By
§
§
§
§
§
§
§
From the * Amgen Institute, Ontario Cancer Institute, § Department of Medical Biophysics and
Department of Immunology, and
Department of Medical Genetics, University of Toronto, Toronto,
Ontario, Canada M5G 2C1; the ¶ Institute for Radiation and Cell Research, University of
Würzburg, D-97078 Würzburg, Germany; the ** Basel Institute for Immunology, CH 4005 Basel,
Switzerland; and the
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
Ontario, Canada M5G 1X5
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Abstract |
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The protooncogene Vav functions as a GDP/GTP exchange factor (GEF) for Rho-like small
GTPases involved in cytoskeletal reorganization and cytokine production in T cells. Gene-targeted mice lacking Vav have a severe defect in positive and negative selection of T cell antigen
receptor transgenic thymocytes in vivo, and vav/
thymocytes are completely resistant to
peptide-specific and anti-CD3/anti-CD28-mediated apoptosis. Vav acts upstream of mitochondrial pore opening and caspase activation. Biochemically, Vav regulates peptide-specific Ca2+ mobilization and actin polymerization. Peptide-specific cell death was blocked both by
cytochalasin D inhibition of actin polymerization and by inhibition of protein kinase C (PKC).
Activation of PKC with phorbol ester restored peptide-specific apoptosis in vav
/
thymocytes. Vav was found to bind constitutively to PKC-
in thymocytes. Our results indicate
that peptide-triggered thymocyte apoptosis is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform.
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Introduction |
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Engagement of the TCR initiates a cascade of molecular events resulting in tyrosine phosphorylation of cytoplasmic proteins, Ca2+ mobilization, activation of the mitogen-activated protein kinase (MAPK)1 and stress-activated protein kinase (SAPK) pathways, and reorganization of the cytoskeleton. The protooncogene product Vav is expressed in hematopoietic cells and is rapidly phosphorylated after activation of T cells by various growth factors or by cross-linking of antigen receptors (1). Vav contains a collection of structural motifs, including a pleckstrin homology (PH) domain, known to facilitate membrane localization; a calponin homology (CH) domain, involved in actin binding; one Src homology (SH)2 domain and two SH3 domains, known to mediate protein-protein interactions; and a Dbl homology (DH) domain (1, 4). In the protooncogene Dbl, the DH domain confers the capacity for guanine nucleotide exchange for the Rho family of small GTPases, which regulate cytoskeletal organization and SAPK/ JNK signaling (5). Although Vav has been implicated in Ras and MAPK signaling (1, 11, 12), recent biochemical and genetic studies have established a role for Vav in the activation of Rac1 and other members of the Rho family of small GTPases (13).
T cells from vav/
mice exhibit a block in cell cycle
progression and fail to produce IL-2 in response to anti-CD3 cross-linking (16). Although Vav has no apparent
role in TCR-mediated signaling pathways leading to MAPK
or SAPK activation after CD3
cross-linking, Vav has been
shown to regulate TCR-mediated Ca2+ flux and reorganization of the actin cytoskeleton. Consistent with this role,
the functional defects observed in vav
/
T cells can be
mimicked using the actin polymerization inhibitor cytochalasin D (CytD [19, 20]). In addition, Vav has been found to have a crucial role in thymocyte development and positive selection of both MHC class I- and MHC class II-
restricted TCR transgenic (Tg) thymocytes (16, 21).
However, it has been reported that superantigen-reactive
and alloreactive vav
/
thymocytes could still undergo negative selection (21), suggesting a differential requirement
for Vav in positive versus negative thymocyte selection.
To examine the role of Vav in the selection and activation of peptide-specific thymocytes, we introduced the H-Y
TCR (22) and the lymphocytic choriomeningitis virus
(LCMV) p33 peptide-specific P14 TCR transgenes (23)
into a vav/
background. We report that Vav is essential
for peptide-specific clonal deletion and TCR-triggered apoptosis. Vav was found to regulate peptide-specific Ca2+
mobilization and actin polymerization in thymocytes. Peptide-triggered apoptosis could be blocked using the actin
polymerization inhibitor CytD and a global protein kinase
C (PKC) blocker. Among all PKC isoforms tested, only
PKC-
was found to associate with Vav in thymocytes.
