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*
Institute of Biochemistry, University of Lausanne, BIL Research Center, Epalinges, Switzerland;
Parke-Davis Research Institute, Paris, France; and
Institute of Molecular Medicine, Medical College of Georgia, Augusta, GA
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain degradation,
indicating that ZAP-70 is degraded in parallel with TCR internalization
and degradation. Pharmacological activation of protein kinase C (PKC)
does not induce ZAP-70 degradation, which, on the contrary, requires
activation of protein tyrosine kinases. Two lines of evidence indicate
that the Ca2+-dependent cysteine protease calpain plays a
major role in initiating ZAP-70 degradation: 1) treatment of T cells
with cell-permeating inhibitors of calpain markedly reduces ZAP-70
degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show
that, in the course of T cell-APC cognate interaction, ZAP-70 is
rapidly degraded via a calpain-dependent
mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß dimer, the CD3 
chains, and the homodimer. Whereas the ß heterodimer is
responsible for specific Ag recognition, the associated CD3 chains and
homodimer are necessary for receptor complex expression
(2) and signal transduction (3). Following
TCR engagement by the specific ligand, the cytoplasmic domains of the
CD3 subunits and homodimer become rapidly phosphorylated by Lck or
Fyn, two members of the src-family protein tyrosine kinase
(PTK), within 16 aa motifs termed immunoreceptor tyrosine-based
activation motifs (ITAMs)3
(3). A subsequent critical step of TCR-mediated signal
transduction is the recruitment of the PTK ZAP-70 to engaged TCRs via
anchorage of ZAP-70 Src homology 2 (SH2) domains to di-phosphorylated
ITAM motifs present in the cytoplasmic portion of the TCR subunit
(4). After recruitment to triggered TCR/CD3- complexes,
ZAP-70 is activated by tyrosine phosphorylation and in turn activates,
by recruiting and phosphorylating cellular substrates (3),
downstream effectors, such as calcineurin and Ras pathways
(5). The critical role of ZAP-70 in transducing
TCR-generated signals has been proven by several studies showing that,
in conditions in which recruitment and activation of ZAP-70 is
impaired, TCR-mediated signal transduction is aborted
(6, 7, 8). Upon conjugation with APCs, T cells undergo a sustained [Ca2+]i increase (9, 10) that results from the serial engagement and triggering of many TCRs by a small number of peptide-MHC complexes (11). A key feature of T cell Ag recognition is that the process of TCR/peptide-MHC interaction is self limited by the down-regulation and degradation in the lysosomes of triggered TCRs (11, 12). The molecular mechanism of TCR sorting in the endocytic pathway is presently not defined, and in particular the fate of TCR-recruited signaling components is not clear once receptors are removed from the T cell surface and targeted to lysosomes for degradation (12, 13). In the present study, we investigated the fate of ZAP-70 in human T cells interacting with peptide-pulsed APCs. We report that stimulation by the specific Ag results, in parallel with TCR down-regulation and degradation, in a rapid and profound degradation of ZAP-70 via a calpain-dependent mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two DRBI*0101-restricted T cell clones (6396p5.1.2 and SDM3.5) specific for the measles virus fusion protein peptide F254–268 (GDLLGILESRGIKAR) and a DRBI*1104-restricted T cell clone (KS140) specific for the tetanus toxin peptide TT830–843 (QYIKANSKFIGITE) were used. DRI-matched EBV-transformed B cells (LG-2, DRBI*0101; and KS-EBV, DRBI*1104) were used as APCs. T cell clones and EBV-B cell lines were generated and maintained as described (10).
Intracellular staining for CD3, -chain, and ZAP-70
EBV-B cells were pulsed for 2 h at 37°C with various
concentrations of F254–268 or of TT830–843 in RPMI 5% FCS. During
the last 10 min, 1 µM BCECF-AM
(2',7-bis-(carboxyethyl)-5(6')-carboxyfluorescin; Calbiochem, San
Diego, CA) was added, and the cells were washed three times. T cells
were mixed with EBV-B cells at a 1:2 ratio in 200 µl RPMI 5% FCS in
U-bottom microplates, centrifuged 1 min at 1500 rpm to allow conjugate
formation, and incubated at 37°C. In some experiments, T cells were
pretreated for 1 h with 10 µg/ml cycloheximide (CHO); in other
experiments, T cells were pretreated for 10 min with 10 µM PP1
(14); in additional experiments, T cells were pretreated
for 30 min either with 100 µM calpeptin (15) or with 100
µM PD150606 (16). Drugs were purchased from Calbiochem.
