| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Center for Immunology, Departments of
Biochemistry, Molecular Biology and Biophysics, and
Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production are not affected in the AINR state. To characterize the
mechanism(s) responsible for lack of IL-2 production in CD8 T cells in
the AINR state, microspheres bearing immobilized anti-TCR Abs or
peptide-MHC complexes, B7-1, and ICAM-1 were used to provide
well-defined stimuli to the cells. Comparison of normal and AINR cells
revealed that in AINR cells extracellular signal-regulated kinase
(ERK) is upregulated more transiently, Janus kinase activation
is substantially reduced, and activation of p38 is eliminated. PMA and
ionomycin restored proliferation and IL-2 production in AINR cells,
indicating a signaling defect upstream of Ras and protein kinase C.
Inhibitors of ERK (PD98059) and of p38 kinase (SB202190) blocked IL-2
mRNA expression and proliferation of both
peptide-MHC/B7-1/ICAM-1-stimulated normal cells and
PMA/ionomycin-stimulated AINR cells. Together these results demonstrate
that activation of at least ERK and p38 is essential for IL-2
production by CD8 T cells and that up-regulation of these
mitogen-activated protein kinases, along with Janus kinase, is
defective in AINR cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have demonstrated that CD8 T cells that were stimulated in vitro by
microspheres bearing anti-TCR Abs and coimmobilized B7-1 and ICAM-1
or by Ag-bearing cells as APC become nonresponsive, or anergic, after
an initial response (4). We have further shown that this
activation-induced nonresponsiveness
(AINR)3 occurs following an in vivo
response to allogeneic tumor, even when the tumor cells express B7-1
(4) and following an in vivo response to a syngeneic tumor
(5). AINR is characterized by a specific defect in IL-2
production since the activated cells can still respond to exogenous
IL-2, and in response to TCR engagement can produce IFN- and lyse
Ag-bearing target cells (4, 5). The surface levels of TCR,
CD8, CD28, and LFA-1 on AINR cells are the same or higher than on naive
cells. In addition, CTLA-4 does not appear to be involved in the
induction or maintenance of AINR in CD8 T cells. Addition of
anti-CTLA-4 mAb during primary in vitro stimulation did not prevent
development of AINR, nor did it reverse nonresponsiveness when added to
the cells following the primary response (4). Similarly,
in vivo administration of anti-CTLA-4 mAb did not overcome CD8
nonresponsiveness following a response to syngeneic tumor, whereas the
same treatment did overcome nonresponsiveness of CD4 Th cells in this
system (6).
The AINR that develops in CD8 T cells is distinctively different from the classical anergy described for CD4 T cell clones. Both are characterized by impaired ability to proliferate and produce IL-2 upon rechallenge, but AINR develops after full stimulation with Ag and costimulation, whereas clonal anergy in CD4 T cells results when the cells are stimulated through just the TCR. Clonal anergy in CD4 T cells has been extensively studied but the physiological relevance of this phenomenon remains unclear. It may be a mechanism for rendering peripheral self-reactive T cells unresponsive (reviewed in Ref. 7).
CD8 T cells become AINR following an initial in vitro or in vivo response (4, 5, 6), and this appears likely to provide a regulatory checkpoint for converting an initially helper-independent CTL response to one that requires help in the form of IL-2 produced by CD4 T cells in order for the response to be maintained (6). The biochemical basis for this potentially important biological nonresponsiveness has not been defined and we therefore sought to understand how CD8 T cells in the AINR state selectively uncouple TCR and/or costimulatory-mediated signals involved in IL-2 production while leaving other TCR-mediated functions intact. Results of studies of anergic CD4 T cell clones suggested that this might involve defects in activation of mitogen-activated protein kinases (MAPKs) that are important in signaling leading to IL-2 production (8, 9, 10). The results reported here demonstrate that AINR CD8 T cells are deficient in up-regulation of extracellular signal-regulated kinase (ERK), Janus kinase (JNK) and p38 MAPKs, and that at least ERK and p38 are critical for IL-2 production by CD8 T cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CD8 T cells used for experiments were obtained from either C57BL/6 mice (Charles River Breeding Laboratories, Raleigh, NC) or OT-I TCR-transgenic mice specific for Kb/OVA257–264 peptide (11) (originally a gift from Dr. F. Carbone, Monash Medical School, Victoria, Australia). All animals were maintained in the specific pathogen-free facility at the University of Minnesota and were used at 6–12 wk. Lymph nodes were harvested, washed, and homogenized with a tissue homogenizer. The resulting single-cell suspension was treated with 11 mM KHCO3/152 mM NH4Cl to lyse RBC and then adherent cell depleted for 1 hr. CD8 T cell enrichment was carried out on negative selection columns (Cytovax Biotechnologies, Edmonton, Alberta, Canada) according to the protocol provided by the suppliers. The purity of CD8 T cells was assessed by flow cytometry and was typically 90–95% with <2% CD4 T cells.