These results suggest that TCR-mediated changes in the
actin cytoskeleton and PKC-
are crucial prerequisites for negative selection and peptide-triggered apoptosis.
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Materials and Methods |
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Mice.
Gene-targeted mice made deficient in Vav by homologous recombination (19), and H-Y and P14 TCR Tg, and P14 TgReagents.
The PKC blockers RO-31-8220 (which blocks all PKC isoforms) and GF109023X (which inhibits the activity of only the Ca2+-dependent PKC isoforms PKC-Induction of Apoptosis.
Freshly isolated thymocytes from C57BL/6 mice were cultured in RPMI 1640 medium containing 10% FCS and 10Immunocytometry.
Blood samples (20 µl) were collected in heparinized capillary tubes and washed once in immunofluorescence staining buffer (1% FCS, 0.01% NaN3 in PBS). Single cell suspensions of thymocytes, spleen cells, and mesenteric lymph node cells were prepared as described (29), resuspended in PBS, and incubated with the appropriate mAbs for 30 min at 4°C. The following mAbs were used: anti-CD4 (FITC- or PE-labeled; PharMingen); anti-CD8 (FITC- or PE-labeled, or biotinylated; Serotec, Inc., Raleigh, NC); anti-pan TCR-Negative Selection In Vitro.
Thymocytes were purified from vav+/+, vav+/Mitochondrial Permeability Transition (m Disruption).
Signal Transduction.
Thymocytes were isolated from nonselectingImmunoprecipitation.
Thymocytes and peripheral lymph node T cells were activated with anti-CD3Peptide-specific Ca2+ Mobilization.
Peptide-specific Ca2+ mobilization ([Ca2+]i) in P14 thymocytes was determined as described (35). In brief, freshly isolated P14 Tg thymocytes (2 × 106) were loaded with 3 mM INDO-1 (Molecular Probes, Inc., Eugene, OR) in IMDM (pH 7.4) for 1 h at 37°C. Thymocytes were then incubated with peptide-presenting EL4 cells loaded with either p33 or AV peptide and centrifuged (1,500 rpm, 4°C) to allow conjugate formation between thymocytes and EL4 cells. Increases in intracellular free Ca2+ were recorded in real time on live-gated thymocyte-EL4 conjugates using a FACScalibur® (Becton Dickinson).Actin Polymerization.
Thymocytes were preincubated with PMA (12.5 ng/ml), and EL4 cells were loaded with either p33 or AV peptide (10 ![]() |
Results |
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To analyze the role in vivo of Vav in thymocyte
selection, we introduced two rearranged TCR-/
transgenes, H-Y and P14, into a vav
/
background. The H-Y
TCR recognizes a male-specific peptide in the context of
MHC class I. Thymocytes expressing the H-Y TCR are
positively selected in female H-2b mice (22, 24, 25). The
P14 TCR is specific for a peptide epitope of the LCMV
glycoprotein p33 in the context of the MHC haplotype
H-2Db (23). In positively selecting vav+/
mice, the development of P14+ and H-Y+ thymocytes is skewed towards the
CD8+ lineage (Fig. 1, A and B). In vav
/
mice, this bias in
favor of mature CD8+ thymocytes did not occur in either
H-Y Tg or P14 Tg thymocytes (Fig. 1, A and B), and development was in fact blocked at the immature CD4+
CD8+ stage of differentiation. Consistent with a block in
positive selection, TCR Tg vav
/
thymocytes expressed
high levels of HSA and did not upregulate surface expression of the maturation and selection markers CD69, CD5
(Fig. 1 C), H-2Kb, or CD45RB (not shown). Expression of
the P14 Tg TCRV
8 and TCRV
2 chains was significantly lower in immature CD4+CD8+ thymocytes of vav
/
mice compared with CD4+CD8+ thymocytes from vav+/
mice (Fig. 1 C). Similarly, expression of the H-Y Tg-
specific TCRV
8 and TCRV
3 chains detected by the
T3.70 Ab was significantly lower in H-Y Tg vav
/
mice
(not shown). Importantly, P14 Tg vav
/
(and H-Y Tg
vav
/
[not shown]) thymocytes displayed a phenotype that
is similar to that of P14 Tg (and H-Y) vav+/+
2m
/
mice, which have a defect in positive selection of MHC
class I-restricted thymocytes (Fig. 1 C). These results show
that Vav regulates the positive selection of MHC class
I-restricted thymocytes and confirm previous data demonstrating the crucial role of Vav in positive thymocyte selection (16, 21).
