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Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, Copenhagen, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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leucine-based (L-based)
internalization motif. In contrast, PKC-induced TCR down-regulation is
dependent on the CD3 L-based internalization motif but independent
of p56lck and p59fyn.
Finally, our data indicate that in the absence of TCR ligation, TCR
expression levels can be finely regulated via the CD3 L-based motif
by the balance between PKC and serine/threonine protein phosphatase
activities. Such a TCR ligation-independent regulation of TCR
expression levels could probably be important in determining the
activation threshold of T cells in their encounter with APC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR down-regulation may also play a role in induction of tolerance. Transgenic models of peripheral nonresponsiveness have demonstrated that T cell tolerance can be maintained by down-regulation of the TCR (20, 21, 22, 23). In addition to the process of tolerance induction, TCR down-regulation has been observed during T cell activation, and it has been proposed that TCR down-regulation might be involved in T cell activation by allowing serial triggering of many TCR by few peptide-MHC complexes (3, 4, 24).
Although p56lck seems to play a central role in regulation of the TCR expression levels on CD4+CD8+ thymocytes, the role of p56lck in ligand-induced TCR down-regulation in mature T cells is not clear. In one study, TCR down-regulation caused by superantigens was not prevented by the protein tyrosine kinase (PTK) inhibitors genistein, tyrphostin, or herbimycin, and furthermore, superantigen-induced TCR down-regulation was observed in the p56lck deficient cell line J.CaM. The authors concluded that ligand-induced TCR down-regulation can occur in the absence of PTK activation including activation of p56lck (25). In contrast, a very recent paper found that ligand-induced TCR down-regulation is dependent on p56lck activation (26).
It is known that during physiologic T cell stimulation, PKC is
activated (27) and CD3 becomes phosphorylated (28). The molecular
mechanisms behind PKC-mediated TCR down-regulation have recently been
characterized in detail. Thus, PKC-mediated TCR down-regulation is
absolutely dependent on the CD3 leucine-based (L-based) (SDKQTLL)
internalization motif and can be described as a two-step process: 1)
recognition of the TCR subunit CD3 by PKC with subsequent
phosphorylation of CD3 S126 (12), in which basic amino acids
surrounding S126 are important (29); and 2) exposure of the CD3
L-based motif following S126 phosphorylation with subsequent binding of
clathrin-coated vesicle adaptor proteins and internalization of the TCR
(30). The role of PKC and the CD3 L-based motif in ligand-induced
TCR down-regulation is still not clear. A recent study has indicated
that phosphorylation of S126 in the CD3 L-based motif is not
required for ligand-induced TCR down-regulation (25). However, another
study has indicated that the cytoplasmic tail of CD3 is required for
ligand-induced TCR down-regulation (31). Thus, it still remains an open
question as to how and if ligand- and PKC-induced TCR down-regulation
are interrelated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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JGN, a TCR cell surface negative variant of the human T cell
line Jurkat that synthesizes no CD3 was produced in our own
laboratory (32). The transfectant JGN-S126V expresses CD3 with a
serine 126 to valine mutation. Neither CD3 phosphorylation nor TCR
down-regulation is seen in this transfectant following PKC activation
(12). JGN-LLAA cells express CD3 with leucine 131 and 132 to alanine
mutations. In this transfectant, CD3 is phosphorylated at S126
following PKC activation, but the TCR is not down-regulated (12).
