteins to the plasma membrane. J. Exp. Med. 188:561.[Abstract/Free Full Text]
- Kirchgessner, H., Dietrich, J., Scherer, J., Isomäki, P., Hilgert, I., Bruyns, E., Leo, A., Cope, A. P. and Schraven, B. 2001. The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR
chain. J. Exp. Med. 11:1269.
- Caplan, S. and Baniyash, M. 2000 Searching for significance in TCR-cytoskeleton interactions. Immunol. Today 21:223.
- Dustin, M. L. and Cooper, J. A. 2000. The actin cytoskeleton and the immunological synapse: molecular hardware for T cell signalling. Nat. Immunol. 1:23.[ISI][Medline]
- Soede, R. D. M., Wijnands, Y. M., van Kouteren-Cobzarn, I. and Roos, E. 1998. Zap-70 tyrosine kinase is required for LFA-1 dependent T-cell migration. J. Cell Biol. 442:1371.
- Gallego, M. D., Santamaría, M., Peña, J. and Molina, I. 1997. Defective actin reorganization and polymerization of WiskottAldrich T cells in response to CD3-mediated stimulation. Blood 8:3089.
- Arnáiz-Villena, A., Pérez-Aciego, P., Ballestin, C., Sotelo, T., Pérez-Seoane, C., Martín-Villa, J. M. and Regueiro, J. R. 1991. Biochemical basis of a novel T-lymphocyte receptor immunodeficiency by immunohistochemistry: a possible CD3
abnormality. Lab. Invest. 64: 675.