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To examine the role of Vav in peptide-specific clonal deletion in vivo and in vitro, we investigated the negative selection of H-Y and LCMV TCR Tg thymocytes. In the
H-Y Tg system, thymocytes expressing the H-Y TCR are
positively selected in female H-2b mice but negatively selected in male H-2b mice. Negative selection in male H-Y
Tg mice results in a small thymus due to deletion of
CD4+CD8+ thymocytes (24; Fig. 1 B). However, male
vav/
mice contained a large number of CD4+CD8+ thymocytes, indicating that negative selection of H-Y Tg thymocytes was severely impaired in the absence of Vav.
To further elucidate the requirement for Vav in negative
selection, an in vitro model of peptide-specific negative selection was used (30). In the LCMV-p33 peptide system,
APCs are loaded with different concentrations of the
LCMV glycoprotein strong agonist peptide p33 or the
weak agonist p33 peptide analogue 8.1. Although vav+/
P14 Tg thymocytes readily underwent apoptosis in a dose-dependent manner in response to treatment with either the
p33 or 8.1 peptide (Fig. 2 A), vav
/
P14 Tg thymocytes
were completely resistant to peptide-mediated apoptosis,
even at very high peptide concentrations (Fig. 2 B). Since
P14 vav
/
thymocytes are blocked in positive T cell selection, we used
2m
/
vav+/+ P14 TCR Tg mice (H-2b/b)
as an additional control since these mice have a block in the positive selection of the P14 TCR due to deletion of the
MHC class I ligand (
2m
/
). The kinetics and extent of
p33-induced apoptosis of
2m
/
CD4+CD8+ P14 Tg
thymocytes were similar to those of vav+/
P14 Tg mice,
indicating that the defect of P14 Tg vav
/
thymocytes to
undergo peptide-specific apoptosis was not due to differences in the composition of thymocyte populations (Fig. 2
C). These results show that Vav is required for the negative selection of peptide-specific thymocytes.
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Immature CD4+CD8+ thymocytes are highly
susceptible to cell death induced by many apoptotic stimuli
(36). We evaluated the ability of vav+/ and vav
/
thymocytes to undergo apoptosis in response to the following stimuli: dexamethasone, PMA/Ca2+ ionophore, anti-CD95
(FAS), anti-CD3
, and anti-CD3
/anti-CD28 (29, 37).
Although treatment with anti-CD3
/anti-CD28 induced the cell death of vav+/
CD4+CD8+ thymocytes, vav
/
thymocytes were strikingly resistant to anti-CD3
/anti-CD28-triggered apoptosis (Fig. 3). No significant differences were observed between vav
/
, vav+/
, and vav+/+
thymocytes in the extent or kinetics of cell death in response to anti-CD95 (FAS), PMA plus Ca2+ ionophore
(Fig. 3), or dexamethasone (not shown), implying that the
cellular apoptotic machinery is functional in the absence of
Vav. These data indicate that Vav is a crucial signal transduction molecule involved in TCR-mediated thymocyte
apoptosis.