Anti-TCR Abs 145-2C11 and B20.1.1, specific for CD3
and V
2 TCR
chains, respectively, were purchased from BD PharMingen (San Diego,
CA). B7-1 and ICAM-1 proteins were purified as previously described
(12, 13). H-2 Kb-OVA257–264
peptide complexes were prepared using standard protocols
(14, 15, 16). Briefly, constructs encoding H-2 Kb
H chain fusion protein with a biotinylation site and human
2-microglobulin were expressed in Escherichia
coli. The H chain fusion and 2-microglobulin
proteins were purified from inclusion bodies and refolded in the
presence of excess OVA257–264 peptide to form complexes.
Biotinylation of the complexes was then done using BirA ligase
(Avidity, Denver, CO).
Recombinant murine IL-2 (R&D Systems, Minneapolis, MN) and human IL-2 were used at final concentrations of 50 and 2.5 U/ml, respectively. PMA (Molecular Probes, Eugene, OR) and ionomycin (Calbiochem, La Jolla, CA) were used at 10 ng/ml and 1 µM, respectively.
Protein immobilization on latex microspheres
Microspheres used for stimulating responder cells were prepared by incubating 107 5-µm sulfate polystyrene latex microspheres (Interfacial Dynamics, Portland, OR) with the relevant proteins in 1 ml PBS for 20 min at 4°C on a rotator. 2C11/B7-1/ICAM-1 microspheres were prepared by the sequential addition and incubation of 145-2C11, ICAM-1, and B7-1 for 20 min each. The final concentrations of 145-2C11, ICAM-1, and B7-1 used to prepare 107 microspheres were 0.75, 0.25, and 0.35 µg/ml, respectively. The microspheres were subsequently blocked by adding 0.5 ml 1% tissue culture grade BSA (Sigma, St. Louis, MO) before being used. To prepare microspheres bearing Kb/OVA or Kb/OVA, B7-1, and ICAM-1, the microspheres were first coated with streptavidin-APC (BD PharMingen) at 0.2 µg/ml, followed by the other ligands. The biotinylated form of Kb/OVA was added at 0.25 µg/ml after the microspheres were blocked with 1% BSA and washed once with PBS.
The levels of 2C11, B7-1, and ICAM-1 on each microsphere preparation were monitored using the FACsCalibur flow cytometer with the CellQuest software (BD Biosciences, San Jose, CA). The Abs used were goat anti-Armenian hamster Ig-FITC (Jackson ImmunoResearch, West Grove, PA), anti-mouse B7-1-FITC, and anti-mouse ICAM-1-FITC (BD PharMingen). The levels of Kb/OVA were determined by staining microspheres with Y3 (anti-Kb) mAb followed by goat anti-mouse Ig-FITC (Jackson ImmunoResearch).
Proliferation and Induction of AINR in CD8+ T cells
Unless indicated otherwise, 5 x 104 responder cells were cultured in triplicate in 96-well flat-bottom culture plates (Falcon; BD Biosciences) with 1 x 105 microspheres in a total volume of 0.2 ml medium. Cells were pulsed with 1 µCi [3H]thymidine/well for the last 8 h of the indicated time and were subsequently harvested using a LKB-Wallac cell harvester (Wallac, Turku, Finland). Proliferation was assessed by measuring [3H]thymidine incorporation using a Betaplate liquid scintillation counter (Wallac). Results are expressed as the average (±SD) of triplicate samples.
When CD8 T cells were stimulated with 2C11/B7-1/ICAM-1 (or Kb/OVA/B7-1/ICAM-1) microspheres, proliferation peaked on day 2 or 3 and then rapidly declined as the cells lost their ability to respond to the initial stimulus. Between day 2.5 and day 3.5, cells become AINR; these cells were pooled, treated on Lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) gradient centrifugation and subjected to further analyses. In each experiment, a secondary proliferation assay was set up in parallel to confirm that the cells were no longer responsive to the initial stimulus, 2C11/B7-1/ICAM-1 or Kb/OVA/B7-1/ICAM-1 microspheres.
IL-2 protein and mRNA measurements
The amounts of murine IL-2 in 50-µl culture supernatants were determined by sandwich ELISA using capture and detection Abs purchased from BD PharMingen according to the protocol provided by the supplier. The Abs were specific for murine IL-2 and were not reactive toward the recombinant human IL-2 in the culture. Results are expressed as the average (±SD) of triplicate samples. The detection limit of the assay was 0.04 ng/ml.
For determination of IL-2 mRNA by RT-PCR analysis, normal or AINR CD8 T cells and 2C11/B7-1/ICAM-1 microspheres were pelleted in 96-well V-bottom plates (1.5 x 105 cells and 3 x 105 microspheres/well) by centrifugation at 2000 rpm for 10 min at 4°C. The pellets were subsequently incubated at 37°C for 8 h. No significant change in cell viability was observed at the end of the incubation period. Total RNA was isolated from the cells using the GlassMAX RNA Microisolation kit (Life Technologies, Gaithersburg, MD). All RNA samples were treated with DNase I (Life Technologies) for 15 min. One microgram of RNA was reverse transcribed to cDNA using 2 µg oligo(dT)12–18 primers and 200 U SuperScript II following the protocol provided by the supplier (Life Technologies). RNA was subsequently removed by the addition of 2 U RNaseH.
