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Department of Cellular and Developmental Biology, "La Sapienza" University, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4 is one of the receptors on T cells that can both potentiate or
suppress T cell activation. The ability of CD4 to enhance or alter
signals generated by the TCR-CD3 complex has been attributed to its
cytoplasmic tail that interacts with the Src family tyrosine
kinase p56lck. Indeed, following recognition of
peptide-MHC complexes by TCR and CD4, Lck is activated and
phosphorylates the tyrosine residue within the immunoreceptor
tyrosine-based activation motif of the CD3 and 
-chains, which is the
earliest known event in T cell signaling (3). It has been
suggested that CD4 binds MHC class II simultaneously or perhaps shortly
after TCR-peptide-MHC engagement (4, 5), stabilizing the
interactions between peptide-MHC and TCR (6, 7). The use
of new technologies such as the multidimensional imaging of T cells
interacting with APC has afforded new insights into T cell activation.
Grakoui et al. (8) have shown the immunological synapse
formation as a dynamic mechanism that allows T cells to distinguish
potential antigenic ligands and the recruitment of
p56lck to the engaged TCR by CD4 may contribute
to this kinetic discrimination. More recently, Krummel et al.
(9) showed unexpected CD4 engagement dynamics. The authors
found that immediately after cell contact and coincident with the
increase in intracellular calcium, CD4 and TCR-associated CD3
clustered in the interface. However, following signaling-,
costimulation-, and cytoskeleton-dependent processes, CD3 chains
were redistributed to central clusters while CD4 moved to the
periphery. It is interesting to note that both papers suggest that CD4
participates in the triggering of early signals necessary in
immunological synapse formation and may not be required once activation
has been initiated.
In the context of the negative signals delivered by CD4, the quality of the Ag, cell cycle phase, decreased availability of Lck by TCR, defective transduction pathways, cytokine availability, and lack of costimulatory signals have been described as apoptosis inducers (10, 11, 12). However, many of these results have been obtained when the obligatory interaction between TCR and CD4 was interrupted by sequential stimulation of CD4 and TCR. Since this kind of dissociation is a rare event in physiological conditions in vivo, the role of CD4 in mediating apoptotic programs in T cells remains still unclear. The observation that CD4 should serve primarily to "boost" recognition of ligand by the TCR (9) prompted us to reconsider the role of CD4 in programmed cell death, one of the apoptotic pathways operative in T lymphocytes (13). We hypothesized that if CD4 is able to transduce both survival and apoptotic programs, both programs must be prepared at the first stage of Ag recognition and in concert with TCR. Indeed, once the engaged TCR are invested in building the stable central cluster and other costimulatory molecules are moved into the immunological synapse by cytoskeleton-mediated membrane raft movement, the apoptotic programs will be deleted and the survival programs executed.
An essential regulator of cytoskeletal rearrangements during T cell activation is Vav (14). Vav functions as a guanine nucleotide exchange factor for the Rho/Rac family of protein G (15, 16) and plays a critical role in both thymocyte development (17) and T lymphocyte activation (18). More recently, data from Wulfing et al. (19) support the role of Vav as a central regulator of T cell activation-induced actin cytoskeleton rearrangements, necessary for an organized accumulation of signaling molecules at the interface of APC and responding cells. Moreover, Vav overexpression has been suggested to mimic the effects of costimulatory molecules, such as CD28, in potentiating TCR signaling (20, 21). Altogether these data strongly support the hypothesis that Vav may play a role in blocking CD4-mediated apoptosis.
The present study aimed to explore the possibility that CD4 engagement, without modifying the physical association with the TCR-CD3 complex (22), induces both apoptotic and survival programs in T cells and to clarify the involved molecular mechanisms. Our results show that suicide signals, triggered in Jurkat T cells by Leu3a mAb-mediated engagement of CD4, crucially depend on CD4-TCR/CD3 coassociation. The apoptotic features observed are related to an increase of Bax expression and mitochondrial damages. Overexpression of Vav rescues T cells from CD4-mediated apoptotic programs by inhibiting up-regulation of Bax expression mediated by CD4/Lck observed in apoptotic cells. These data suggest that signaling effectors, which contribute in facilitating TCR and costimulatory molecules signaling, can neutralize CD4-triggered suicide programs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat (clone J20) and its derivative Jurkat 31.13 defective for TCR/CD3 surface expression (23) were maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine, penicillin, and streptomycin (Life Technologies, Milan, Italy). Staining of 31.13 cells with anti-CD4 Ab revealed that only 10–20% of the overall population expresses high levels of CD4. Thus, cells expressing CD4 molecules were enriched by magnetic cell sorting using beads coated with anti-CD4 Ab and following the manufacturer’s recommended procedure (Miltenyi Biotec, Bologna, Italy). The Jurkat cell lines overexpressing Vav (CL9) or Bcl-2 were grown in the same culture medium supplemented with 2 mg/ml G418 (Life Technologies) or 500 ng/ml puromycin (Sigma-Aldrich, St. Louis, MO), respectively.