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Disruption of the mitochondrial transmembrane potential
(m) due to the opening of mitochondrial membrane pore
complex has been identified as the earliest common denominator of apoptosis (36). Alterations in mitochondria lead to
the release of "apoptosis-inducing factor" (AIF) or cytochrome
c, resulting in the activation of effector caspases such as
caspase 3 (Cpp32) (32, 38). To assess whether Vav links
TCR signaling to the opening of mitochondrial pores, we
determined the mitochondrial transmembrane potential (
m) by cytometry using the fluorochromic dye DiOC6(3)
(33). Stimulation of P14 Tg vav+/
thymocytes with 10
6 M
p33 peptide led to measurable changes in
m compared
with P14 Tg vav+/
thymocytes treated with similar concentrations of the nondeleting control AV peptide (compare Fig. 4, A and C). However, p33 peptide-triggered disruption of
m did not occur in vav
/
thymocytes
(compare Fig. 4, B and D). Fig. 4 E shows that this effect
was dose dependent only in P14 Tg vav+/
thymocytes.
Moreover, peptide-specific activation of the downstream effector caspase 3 (42) did not occur in P14 Tg vav
/
thymocytes even when very high concentrations of the deleting p33 peptide were used (not shown). Disruption of
m and caspase 3 activation occurred normally in vav
/
thymocytes treated with dexamethasone or CD95 (not
shown).
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We (19) and other groups (17, 20, 21) have used
anti-CD3 cross-linking to establish that Vav links TCR/
CD3 activation to the actin cytoskeleton and Ca2+ flux.
However, these experiments showed that Vav had no apparent role in phosphotyrosine signaling, or in the activation of MAPK, SAPK, or NF-B. However, the role of
Vav in peptide-MHC-mediated physiological responses
and peptide-MHC-specific signaling remained obscure. To
determine the biochemical defect in vav
/
T cells, we analyzed peptide-triggered signaling pathways in P14 TCR
Tg thymocytes from vav+/+, vav
/
, and various control
strains. Since P14 vav
/
thymocytes are blocked in positive T cell selection, we used
2m
/
vav+/+ P14 TCR Tg
mice (H-2b/b) as an additional control. These mice have a
block in the positive selection of the P14 TCR due to the
deletion of the MHC class I ligand (
2m
/
[27, 43]). The
use of
2m
/
vav+/+ P14 Tg mice ensured that thymocytes expressing the Tg TCR chain had not been previously activated by the selecting MHC ligand. TCR levels
and expression of maturation markers were similar on the
thymocytes of P14 vav
/
and P14
2m
/
vav+/+ mice
(Fig. 1 C).
P14 vav/
, P14 vav+/
, and P14
2m
/
vav+/+ thymocytes were cultured with the deleting p33 peptide or the
nondeleting control AV peptide for different time periods.
No apparent differences were observed between vav
/
,
vav+/
, and
2m
/
vav+/+ P14 Tg thymocytes in either
the extent or kinetics of phosphotyrosine signaling, MAPK
activation, or NF-
B activation after p33 peptide-specific activation (not shown). Intriguingly, p33 peptide-MHC-mediated activation of either vav
/
, vav+/
, or
2m
/
vav+/+ P14 Tg thymocytes did not lead to any detectable
SAPK/JNK activity (not shown). However, p33 peptide-
specific Ca2+ mobilization was significantly decreased in
vav
/
P14 thymocytes compared with P14
2m
/
vav+/+
thymocytes (compare Fig. 5, C and D) and P14 vav+/
thymocytes (not shown). Moreover, phalloidin staining experiments showed that vav
/
thymocytes exhibited a decrease
in actin polymerization and the formation of F-actin after
activation with the deleting p33 peptide (Fig. 6). Peptide-
MHC-triggered Ca2+ mobilization and actin polymerization were comparable between P14 vav+/
and P14
2m
/
vav+/+ thymocytes (not shown). These results provide the
first genetic evidence that Vav links peptide-specific activation of the TCR to cytoskeletal reorganization and Ca2+
mobilization.
