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9V
2 T Cells to Phosphate Antigens1
Departments of
* Pathology and
Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461; and
Istituto di Ricovero e Cura a Carattere Scientifico, Fondazione Santa Lucia, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells expressing the V9V2 gene segments
are activated polyclonally by phosphoantigens found on a wide variety
of pathogenic organisms. After ligand exposure, V9V2 T cells
proliferate and rapidly secrete large amounts of cytokines and
chemokines that contribute to the innate immune response to these
pathogens. Neither APCs nor costimulatory molecules are required. In
this study we examined whether these phosphoantigens activate protein
kinase C
(PKC). This novel PKC isoform is essential for Ag
signaling through the 
TCR in a costimulation-dependent fashion.
The results showed that isopentenyl pyrophosphate (IPP), a soluble
phospholigand released by mycobacteria, led to the rapid and persistent
activation of PKC in T cells, as determined by evidence of
translocation and phosphorylation. In contrast, no ligand-dependent
response was detected for PKC/ or PKC. Using the inhibitors
Gö6976 and rottlerin, a role for both conventional and novel PKC
isoforms in IPP-induced proliferation, CD25 expression, and cytokine
and chemokine production was demonstrated. Gel-shift assays indicated
that the transcription factors NF-
B and AP-1 were downstream targets
of PKC activation. IPP also induced the rapid and persistent
phosphorylation of extracellular signal-regulated kinases 1 and 2, p38
mitogen-activated kinase, and stress-activated kinase/c-Jun N-terminal
kinase, but only an inhibitor of conventional PKCs blocked these
responses. We conclude that the T cell response to
phosphoantigens is regulated by both novel and conventional PKC
isoforms, with PKC being more responsive to ligand stimulation and
PKC/ to growth-factor availability. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9 (also known as V2)/V2 TCR are the
major population of human T cells in the peripheral blood and
account for 1–10% of the total lymphocyte population in adults
(1). In common with other lymphocytes, T cells
develop a highly diverse Ag recognition receptor (2), but
the nature of the Ag(s) that activate these cells remain poorly
defined. In humans and primates, mycobacteria selectively activate
T cells that use the V9V2 TCR via low-m.w.
protease-resistant and phosphatase-sensitive ligands, which have been
identified as monoalkyl phosphates such as isopentenyl pyrophosphate
(IPP)3 and related
prenyl phosphates (3, 4, 5, 6). They have been detected in other
potential pathogens including Escherichia coli,
Plasmodium falciparium, and Francisella
tularensis and are collectively referred to as phospholigands
(reviewed in Ref. 7). Activation of V9V2 T cells by
these ligands is polyclonal and does not require Ag processing or APCs,
although cell-cell contact enhances the response
(4).
Gene transfer experiments into TCR-negative Jurkat cells demonstrated
that phosphate Ag recognition occurs via the Ag-combining site of the
TCR and involves both the V9 and V2 gene segments
(8). Structural analysis of the
complementarity-determining region 3 formed by this combination
shows a very small angle between the and chain, exhibiting a
chemically feasible binding site for phosphorylated Ags (9, 10). Additional studies demonstrated a critical role for lysine
residues encoded by the J1.2 segment (11, 12). Because
these sequences are not found in other human or mouse J segments,
this observation would account for the unique restriction of this
response to this subset of T cells.
The structural features of the TCR, as well as the observation
that responses to phosphoantigens do not require costimulatory
molecules or APCs, suggest that the mechanisms involved in TCR
activation differ from that used by T cells. This would further
suggest that after activation, the TCR forms different
recognition/signaling complexes than that found in other lymphocytes.
However, relatively little is known about the signaling events
initiated by mycobacterial phosphoantigens such as IPP. Lafont et al.
(13) showed that IPP binding to the V2 TCR induces the
early activation of p56lck and a delayed and
sustained activation of TNF- secretion. They also showed that
activation with IPP does not lead to down-regulation of the TCR, in
contrast with cross-linking with Abs to CD3. This observation may
explain the sustained activation of these cells elicited by IPP.
Additional studies by Lafont et al. (14, 15) showed that
optimal cytokine release mediated by cross-linking CD3 is dependent
upon the p38 mitogen-activated protein kinase (MAPK) and extracellular
signal-regulated kinases 1 and 2 (ERK1/2). In our own studies, we
showed that stimulation with IPP led to the rapid activation of the
transcription factors NF-B and AP-1 and that inhibition of NF-B
signaling blocked IPP-induced production of TNF-,
macrophage-inflammatory protein 1 (MIP-1), and RANTES
(16).