Mouse anti-CD4 mAb (Leu3a) was purchased from BD Biosciences (Mountain View, CA). Rabbit anti-human Bax and Bcl-2 Abs were obtained from BD PharMingen (San Diego, CA). Mouse (3A5) and rabbit anti-p56lck Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-c-myc (9E10) Ab was obtained from Boehringer Mannheim (Monza, Italy).
PP2, a selective inhibitor of the Src family of tyrosine kinases, was provided by Calbiochem (La Jolla, CA). Bax antisense (24) and unrelated oligodeoxynucleotides with a natural phosphodiester backbone were synthesized by M-Medical Genenco (Florence, Italy). Sequences used were: Bax antisense oligonucleotides 5'-TCG ATC CTG GAT GAA ACC CT-3' and 5'-TCC CCC CCC ATT CGC CCT GC-3', and unrelated oligonucleotides 5'-GGT ATG GCT AGC ATG ACT GG-3' and 5'-GTC GTA CTC TTC TGC ATT GA-3'.
Plasmid and cell transfection
pEF-Bos-expressing N-terminal myc-tagged Vav was a kind gift from Dr. A. Weiss (University of California, San Francisco, CA). pEF-pGKpuro-expressing human Bcl-2 (25) was kindly provided by Dr. A. Strasser (Walter and Eliza Hall Institute, Melbourne, Australia).
Stable transfectants overexpressing Vav wild-type (Vav CL9) was obtained by electroporating (at 260 V, 960 F) 107 Jurkat cells in 0.5 ml of RPMI 1640 containing 20% FCS with 30 µg of pEF-Bos-expressing N-terminal myc-tagged Vav. After 48 h, cells were placed in 96-well flat-bottom culture plates in RPMI 1640 medium plus G418. The Jurkat cell line, overexpressing Bcl-2, was generated by transfecting Jurkat with 30 µg of pEF Bcl-2-pGKpuro. After 24 h, cells were cultured for 3 wk with RPMI 1640 medium containing 500 ng/ml puromycin. Transfectants were analyzed for CD3, CD4, and CD28 expressions by FACS analysis (FACSCalibur; BD Biosciences) and for myc-Vav and Bcl-2 expression by Western blotting.
Cell stimulation, immunoprecipitation, and in vitro kinase assay
Jurkat cells were washed twice, resuspended in medium
(108/ml), and incubated for different times at
37°C in the presence or absence of Leu3a mAb. At the end of
incubation, cells were harvested and lysed for 30 min on ice in 1%
Nonidet P-40 lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM
NaCl, and 1 mM EGTA in the presence of inhibitors of proteases and
phosphatases: 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM
Pefabloc-sc, 50 mM NaF, 10 mM
Na4P2O7,
and 1 mM NaVO4. The kinase activity of
immunoprecipitated p56lck was assayed at 30°C
for 15 min in 25 µl of kinase buffer (1 mM Tris-HCl (pH 7.5), 7.5 mM
NaCl, 25 mM HEPES (pH 7.3), 10 mM MnCl2, and
0.05% Nonidet P-40) in the presence of 10 µCi of
[
-32P]ATP (10 Ci/mmol). The reactions were
terminated with 2x Laemmli sample buffer. Samples were analyzed by
SDS-PAGE gel. Gels were fixed, treated with 1 M of KOH for 1 h at
55°C to remove the alkali-labile phosphate groups from serine- and
threonine-phosphorylated proteins, dried, and autoradiographed.
Radioactivity in the phosphorylated proteins was quantitated by a
phosphoimager.