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Treatment with the actin polymerization blocker CytD
is known to mimic the functional and biochemical defects
observed in peripheral vav/
T cells after CD3 cross-linking (20). It has also been reported that CytD addition impairs peptide-induced Ca2+ flux in human CD4+ T cell
clones (35). Therefore, we analyzed whether CytD treatment could abrogate Ca2+ mobilization in freshly isolated,
wild-type P14 thymocytes. Incubation of P14 thymocytes
with CytD (Fig. 5 E) or chelation of extracellular Ca2+ by
EGTA (Fig. 5 F) significantly decreased p33 peptide-triggered Ca2+ mobilization. The extent of the CytD-mediated decrease in peptide-specific Ca2+ flux was dependent
on the concentration of the p33 peptide (not shown).
Treatment of P14 thymocytes with CytD had no apparent effect on the extent of tyrosine phosphorylation, NF-
B
activation, or MAPK activation (not shown). These results
suggest that peptide-specific actin polymerization has an
important role in the extent and duration of TCR-mediated Ca2+ mobilization in thymocytes.
To further elucidate the role of Vav-regulated changes in the actin cytoskeleton during negative selection, we tested whether the actin polymerization inhibitor CytD could interfere with peptide-specific apoptosis of P14 TCR Tg thymocytes. Interestingly, CytD blocked the peptide-specific cell death of vav+/+ P14 thymocytes in a dose-dependent manner (Fig. 7). Similarly, anti-CD3/anti-CD28-mediated, but not dexamethasone- or CD95-mediated, apoptosis of vav+/+ thymocytes could be inhibited by CytD (not shown). These data suggest that TCR-mediated actin polymerization plays a role in peptide-specific apoptosis of thymocytes.
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The primary biochemical defects associated with signaling
in peptide-specific vav/
thymocytes were impaired Ca2+
flux and reduced actin polymerization. Addition of Ca2+
ionophore, which restores Ca2+ flux in P14 vav+/
and
vav
/
thymocytes (not shown), did not restore p33 peptide-mediated apoptosis even when used at very high concentrations (100 ng/ml; Fig. 8 A). In contrast, activation of
vav
/
P14 Tg cells with the deleting p33 peptide plus activation of PKC via the phorbol ester PMA significantly
shifted the dose-response curve of cell death in a dose-
dependent manner (Fig. 8 B). Thus, activation of PKC via
PMA, but not rescue of Ca2+ flux by Ca2+ ionophore, was
able to restore peptide-specific apoptosis in P14 vav
/
thymocytes, suggesting that the Vav and PKC signaling cascades cooperate in the induction of TCR-mediated thymocyte apoptosis. However, it should be noted that our
results do not preclude a role for Ca2+ elevation in thymocyte selection and clonal deletion in vivo.
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Recently, it has been shown that in order for Vav to act as a GDP/GTP exchange factor (GEF) for Rac, RhoA, or CDC42, PI3'K activity and the binding of PI3'K-generated phospholipid products to the PH domain of Vav are required (44). Therefore, we investigated whether the specific PI3'K inhibitor Wortmannin could block peptide-specific apoptosis. As shown in Fig. 8, C and D, the inhibition of PI3'K did not affect the apoptosis of P14 thymocytes triggered by the deleting p33 peptide, suggesting that the role of Vav in thymocyte apoptosis is independent of PI3'K phospholipid-dependent Vav activation. However, inhibition of PKC by the compound RO-31-8220 (which blocks all PKC isoforms) prevented peptide-specific apoptosis in wild-type thymocytes (Fig. 8, C and D). Peptide-specific apoptosis of P14 thymocytes was not blocked by the pharmacological inhibitor GF109203X, which prevents activation of Ca2+-dependent PKC isoforms (Fig. 8 C). PMA-triggered apoptosis was blocked by the global PKC blocker RO-31-8220 but not by CytD or Wortmannin (Fig. 8 E). These data indicate that Vav links TCR signaling to activation of a Ca2+-independent PKC isoform required for the induction of peptide-specific thymocyte apoptosis.