JGN-tP133 cells express CD3 truncated at proline 133. CD3
phosphorylation and TCR down-regulation is observed in this
transfectant following PKC activation (12). JGN-tQ117 cells express a
cytoplasmic tail-less CD3 truncated at glutamine 117 (33) and do not
down-regulate the TCR subsequent to PKC activation. The Jurkat clones
E6-1 (E6), J.CaM1.6 (J.CaM) (a p56lck-deficient
variant of E6 (34)), J.45.01 (J.45) (a CD45-deficient variant of E6
(35)), and the Burkitt’s lymphoma cell line Raji were from American
Type Culture Collection (Manassas, VA). Cells were cultured in RPMI
1640 medium supplemented with penicillin, 2 x 105 U/L
(Leo Pharmaceutical Products, Ballerup, Denmark), streptomycin, 50 mg/L
(Merck, Darmstadt, Germany), and 10% (v/v) FCS (Life Technologies,
Paisley, U.K.) at 37°C in 5% CO2. The anti-TCR mAb
F101.01 was produced in our own laboratory (36). Phycoerythrin
(PE)-conjugated anti-CD3
mAb UCHT1, FITC-conjugated
anti-transferrin receptor (TfR) mAb, and FITC-conjugated
anti-CD45 mAb were from PharMingen (San Diego, CA). The
anti-phosphotyrosine mAb 4G10 was from United States Biochemicals
(Lake Placid, NY). The FITC-conjugated goat anti-mouse Ig Ab was
from Jackson ImmunoResearch (West Grove, PA). The phorbol ester phorbol
12,13-dibutyrate (PDB) was from Sigma Chemical (St. Louis, MO); the
superantigen staphylococcal enterotoxin E (SEE) from Toxin Technology
(Sarasota, FL); and the PKC inhibitor Ro 31-8220 was a kind gift from
Dr. D. Bradshaw (Roche Research Centre, Welwyn Garden City, U.K.).
Brefeldin A (BFA) was from Boehringer Mannheim (Mannheim, Germany), and
the serine/threonine protein phosphatase inhibitor calyculin A was from
Biomol (Plymouth Meeting, PA).
TCR down-regulation, phosphotyrosine blots, and measurement of endocytic rates
For TCR down-regulation, cells were adjusted to 1 x
106 cells per ml of medium and incubated at 37°C
with various concentrations of PDB, the anti-TCR mAb F101.01, or
Raji cells pulsed for 1 h with different concentrations of SEE. At
the indicated time, cells were transferred to ice-cold PBS containing
2% FCS and 0.1% NaN3 and washed twice. The cells were
stained directly with PE-conjugated anti-CD3 and analyzed using
a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA). In
experiments using the anti-TCR mAb F101.01 for TCR down-regulation,
TCR expression was measured by staining the cells with saturating
amounts of F101.01 followed by FITC-conjugated goat anti-mouse Ig
Ab to avoid false negative results, as F101.01 inhibits binding of
anti-CD3 mAb and vice versa (36). Likewise, in experiments using
the PKC inhibitor Ro 31-8220, TCR expression was measured by staining
the cells with saturating amounts of F101.01 followed by
FITC-conjugated goat anti-mouse Ig Ab, as Ro 31-8220 disturbs
measurements of PE fluorescence. Mean fluorescence intensity (MFI) was
recorded and used in the calculation of percentage anti-CD3/TCR
binding: (MFI of treated cells)/(MFI of untreated cells) x 100%. For
each construct, at least three independent clones were analyzed.
Phosphotyrosine blots were performed as previously described (37). To
determine the endocytic rate of the TCR, cells were incubated at a cell
density of 2 x 105 cells per ml medium at 37°C or
4°C with PE-conjugated anti-CD3 mAb. At the indicated time,
aliquots of cell suspension were washed in ice-cold PBS containing 2%
FCS and 0.1% NaN3 and immediately treated with 300 µl
0.5 M NaCl, 0.5 M acetic acid, pH 2.2, for 10 s. The
acid-resistant fluorescence of the cells (representing internalized
anti-CD3 mAb) was measured in the FACScalibur. The percentage of
internalized anti-CD3 mAb to cell surface-bound anti-CD3 mAb
was subsequently calculated using the equation: ((HAR - CAR)/CT)
x 100%, where HAR is the MFI of acid-treated cells incubated at
37°C, CAR is the MFI of acid-treated cells incubated at 4°C, and CT
is the MFI of untreated cells incubated at 4°C.
Transfection
The plasmid pRep-lck, containing mouse p56lck cDNA (38), was transfected into J.CaM cells, and the plasmid pAW-HLCA, containing cDNA coding for the human 180 kDa isoform of CD45 (39), was transfected into J.45 cells. Transfections were performed using the Bio-Rad (Hercules, CA) Gene Pulser at a setting of 260 V, 960 µF with 40 µg of plasmid per 2 x 107 cells. After 3 to 4 wk of selection, hygromycin B (pRep-lck)- and G418 (pAW-HLCA)-resistant clones were expanded.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-based internalization motif
To analyze whether ligand-induced TCR down-regulation was
dependent on the CD3 L-based internalization motif, transfectants
expressing either wild-type CD3 (JGN-WT) or CD3 with mutations in
the L-based motif that completely abolish PKC-mediated TCR
down-regulation (JGN-S126V and JGN-LLAA) (Fig. 1
A) were incubated with
the anti-TCR mAb F101.01, SEE-pulsed Raji cells, or the phorbol
ester PDB. In contrast to PKC-mediated TCR down-regulation,
ligand-induced TCR down-regulation following incubation with
anti-TCR mAb or SEE-pulsed Raji cells was as efficient in JGN-S126V
and JGN-LLAA cells as in JGN-WT cells (Fig. 1, B–D).