In T cells that express the TCR, an important role for protein
kinase C (PKC), a member of the novel family of PKCs that is
diacylglycerol-dependent but Ca2+-independent for
catalytic activity, has recently been recognized for the activation of
NF-B and AP-1 after cross-linking of the TCR (17, 18, 19, 20, 21).
However, whether T cells use a similar signaling pathway has not
yet been determined. In mouse intraepithelial T cells, Fahrer et
al. (22) failed to detect the presence of PKC using
transcriptional profiling, suggesting that at least in this subset of
T cells this pathway is not involved. In this study, we asked
whether activation of V9V2+ T cells with
IPP led to the activation of PKCs and, if so, whether these kinases
were involved in IPP-induced signaling. The data show that human
peripheral blood T cells express both classical and novel PKC
isoforms, including PKC, and demonstrate that they are involved in
IPP-induced proliferation, CD25 expression, and cytokine and chemokine
production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMCs from healthy donors were isolated by Ficoll-Hypaque
gradient centrifugation (Pharmacia, Uppsala, Sweden). Use of human
tissues was approved by the Committee on Clinical Investigation of the
Albert Einstein College of Medicine. Long-term cultures of
V2+ cells were established by stimulating once
with IPP (30 µM; Sigma-Aldrich, St. Louis, MO) and were maintained
with 50 U/ml IL-2 (National Cancer Institute, Frederick, MD) as
described (16). V2 expression was determined by FACS
(clone B6 mAb; BD PharMingen, San Diego, CA). At the time of testing,
V2+ T cells were restimulated with IPP
(IPP2X, 3 or 30 µM). In some experiments, cells were
pretreated with Gö6976 (250 nM) as an inhibitor of the
Ca2+-dependent PKC or with rottlerin (1–5 µM)
as an inhibitor of the Ca2+-independent PKC
(Calbiochem, La Jolla, CA).
Abs and reagents
Abs specific for PKC, PKC/II and PKC total (BD
Transduction Laboratories, Lexington, KY), and phospho-PKCs (New
England Biolabs, Beverly, MA) and for total and phospho-specific
ERK1/2, p38, and c-Jun N-terminal kinase (JNK) (Cell Signaling) were
used. Blocking peptide for the phosphospecific PKC Ab was used
according to the manufacturer’s instructions (New England Biolabs).
Protein and phosphatase inhibitors were purchased from Sigma-Aldrich.
For the immunoprecipitation assays, a mAb for PKC (BD Transduction
Laboratories) was used for the pull-downs, and a polyclonal Ab for
total PKC was used for the immunoblotting control (Santa Cruz
Biotechnology, Santa Cruz, CA).
Protein extraction and subcellular fractionation
V2+ T cells (40–50 x
106) were washed in PBS/PMSF, pelleted by
microcentrifugation (4,000 rpm/2 min/room temperature (RT)),
resuspended in 250 µl of ice-cold hypotonic buffer (10 mM HEPES (pH
7.2), 42 mM KCl, 5 mM MgCl2, 5 mM NaF, 5 mM
Na3VO4, 1 mM PMSF, 10
µg/ml aprotinin and leupeptin), incubated on ice for 20 min,
sonicated, and centrifuged at 12,000 rpm/15 min/4°C. Supernatants
(cytosolic fraction) were collected, and pellets were resuspended (50
µl of hypotonic buffer/1% Nonidet P-40) and incubated on ice for 20
min and centrifuged (12,000 rpm/15 min/4°C). Supernatants
(particulate membrane fraction) were collected and pellets
(cytoskeleton) were resuspended in 1x Laemmli buffer. Fractions were
processed for Western blotting using chemiluminescence and ECL (Pierce,
Rockford, IL) and blots quantitated using NIH Image.
Flow cytometry
Cells (1 x 106) were incubated with
anti-V2 FITC conjugated on ice for 10 min, washed, and
resuspended in 500 µl of PBS. Propidium iodide (PI; 2 µM/ml) was
added immediately before data acquisition (10,000 cells) and results
were analyzed using CellQuest software (BD Biosciences, Franklin Lakes,
NJ). For CD25 expression, cells were double labeled with
anti-CD25-PE (clone M-A251; BD PharMingen) and anti-V2
FITC.