The drugs were present throughout the assay. At different times, the
cells were resuspended, washed in PBS 0.5 mM EDTA, and fixed for 10 min
with 3% paraformaldehyde. After three washings with PBS containing 3%
BSA, the cells were permeabilized for 10 min at room temperature with
washing buffer (HEPES-buffered PBS containing 0.1% saponin, 3% BSA)
and stained with anti-CD3 (TR66; 10), anti- (6B10.2,
Santa Cruz Biotechnology, Santa Cruz, CA), or anti-ZAP-70 mAb
(2F3.2, directed against the SH2 portion of ZAP-70; 17) in
HEPES-buffered PBS containing 0.1% saponin and 5% BSA (or 5% FCS),
followed by a PE-labeled goat anti-mouse Ab (Southern Biotechnology
Associates, Birmingham, AL). The CD3, , and ZAP-70 fluorescence were
analyzed on a FACScan (Becton Dickinson, Mountain View, CA). EBV-B
cells were gated out using both forward and side scatter (FSC/SSC)
parameters and green BCECF fluorescence.
In some experiments T cells were conjugated with peptide-pulsed or unpulsed EBV-B cells previously loaded for 10 min at 37°C with 0.5 µM Orange-CMTMR (Molecular Probes, Leiden, The Netherlands); after 2 h incubation at 37°C, the cells were gently resuspended and laid on poly-L-lysine-coated slides for 10 min at 37°C. The cells were either untreated or treated with 100 µM calpeptin (Calbiochem); the drug was present in the culture throughout the assay. The cells were fixed for 10 min with 3% paraformaldehyde, permeabilized for 10 min with washing buffer and stained with two different anti-ZAP-70 rabbit Ab (either ZAP-70 LR (Santa Cruz) or ZAP-4 (kindly provided by Dr. S. Ley, Division of Cellular Immunology, NIMR, London, U.K.)), followed by FITC-labeled goat anti-rabbit Ab (SBA). In some experiments, the cells were treated with 1 µM TOTO-3 (Molecular Probes) to stain cell nuclei. The samples were mounted in 90% glycerol-PBS containing 2.5% 1-4-diazabicyclo (2.2.2) octane (DABCO; Fluka, Buchs, Switzerland) and were examined using a Carl Zeiss LSM 410 confocal microscope (Carl Zeiss, Oberkochen, Germany).
ZAP-70 detection by Western blot
EBV-B cells were pulsed with various concentrations of F254–268 or of TT830–843. T cells (5 x 105) were mixed with 106 EBV-B cells in 200 µl RPMI 5% FCS in U-bottom tubes, centrifuged to allow conjugate formation, and incubated at 37°C for 2 h. In some experiments, T cells alone were either untreated or treated with 100 nM PMA, or 1:50 pervanadate (0.035% H2O2 + 100 mM Na3VO4, 1:1 ratio), or with 1 µg/ml ionomycin. In some experiments, T cells were pretreated with 100 µM calpeptin for 30 min before stimulation. The drug was present in the culture throughout the assay. The cells were washed twice, lysed in prewarmed Laemmli buffer, sonicated, and boiled. After separation on a 12.5% SDS-PAGE and transfer to nitrocellulose, membranes were blocked for 1 h at room temperature with blocking buffer (5% nonfat dry milk, 0.05% Tween 20 in Tris-buffered saline) and incubated for 1 h with 2F3.2 mouse mAb. Similar results were obtained when blots were reprobed with a different mouse mAb directed against the kinase domain of the ZAP-70 (Transduction Laboratories, Lexington, KY) (not shown). After washing, the membranes were incubated for 1 h with HRP-labeled goat anti-mouse Ab (SBA) in blocking buffer. Filters were developed using an enhanced chemiluminescence detection system (Pierce, Rockford, Illinois). Densitometric analysis was performed using a Hewlett Packard ScanJet 4C/T (Hewlett Packard, Palo Alto, CA) equipped with DeskScan II and Image 1.40 programs.