This demonstrated that ligand-induced TCR down-regulation was not
dependent on the presence of an intact CD3 L-based motif. In
addition to the L-based motif, a tyrosine-based (Y-based) motif
involved in receptor internalization and sorting has been described in
the cytoplasmic tail of CD3 (40). To test whether this motif was
required for ligand-induced TCR down-regulation, the transfectant
JGN-tP133 that expresses a truncated CD3 lacking the Y-based motif,
was analyzed. Ligand-induced TCR down-regulation was as efficient in
JGN-tP133 cells as in JGN-WT cells, demonstrating that the Y-based
motif of CD3 is not required for ligand-induced TCR internalization.
Finally, to test whether a still unidentified motif in the CD3
cytoplasmic tail required for ligand-induced TCR down-regulation
exists, the CD3 tail-less transfectant JGN-tQ117 was analyzed. Even
JGN-tQ117 cells down-regulated the TCR as efficiently as JGN-WT cells
following TCR ligation, demonstrating that the CD3 cytoplasmic tail,
inclusive of the L- and Y-based sorting motifs, was dispensable for
ligand-induced TCR down-regulation.
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The studies described above demonstrate that ligand-induced TCR
down-regulation is independent of the CD3 L-based internalization
motif. The TCR is associated with nonreceptor PTK that become activated
following receptor ligation, which leads to activation of a range of
intracellular molecules including PKC (27). As a consequence, PKC was
most probably activated in all of the methods used to induce TCR
down-regulation in the present study. Thus, the possibility existed
that PKC activity was required for ligand-induced TCR down-regulation
via a mechanism other than CD3 S126 phosphorylation. To test this
hypothesis, TCR down-regulation was induced in JGN-WT cells by
incubation with anti-TCR mAb, SEE-pulsed Raji cells, or PDB in the
presence or absence of the PKC inhibitor Ro 31-8220. Ro 31-8220 clearly
inhibited PDB-mediated TCR down-regulation, whereas ligand-induced TCR
down-regulation was unaffected by the presence of the PKC inhibitor
(Fig. 2). These results indicated that
ligand-induced TCR down-regulation, in addition to being independent of
the CD3 L-based motif, is independent of PKC activity.
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chain and activation of
p56lck and p59fyn in
Jurkat cells (45, 46, 47, 48). Treatment of JGN-WT cells with pervanadate
induced strong protein tyrosine phosphorylation of several substrates
including the chain and an efficient down-regulation of the TCR
(Fig. 2
and data not shown). JGN-S126V cells down-regulated the TCR as
efficiently as JGN-WT cells following pervanadate treatment and Ro
31-8220 did not inhibit pervanadate-induced TCR down-regulation in
JGN-WT or JGN-S126V cells (Fig. 2). Thus, these experiments indicated
that protein tyrosine phosphorylation per se is able to mediate TCR
down-regulation independently of PKC and CD3 S126
phosphorylation. Ligand-induced TCR down-regulation is dependent on protein tyrosine phosphorylation mediated by p56lck and p59fyn
To further examine the requirement of protein tyrosine
phosphorylation in ligand- and PKC-induced TCR down-regulation, the
cell lines J.CaM and J.45 were included in the study. J.CaM is a mutant
of the Jurkat E6 line that lacks functional
p56lck (38). J.45 is also a mutant of E6 and has
markedly reduced amounts of CD45 resulting in an inability to activate
p56lck and p59fyn
following TCR stimulation (35). Both J.CaM and J.45 show very reduced
protein tyrosine phosphorylation following TCR ligation (Refs. 35 and
38; and data not shown). Compared with E6 cells, anti-TCR-induced
TCR down-regulation was markedly reduced in J.CaM cells and almost
completely abolished in J.45 cells (Fig. 3A). SEE-induced TCR
down-regulation was reduced in both variants compared with SEE-induced
TCR down-regulation in E6 cells (Fig. 3B). In
contrast, PKC-mediated TCR down-regulation was as efficient in both
mutants as in the E6 cell line (Fig. 3C). These data
indicate that both p56lck and
p59fyn-mediated protein tyrosine phosphorylation
is required for ligand-induced TCR down-regulation but is dispensable
for PKC-mediated TCR down-regulation. To exclude the possibility that
some unidentified mechanisms should be responsible for the
reduced/abolished ligand-induced TCR down-regulation in J.CaM and J.45
cells, these mutants were transfected with
p56lck and CD45, respectively. In the
transfectants J.CaM-lck and J.45-CD45 TCR down-regulation and protein
tyrosine phosphorylation patterns following anti-TCR stimulation
were restored (Fig. 3D and data not shown). This indicated
that the defect in ligand-induced TCR down-regulation in J.CaM and J.45
was directly correlated with the defect in
p56lck and
p59fyn-mediated protein tyrosine phosphorylation
and was not caused by other defects.