Detection of chemokine production by sandwich ELISA
Supernatants from V2+ T cell lines
(2 x 105 cells/well) were harvested at
72 h poststimulation, and chemokine production was quantified by
sandwich ELISA as described (23).
EMSA
Nuclear extracts were prepared using the NE-PER Nuclear
and Cytoplasmic Extraction Reagents (Pierce). EMSA was performed using
15 µg of total nuclear extract and the LightShift Chemiluminescent
EMSA kit (Pierce). Single-strand oligonucleotides containing the
NF-B consensus binding sequence (5'-AGT TGA GGG GAC TTT CCC AGG
C-3') or the AP-1 consensus binding sequence (5'-CGC TGG ATG AGT CAG
CCG GAA-3') were biotin labeled using the Biotin 3' End DNA Labeling
kit (Pierce) and visualized using a Streptavidin-HRP-conjugate and ECL.
Blots were exposed to Kodak BioMax Film (Eastman Kodak, Rochester, NY)
and quantitated by densitometry.
Confocal imaging
V2 T cells (5 x 105) were
activated with IPP (10 µM) or subjected to a medium change, incubated
at 37° for 10 min, and then fixed with 4% paraformaldehyde for 30
min. The cells were washed twice, permeabilized with 0.3% Triton X-100
in 10% normal goat serum in PBS for 5 min at 4°C, and blocked in
10% goat serum/PBS for 1 h at RT. Cells were incubated with Abs
specific for phospho-PKC (New England Biolabs) or phospho-PKC/
(BD Transduction Laboratories) in 5% goat serum/PBS overnight at
4°C, washed, and incubated with secondary Abs coupled to AlexaFluor
594 (Molecular Probes, Eugene, OR). After washing, cells were stained
with FITC-coupled cholera toxin (FITC-Ctx; 15 µg/ml; Molecular
Probes) for 15 min at RT. Control cells were incubated with
isotype-matched irrelevant primary Abs. Cells were adhered to
poly-L-lysine-coated slides, mounted in aqueous mounting
medium (Gel mount; Biomedia, Foster City, CA), and viewed using an
Eclipse epifluorescent microsope (Nikon, Melville, NY). A complete Z
series of images was taken using a Radiance 2000 Laser Scanning
Confocal System (Bio-Rad, Hercules, CA).
Data analysis
Results are expressed as mean ± SD. Statistical analysis was performed using Excel (Microsoft, Redmond, WA) and was calculated using a two-samples t test assuming an unequal variance with 99% confidence levels and p values <0.05 considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2+ T cells, we first tested Western blots of
V2 cell extracts using a panel of PKC isoform-specific Abs.
V2+ T cells expressed the classical isoform
PKC/II but not ; the novel isoforms PKC , 
, and ; and
the atypical isoforms 
and 
(data not shown). Phosphate Ag-induced translocation of novel and classical PKC
In TCR+ T cells or Jurkat T cells,
PKC becomes phosphorylated on various sites within the C-terminal
catalytic domain after cooperative activation via CD3 and CD28 and
translocates rapidly from the cytosol to the more detergent-insoluble
fractions (cell membrane and cytoskeleton), particularly at sites of
interaction between T cells and APCs (21). However,
activation of cytokines and chemokines in V2+
T cells by IPP or Abs to CD3 does not require costimulatory molecules
or APCs, although cell-cell contact enhances this response (4, 24). To determine whether IPP-induced activation of V2 T
cells caused a similar translocation of PKC, we used a combination
of subcellular fractionation and Western blotting for total PKC
(Fig. 1
). Parallel blots were probed for
total PKC/. In control unstimulated samples, essentially no
PKC was found in the membrane fractions, whereas 
20% of total
PKC/ was present in these same samples.
V2+ T cells were then harvested 5, 15, and 45
min postchallenge with IPP + IL-2. Control cultures were subjected to a
medium change with fresh IL-2 and harvested in parallel. After
stimulation with IPP (30 µM), we observed a progressive enrichment of
PKC in the membrane fraction that was markedly higher than that
found in the control cultures (Fig. 1A). Translocation of
PKC/ to the membrane was also noted, but no differences were
observed between cells treated with IPP + IL-2 and those treated with
IL-2 alone. The experiment was then repeated to compare the
distribution of PKC between the cytosolic and cytoskeletal
fractions. Consistent with previous data, IPP induced a rapid
translocation of PKC to the cytoskeletal fraction that continued to
increase over time (Fig. 1B), whereas in control cells
PKC remained in the cytosolic fraction. Taken together, these data
show that IPP induces a rapid translocation of PKC to the
particulate fraction, consistent with a stimulus-induced change in its
subcellular localization.