Immunoblotting
Jurkat cells were washed twice, resuspended in medium (108/ml), and incubated at 37°C in the presence or absence of Leu3a mAb. At the end of incubation, cells were lysed for 30 min on ice and proteins were resolved by 10% SDS-PAGE and blotted onto nitrocellulose membranes. Blots were incubated with anti-Bax, or anti-Bcl-2, or anti-Vav, or anti-Lck Abs, extensively washed, and after incubation with HRP-labeled goat anti-rabbit or HRP-labeled goat anti-mouse (Amersham International, Amersham, U.K.) Abs developed with the ECL detection system (Amersham International).
Apoptosis analysis
Jurkat T cell lines were incubated for 48 h in the presence or absence of Leu3a (1:50). Apoptosis was measured by a BD Biosciences FACSCalibur flow cytometer by evaluating changes in cell dimensions measured by forward scatter height (FSC-H)4 vs cell condensation and granularity measured by side scatter height (SSC-H), as previously described (26). The percentage of specific apoptosis was calculated as follows: percentage of specific apoptosis = 100 x (percent Leu3a-treated apoptotic cells - percent spontaneous untreated apoptotic cells)/(100 - percent spontaneous untreated apoptotic cells).
Analysis of variation in the mitochondrial transmembrane electrical
potential (
m)
Variation in m at the single
mitochondrial level was detected by using the lipophilic cation
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine
iodide (JC-1). Briefly, cells were incubated at
106 cells/ml in RPMI 1640 with 10% FCS for 15
min at 37°C with 10 µg/ml JC-1 (Molecular Probes, Eugene, OR). At
the end of incubation, cells were washed twice, resuspended in PBS, and
m was analyzed by a BD Biosciences
FACSCalibur flow cytometer as described previously (27).
Depolarization of mitochondrial membrane is accompanied by a change of
JC-1 color from greenish orange (analyzed in FL-2) to green (analyzed
in FL-1).
Flow cytometric analysis of Bax and Bcl-2
Cells were fixed with PBS containing 2% paraformaldehyde and subsequently permeabilized in PBS containing 0.5% BSA, 0.02% sodium azide, and 0.5% saponin. Then cells were incubated for 15 min at room temperature with rabbit anti-human Bax or Bcl-2 Abs (BD PharMingen) or isotype-matched rabbit IgG Abs (Sigma-Aldrich), washed, and stained with FITC-labeled anti-rabbit IgG (Sigma-Aldrich). Flow cytometric analysis was performed on a BD Biosciences FACSCalibur flow cytometer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been reported that in Jurkat T cells CD4 functionally
interacts with the TCR-CD3 complex even in the absence of TCR
engagement (28). This could be related to a high
background of tyrosine phosphorylation compared with normal T cells and
could mimic a low activation threshold. Thus, the engagement of CD4 in
Jurkat cells could mimic the boost effect, described by Krummel et al.
(9), that normally occurs when T lymphocytes interact with
APC. If the early signals mediated by the interaction of CD4 with
TCR/CD3 are not sustained by other costimulatory signals such as those
supported by APC, the activation pathways may be switched off in favor
of apoptotic pathways. To test this hypothesis, we cultured Jurkat
cells in the presence or absence of Leu3a, and both Lck kinase activity
and apoptotic features were analyzed. To evaluate Lck activation, cells
were treated with Leu3a for different times and in vitro kinase assays
were performed in anti-Lck immunoprecipitates. Fig. 1
A (upper panel)
clearly shows that cross-linking of CD4 by Leu3a results in a rapid and
strong increase of Lck kinase activity that returns to the basal level
after 15 min of stimulation. No induction of Lck kinase activity was
observed following immunoprecipitation of CD4 itself in unstimulated
cells (data not shown). Lck protein content in each sample was also
checked by Western blotting using anti-Lck Ab (Fig. 1A,
lower panel). Apoptosis was analyzed on the same cells after
48 h of treatment with Leu3a by FACS analysis (Fig. 1,
B and C). Cell death was evaluated by FACS
analysis measuring either changes in cell dimension vs cell granularity
(Fig. 1B) or changes in the m
(Fig. 1C) after staining with the lipophilic cationic probe
JC-1 (27). The results obtained show that the activation
of Lck by Leu3a in the absence of other signals results in a massive
apoptosis of Jurkat cells, and almost all apoptotic cells showed
significant alterations of the mitochondrial membrane potential. No
differences in both basal apoptosis and mitochondrial membrane
depolarization were observed when an isotype-matched mAb was used as
control (data not shown).
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To assess whether the kinase activity of Lck was responsible of
the apoptotic features observed in Leu3a-treated Jurkat cells, we used
PP2, a potent selective inhibitor of the Src family tyrosine kinases.