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L-based
motif by the balance between PKC and serine/threonine protein
phosphatase activities in the absence of TCR ligation
As the CD3 L-based motif was dispensable for ligand-induced TCR
down-regulation, we speculated as to what role(s) this motif could play
in TCR function. It may be suggested that in the absence of TCR
ligation, TCR expression is regulated via the CD3 L-based motif by
environmental factors such as cytokines influencing the balance between
PKC and serine/threonine protein phosphatase activities (49, 50). Thus,
tuning the balance in favor of PKC activity should result in activation
of the CD3 L-based motif, leading to reduced levels of TCR
expression, whereas tuning the balance in favor of serine/threonine
protein phosphatase activity should result in inactivation of the
CD3 L-based motif, leading to increased levels of TCR expression.
Such a cytokine-mediated, TCR ligation-independent regulation of TCR
expression levels could probably be important in determining the
activation threshold of T cells in their encounter with APC
(51).
We wanted to determine first whether different levels of PKC activity
would result in different stable levels of TCR expression. Previous
studies have demonstrated that PKC is activated by phorbol esters in a
dose-dependent manner (52, 53). Up-regulation of PKC activity to
different levels clearly influenced TCR expression in JGN-WT cells
(Fig. 4A) but not in
cells with a mutated CD3 L-based motif (data not shown). By varying
the amount of PDB, new levels of TCR expression (between 80 and 30% of
untreated cells) were obtained (Fig. 4A).
Importantly, these new levels of TCR expression were stable following
incubation with PDB for 30 to 60 min and correlated with the
concentration of PDB. In agreement with other studies (Refs. 54 and 55;
and J. Dietrich et al.4), we
found that following PKC-mediated TCR internalization, the TCR was not
degraded but was recycled back to the cell surface (data not shown).
This indicated that different stable TCR expression levels were
obtained by changing the endocytic rate of the TCR. To examine whether
the reduced but stable levels of TCR expression obtained by
up-regulation of PKC activity were maintained by an increased endocytic
rate, TCR internalization was measured in untreated cells and in cells
pretreated with PDB for 60 min. Although the concentration of
anti-CD3 mAb used to measure the endocytic rate induced some TCR
internalization, the endocytic rate of the TCR was clearly increased in
PDB-pretreated JGN-WT cells as compared with untreated cells (Fig. 4B). In contrast, the endocytic rate of the TCR in
JGN-LLAA cells was unaffected by the presence of PDB (Fig. 4B).
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L-based motif,
the amount of recycling TCR in PDB-untreated cells should be higher in
cells with an intact CD3 L-based motif than in transfectants with a
mutated CD3 L-based motif. This implies that a reduction in
exocytosis should result in a greater decrease in TCR expression in
JGN-WT cells than in cells with a mutated CD3 L-based motif. The
fungal metabolite BFA has been demonstrated to reduce the exocytic rate
of the TfR with little or no effect on the endocytic rate (56). The TfR
is a spontaneously recycling receptor with high endocytic and exocytic
rates and was included as a control in the following experiments.
Treatment of JGN-WT cells with BFA caused a down-regulation of TCR and
TfR expression to stable levels of approximately 90 and 55%,
respectively, as compared with untreated cells. In contrast, BFA
treatment of cells with a disrupted CD3 L-based motif did not cause
a reduction in TCR expression but only in TfR expression (Fig. 5, A and B).