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To determine whether the various isoforms of PKC were
phosphorylated in response to IPP, we used phosphospecific Abs for
PKC, PKC/II, and PKC and probed total cell homogenates
harvested at varying times postactivation with IPP (Fig. 2A). IPP induced a rapid and
persistent increase in the phosphorylation state of PKC. In
addition, phosphospecific PKC Abs identified the formation of an
50-kDa fragment that also increased with time, reflecting the
cleavage of PKC and the formation of the catalytically active
fragment. In contrast, no IPP-specific phosphorylation of PKC/ or
PKC was observed (Fig. 2A). However, stimulation with Abs
to CD3 induced a slight increase in this phosphorylation state, as well
as the formation of an 50-kDa fragment for both PKC/ and
PKC in a time-dependent manner (data not shown), indicating that
additional phosphorylation of these isoforms could be induced by other
stimuli.
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in these
cultures, cells were stimulated for 15 min, homogenates were
immunoprecipitated with Abs to PKC, and immunoblots were probed with
phosphospecific PKC Abs (Fig. 2
B). The data indicated
that PKC was prominently phosphorylated after activation with IPP (3
or 30 µM), Abs to CD3, or PMA. Densitometric analysis indicated that
the extent of phosphorylation induced by IPP was concentration
dependent, elevated over that induced by IL-2, and equivalent to that
induced by stimulation with Abs to CD3 or PMA (Fig. 2B).
To assess whether there was an enrichment of the phosphorylated form of
PKC at the cell membrane, cells were activated with IL-2, IPP (30
µM), or IPP + IL-2 for 5 min and membranes were prepared and probed
as before. Higher levels of phospho-PKC were noted in the
membrane fraction after activation with IPP or IPP + IL-2 than were
found in control cells or cells activated with IL-2 alone (Fig. 2C). Activation with IPP and IPP + IL-2, in addition to the
expected 79-kDa band (Fig. 2C, arrow), also led to the
formation of a phosphorylated fragment that ran at 50 kDa, as well
as a higher-molecular mass form (150 kDa) that was only
detected in membrane fractions. These bands were not detected after
adsorption with a blocking peptide for the phosphospecific PKC
Ab.
Finally, we used confocal imaging to assess the expression and
distribution of p-PKC and p-PKC/ in response to IPP
stimulation. After activation with medium alone or IPP for 15 min,
cells were permeabilized and stained with phosphospecific Abs plus
FITC-Ctx and imaged by confocal laser microscopy to assess
cell-membrane staining. In cells cultured with medium alone,
immunoreactivity for p-PKC was only detected at very low levels
(Fig. 2Da), whereas cells that had been activated by IPP
showed prominent immunoreactivity for p-PKC (Fig. 2Dd),
particularly at sites of cell-cell contact as demonstrated by merging
of the FITC-Ctx staining and the p-PKC immunoreactivity (Fig. 2Df). In contrast, p-PKC/ was readily detected in the
cells activated with medium alone (Fig. 2D, g and
i), and in cells activated with IPP it was not focused at
sites of cell-cell contact (Fig. 2D, j and l).
Novel and classical isoforms are required for IPP-induced expansion
of V2 cells
The data presented above show that activation of
V2+ T cells with IPP led to the specific
translocation and phosphorylation of PKC, suggesting a role for this
PKC isoform in the functional properties of T cells activated by
phospholigands. To test for this, we determined the effect of
rottlerin, a putative inhibitor of PKC and PKC, on IPP-induced
expansion and CD25 expression using FACS analysis.
Freshly isolated PBMCs were stimulated with 30 µM IPP in the absence
or presence of rottlerin at 1 µM, harvested on days 6, 9, 15, and 21,
and stained with a FITC-conjugated anti-V2 TCR Ab. Cell
viability was determined with PI. In control cultures, V2 cells
expanded efficiently in response to IPP from 2% on day 1 to 87% of
the total lymphocyte population after 21 days (Fig. 3). However, in cells that were
pretreated with rottlerin (1 µM), the expansion was inhibited but no
cell death was detected by PI in these cultures (Fig. 3A).