Detection of -chain phosphorylation by Western blot
T cells (5 x 105) were mixed with
106 EBV-B cells in 200 µl RPMI 5% FCS in
U-bottom tubes and centrifuged to allow conjugate formation. In some
experiments, T cells were pretreated with either 100 µM calpeptin or
100 µM PD150606 for 30 min or with 10 µM PP1 for 10 min before
conjugate formation. The drugs were present in the culture throughout
the assay. After different times of conjugation, the cells were washed
in ice cold PBS containing 10 mM
Na3VO4 (Sigma, St. Louis,
MO), lysed in ice cold lysis buffer (PBS containing 1% Nonidet P-40,
10 mM Na3VO4 and a mixture
of protease inhibitors from Boehringer Mannheim (Mannheim, Germany).
Post nuclear supernatants were mixed with sample buffer, sonicated,
separated on a nonreducing 12.5% SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with an anti- mAb (6B10.2,
Santa Cruz) directed against the transmembrane domain of human
-chain. Filters were developed using an enhanced chemiluminescence
detection system (Pierce).
In vitro transcription and translation of expression constructs
The rabbit reticulocyte-coupled transcription/translation system TnT T7 (Promega, Madison, WI) was used for expression of proteins in vitro, according to the manufacturer’s instructions. Plasmid DNAs of ZAP-70 and receptor interacting protein (RIP) (1 µg/reaction) were used for the TnT reactions conducted at 30°C for 90 min in a total volume of 50 µl. T7 RNA polymerase, trans-[35S]methionine label (9 µCi) (Amersham, Arlington Heights, IL) and one unit RNase inhibitor (Boehringer Mannheim) were added to the TnT reactions.
In vitro proteolysis assay
The [35S]methionine-labeled ZAP-70 and RIP in vitro-translated products were suspended in a proteolysis buffer (20 mM Tris-HCl (pH 7.4), 10 mM 2-ME) on ice, and then incubated with 0.1 units of purified m-calpain (Sigma) at various concentrations of CaCl2 at 30°C for 30 min. Samples were resolved on 12% SDS-PAGE and transferred to nitrocellulose. Blots were subjected to autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells conjugated with peptide-pulsed APCs undergo rapid
down-regulation and degradation of triggered TCR/CD3- complexes that
plateau within 2 h (11, 12). To investigate whether
ZAP-70, which is known to bind to the -chain with high affinity upon
receptor triggering (18), might share the fate of
triggered and internalized TCRs, ZAP-70 levels were measured by FACS
analysis in fixed and permeabilized T cells following Ag
stimulation.
In T cells conjugated with peptide-pulsed APCs, the content in ZAP-70
decreased, in parallel with CD3 and -chain internalization and
degradation, with increasing doses of Ag (Fig. 1
, A–C). Treatment of
T cell-APC conjugates with the src-kinase inhibitor PP1 (10
µM) resulted in a marked inhibition of TCR/CD3- internalization
and degradation, as well as inhibition of ZAP-70 degradation (Fig. 1, B and C). On the other hand, CHO-mediated
inhibition of protein synthesis did not affect Ag-induced ZAP-70 loss,
indicating that disappearance of this signaling component was not due
to a decreased synthesis (Fig. 1D). Interestingly, the time
kinetics of ZAP-70 consumption overlapped with that of -chain
degradation, since both reached a plateau in about 120 min (Ref. 12 and
Fig. 1E).
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, a dose-dependent
decrease of ZAP-70 expression was observed in T cells conjugated with
peptide-pulsed APCs. Reexpression of basal levels of ZAP-70 in T cells
was observed about 48–72 h after conjugate formation (not shown).