In JGN-WT cells, in which the endocytic rate was further increased by
pretreatment with PDB for 60 min, the effect of BFA was even more
obvious. BFA treatment of cells pretreated with PDB thus resulted in a
TCR down-regulation to 66% as compared with 90% for nonpretreated
cells (Fig. 5C). These analyses indicated that TCR
expression levels in cells with an intact CD3 L-based motif were
determined by the degree of PKC activity, and furthermore, that a basal
PKC-mediated phosphorylation of the CD3 L-based motif existed. In
addition, a basal serine/threonine protein phosphatase-mediated
dephosphorylation of the CD3 L-based motif probably also existed in
the cells, as incubation of JGN-WT cells with the serine/threonine
protein phosphatase inhibitor calyculin A caused a down-regulation of
the TCR to new stable levels, whereas TCR expression levels on JGN-LLAA
cells were unaffected by this inhibitor (Fig. 5D and data
not shown).
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L-based motif by the balance between PKC and serine/threonine
protein phosphatase activities. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-based motif but
independent of p56lck and
p59fyn, and leads to TCR recycling (54, 55).4 The other pathway is induced by TCR ligation, is
dependent on p56lck- and
p59fyn-mediated protein tyrosine phosphorylation
but independent of the CD3 L-based motif and PKC, and leads to TCR
degradation (26, 57, 58). Both p56lck and
p59fyn probably play a role in ligand-induced
TCR down-regulation. Thus, although markedly reduced, the anti-TCR
mAb-induced TCR down-regulation was not abolished in J.CaM cells that
lack functional p56lck but express
p59fyn (35, 38). In contrast, anti-TCR
mAb-induced TCR down-regulation was almost completely abolished in J.45
cells, which are unable to activate both p59fyn
and p56lck following TCR ligation (35). A role
of p56lck in TCR down-regulation is in agreement
with previous studies describing involvement of
p56lck in TCR down-regulation in
CD4+CD8+ thymocytes (13, 14, 15) and with a very
recent study demonstrating that a constitutive, active form of
p56lck induces rapid internalization of the TCR
and that p56lck is required for anti-TCR mAb
induced TCR down-regulation (26). In contrast, another study found that
ligand-induced TCR down-regulation did occur in the absence of
p56lck (25). In that study, SEE-induced TCR
down-regulation in J.CaM cells was found to be comparable with that of
wild-type cells; furthermore, the PTK inhibitors genistein, tyrphostin,
and herbimycin did not inhibit SEE-induced TCR down-regulation.
Anti-TCR mAb-induced TCR down-regulation was not examined in that
study. We found that although anti-TCR mAb-induced
TCR-down-regulation was markedly reduced in J.CaM cells and almost
completely abolished in J.45 cells, SEE-induced TCR-down-regulation was
reduced to a lesser degree in these cells. It is possible that
SEE-induced TCR-down-regulation is mediated by protein tyrosine
phosphorylation independently of p56lck, caused
by signaling through several costimulatory molecules, as recently
described (59). The exact substrate(s) needed to be tyrosine
phosphorylated for ligand-induced TCR down-regulation to take place
still remains to be identified. Furthermore, the exact mechanisms
behind ligand-induced TCR down-regulation remain to be determined.
Several, not mutually exclusive mechanisms can be proposed. 1)
Ligand-mediated protein tyrosine phosphorylation induces a
conformational change that exposes a Y-based internalization motif for
clathrin-coated vesicle adaptor protein binding. In line with this
proposal, a direct interaction between adaptor proteins and Y-based
internalization motifs has been described for several receptors
(60, 61, 62, 63, 64, 65, 66). The present study demonstrated that the Y-based
internalization motif in CD3 (40) is not required for ligand-induced
TCR down-regulation, as ligand-induced TCR down-regulation was intact
in JGN-tP133 and JGN-tQ117 cells lacking the Y-based motif of CD3.
2) Ligand-mediated protein tyrosine phosphorylation induces a
conformational change that exposes the DxxxLL internalization motif of
CD3 and CD3
for adaptor protein binding. In line with this
proposal, we have recently described both the CD3 and CD3 L-based
motifs as having the potential to function as internalization motifs
and have shown that adaptor proteins can bind to such motifs (30). The
present study does not rule out this possibility, as all of the
transfectants analyzed contained an intact CD3 DxxxLL motif.