For each sample, PCRs were carried out in the presence of IL-2 primers
(5' primer, 5'-ATGTACAGCATGCAGCTCGCATCCTGTGTC-3'; 3' primer,
5'-AGTCAAATCCAGAACATGCCGCAGAGGTCC-3') or -actin primers (5' primer,
5'-CCTAAGGCCAACCGTGAAAAG-3'; 3' primer, 5'-TCTTCATGGTGCTAGGAGCCA-3'),
PCR buffer, 50 mM MgCl2, dNTP mix (10 mM each), and 1 U DNA
polymerase (Bioline, Reno, NV) and water. The PCR mixtures were heated
to 95°C for 10 min followed by 27 cycles of the following conditions:
95°C for 1 min, 63°C for 2 min, and 72°C for 3 min. This number
of cycles was determined to be in the linear amplification range under
these conditions. PCR products were resolved on a 1% agarose and RNA
quantitation was performed using IQMac software (Molecular Dynamics,
Sunnyvale, CA).
ERK and JNK assays
For kinase assays, 1 x 106 normal or AINR CD8 OT-1 cells were pelleted with 2 x 106 ligand-bearing microspheres by centrifugation at 1250 x g in a swinging bucket rotor for 10 min at 4°C. The pellets were then incubated in a 37°C water bath for the indicated times and the cells were then lysed by addition of ice-cold buffer containing 25 mM HEPES (pH 7.7), 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5% Triton X-100, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. Cell lysates were precleared by centrifugation at 14,000 rpm for 10 min at 4°C. Inhibition of the MAPKs was examined using PD98059 and SB202190 (Calbiochem) dissolved in DMSO. Cells were preincubated in the presence of the inhibitor, or DMSO alone for controls, for 45 min at 37°C prior to stimulation with microspheres. Additional inhibitor, at the same final concentration, was added to the cell lysate before assaying enzyme activity.
ERK activity was assayed as previously described following
precipitation with GST-Elk-1 (aa 307–428) (17). The
plasmid encoding the GST-Elk-1 fusion protein was a kind gift from Dr.
R. Hipskind (Institut de Genetique Moleculaire, Montpellier, France).
Briefly, precipitation was carried out by incubating cell lysates in
HEPES binding buffer (20 mM HEPES (pH 7.7), 50 mM NaCl, 0.1 M EDTA 2.5
mM MgCl2, 0.05% Triton X-100, 20 mM -glycerophosphate,
1 mM Na3VO4, 2 mM PMSF, 10 mM benzamidine, 0.1
µg/ml aprotinin, 0.1 µg/ml leupeptin, and 0.1 µg/ml pepstatin)
with recombinant GST-Elk-1 coupled to glutathione-agarose beads on a
rotator for 4 h at 4°C. The precipitates were then washed three
times with kinase buffer (20 mM HEPES (pH 7.7), 20 mM
MgCl2, 20 mM -glycerophosphate, 0.1 mM
Na3VO4, 2 mM DTT, and 20 µM ATP). Three
microcuries of [-32P]ATP was then added and the
samples were incubated at 30°C for 20 min. Reactions were then
stopped by the addition of 6x Laemmli loading buffer and the
reaction mixtures were resolved by SDS-PAGE and analyzed by
autoradiography. Densitometry was performed using a personal SI
densitometer with the IQMac software (Molecular Dynamics). JNK
activity was assayed in the same way following precipitation with
GST-c-jun (aa 1–69) fusion protein bound to glutathione-agarose beads
(17) using lysate prepared from 2 x 106
normal or AINR cells.
Western blot analysis to detect phosphorylated p38
Normal (2 x 106) and AINR CD8 OT-1 cells were stimulated as described above for ERK and JNK assays and then lysed by addition of ice-cold 1% Triton X-100 lysis buffer (1% Triton X-100, 20 mM Tris (pH 7.6), 150 mM NaCl, 1 mM Na3VO4, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin). Laemmli loading buffer (6x) was then added, the samples were then boiled for 5 min, and resolved on a 12% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane (Amersham, Little Chalfont, U.K.) which was subsequently blocked with 5% milk powder. The membrane was probed with an Ab specific for phosphorylated p38 (Cell Signaling Technology, Beverly, MA) followed by HRP-conjugated sheep anti-mouse IgG (Jackson ImmunoResearch) and developed using the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). In experiments examining inhibition of phosphorylation of p38, cells were preincubated in the presence of the inhibitor, or DMSO alone for controls, for 45 min at 37°C before stimulation with microspheres and preparation of the cell lysate for Western blotting.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our previous work showed that coimmobilizing B7-1 and ICAM-1 on
microspheres along with an anti-TCR mAb provided potent
costimulation to CD8 T cells, stimulating IL-2 production and
proliferation. However, within 3 days the cells stopped proliferating
and became refractory to further costimulation. To determine whether
this AINR involved CD28- or LFA-1-dependent signals, we examined
whether AINR was induced when the individual costimulatory ligands were
present along with the TCR stimulus. When OT-I CD8 lymph node T cells
were stimulated with microspheres having B20 anti-V2 mAb
immobilized along with B7-1 (B20/B) or ICAM-1 (B20/I), significant
proliferative responses were observed (Fig.