PP2 is equivalent to PP1 that has been described as a tool for
examining the role of Lck and FynT tyrosine kinases vs ZAP-70 in T cell
activation (29). As shown in Fig. 2A, the addition of PP2 to
Leu3a-treated cells induced a significant reduction of specific
apoptosis, supporting the need of Lck-mediated tyrosine phosphorylation
in inducing apoptosis. However, the evidence that the PP2, when added
to kinase assay, did not completely abolish Lck kinase activity (data
not shown) and did not affect totally the induction of apoptosis
suggested to us that kinases different from Lck or other signals could
be involved in CD4-mediated apoptosis. Therefore, we analyzed the need
of the TCR-CD3 complex in favoring the execution of apoptotic pathways
triggered by CD4. To do that, we performed apoptosis experiments in the
Jurkat derivative subline 31.13. These cells are defective for the
expression of the TCR 
-chain, an event that blocks the assembly of
the TCR-CD3 complex at the cell surface (23). Although
both cells expressed similar levels of Lck (Fig. 2B,
inset) and similar increases of Lck kinase activity
following Leu3a treatment (data not shown), Leu3a-mediated Lck
activation in the absence of TCR/CD3 failed to induce significant
apoptosis in 31.31 cells (Fig. 2B). Hence, surface
expression of the TCR-CD3 complex is a prerequisite not only for
propagating the early tyrosine phosphorylation cascade induced by CD4
engagement (30), but also for triggering apoptotic
signals.
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By using Ag-specific memory T cells, we have recently
demonstrated that CD4 cross-linking before activation via TCR results
in the up-regulation of the proapoptotic protein Bax, without modifying
the levels of the anti-apoptotic protein Bcl-2 and in mitochondrial
damage (11). The evidence that apoptosis observed in
Leu3a-treated Jurkat cells was mitochondrion-dependent prompted us to
analyze in these cells the level of Bax expression. Data reported in
Fig. 3A clearly
demonstrate that CD4/Lck activation results in up-regulation of
Bax. To further confirm that a Bax increase in Leu-3a-treated cells was
responsible for mitochondrion-dependent apoptosis, Jurkat cells were
incubated for 48 h with medium or Leu3a in the presence of 5 µM
Bax antisense oligonucleotides or unrelated oligonucleotides. The
initial first hour of incubation was performed in medium without serum
to increase the uptake of oligonucleotides. At the end of incubation,
mitochondrial membrane depolarization was evaluated by JC-1 staining.
As shown in Fig. 3B, Bax antisense oligonucleotides
significantly inhibited Leu3a-induced mitochondrial damage. These data,
along with our previous results (11), confirm that Bax can
be considered an important mediator of CD4-mediated apoptosis.
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The Bcl-2 family comprises death-inducing and death-inhibitory
members that differ in their tissue- and activation-dependent
expression patterns (31). Many of these proteins are
predominantly located in the outer mitochondrial membrane regulating
the mitochondrial transmembrane potential; this applies to Bcl-2,
Bcl-xL, and Bax. The ratio of death-inducing
(Bax) and death-inhibitory members (Bcl-2,
Bcl-xL) determines whether a cell will respond to
an apoptotic signal by mediating the disruption of the mitochondrial
membrane and the release of protease activators (32, 33).
Our evidence that the activation of Lck resulted in the up-regulation
of Bax without modifying Bcl-2 levels (see Figs. 3
A and 6)
prompted us to verify whether the overexpression of Bcl-2 in
Leu3a-treated cells could abolish mitochondrial membrane
depolarization. Therefore, we stably transfected Jurkat cells to
overexpress Bcl-2 (JBcl-2). Both Jurkat and JBcl-2 cells were incubated
for 48 h, in the presence or absence of Leu3a, and apoptosis was
evaluated by FACS analysis, measuring the variation of cell dimension
(FSC-H) vs cell granularity (SSC-H). As shown in Fig. 4A, Bcl-2 overexpression
reduced the percentage of apoptotic cells in Leu3a-treated cells.
Leu3a-induced mitochondrial membrane depolarization was also reduced to
41% in Bcl-2-overexpressing cells (data not shown). These results
evidence that the overexpression of Bcl-2 contrasts the apoptotic
pathways activated by CD4/Lck.