Furthermore, this proposal is supported by a recent study describing
the inability to down-regulate the TCR after ligand binding in a T cell
variant with deletion of the cytoplasmic tails of both CD3 and
CD3 (31). 3) Ligand-mediated protein tyrosine phosphorylation leads
to ubiquitination of the TCR with subsequent endocytosis. This
suggestion is supported by the observation that the TCR is
ubiquitinated, in a PTK-dependent way, following ligation (47, 67) and
that receptor ubiquitination can lead to internalization (68, 69, 70). 4)
Ligand-mediated tyrosine phosphorylation of the chain creates
docking sites for src homology domain 2-containing proteins
leading to recruitment of several other molecules including the adaptor
molecule Grb2 (71), which might play a role in receptor
internalization (72).
It is known that during physiologic T cell stimulation, PKC is
activated (27) and CD3 becomes phosphorylated (28). In the present
study, the PKC inhibitor Ro 31-8220 did not inhibit ligand-induced TCR
down-regulation, indicating that non-PKC-dependent pathways are
dominant during ligand-induced TCR down-regulation. Alternatively, TCR
ligation may activate PKC isoforms distinct from those stimulated by
PDB, and only those induced by PDB promote TCR internalization.
Treatment of cells with PDB is likely to have pleiotropic effects,
including the potential activation of some serine/threonine protein
phosphatases, which could complicate the interpretation of our data.
However, we find that treatment of Jurkat cells with 111 nM of PDB for
1.5 h does not influence the activity of PP1 and PP2A, which are
the phosphatases most likely involved in dephosphorylation of CD3
(Lauritsen et al., unpublished data).
The present study strongly indicates that the CD3 L-based motif is
not required in ligand-mediated TCR down-regulation. However, the
CD3 L-based motif has been extremely conserved during evolution and
is even found in the common CD3/ chain from the chicken (73) and
the frog (GenBank accession number U78290), indicating that the motif
must play some important physiologic role(s). One role of the CD3
L-based motif could be in TCR quality control, by targeting
incompletely assembled TCR complexes to the endosomes/lysosomes for
degradation, as previously described (30). In line with this
possibility, it has been demonstrated that TCR complexes lacking the
chain are mainly transported to the late endosomes/lysosomes for
degradation (74). These observations suggest that normally cover
the L-based TCR internalization motifs, which could explain why only
completely assembled TCR are allowed to be normally expressed at the T
cell surface. Another role of the CD3 L-based motif, as suggested by
the present study, could be in finely tuning the TCR expression levels
as a response to environmental factors such as cytokines that influence
the activities of PKC and serine/threonine protein phosphatases.
Further studies are in progress to evaluate these potential roles of
the CD3 L-based motif.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carsten Geisler, Institute of Medical Microbiology and Immunology, University of Copenhagen, The Panum Institute, Building 18.3, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark. E-mail address:
3 Abbreviations used in this paper: PKC, protein kinase C; PTK, protein tyrosine kinase; PE, phycoerythrin; TfR, transferrin receptor; PDB, phorbol 12,13-dibutyrate; SEE, staphylococcal enterotoxin E; BFA, brefeldin A; MFI, mean fluorescence intensity; L-based, leucine-based; Y-based, tyrosine-based.
4 J. Dietrich, T. Backstrom, J. P. H. Lauritsen, J. Kastrup, M. D. Christensen, F. von Bulow, E. Palmer, and C. Geisler. The phosphorylation state of CD3 influences T cell responsiveness and controls T cell receptor cycling. Manuscript submitted for publication.
Received for publication December 26, 1997. Accepted for publication March 6, 1998.