The inhibitory effect of rottlerin persisted through day 15, even
though no further addition of either inhibitor or IPP was made to these
cultures and fresh medium was provided on a 50% v/v basis twice
weekly. After this time, T cells in the culture expanded,
indicating that the inhibitory effect of rottlerin was not toxic for
these cells. The experiment was then repeated with an additional donor,
and the effect of rottlerin was compared with varying doses of
Gö6976 (250 nM). Gö6976 also showed a dose-dependent
inhibitory effect on IPP-induced expansion of
V2+ T cells (Fig. 3A). Rottlerin (1
µM) and Gö6976 (250 nM) also effectively blocked the expansion
of established V2+ T cell lines after
reactivation with IPP without inducing toxicity (data not shown). These
data implicate a role for both novel and classical PKC isoforms in the
proliferative response of V2 cells to IPP, although it should be
noted that concerns have been raised about the specificity of rottlerin
for novel PKCs (see Discussion).
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T cells, we examined
whether expression of CD25, the low-affinity IL-2 receptor, was
modulated. The T cells require IL-2 for survival in vitro, and
in T cells, PKC is required for TCR-dependent expression of
CD25 and CD69 (20). V2 T cell lines were stimulated
with IL-2 or IPP + IL-2 in the absence or presence of rottlerin at 2
µM or 5 µM, Gö6976 at 250 nM, or both for 18 h and were
analyzed for expression of CD25 by FACS. Resting
V2+ T cells did not express CD25 (Fig. 3B, black trace). However, a second stimulation with IPP
induced a marked up-regulation of CD25 expression at the cell surface
(Fig. 3B, orange trace). Pretreatment with rottlerin at
either 2 µM or 5 µM (Fig. 3B, blue trace and green
trace, respectively) completely inhibited the IPP-induced CD25
expression. Gö6976 also inhibited, although to a lesser extent,
whereas the combination of these two compounds totally blocked
IPP-induced up-regulation of this receptor. These results indicate that
T cells are highly dependent on both novel and classical PKCs
for their expansion in response to phosphoantigens and suggest that
this effect is mediated through regulation of the receptor for
IL-2. Rottlerin and Gö6976 differentially regulate IPP-induced cytokine and chemokine expression
We then investigated the role of rottlerin and Gö6976 on
IPP-induced cytokine and chemokine production. V2 T cell lines from
three different donors were stimulated with IPP (3 or 30 µM) with or
without IL-2, in the absence or presence of rottlerin (2, 1, and 0.5
µM; data shown only for 2 µM) or Gö6976 (250 nM), and
supernatants were harvested on day 3. As expected (23),
IPP strongly induced the release of MIP-1, MIP-1, IFN-, and
TNF-, with the addition of IL-2 increasing the levels of MIP-1,
TNF-, and IFN- but having relatively little effect on MIP-1
(Fig. 4). Rottlerin at 2 µM
significantly inhibited the production of MIP-1, MIP-1, and
IFN-. No significant effect of rottlerin on TNF- release was
observed. In contrast, Gö6976 at 250 nM caused a more potent
down-regulation of both cytokines and chemokines.
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Given the complex results shown above, we speculated that the
different isoforms of PKCs might be involved in differentially
regulating downstream signaling molecules such as transcription factor
activation. Because we showed previously that IPP at 30 or 3 µM
induced activation of both NF-B and AP-1 (16), we first
tested the effects of rottlerin and Gö6976 on IPP-induced DNA
binding by EMSA. The results confirmed our previous data that IPP
induces a strong nuclear translocation and DNA binding activity of both
AP-1 and NF-B at 1 h postchallenge (Fig. 5A, compare lanes 2
and 3, upper panel and lower panel,
respectively). In addition, they revealed that Gö6976 (Fig. 5A, lane 4) inhibited activation of AP-1 (Fig. 5A, upper panel) and NF-B (Fig. 5A,
lower panel). With rottlerin, the data from donor to donor
were more variable, but densitometric analysis of data from three
independent experiments using cells from different donors showed that
rottlerin reduced binding by 30% and Gö6976 by 50% of the
control values (Fig. 5B). Interestingly, these inhibitors
did not block AP-1 and NF-B activation and binding induced by
anti-CD3 Abs (Fig. 5B, compare lanes
5 and 6, lanes 13 and 14,
and upper and lower panels, respectively) or
after stimulation with TNF- (data not shown). Analysis of
IPP-induced degradation of IB further supported these results
because, in the presence of rottlerin and Gö6976, IPP-induced
IB degradation was partially blocked at both 15 min and 1 h
(Fig. 5C).