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complex
and ZAP-70. Dissociation between TCR down-regulation and ZAP-70 degradation in PMA-stimulated T cells
Several lines of evidence indicate that two different pathways
exist for regulation of TCR surface levels: 1) TCR internalization can
be induced by pharmacological activation of protein kinase C (PKC),
which leads to phosphorylation of a target serine residue
(Ser126) within the CD3 leucine-based
internalization motif (19); 2) ligation of TCR by the Ag
or by anti-TCR/CD3 Abs results in a TCR down-regulation that
requires Lck- and Fyn-mediated tyrosine phosphorylation
(20). The first internalization pathway via PKC leads to
rapid TCR recycling and reexpression at the T cell surface
(21), whereas the second pathway mediated by tyrosine
phosphorylation leads to TCR degradation in the lysosomes (12, 13). To investigate which of these two pathways might be
responsible of ZAP-70 degradation, surface levels of CD3, as well as
total cellular levels of CD3, -chain, and ZAP-70, were measured
in T cells stimulated with 100 nM PMA. As shown in Fig. 3, treatment with PMA induced only TCR/CD3
complexes internalization in the absence of TCR/CD3 and ZAP-70
degradation.
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, B and
C, the above results indicate that ZAP-70 degradation is not
induced by pharmacological activation of PKC, but occurs in parallel
with TCR/CD3 degradation, via an src-kinase-dependent
mechanism. Degradation of ZAP-70 via a calpain-dependent mechanism
We next investigated the mechanisms by which ZAP-70 could be
degraded in Ag-stimulated T cells. We have previously shown that
treatment of T cell-APC conjugates with bafilomycin A1, a powerful and
selective inhibitor of lysosome function, inhibits -chain
degradation in Ag-stimulated T cells (12). However,
treatment of Ag-stimulated T cells with this drug failed to block
degradation of ZAP-70, indicating that ZAP-70 is not degraded, in
association with TCR, in the lysosomal compartment (data not
shown).
Since the calcium-dependent neutral protease calpain has been reported to play a role in the T cell activation process (15, 22), we investigated whether ZAP-70 degradation might result from activation of this proteolytic pathway. T cells were stimulated with peptide-pulsed APCs in the presence of 100 µM calpeptin, a specific inhibitor of calpain proteolytic activity (15).
Treatment with this drug inhibited ZAP-70 degradation as detected by
Western blot analysis on total cell lysates and FACS analysis on fixed
and permeabilized T cells (Fig. 4,
A–C). Interestingly, in parallel experiments,
treatment with calpeptin also inhibited Ag-induced -chain
degradation to a similar extent, indicating that ZAP-70 degradation is
mandatory for -chain targeting to lysosomes. To investigate whether
calpeptin could be toxic for T cells, we tested whether it could
interfere with Ag-induced -chain phosphorylation. To this end,
-chain phosphorylation was detected as a m.w. shift in Western blot
analysis of T cell/APC total cell lysates probed with an anti-
chain Ab directed against the transmembrane domain of -chain.
Treatment of T cells with 100 µM calpeptin did not affect the
Ag-induced -chain m.w. shift, indicating that this drug does not
inhibit T cell/APC conjugate formation and TCR engagement (data not
shown).
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C) and by Western blot analysis (data
not shown). It should be noted that calpeptin and PD150606 inhibit
calpain function by acting at two different levels (calpeptin blocks
the proteolytic domain of calpain, whereas PD150606 acts at the level
of the calcium-binding regulatory domain of calpain)
(23).
Since calpains are
[Ca2+]i-dependent enzymes
(23), we tested whether ZAP-70 degradation could be simply
achieved in T cells by inducing
[Ca2+]i increase with
ionophores. As shown in Fig. 4D, treatment of T cells with
ionomycin did not induce a strong ZAP-70 degradation. Conversely,
treatment with pervanadate (a powerful and not specific stimulus for
tyrosine phosphorylation), induced a profound degradation of ZAP-70,
which was blocked by calpeptin (Fig. 4D). This indicates
that, in T lymphocytes,
[Ca2+]i rise is not
sufficient by itself to induce ZAP-70 degradation, yet activation of
PTKs is required.
Taken together, these results indicate that, in Ag-stimulated T cells, cleavage of ZAP-70 by the neutral protease calpain leads to ZAP-70 degradation.
In vitro proteolysis of ZAP-70 by calpain
To better define whether ZAP-70 is substrate for calpain, we incubated in vitro-translated, [35S]methionine-labeled ZAP-70 with purified calpain in the presence of different concentrations of CaCl2.