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DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro. J. Cell. Biol. 138:271. and CD3 cytoplasmic domains in cytolytic T lymphocyte functions and TCR/CD3 down-modulation. J. Immunol. 158:4162.[Abstract]
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C. M. Bonefeld, A. B. Rasmussen, J. P. H. Lauritsen, M. von Essen, N. Odum, P. S. Andersen, and C. Geisler TCR Comodulation of Nonengaged TCR Takes Place by a Protein Kinase C and CD3{gamma} Di-Leucine-Based Motif-Dependent Mechanism J. Immunol., September 15, 2003; 171(6): 3003 - 3009. [Abstract] [Full Text] [PDF]
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P. S. Torres, A. Alcover, D. A. Zapata, J. Arnaud, A. Pacheco, J. M. Martin-Fernandez, E. M. Villasevil, O. Sanal, and J. R. Regueiro TCR Dynamics in Human Mature T Lymphocytes Lacking CD3{gamma} J. Immunol., June 15, 2003; 170(12): 5947 - 5955. [Abstract] [Full Text] [PDF]
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 B. M. Badran, S. M. Wolinsky, A. Burny, and K. E. Willard-Gallo Identification of Three NFAT Binding Motifs in the 5'-Upstream Region of the Human CD3gamma Gene That Differentially Bind NFATc1, NFATc2, and NF-kappa B p50 J. Biol. Chem., November 27, 2002; 277(49): 47136 - 47148. [Abstract] [Full Text] [PDF]
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 P. S. Torres, D. A. Zapata, A. Pacheco-Castro, J. L. Rodriguez-Fernandez, C. Cabanas, and J. R. Regueiro Contribution of CD3{gamma} to TCR regulation and signaling in human mature T lymphocytes Int. Immunol., November 1, 2002; 14(11): 1357 - 1367. [Abstract] [Full Text] [PDF]
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C. Dumont, N. Blanchard, V. Di Bartolo, N. Lezot, E. Dufour, S. Jauliac, and C. Hivroz TCR/CD3 Down-Modulation and {zeta} Degradation Are Regulated by ZAP-70 J. Immunol., August 15, 2002; 169(4): 1705 - 1712. [Abstract] [Full Text] [PDF]
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J. Dietrich, C. Menne, J. P. H. Lauritsen, M. von Essen, A. B. Rasmussen, N. Odum, and C. Geisler Ligand-Induced TCR Down-Regulation Is Not Dependent on Constitutive TCR Cycling J. Immunol., June 1, 2002; 168(11): 5434 - 5440. [Abstract] [Full Text] [PDF]
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R. Zaru, T. O. Cameron, L. J. Stern, S. Muller, and S. Valitutti TCR Engagement and Triggering in the Absence of Large-Scale Molecular Segregation at the T Cell-APC Contact Site J. Immunol., May 1, 2002; 168(9): 4287 - 4291. [Abstract] [Full Text] [PDF]
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M. von Essen, C. Menne, B. L. Nielsen, J. P. H. Lauritsen, J. Dietrich, P. S. Andersen, K. Karjalainen, N. Odum, and C. Geisler The CD3{gamma} Leucine-Based Receptor-Sorting Motif Is Required for Efficient Ligand-Mediated TCR Down-Regulation J. Immunol., May 1, 2002; 168(9): 4519 - 4523. [Abstract] [Full Text] [PDF]
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M. D. Puente Navazo, D. Valmori, and C. Ruegg The Alternatively Spliced Domain TnFnIII A1A2 of the Extracellular Matrix Protein Tenascin-C Suppresses Activation-Induced T Lymphocyte Proliferation and Cytokine Production J. Immunol., December 1, 2001; 167(11): 6431 - 6440. [Abstract] [Full Text] [PDF]
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B. Barbeau, G. A. Robichaud, J.-F. Fortin, and M. J. Tremblay Negative Regulation of the NFAT1 Factor by CD45: Implication in HIV-1 Long Terminal Repeat Activation J. Immunol., September 1, 2001; 167(5): 2700 - 2713. [Abstract] [Full Text] [PDF]
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 S. Krishnan, V. G. Warke, M. P. Nambiar, H. K. Wong, G. C. Tsokos, and D. L. Farber Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3{zeta} expression Blood, June 15, 2001; 97(12): 3851 - 3859. [Abstract] [Full Text] [PDF]
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H. Kirchgessner, J. Dietrich, J. Scherer, P. Isomaki, V. Korinek, I. Hilgert, E. Bruyns, A. Leo, A. P. Cope, and B. Schraven The Transmembrane Adaptor Protein TRIM Regulates T Cell Receptor (TCR) Expression and TCR-mediated Signaling via an Association with the TCR {zeta} Chain J. Exp. Med., June 4, 2001; 193(11): 1269 - 1284. [Abstract] [Full Text] [PDF]