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B were
blocked by inhibitors of the PKC family, we investigated whether PKCs
could be mediating the activation of MAPKs. First, we tested whether
these MAPKs were activated by IPP in V2+ T
cells (Fig. 6). Cells were stimulated
with IPP for the times indicated, and phosphorylation of ERK1/2, p38,
and JNK was determined by Western blotting. In agreement with previous
data (15), we detected rapid (within 5 min) and persistent
activation of p38 MAPK, ERK 1 and 2, and JNK (Fig. 6A).
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2 T cells for 15 min or 1 h with 2 µM rottlerin. No affect
on the IPP-induced activation of any of these kinases by rottlerin was
observed (Fig. 6B). Gö6976 (250 nM) down-regulated the
signal for phospho-p38 as well as phospho-JNK at both 15 and 60 min,
whereas down-regulation of phospho-ERK1/2 was only noted at 60 min. We
then tested the effect of higher concentrations of the PKC inhibitors
on MAPKs. Rottlerin (5 µM) had no effect on IPP-induced activation of
phospho-ERK1/2, p38, and JNK (Fig. 6C). In contrast,
Gö6976 (500 nM) totally blocked the activation of ERK1/2, p38,
and JNK (Fig. 6C). Interestingly, when these two inhibitors
were used in combination, the levels of phospho-ERK and phospho-p38
were higher than the levels detected in the Gö6976 treatment
alone, whereas phospho-JNK was equally potently inhibited. These data
indicate that IPP-induced phosphorylation of these MAPK pathways
involves the classical but not the novel PKC isoforms. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2V9 T
cells with IPP, a phospholigand that is secreted by mycobacteria, leads
to the rapid and persistent activation of PKC, as determined by
evidence of translocation to the cell membrane and cytoskeletal
fractions, and phosphorylation of threonine 538 within the catalytic
domain. In contrast, no phospholigand-specific phosphorylation or
translocation of PKC or PKC was detected. The inhibitors
rottlerin and Gö6976, which have been reported to be specific
inhibitors of novel and classical PKC isoforms, respectively,
demonstrated a role for these enzymes in IPP-induced proliferation and
CD25 expression, as well as cytokine and chemokine production.
Gel-shift assays indicated that the transcription factors NF-B and
AP-1 were downstream targets of PKC activation. Although IPP also
induced the rapid and persistent phosphorylation of ERK 1 and 2, p38
MAPK, and SAPK/JNK, only an inhibitor of classical PKCs blocked this
response. From these data, we conclude that both the novel and
classical PKC isoforms are required for phosphoantigen-induced
activation of human V2V9 T cells, but that activation of PKC
appears to be ligand dependent, whereas activation of PKC/
appears to be more growth-factor dependent.
The observation that PKC is activated in T cells by phosphate
Ags would be consistent with the important role for PKC that has
been defined in T cell activation. So, for example, Sun et al.
(20) showed that in mice with a targeted disruption of the
gene for PKC, mature T cells stimulated through the TCR demonstrated
a defect in NF-B activation, a low-level of IL-2 synthesis, and a
low proliferative response. Interestingly, the NF-B pathway could
still be activated in these cells by cytokines such as TNF- or IL-1,
implying a specific role for PKC in NF-B activation induced by
TCR triggering (20). The defect in the proliferative
response coincided with an inability of the
TCR-/- T cells to
up-regulate CD25 expression after TCR stimulation. Freshly isolated
human V2 T cells also up-regulate CD25 expression in response to
phosphoantigens or aminobiphosphonates (25, 26), but
whether this occurs after restimulation with phosphoantigens has not
been determined. We now show that CD25 is up-regulated in V2 T cell
lines restimulated with IPP and, in agreement with the results of Sun
et al. (20), find that when V2 T cells were pretreated
with the putative novel PKC inhibitor rottlerin (2 and 5 µM), the
phosphoantigen-induced CD25 up-regulation was completely blocked. This
may account for the inhibitory effect of pretreatment with rottlerin on
IPP-induced expansion of V2+ T cells, even in
the presence of IL-2. Although concerns have been raised regarding the
specificity of rottlerin for PKC (27) and have
suggested that it may indirectly block PKC by lowering intracellular
levels of ATP (28), this inhibitor has been found to block
kinase activity in vitro of both PKC and PKC (for example, see
Refs. 29 and 30). The classical PKC inhibitor
Gö6976 also blocked IPP-induced CD25 expression and proliferation
of V2 T cells. A requirement for both PKC and PKC in the
up-regulation of CD25, CD69, and IL-2 production after T cell
activation in T cells has been noted previously
(31), whereas PKCII was found to be more critical for
IL-2 secretion (32). Although we failed to detect
ligand-specific activation of classical PKCs in response to IPP, these
kinases were constitutively activated in V2 T cell lines maintained
in IL-2. In contrast to T cells in which appropriate activation
of PKC requires the formation of an immunological synapse with an
APC and costimulation through CD28 (21), these components
are not required to achieve PKC activation in response to IPP in
V2+ cells (4). Nevertheless,
recent studies have shown that T cells do conjugate with, and
demonstrate membrane transfer with, tumor cell targets or accessory
cells used in conjunction with soluble Ag, consistent with
immunological synapse formation (33).