As shown in Fig. 5A, a
Ca2+-dependent proteolysis of ZAP-70 was
observed, resulting in the appearance of an 
35-kDa proteolytic
fragment. ZAP-70 cleavage was not observed, at high
Ca2+ concentrations, in the absence of calpain.
In addition, calpain-induced ZAP-70 proteolysis was inhibited by three
different inhibitors of calpain (20 µM calpastatin, 100 µM
calpeptin, and 100 µM PD150606). Finally, to verify the specificity
of the in vitro proteolysis of ZAP-70 by calpain, we incubated an in
vitro-translated, [35S]methionine-labeled
unrelated protein (RIP) with calpain in the presence or in the absence
of high Ca2+ concentrations. As shown in Fig. 5B, incubation of RIP with calpain did not result in the
appearance of a major proteolytic fragment.
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Morphological evidence of ZAP-70 degradation in Ag-stimulated T cells
T cells were conjugated with peptide-pulsed or unpulsed APCs,
stained with two different anti-ZAP-70 Abs, ZAP-70 LR (Fig. 6, a, c, e,
and g) or ZAP-4 (Fig. 6, b, d,
f, h, i, and j), and
examined by confocal microscopy. In unstimulated human T cell clones
(Fig. 6, a and b), ZAP-70 does not appear to be
localized at the cell cortex, as described for Jurkat cells
(24), but is mostly diffusely distributed throughout the
cytosol. By contrast, staining for ZAP-70 was strongly reduced in T
cells that had been conjugated for 2 h with peptide-pulsed APCs
(Fig. 6, c and d). Pretreatment of T cells with
calpeptin did not affect ZAP-70 expression in unstimulated T cells
(Fig. 6, e and f) but markedly reduced Ag-induced
ZAP-70 degradation (Fig. 6, g and h).
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, g and h). The localization in
the nucleus was equally detectable with both the Abs but was in some
conjugates only partial, whereas in others more pronounced. This
observation was confirmed by evidencing T cell nucleus in a three color
staining (Fig. 6, i and j). A constitutive
localization of ZAP-70 in the nucleus has been previously reported in
GFP/ZAP-70-transfected Jurkat cells (25). Here, we show
that, in unstimulated human T cell clones, ZAP-70 does not reside in
the T cell nucleus but appears to be shifted following antigenic
stimulation. Taken together, the above results confirm those obtained by FACS and Western blot analysis and provide a morphological evidence of ZAP-70 degradation in Ag-stimulated T cells via a calpain-dependent mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thus, T cell Ag recognition appears to be a peculiarly regulated process, since, on the one hand, sustained signaling is required for full T cell activation (9), whereas, on the other hand, maintenance of signaling itself progressively reduces T cell responsiveness.
In the present work, we focused on the fate of ZAP-70 in T cells stimulated for prolonged times by the Ag displayed on the APC surface. We report that sustained T cell-APC interaction results in Ag dose- and time-dependent degradation of ZAP-70 via a calpain-dependent mechanism.
Calpains are a family of ubiquitous, Ca2+-dependent, cytosolic cysteine proteases involved in the cleavage of several cellular substrates, including cytoskeletal elements, transcription factors, and signaling components (23). Calpain is highly expressed in T cells, and its expression and proteolytic activity are increased following TCR/CD3 triggering (22, 28). In T cells, calpain has been reported to play a role in the induction of apoptosis (16, 29) and in cell-cell adhesion (15), but its role in the early TCR-mediated signal transduction has not been elucidated.
Here, we propose that calpain interferes with the earliest steps of T cell activation as indicated by two independent experimental observations: 1) the inhibition of ZAP-70 degradation by calpain inhibitors in Ag-stimulated T cells and 2) the cleavage of ZAP-70 by calpain in vitro.
The molecular mechanisms of ZAP-70 degradation in T lymphocytes, and the role played by calpain in initiating this process, are presently not clear. We propose that cleavage of ZAP-70 by calpain could result in the production of unstable fragments, promptly degraded by cellular degradative pathways.
Accordingly, we could not detect degradation fragments of ZAP-70 by
Western blot analysis in Ag-stimulated T cell clones, whereas
overexpression of ZAP-70 in Jurkat cells resulted in the formation of a
35-kDa fragment (F. Martinon and D. Penna, unpublished observations),
corresponding to that observed in the in vitro proteolysis assay
(Fig. 5).