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J. Dietrich, M. Cella, and M. Colonna Ig-Like Transcript 2 (ILT2)/Leukocyte Ig-Like Receptor 1 (LIR1) Inhibits TCR Signaling and Actin Cytoskeleton Reorganization J. Immunol., February 15, 2001; 166(4): 2514 - 2521. [Abstract] [Full Text] [PDF]
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M. C. Haks, T. A. Cordaro, J. H. N. van den Brakel, J. B. A. G. Haanen, E. F. R. de Vries, J. Borst, P. Krimpenfort, and A. M. Kruisbeek A Redundant Role of the CD3{{gamma}}-Immunoreceptor Tyrosine-Based Activation Motif in Mature T Cell Function J. Immunol., February 15, 2001; 166(4): 2576 - 2588. [Abstract] [Full Text] [PDF]
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C. Menne, J. P. H. Lauritsen, J. Dietrich, J. Kastrup, A.-M. K. Wegener, N. Odum, and C. Geisler Ceramide-Induced TCR Up-Regulation J. Immunol., September 15, 2000; 165(6): 3065 - 3072. [Abstract] [Full Text] [PDF]
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A. Qadri, C. G. Radu, J. Thatte, P. Cianga, B. T. Ober, R. J. Ober, and E. S. Ward A Role for the Region Encompassing the c'' Strand of a TCR V{alpha} Domain in T Cell Activation Events J. Immunol., July 15, 2000; 165(2): 820 - 829. [Abstract] [Full Text] [PDF]
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A. G. Schrum, A. D. Wells, and L. A. Turka Enhanced surface TCR replenishment mediated by CD28 leads to greater TCR engagement during primary stimulation Int. Immunol., June 1, 2000; 12(6): 833 - 842. [Abstract] [Full Text] [PDF]
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 L. Bouhdoud, P. Villain, A. Merzouki, M. Arella, and C. Couture T-Cell Receptor-Mediated Anergy of a Human Immunodeficiency Virus (HIV) gp120-Specific CD4+ Cytotoxic T-Cell Clone, Induced by a Natural HIV Type 1 Variant Peptide J. Virol., March 1, 2000; 74(5): 2121 - 2130. [Abstract] [Full Text]
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 J. E. M. van Leeuwen, P. K. Paik, and L. E. Samelson The Oncogenic 70Z Cbl Mutation Blocks the Phosphotyrosine Binding Domain-Dependent Negative Regulation of ZAP-70 by c-Cbl in Jurkat T Cells Mol. Cell. Biol., October 1, 1999; 19(10): 6652 - 6664. [Abstract] [Full Text] [PDF]
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N. Bronstein-Sitton, L. Wang, L. Cohen, and M. Baniyash Expression of the T Cell Antigen Receptor zeta Chain following Activation Is Controlled at Distinct Checkpoints. IMPLICATIONS FOR CELL SURFACE RECEPTOR DOWN-MODULATION AND RE-EXPRESSION J. Biol. Chem., August 13, 1999; 274(33): 23659 - 23665. [Abstract] [Full Text] [PDF]
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B. Fernandez, M. P. Czech, and H. Meisner Role of Protein Kinase C in Signal Attenuation following T Cell Receptor Engagement J. Biol. Chem., July 16, 1999; 274(29): 20244 - 20250. [Abstract] [Full Text] [PDF]
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D. Penna, S. Muller, F. Martinon, S. Demotz, M. Iwashima, and S. Valitutti Degradation of ZAP-70 Following Antigenic Stimulation in Human T Lymphocytes: Role of Calpain Proteolytic Pathway J. Immunol., July 1, 1999; 163(1): 50 - 56. [Abstract] [Full Text] [PDF]
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A. N. Vallejo, J. C. Brandes, C. M. Weyand, and J. J. Goronzy Modulation of CD28 Expression: Distinct Regulatory Pathways During Activation and Replicative Senescence J. Immunol., June 1, 1999; 162(11): 6572 - 6579. [Abstract] [Full Text] [PDF]
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J. Dietrich, T. Backstrom, J. P. H. Lauritsen, J. Kastrup, M. D. Christensen, F. von Bulow, E. Palmer, and C. Geisler The Phosphorylation State of CD3gamma Influences T Cell Responsiveness and Controls T Cell Receptor Cycling J. Biol. Chem., September 11, 1998; 273(37): 24232 - 24238. [Abstract] [Full Text] [PDF]
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