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Vav is an intracellular signaling molecule involved in the
regulation of TCR-initiated calcium signaling and gene transcription
(18, 34) and in the control of actin cytoskeleton
(19). We hypothesized that the apoptotic signals triggered
by CD4-mediated up-regulation of Bax should be neutralized following
Vav overexpression. Jurkat- and Vav-overexpressing (Vav CL9)
cells were treated with Leu3a and
apoptosis (Fig. 5) as well as Bax (Fig. 6A), and Bcl-2 (Fig. 6B) expressions were analyzed. As reported in Fig. 5A, Vav overexpression reduced the percentage of apoptotic
cells induced by Leu3a treatment. The inhibition of Leu3a-induced
apoptosis in Vav-overexpressing cells was also associated with reduced
mitochondrial damage (data not shown). Fig. 5B shows the
expression of Vav-myc in Vav CL9 cells. We next examined Bax
and Bcl-2 expression by FACS analysis. As shown in Fig. 6A,
Jurkat cells expressed a basal level of Bax (mean fluorescence
intensity (MFI), 9.8) that was significantly increased following Leu3a
treatment (MFI, 20.4). Vav overexpression significantly inhibited
CD4/Lck-induced Bax up-regulation (MFI of untreated cells = 8.9 vs
MFI of Leu3a-treated cells = 12.4). Calling
1 the percentage of Bax increase observed in
Jurkat cells with respect to basal level (mean ± SD, 47.5 ±
15) and 2 the percentage of Bax increase
observed in Vav CL9 (mean ± SD, 15.5 ± 8),
2 appeared to be systematically lower. Indeed,
the reduction factor (r = 1 -
(2/1)) on three
independent experiments was 0.68. Similar to that observed in Jurkat
cells, Leu3a did not affect Bcl-2 levels in Vav CL9 cells (Fig. 6B). Our evidence that Vav, a signaling molecule that
cooperates with CD28 to boost TCR signals (20),
counteracts CD4/Lck-mediated apoptotic signals indicates that the
activation of Lck in the absence of costimulatory signals may activate
in T cells apoptotic programs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is clear that when Th cells recognize the peptide-MHC complexes
through their TCRs, CD4 binds to an Ag-independent region of the MHC
(37). These interactions result in the tyrosine
phosphorylation of the TCR-associated CD3 -, -, 
-, and
-chains, an event that precedes all cellular changes accompanying T
cell activation. The earliest step in this process is the activation of
the Src and Syk families of tyrosine kinases, such as Lck and Zap-70,
respectively (3). In CD4+ T cells,
75–95% of cellular Lck associates with the cytoplasmic tail of
CD4, involving 85–95% of CD4 (38). Thus, CD4
cross-linking results in the activation of Lck kinase activity and,
even in the absence of TCR engagement, supports the initiation and
propagation of TCR signaling cascade (30). It is
interesting to note that all of this machinery, necessary to lead to
IL-2 production and to cellular growth, is also required for delivering
apoptotic signaling. Indeed, our data show that Lck activity as well as
TCR/CD3 expression and tyrosine phosphorylation events are necessary
for inducing T cell suicide.
That CD4 signaling induces apoptosis in T cells has been known since
1990 (39). However, the large amount of evidence
demonstrating that CD4 regulates apoptosis has been obtained after
separate ligation of CD4 and TCR (11, 40, 41, 42, 43). This has
been a good approach to analyze the effects of CD4 antagonist ligands
such as anti-CD4 Abs (44) or HIV gp120 (45, 46). Indeed, this approach has provided useful information to
control autoimmune diseases and organ graft rejection and to explain
CD4+ T cell deletion in AIDS. However, it does
not resemble the physiological role of CD4 following Ag recognition. We
reasoned on the possibility that an incorrect activation signal, such
as that mediated by altered peptide ligands, must be repaired through
the activation of cell suicide. It has been reported that partial
agonist or antagonist ligands induce a distant biochemical profile
characterized by partial -chain phosphorylation without activation
of the associated ZAP-70 kinase (47, 48). The CD4-Lck
activation in the absence of a concomitant increase in ZAP-70
phosphorylation has been considered a dominant-negative signal
(49). Our evidence that Lck kinase activity is strongly
induced by CD4 engagement in the absence of TCR/CD3-mediated
phosphorylation events (data not shown) suggests that our system can
mimic a partial activation signal.