Interestingly, using the PEST-SEARCH program, we found that ZAP-70 contains one region rich in proline (P), glutamic acid (E), serine (S) and threonine (T), which is considered to be typical of proteins that are substrate of calpain (30) and is located in a position compatible with the dimension of the fragment obtained in the in vitro proteolysis experiments (ZAP-70 306–326).
An intriguing question that derives from our observation of a parallel
degradation of -chain and ZAP-70 proteins in Ag-stimulated T cells
concerns the quantitative relationship between the degradation of these
two critical signaling components. The results presented here do not
allow a comprehensive understanding of this issue since an accurate
quantification of the number of ZAP-70 and -chain molecules per T
cell is required to define the stoichiometry of interaction between
these two molecules.
Nevertheless, it is tempting to speculate that our results are
compatible with the following model of -chain/ZAP-70 interaction and
degradation in Ag stimulated T cells. Engagement of any individual TCR
by the Ag would result in several rounds of ligation and detachment of
ZAP-70 with the same phosphorylated -chain. Phosphorylated ZAP-70
would be promptly degraded by calpain, and the number of ZAP-70
molecules present in the T cell/APC contact area would rapidly
decrease. In addition, calpain proteolytic action would also be exerted
at the level of ZAP-70 -chain complexes to rapidly release recently
engaged ZAP-70 from -chain and unmask the -chain-phosphorylated
ITAMs, which are considered to be the lysosome-targeting motifs
for -chain degradation (20).
What could be the physiological relevance of Ag-induced ZAP-70 degradation? One possibility is that degradation of ZAP-70, together with TCR down-regulation (11) and Lck inactivation (27), could contribute to terminate the signal transduction process and to control the extent of T cell activation by the Ag (26).
Alternatively, degradation of ZAP-70 could effectively favor Ag-mediated T cell activation, avoiding excessive usage of TCRs in the early phase of conjugate formation and thereby permitting sustained signaling. Since TCR engagement is a localized process linked to a relatively small two-dimensional area of cell contact (31, 32), supply of signal-transducing proteins to engaged TCRs from the three-dimensional T cell interior may represent a limiting step. Rapid degradation of ZAP-70 would then affect its availability at the T cell-APC contact area and thereby control signaling.
In agreement with this hypothesis, we have previously shown that, during sustained T cell-APC interaction, TCR triggering occurs at a relatively low rate and for a sustained time at low as well as at very high Ag concentrations (26). ZAP-70 degradation would therefore favor T cell activation by controlling the rate of TCR engagements and allowing sustained signaling even in conditions in which a massive amount of Ag is displayed on the APC surface. In other words, Ag-dependent loss of this critical signaling component would act as a pace-maker of TCR serial engagement.
In conclusion, in the present work we provide new insights to the
understanding of signaling dynamics in the T cell-APC contact area.
Large amounts of information are available concerning the molecular and
structural aspects of ZAP-70/-chain interaction. By contrast,
mechanisms that regulate and eventually terminate this process have not
been investigated.
Our results show that -chain and ZAP-70 share a common fate in the
course of T cell activation, in that both are rapidly degraded,
although via different mechanisms, following TCR engagement by the
specific Ag.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Salvatore Valitutti, Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland. E-mail address:
3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; RIP, receptor interacting protein; MFI, median fluorescence intensity; CHO, cycloheximide; PTK, protein tyrosine kinase; PKC, protein kinase C.
Received for publication February 26, 1999. Accepted for publication April 23, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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complexes after antigenic stimulation. J. Exp. Med. 185:1859. contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor. EMBO J. 13:2156.[Medline]
influences T cell responsiveness and controls T cell receptor cycling. J. Biol. Chem. 273:24232.-actinin by calpain in T cells stimulated with anti-CD3 monoclonal Ab. J. Immunol. 156:3215.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), total CD3
,
) total 
, 
) and ZAP-70
(
, 
) as a function of Ag concentration. T cells were either
untreated (filled symbols) or pretreated for 10 min with 10 µM PP1
(open symbols). D, T cells were conjugated with APCs
either unpulsed (black) or pulsed with 10 µM (diagonal stripes) or
100 µM (white) peptide. The total level of 