1A). As expected
(12), optimal cell proliferation was achieved when both
B7-1 and ICAM-1 were coimmobilized with B20 (Fig. 1A). In
all cases, the proliferative responses were transient (Fig.
1A), and proliferation and clonal expansion declined after
day 2.
The decline in responses after day 2 suggested that the cells become AINR with either costimulatory ligand, and this was directly examined. Shortly after the peak of proliferation (at 60 h), cells stimulated with B20/B/I, B20/B and B20/I were harvested, washed, and restimulated, and the secondary responses were assessed 2 days later. Cells from all three groups failed to respond when restimulated with either BSA-blocked microspheres or B20/B/I microspheres (Fig. 1B). In all three cases, however, the AINR cells were able to respond to exogenous IL-2 (Fig. 1B).
The requirement for B7-1 or ICAM-1 in the primary stimulation can be bypassed by the addition of IL-2 to cells stimulated with microspheres having just anti-TCR mAb on the surface. CD8 T cells that were activated in this way using B20 on microspheres and exogenously added IL-2 (Fig. 1C) also became nonresponsive to subsequent restimulation with B20/B/I microspheres, but still proliferated in response to exogenous IL-2 in the secondary stimulation (Fig. 1D). These results demonstrate that AINR develops in CD8 T cells within 3 days of initial stimulation, irrespective of the nature of the primary stimulus. Thus, stimulation through just the TCR and IL-2R are sufficient; signaling through CD28 and/or LFA-1 is not required to induce AINR (Fig. 1D), nor does it prevent establishment of AINR (Fig. 1B).
In other experiments, we have stimulated CD8 T cells from OT-I TCR-transgenic mice with microspheres having immobilized with H-2 Kb/OVA257–264 peptide complexes, B7-1, and/or ICAM-1 and have observed the induction of AINR with a similar time course. The differences between using anti-TCR mAb or Kb/OVA257–264 for signal 1 were quantitative, rather than qualitative, with the latter giving stronger proliferative response and enhanced biochemical signals (data not shown, and see below).
AINR CD8 T cells display decreased up-regulation of IL-2 mRNA levels
AINR cells fail to produce IL-2 in response to TCR engagement and
costimulation (4). To determine whether the defect was at
the level of IL-2 message, we used RT-PCR to compare the ability of
naive and AINR cells to increase IL-2 mRNA levels upon stimulation.
Naive and AINR cells were stimulated by centrifuging the cells and
microspheres bearing the 2C11 anti-TCR mAb along with B7-1 and
ICAM-1 in V-bottom 96-well plates at 2000 rpm for 10 min and then
incubating the plates at 37°C. The cells can be incubated in the
pellet form for up to 8 h without significant changes in cell
viability (data not shown). RNAs isolated from naive and AINR cells,
both stimulated and unstimulated, were reverse transcribed and
amplified by PCR. In unstimulated naive and AINR cells, IL-2 mRNA was
not detectable (Fig. 2
). Upon
stimulation, a large increase in IL-2 mRNA was observed in the naive
cells whereas the amount of IL-2 mRNA in AINR cells was minimal (5-fold
less than in normal) (Fig. 2). In a similar experiment, a 10-fold
difference in IL-2 mRNA was observed between naive and AINR cells
simulated with Kb/OVA/B7-1/ICAM-1. Thus, cells that have
undergone initial stimulation and become AINR display a profound defect
in their ability to increase levels of IL-2 mRNA in response to TCR
engagement and costimulation.
|
MAPKs are involved in the regulation of transcription factors required for IL-2 gene expression in CD4 T cells. Anergized CD4 T cells clones show a marked reduction in ERK and JNK activities upon stimulation and are unable to produce IL-2 (8, 9, 10). ERK is fully activated by TCR engagement whereas the activation of JNK requires the engagement of both TCR and CD28 (18). These observations suggested that the AINR state induced in CD8 T cells might involve one or more defects in activation of MAPKs.