The duration of antigenic stimulation determines the fate of naive T
cells and costimulation via CD28 facilitates T cell activation by
decreasing the time of commitment and by protecting T lymphocytes from
cell death (50). Ag-stimulated Jurkat T cells in the
absence of CD28/B7 interaction present a strong inhibition of tyrosine
phosphorylation of -chain and ZAP-70, confirming the need of CD28 in
the earliest events of tyrosine phosphorylation in these cells
(51). CD28 engagement in the same cells activates Vav to
boost TCR signaling and to enhance TCR proximal signaling
(20). Moreover, recent data in vivo demonstrated that Vav
is an important regulator of both TCR proximal signaling events
(18) and cytoskeleton rearrangements (19). In
this context, Jurkat cells overexpressing Vav display cellular
processes identifiable as typical lamellipodia and microspikes
(20). These findings suggest that activating signals such
as those mediated by the engagement of TCR/CD3 with peptide-MHC and CD4
with MHC in the absence of CD28 can represent an obstacle in the
process of immunological synapse formation, and consequently the cells
may be prone to apoptosize. If this obstacle is overcome by the
recruitment of costimulatory signals and active cytoskeletal
organization forms a scaffold for signaling components
(1), the cells are rescued and the events for cell
proliferation occur. Our evidence that Vav overexpression rescues T
cells from the CD4-activated apoptotic pathway supports the role of
cytoskeletal organization in mediating T cell commitment
(1) and suggests that Vav may represent a critical element
in counteracting the activation of apoptotic pathways.
It has been proposed that Bcl-2 family members regulate apoptosis like a rheostat (52). If proapoptotic factors such as Bax prevail, they form homodimers and cell death ensues. In contrast, cell survival is ensured when antiapoptotic factors such as Bcl-2 are more abundant, eventually leading to the formation of Bcl-2/Bax heterodimers (53). It has been reported that Bax, when overexpressed, translocates to the mitochondria and produces cell death (54). Activation and oligomerization of Bax results in formation of a homomultimeric pore to initiate cytochrome c release (55), an event that is accompanied by typical features of apoptosis. More recently, the role of Bax, as an essential gateway to mitochondria dysfunction, has been described in vivo (56). Very little is known about the molecular mechanisms that control mitochondrion-dependent apoptosis in T cells after TCR triggering. A described physiological mechanism is the absence of costimulatory signals that influence the expression of the anti-apoptotic protein Bcl-xL (57). No data are available on the possibility that other physiological mechanisms could be involved. Our evidence that in T lymphocytes CD4-Lck through TCR up-regulates Bax and induces mitochondria dysfunction, defines a new mechanism. Moreover, the observation that Vav overexpression blocks the execution of apoptotic programs by interfering with CD4-Lck-mediated signaling that leads to Bax up-regulation suggests a novel role for costimulation in protecting T cells from cell death.
In conclusion, our studies have identified the molecular mechanisms that allow CD4 to cooperate with TCR/CD3 in mediating apoptotic signals and suggest a new critical role of Vav in mediating survival signals.
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Acknowledgments |
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Footnotes |
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2 L.T. and B.M. have equally contributed to this work.
3 Address correspondence and reprint requests to Dr. Enza Piccolella, Department of Cellular and Developmental Biology, University of Rome "La Sapienza," Via dei Sardi, 70-00185, Rome, Italy. E-mail address: enza.piccolella{at}uniroma1.it
4 Abbreviations used in this paper: FSC-H, forward scatter height; SSC-H, side scatter height; m, mitochondrial transmembrane electrical potential; MFI, mean fluorescence intensity.
Received for publication September 17, 2001. Accepted for publication April 5, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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during T cell recognition. Science 289:1349.
B pathways. Proc. Natl. Acad. Sci. USA 96:3035. chain is necessary for its assembly with the CD3 complex but not for a/b dimerization. J. Biol. Chem. 265:4131. phosphorylation. Science :281.
2 domain of HLA-DR molecules. Nature 356:799.[Medline]
receptor mediates apoptosis. Eur. J. Immunol. 23:2676.[Medline]
and tumor necrosis factor-
secretion. Blood 84:2622. phopshorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 267:515. and lack of Zap-70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
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10%. B, Jurkat cells and
TCR/CD3-negative Jurkat cells (31.13) were cultured for 48 h with
or without Leu3a and apoptosis was analyzed as in A. The
results, expressed as percentage of specific apoptosis, represent the
mean ± SD of three independent experiments.
p56lck expression in Jurkat and 31.13 cells was
evaluated by Western blotting (inset).