To further evaluate the link between Vav and PKC, we
immunoprecipitated Vav from wild-type thymocytes and
analyzed the binding of various PKC isoforms to Vav. Surprisingly, Vav coimmunoprecipitated with the Ca2+-independent PKC isoform PKC- but not with any other PKC
isoform tested (Fig. 9, left). The association between Vav
and PKC-
did not change after CD3 cross-linking, suggesting that Vav/PKC-
binding is constitutive in thymocytes. Constitutive Vav/PKC-
association was also observed in mature peripheral T cells (not shown). Our
results do not preclude associations between Vav and low
abundance PKC isoforms.
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Discussion |
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Our genetic and functional analyses of vav/
mice show
that Vav is a crucial regulator both of positive and negative
selection of CD8+ thymocytes and the peptide-triggered
apoptosis of developing T cells. Vav is involved as a regulator of TCR-mediated cytoskeletal reorganization and Ca2+
mobilization after peptide-specific activation by APCs. Our
results suggest that peptide-triggered thymocyte apoptosis
is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform. This
hypothesis is based on the following findings: (a) Vav-deficient thymocytes do not undergo peptide-MHC-mediated cell death in vitro or in vivo; (b) vav
/
thymocytes exhibit
a defect in actin polymerization, and inhibition of cytoskeletal changes by CytD blocks peptide-MHC-mediated and anti-CD3/anti-CD28-mediated thymocyte apoptosis. However, inhibition of actin polymerization does not inhibit
dexamethasone-, CD95-, or PKC-mediated thymocyte apoptosis; (c) activation of PKC restores the susceptibility to
apoptosis of p33 peptide-triggered P14 Tg vav
/
thymocytes, and inhibition of a Ca2+-independent PKC isoform inhibits TCR-mediated thymocyte apoptosis; and (d)
Vav associates constitutively with PKC-
but not with any
other PKC isoform. Thus, the PKC isoform PKC-
is a
good candidate for the effector kinase of negative thymocyte selection.
The PKCs are a family of 11 phospholipid-dependent
serine/threonine kinases. These closely related isoenzymes
differ in their structural and biochemical properties, tissue
distribution, subcellular localization, and substrate specificity (45, 46). According to primary structure and binding to
Ca2+ or phorbol esters, different PKC subgroups exist:
conventional PKCs (,
I,
II, and
) bind Ca2+ and are
activated by PMA; novel PKCs (
,
,
, and
) are activated by PMA but do not bind Ca2+; atypical PKCs (
,
,
, and µ) bind to diacylglycerol (DAG) but are not activated by PMA or Ca2+ ionophores. Activation of PKC
molecules by lipid second messengers requires membrane
recruitment (47). T cell activation requires PKC activity.
Previously, it has been reported using PCR and Northern blotting that PKC-
, -
, -
, -
, -
, and -
are expressed in
thymocytes (48). Our study shows for the first time that
all PKC isoforms are expressed in thymocytes, albeit at different levels. Although most PKC isoforms are expressed
ubiquitously, the isoform PKC-
is predominantly expressed in the hematopoietic system, particularly in T cells
(48), and has been placed upstream of IL-2 transactivation and AP1 (Fos/Jun) activity in T lymphoma cells (51,
52). Recently, it has been shown in Jurkat cells that calcineurin and PKC-
cooperate in inducing IL-2 gene transcription (53), a function that is reminiscent of the coordination of IL-2 expression by calcineurin and Vav/Rac1
(20, 54). Interestingly, the small Ras-like molecules Rac1, CDC42, and Rho, whose activation is regulated by
Vav, may act upstream of PKC (58, 59), suggesting that Vav,
Rac1, and PKC-
may form a complex. Although Vav and
PKC-
clearly associate in thymocytes and peripheral T
cells, Rac1, CDC42, and RhoA do not coimmunoprecipitate with these complexes.