The probable involvement of both novel and classical PKC isoforms in
the activation of T cells was also observed in the IPP-induced
release of cytokines and chemokines. The fact that both rottlerin and
Gö6976 markedly inhibited MIP-1 and IFN- demonstrates for
the first time the importance of these PKC isoforms in the induction of
soluble factors other than IL-2 in T cells. In general, in the absence
of APCs, IPP does not induce IL-2 in T cell lines, whereas
Jurkat cells transfected with a phosphoantigen-responsive V2V9
TCR readily release IL-2 in response to IPP (8). In
contrast, TNF- production was not significantly affected by
rottlerin, whereas Gö6976 potently inhibited the production of
this cytokine. Interestingly, the production of MIP-1 was less
potently inhibited by Gö6976 and rottlerin than by MIP-1. It
is also of interest to note that IPP-induced MIP-1 production was
less dependent on IL-2 in the medium. These data would suggest that the
combination of transcription factors required for optimal
cytokine/chemokine expression differs for each of these factors. The
stimulatory effect of IL-2 would further imply a role for NFAT, a
transcription factor that is known to act cooperatively with other
transcription factors in the expression of several proinflammatory
factors (reviewed in Ref. 34). EMSA demonstrated that IPP
did not stimulate nuclear translocation and DNA binding of NFAT in
these cultures, although transient increased DNA binding was noted in
response to IL-2 (data not shown).
The transcription factors AP-1 and NF-B are essential for cytokine
production in lymphocytes. The inhibitory effect of rottlerin and
Gö6976 on IPP-induced activation of AP-1 and NF-B in V2 T
cell lines suggests that both novel and classical PKC isoforms function
upstream of these transcription factors. As reported previously for
T cells (20), TNF activation of NF-B was not
affected by either of these inhibitors in T cells. However, it
was surprising to note that NF-B signaling induced by Abs to CD3 was
also unaffected in these cells. This may reflect a lack of requirement
for CD28 costimulation in this response. MAPK also mediates the
activation of transcription factors in response to a variety of
extracellular stimuli, so we examined the effect of rottlerin
and Gö6976 on IPP-induced activation of the major MAPK pathways.
In agreement with the data of Lafont et al. (14), IPP
rapidly and persistently induced the phosphorylation of ERK1/2, p38,
and SAPK/JNK in V2 T cell lines. However, our data show that only
Gö6976 inhibited this response. Because these pathways, in
addition to NF-B (16), have also been implicated in
regulating IPP-induced TNF- production (14, 15), this
may explain why Gö6976 efficiently inhibited the production of
this cytokine. The fact that rottlerin was without effect was
unexpected, particularly for JNK given the AP-1 data. These data imply
that PKC, once activated by IPP, does not promote chemokine and
cytokine production by activating AP-1 through JNK as described for
T cells (reviewed in Refs. 35 and 36).
However, in the PKC-/-
mice, JNK activation was normal after T cell activation with CD3/CD28
(20), indicating the presence of a JNK-independent but
PKC-dependent pathway for AP-1 activation, and it is possible that
integrins function in this regard (reviewed in Ref. 36).
Both LFA-1 and LFA-3 are expressed on
V2+ T cells in response to IPP, with blocking
of the LFA-3/CD2 interaction inhibiting IPP-induced TNF- production,
but not cytotoxicity, and blocking of LFA-1/ICAM-1 interaction
inhibiting cytotoxicity, but not TNF- production (37).