Using microspheres having Kb/OVA alone, or coimmobilized
B7-1, ICAM-1, or both, we assessed the contributions of TCR engagement
and costimulation to the activation of ERK and JNK in naive and AINR
CD8 T cells. We also examined a third MAPK, p38, since recent data have
indicated that it is involved in signal integration during
costimulation of primary murine T cells (19). ERK and JNK
activities were determined in lysates of stimulated cells using GST
fusion proteins as substrates. Initial experiments showed that ERK
activity in normal cells peaked between 2.5 and 5.0 min and JNK
activity at 10.0 min (data not shown). The costimulation requirement
for ERK and JNK activation in CD8 T cells was therefore determined at
2.5 and 10 min, respectively (Fig. 3, A and B). As shown in Fig. 3A, ERK
activity in both naive and AINR cells increased in response to
Kb/OVA alone and the presence of B7-1 and/or ICAM-1 did not
further increase the activity, demonstrating that costimulation plays a
minimal role in the activation of ERK in CD8 T cells. Unlike ERK
activation, optimal activation of JNK in naive cells required B7-1
and/or ICAM-1 (Fig. 3B). Furthermore, AINR cells showed a
significant defect in activating JNK in response to either B7-1 or
ICAM-1, although substantial up-regulation of JNK was seen when both
costimulatory ligands were provided (Fig. 3B). The levels of
ERK and JNK protein were found to be essentially identical in naive and
AINR cells, as assessed by Western blotting using specific Abs (data
not shown).
|
ERK and p38 are required for costimulation-dependent IL-2 production and proliferation by CD8 T cells
To examine the requirement for the MAPKs in stimulating production of IL-2, specific inhibitors of ERK and p38 were examined for their effects on up-regulation of IL-2 mRNA. PD98059 is a specific inhibitor of mitogen-activated protein/ERK kinase 1, the upstream kinase required for ERK activation, and does not inhibit JNK or p38 (20). Treatment of cells with PD98059 before stimulation with Kb/OVA/B7-1/ICAM-1 inhibited up-regulation of ERK activity (Fig. 3D) and blocked IL-2 mRNA up-regulation (Fig. 3E) with the same dose response.
Recent studies have shown that pyridinylimidazole inhibitors such as
SB202190 compete for the ATP binding site and inhibit the
phosphorylation of p38 that is required to activate the enzyme
(21), but do not inhibit ERK (22). Treatment
of cells with SB202190 before stimulation with
Kb/OVA/B7-1/ICAM-1 inhibited p38 phosphorylation in the
10–50 µM range (Fig. 3F) and blocked IL-2 mRNA
up-regulation in the same concentration range (Fig. 3G). In
contrast, SB202190 inhibited JNK activity only at high concentrations
(
100 µM) and at lower concentrations potentiated JNK activity
(Fig. 3F). Thus, at 10 and 50 µM concentrations where IL-2
mRNA up-regulation was substantially reduced, JNK activity was as high
or higher than in cells that were not treated with the inhibitor.
Together these results suggest that the activation of ERK and p38 is
required for IL-2 mRNA transcription in CD8 T cells stimulated via the
TCR and costimulatory receptors. Whether JNK activity is required
cannot be determined from these experiments, but activation of this
enzyme does not appear to be sufficient for IL-2 mRNA
up-regulation.
As predicted by the results examining IL-2 mRNA up-regulation, PD98059 and SB202190 also inhibited proliferation of CD8 T cells stimulated with Kb/OVA/B7-1/ICAM-1. Lymph node OT-I CD8 T cells were stimulated with Kb/OVA/B7-1/ICAM-1 microspheres in the presence of the inhibitors and cell proliferation was determined after 48 h by [3H]thymidine incorporation. PD98059 inhibited proliferation with a dose dependence that correlated closely with its capacity to inhibit ERK activation and IL-2 mRNA up-regulation (Fig. 4A). Similarly, the ability of SB202190 to inhibit proliferation also correlated closely with its inhibition of phosphorylation of p38 and IL-2 mRNA up-regulation, but not with JNK activation (Fig. 4B). These results further confirmed that up-regulation of ERK and p38 is required for IL-2 gene expression and proliferation in CD8 T cells.
Up-regulation of ERK, JNK, and p38 activities is reduced in AINR cells at all times
The above results strongly suggested that the inability of AINR cells to respond to TCR engagement and costimulation involves, at least in part, a defect in up-regulation of activity of the MAPKs. To confirm that up-regulation of these enzymes is defective over the entire course of the response, we examined the time course for up-regulation of ERK and JNK activities and p38 phosphorylation in normal and AINR cells. ERK kinase activity was activated early (within 1 min) in both naive and AINR cells but decreased much more rapidly in AINR cells than in naive cells at later time points (Fig. 5A). In four experiments, at the time that ERK activity peaked in the naive cells, AINR cells had 41–53% less activity. In contrast to ERK, JNK activity increased more slowly upon stimulation, peaking between 10 and 15 mins, and activity in AINR cells was substantially reduced at all time points in comparison to naive cells (Fig. 5B). In four experiments, at the time that JNK activity peaked in the naive cells, AINR cells had 11–67% less activity. Activation of p38 was most profoundly defective in AINR cells. Activation peaked at 5–10 min in naive CD8 T cells, whereas little or no increase in p38 phosphorylation was detected at any time in AINR cells (Fig. 5C). In four experiments, AINR cells had 74–100% less phospho-p38 than naive cells.