Vav and Vav-regulated Rac1 and CDC42 have been
previously shown to mediate changes in the actin cytoskeleton and to activate SAPK/JNK (13, 60, 61). Similarly,
PKC- has been placed upstream of SAPK/JNK in T cell
lines (53). However, TCR/CD28-mediated SAPK/JNK
activation appears normal in vav
/
thymocytes and mature
T cells after anti-CD3 cross-linking (19, 20). Similarly, a
genetic mutation in the SAPK/JNK signaling pathway had
no effect on the induction of thymocyte apoptosis (29),
suggesting that Vav and perhaps PKC-
-mediated negative selection are independent of SAPK/JNK. Importantly,
although SAPK/JNK activity is readily induced in thymocytes in response to anti-CD3 and anti-CD28 Abs (62),
we failed to detect SAPK/JNK activation in thymocytes
after specific peptide-MHC-mediated stimulation. Thus,
Vav may define a novel signaling pathway that leads to negative thymocyte selection and IL-2 production in peripheral
T cells. This signaling pathway appears to be independent
of MAPK, SAPK/JNK, or NF-
B activation.
Rac1, RhoA, or CDC42 activation by Vav requires
PI3'K activity in vitro (44). However, inhibition of PI3'K
has no apparent effect on the peptide-triggered thymocyte
apoptosis of thymocytes, suggesting either that the role of
Vav in thymocyte apoptosis is independent of its GTP exchange activity for Rac and CDC42, or that Vav can function as an exchange factor even in the absence of PI3'K activity in vivo. Similarly, it has been shown that a pharmacological PKC inhibitor, but not the PI3'K inhibitor Wortmannin, can block anti-CD3/anti-CD28-mediated thymocyte apoptosis (63). These latter results are consistent with our observation that Vav and PKC regulate
anti-CD3/anti-CD28-mediated apoptosis and imply that
PKC activation is a crucial signal in the pathway of TCR-triggered apoptosis in developing thymocytes. Whether
PKC- and/or other PKC isoforms are the crucial downstream kinases in thymocyte selections needs to be determined in genetic experiments.
Both we (19) and Holsinger et al. (20) have previously
shown that Vav regulates Ca2+ mobilization and actin reorganization in peripheral T cells after anti-CD3 Ab cross-linking. The results of the present study provide the first
genetic evidence that Vav is also a crucial regulator of the
TCR-mediated Ca2+ flux and actin polymerization required for thymocyte selection of peptide-MHC-stimulated thymocytes. In particular, actin polymerization was
found to be crucial for the induction of apoptosis. Various cytoskeletal proteins and regulators of cytoskeletal changes, such as gelsolin (64),
-catenin (67), PAK2 (68), actin (67, 69), fodrin (77), and Gas2 (76), are proteolytically cleaved after the induction of apoptosis. Cleavage of cytoskeletal proteins, which occurs after caspase activation,
has an important role in the death effector phase of apoptosis, particularly in the regulation of membrane alterations,
morphological changes, and DNA fragmentation (68, 69).
In contrast, inhibition of actin polymerization by CytD resulted in impaired TCR-mediated apoptosis. These cytoskeletal changes were found to act upstream of the opening
of the mitochondrial membrane pore complex and caspase
activation. However, CytD could not block apoptosis after PKC activation (which induces rapid changes in the actin
cytoskeleton) and CytD could not protect thymocytes
from dexamethasone- or CD95-mediated apoptosis, suggesting that actin changes per se are not the principle mechanism for conveying a cell death signal in thymocytes.
Rather, we propose that TCR-mediated and Vav-regulated changes in the actin cytoskeleton lead to the recruitment and/or assembly of a death effector molecule(s) that
relays the TCR signal to the apoptotic machinery.