Clearly, the role of PKC isoforms in these responses will be
interesting to address in future experiments.
Similarly, the upstream targets of PKC involvement in IPP-induced
NF-B activation remain to be more clearly defined. In
TCR+ T cells stimulated with CD3/CD28, Lin et al.
(38) demonstrated a role for PKC in activation of
inhibitory B kinase (IKK), whereas studies by
McAllister-Lucas et al. (39) demonstrated that two caspase
recruitment domain-containing proteins called Bimp-1 and Bcl-10
interact with PKC to mediate NF-B activation through the
downstream recruitment of MALT1 in T cell lines stimulated by
anti-CD3 or PMA, with NF-B activation occurring through either
IKK or IKK. In our studies, rottlerin had only a minimal effect
on IPP-induced IB degradation, suggesting that other pathways may
also be involved in NF-B activation, perhaps again reflecting the
lack of a need for costimulation through CD28 in this response.
Although we have considered the response to IPP as representing a
TCR-dependent process, the actual nature of Ag recognition by the
TCR, and its relationship to other lymphocyte receptors, remains
enigmatic. In fact, several studies have suggested that the TCR
may have more in common with Ig responses than TCR-dependent responses
(reviewed in Ref. 40). Crystallographic analysis of a
complete phosphoantigen-responsive V9V2 receptor has indicated
that this receptor has both Ab-like and TCR-like features
(10). Although it was initially thought that B cells do
not express PKC, more recent studies demonstrate that rottlerin
inhibits B cell receptor-mediated NF-B and JNK activation in a
PKC-dependent fashion (41). However, this inhibitory
activity was only noted at 30 µM, with lower doses having no effect.
This dose of rottlerin is 10-fold higher than that used in our
experiments, and we found that rottlerin at >5 µM was toxic for
T cells. That inhibition of PKC induces cell death has been
noted previously and has been linked to a role for PKC in the
p90rsk phosphorylation of the BCL2 family member
BAD, inhibiting its survival signals (42, 43).
However, PKC has also been implicated in Ag-induced activation of
Fas (44), indicating that PKC may play a dual
regulatory role in T cell survival (21).
The expression of these phosphoantigens by a wide spectrum of
potentially pathogenic organisms would suggest an important role for
this response in pathogen defense. However, this has been difficult to
document in vivo, because a similar response has not been detected in
mice or rats. Thus, it is particularly exciting to note the recent data
supporting a role for this subset of T cells in mycobacterial
infections in macaques (45). Using infection with bacillus
Calmette-Guérin (BCG), this study showed a major expansion and
development of a memory response in V2V9 T cells in these
animals. Similar responses were noted after BCG vaccination or
infection with Mycobacterium tuberculosis. The authors
further showed that expansion of V2+ cells in
BCG-vaccinated animals coincided with clearance of a BCG bacteremia and
with the development of immunity to fatal tuberculosis. Expansion and
activation of this subset also occurs in rhesus monkeys challenged with
phosphoantigens (46). That this subset expands in the
peripheral blood of humans in association with different diseases is
now well accepted (1). Nevertheless, in chronic
infections, V2 T cells may be lost or anergized (25, 47), suggesting that these cells may make an attractive target
for immunoregulatory therapies that seek to boost the immune response.
Alternatively, V2+ cells may contribute to the
immunopathology associated with chronic inflammatory or autoimmune
disorders through their high and sustained release of inflammatory
cytokines and chemokines. Knowledge of the signaling mechanisms
activated by these Ags, and particularly the role of different PKC
isoforms, will hopefully provide information that may help to fine-tune
this response, permitting selective enhancement or inhibition of
ligand-specific events in the unusual Ag response of this subset of T
cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Celia F. Brosnan, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: Brosnan{at}aecom.yu.edu
3 Abbreviations used in this paper: IPP, isopentenyl pyrophosphate; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MIP, macrophage-inflammatory protein; PKC, protein kinase C; JNK, c-Jun N-terminal kinase; RT, room temperature; PI, propidium iodide; FITC-Ctx, FITC-coupled cholera toxin; SAPK, stress-activated protein kinase; IKK, inhibitory kappa B kinase; BCG, bacillus Calmette-Guérin.
Received for publication May 6, 2002. Accepted for publication September 5, 2002.
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