PMA and ionomycin restore proliferation and IL-2 production in AINR cells
Activation of p21ras and protein kinase C (PKC) leads to activation of the MAPKs and the active form of p21ras plays a key role in coupling signals generated from the TCR and costimulatory receptors to the transactivation of AP-1 and other transcription factors involved in the regulation of IL-2 gene expression (23). PMA, a PKC activator, activates Ras by suppressing the activity of the GTPase-activating protein RasGAP (24). When PMA is used in conjunction with ionomycin, a calcium ionophore, signaling pathways downstream of Ras and PKC can be activated without the activation of proximal TCR-triggered signaling events.
We found that ERK and JNK activities and p38 phosphorylation were
restored when AINR cells were treated with PMA and ionomycin (Fig. 5),
although appearance of phospho-p38 was consistently lower in AINR cells
than in naive cells despite comparable amounts of total p38 protein in
both types of cell. We therefore examined the ability of these agents
to restore proliferation and IL-2 production in AINR cells. When used
at 10 ng/ml and 1 µM, respectively, PMA and ionomycin provided potent
stimulation for proliferation and IL-2 production of naive CD8 T cells,
and the responses were greater than those obtained using
2C11/B7-1/ICAM-1 microspheres as the stimulus (Fig. 6, A and C). Naive
cells stimulated with PMA and 2C11 proliferated to the same extent as
those treated with PMA and ionomycin, although substantially less IL-2
was produced (Fig. 6, A and C). As shown
previously, AINR cells did not proliferate in response to
2C11/B7-1/ICAM-1 (Fig. 6B). However, when these cells were
stimulated with PMA and ionomycin, their ability to proliferate and
produce IL-2 was restored (Fig. 6, B and D), and
PMA alone or along with 2C11 microspheres stimulated some response.
Naive and AINR CD8 T cells did not respond to 2C11 or ionomycin alone
or in combination (data not shown).
|
|
) and restores proliferation and IL-2 production
(Fig. 6). To confirm that the proliferative response of the
PMA/ionomycin-treated cells was dependent on activation of the MAPKs,
AINR cells were stimulated with PMA/ionomycin in the presence of
various concentrations of PD98059 and SB202190. As expected, both
PD98059 and SB202190 inhibited proliferation of the cells in a
dose-dependent manner (Fig. 7). |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, A and B), TCR, and IL-2 (Fig. 1, C and D), and in vivo responses to B7-bearing
tumor (4) and syngeneic tumor (5, 6). The
AINR cells can no longer make their own IL-2, but can still respond to
exogenous IL-2 (Figs. 1, 4, and 6). We would suggest that development
of AINR following full stimulation is an inherent response to CD8 T
cells and serves to provide an important regulatory checkpoint,
converting an initially helper-independent response to a
helper-dependent response. Thus, development of AINR limits an initial
CD8 response, and even when Ag persists a sustained response is only
obtained if IL-2 provided by CD4 helper T cells is available to support
further expansion.
|
|
) or protein upon
restimulation and a resulting failure to proliferate. Development of
AINR does not require signals through CD28, LFA-1, or other
costimulatory receptors, in that it also occurs when the initial
stimulus is just TCR engagement and exogenous IL-2 (Fig. 1, C and D). Failure to increase IL-2 mRNA levels
might result from defective activation of transcription, decreased mRNA
stability, or both (31). MAPK signaling cascades are activated upon stimulation of T cells and are involved in the regulation of numerous transcription factors, including those required for IL-2 mRNA transcription (32, 33). In anergic CD4 clones, the failure to activate p21ras upon TCR and CD28 engagement leads to a profound block in ERK and JNK up-regulation (8, 9, 10) and failure to transactivate AP-1, a transcription factor required for optimal IL-2 production (34). Both ERK and JNK signaling cascades act synergistically to regulate the level and activity of AP-1. There is considerable evidence in support of an important role(s) for JNKs in activating T cell proliferation (35, 36), although there is also some evidence to indicate that they may not be essential in all cases (37). Mutational analysis of CD28-mediated costimulation has shown that optimal IL-2 production requires CD28-mediated signals in addition to those for JNK activation (38). A recent report demonstrated that coengagement of TCR and CD28 also acts synergistically to activate a third member of the MAPK family, p38, in splenic T cells and suggested that activation of this enzyme is necessary for IL-2 production (19).