It has been shown in the D10 T cell line that only PKC-
translocates to the site of contact between T cells and
APCs. All other PKC isoforms (
,
1,
,
, and
) are excluded from the contact site, suggesting that PKC-
has a
specific role in T cell activation (78). Since Vav regulates
TCR clustering and PKC-
is found in these clusters, Vav-regulated actin polymerization may be required for recruitment of PKC-
to the TCR and activation of PKC at the
cell membrane. How Vav and possibly PKC-
link TCR-mediated signals to mitochondrial apoptosis and caspase 3 activation remains to be determined. Importantly, our results show that Vav and actin polymerization also regulate
the extent and duration of peptide-specific Ca2+ mobilization, an event thought to be involved in thymocyte apoptosis and clonal selection (79).
Our data show that Vav regulates positive and negative
selection of MHC class I-restricted thymocytes. We have
previously reported that the progression of CD4CD8
T
cell precursor cells to CD4+CD8+ thymocytes is impaired
in vav
/
mice on a C57BL/6 (H-2b/b) background but not
on a CD1 (H-2q/q) background (19). Thus, Vav clearly has
a role in preTCR-mediated expansion of early thymocytes,
but the absence of Vav can be partially compensated by
other signaling molecules. However, this compensatory mechanism does not appear to be operational or sufficient
to rescue positive and negative selection of TCR Tg thymocytes in vav
/
mice. Thus, Vav either plays a different
role in TCR signaling at different stages of development
and/or different compensatory mechanisms are available to
CD4
CD8
versus CD4+CD8+ thymocytes that make up
for the Vav mutation in the expansion from CD4
CD8
to CD4+CD8+ cells but cannot compensate for selection
of CD4+CD8+ thymocytes.
Our data provide the first genetic evidence
for a role for Vav in peptide-MHC-triggered cytoskeletal
reorganization in vivo. These results indicate that Vav-regulated actin reorganization is a crucial prerequisite for antigen receptor-mediated selection and apoptosis in peptide-specific thymocytes. We have shown that Vav and the actin
cytoskeleton regulate the extent and duration of Ca2+ mobilization after peptide-specific activation, and that Vav functions upstream of mitochondrial pore opening and
caspase activation. The defect in peptide-specific apoptosis
in vav/
thymocytes can be overcome by activation of
PKC, and the inhibition of PKC blocks peptide-specific
cell death in wild-type thymocytes, indicating that PKC
activation is the trigger for thymocyte apoptosis in response
to peptide. Of all PKC isoforms tested, Vav associated only
with the PKC-
isoform expressed primarily in T cells,
suggesting that PKC-
is the crucial kinase involved in thymocyte selection and clonal deletion.
![]() |
Footnotes |
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Address correspondence to Josef M. Penninger, The Amgen Institute, Ontario Cancer Institute, Departments of Medical Biophysics and Immunology, University of Toronto, 620 University Ave., Toronto, Ontario M5G 2C1, Canada. Phone: 416-204-2241; Fax: 416-204-2278; E-mail: jpenning{at}amgen.com
Received for publication 7 July 1998 and in revised form 10 September 1998.
Y.-Y. Kong and K.-D. Fischer contributed equally to this work.We thank K. Bachmaier, K. Tedford, A. Oliveira-dos-Santos, T. Sasaki, H. Nishina, A. Tafuri-Bladt, and L. Zhang for helpful comments, and M. Saunders for scientific editing.
This work was supported by grants from the Medical Research Council of Canada (to J.M. Penninger and A. Bernstein); from the National Cancer Institute of Canada (to A. Bernstein); and from the Deutsche Forschungsgemeinschaft (SFB 465; to K.-D. Fischer).
Abbreviations used in this paper
7-AAD, 7-amino-actinomycin D;
2m,
2-microglobulin;
CytD, cytochalasin D;
F-actin, filamentous actin;
JNK, c-Jun NH2-terminal kinase;
LCMV, lymphocytic choriomeningitis
virus;
MAPK, mitogen-activated protein kinase;
NF-
B, nuclear factor
B;
PI3'K, phosphatidylinositol 3'-kinase;
PKC, protein kinase C;
SAPK, stress-activated protein kinase;
Tg, transgenic.
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