Examination of the MAPKs in normal resting CD8 T cells showed that ERK is activated in response to stimulation through just the TCR, with little or no further increase when costimulation is provided (Fig. 3A). In contrast, JNK activation required costimulation (Fig. 3B), as is seen for Jurkat T cells (18) and CD4 T cell clones (8). Our results with CD8 T cells differ from those of Zhang et al. (19) who found that stimulation of resting murine T cells with anti-TCR and anti-CD28 mAbs did not result in JNK activation. Cross-linking CD28 with an Ab may not provide an identical set of signals to that generated when CD28 binds its native B7 ligand, as was done in our experiments. TCR engagement alone caused a significant increase in the active phosphorylated form of p38 and a further substantial increase occurred in response to costimulation (Fig. 3C), as has been reported for mouse splenic T cells (19). Consistent with their abilities to costimulate CD8 T cell proliferation (13) either B7-1 or ICAM-1 could provide costimulation to up-regulate JNK and p38. Activation of all three MAPKs was defective in AINR cells, but the extent of the defect varied for the different enzymes. ERK was activated to comparable levels in normal and AINR cells at very early times (1 min), but at longer times (2.5 min) was greatly reduced in comparison to normal cells (Fig. 5A). In contrast, JNK activity was substantially reduced in AINR cells at all time points in comparison to normal cells (Fig. 5B). The block in p38 activation was most profound, with little or no phosphorylated p38 being detectable in the AINR cells at any time (Fig. 5C).
To assess the importance of up-regulation of the MAPKs in signaling for
IL-2 production and proliferation, we examined the effects of PD98059,
a specific inhibitor of mitogen-activated-protein/ERK kinase (and thus
ERK), and SB202190, an inhibitor of p38 and, at higher concentrations,
JNK. PD98059 decreased ERK activity in a dose-dependent manner (Fig.
3D), and IL-2 mRNA up-regulation (Fig. 3E) and
proliferation (Fig. 4) were inhibited in the same concentration range.
Thus, a TCR-dependent increase in ERK activity appears to be critical
for IL-2 production and proliferation. Similarly, SB202190 inhibited
p38 phosphorylation, IL-2 mRNA up-regulation, and proliferation, with
substantial inhibition at 10 µM and almost complete inhibition at 50
µM concentrations. SB202190 also inhibited JNK activation, but only
at high concentration (100 µM). At lower SB202190 concentrations that
effectively inhibited p38 activation, IL-2 mRNA up-regulation, and
proliferation, JNK activity was substantially enhanced over the level
of activation seen in cells not treated with the drug (Fig. 4). These
results strongly suggest that activation of p38, like ERK, is critical
for IL-2 production and proliferation. Whether JNK activation is also
required cannot be determined from these experiments. It does appear
that, however, that JNK activation is not sufficient in the absence of
p38 up-regulation since JNK activity remains high at SB202190
concentrations where p38 activation and IL-2 production are inhibited.
Furthermore, JNK can be up-regulated in AINR cells when potent
costimulation is provided by B7-1 and ICAM-1 together (Fig.
3B), but IL-2 production and proliferation do not occur. The
conclusion that p38 is critical for the CD8 T cell response is
consistent with the recent report of Zhang et al. (19)
providing evidence for the importance of p38 in activating murine
splenic T cells.
Defective activation of the MAPKs in AINR cells suggested a defect in a
common upstream signaling pathway, most likely at the level of
p21ras (39, 40). To address this
possibility, we examined the effects of treating AINR cells with PMA,
an activator of ras and PKC, and ionomycin, a calcium ionophore. PMA-
and ionomycin-restored ERK, JNK, and p38 activation (Fig. 5), IL-2
production (Fig. 6D) and proliferation (Fig. 6B)
in the AINR cells. Proliferation of AINR cells in response to
PMA/ionomycin was blocked by both PD98059 and SB202190 (Fig. 7),
indicating that these pharmacological agents were acting, at least in
part, by restoring activation of the MAPKs. An increase in
intracellular Ca2+ concentration occurs upon stimulation
through the TCR and results from release of Ca2+ from
intracellular stores followed by the influx of extracellular
Ca2+ (41). We have not directly examined
whether AINR cells flux Ca2+ in response to TCR ligation,
but would predict that they do given that they retain lytic effector
function, an activity that is dependent on Ca2+ signaling
(42). PMA in conjunction with TCR ligation, but not PMA
alone, could restore IL-2 production (Fig. 6D) and
proliferation (Fig. 6B) by the AINR cells. Thus, some of the
TCR-dependent signals needed to activate these functions appear to
remain intact in the AINR cells.
|
AINR develops in CD8 T cells following an initial response to a full stimulus, both in vitro and in vivo, and appears likely to be an integral part of the developmental program that a CD8 T cell progresses through following TCR engagement and costimulation. There are numerous reports in the literature from a variety of virus and tumor model systems where CD8 T cells are observed to become nonresponsive. Rather than this being a defect in the response or a nonresponsiveness induced in some manner by the disease agent, it may in most cases simply be the result of the normal progression of the CD8 response. AINR would provide a critical checkpoint to convert a helper-independent CTL response to a helper-dependent response, requiring provision of IL-2 by CD4 helper T cells to sustain and further expand the Ag-specific CD8 cells. Understanding the biochemical basis for the AINR state has the potential to suggest ways of reversing the nonresponsiveness to sustain a CTL response in cases where an effective helper response by CD4 T cells cannot be induced. As a first step in developing such an understanding, we have demonstrated in this report that the defect in AINR cells results, at least in part, from an inability to up-regulate MAPK signaling pathways through either the TCR or costimulatory receptors.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence to: Dr. Matthew F. Mescher, Center for Immunology, University of Minnesota, Mayo Mail Code 334, 420 Delaware Street, S.E., Minneapolis, MN 55455. E-mail address: mesch001{at}tc.umn.edu
3 Abbreviations used in this paper: AINR, activation-induced nonresponsiveness; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, Janus kinase; PKC, protein kinase C.
Received for publication March 12, 2001. Accepted for publication June 5, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2. J. Immunol. 157:5403.[Abstract]
-herpesvirus in the absence of CD4+ T cells. J. Exp. Med. 184:863. and IL-4 responses to viral infections: requirements for IL-2. J. Immunol. 160:5007.This article has been cited by other articles:
|
|
 |
|
  S. D. Liu, T. Tomassian, K. W. Bruhn, J. F. Miller, F. Poirier, and M. C. Miceli Galectin-1 Tunes TCR Binding and Signal Transduction to Regulate CD8 Burst Size J. Immunol., May 1, 2009; 182(9): 5283 - 5295. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Renda, S. Baraldo, G. Pelaia, E. Bazzan, G. Turato, A. Papi, P. Maestrelli, R. Maselli, A. Vatrella, L. M. Fabbri, et al. Increased activation of p38 MAPK in COPD Eur. Respir. J., January 1, 2008; 31(1): 62 - 69. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Horton, I. Frank, R. Baydo, E. Jalbert, J. Penn, S. Wilson, J. P. McNevin, M. D. McSweyn, D. Lee, Y. Huang, et al. Preservation of T Cell Proliferation Restricted by Protective HLA Alleles Is Critical for Immune Control of HIV-1 Infection J. Immunol., November 15, 2006; 177(10): 7406 - 7415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Kennedy and E. Celis T Helper Lymphocytes Rescue CTL from Activation-Induced Cell Death. J. Immunol., September 1, 2006; 177(5): 2862 - 2872. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Uhlin, M. G. Masucci, and V. Levitsky Regulation of lck degradation and refractory state in CD8+ cytotoxic T lymphocytes PNAS, June 28, 2005; 102(26): 9264 - 9269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Y. Lin, T. Zal, I. L. Ch'en, N. R. J. Gascoigne, and S. M. Hedrick A Pivotal Role for the Multifunctional Calcium/Calmodulin-Dependent Protein Kinase II in T Cells: From Activation to Unresponsiveness J. Immunol., May 1, 2005; 174(9): 5583 - 5592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. L. Redmond, B. C. Marincek, and L. A. Sherman Distinct Requirements for Deletion versus Anergy during CD8 T Cell Peripheral Tolerance In Vivo J. Immunol., February 15, 2005; 174(4): 2046 - 2053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Boissonnas, C. Combadiere, E. Lavergne, M. Maho, C. Blanc, P. Debre, and B. Combadiere Antigen Distribution Drives Programmed Antitumor CD8 Cell Migration and Determines Its Efficiency J. Immunol., July 1, 2004; 173(1): 222 - 229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. F. Rowley and A. Al-Shamkhani Stimulation by Soluble CD70 Promotes Strong Primary and Secondary CD8+ Cytotoxic T Cell Responses In Vivo J. Immunol., May 15, 2004; 172(10): 6039 - 6046. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Mitchell and B. P. Lawrence Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Renders Influenza Virus-Specific CD8+ T Cells Hyporesponsive to Antigen Toxicol. Sci., July 1, 2003; 74(1): 74 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Curtsinger, D. C. Lins, and M. F. Mescher Signal 3 Determines Tolerance versus Full Activation of Naive CD8 T Cells: Dissociating Proliferation and Development of Effector Function J. Exp. Med., May 5, 2003; 197(9): 1141 - 1151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Myers, C. Takahashi, R. S. Mittler, R. J. Rossi, and A. T. Vella Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering PNAS, April 29, 2003; 100(9): 5348 - 5353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L.-X. Wang, B.-G. Chen, and G. E. Plautz Adoptive Immunotherapy of Advanced Tumors with CD62 L-Selectinlow Tumor-Sensitized T Lymphocytes Following Ex Vivo Hyperexpansion J. Immunol., September 15, 2002; 169(6): 3314 - 3320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Shrikant and M. F. Mescher Opposing Effects of IL-2 in Tumor Immunotherapy: Promoting CD8 T Cell Growth and Inducing Apoptosis J. Immunol., August 15, 2002; 169(4): 1753 - 1759. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Tham and M. F. Mescher The Poststimulation Program of CD4 Versus CD8 T Cells (Death Versus Activation-Induced Nonresponsiveness) J. Immunol., August 15, 2002; 169(4): 1822 - 1828. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Tham, P. Shrikant, and M. F. Mescher Activation-Induced Nonresponsiveness: A Th-Dependent Regulatory Checkpoint in the CTL Response J. Immunol., February 1, 2002; 168(3): 1190 - 1197. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
