From the
Department of Applied Biological
Chemistry, Graduate School of Agricultural and Life Sciences, The University
of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan and the
||Department of Food Science and Technology,
College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi,
Kanagawa 252-8510, Japan
Received for publication, December 17, 2002 , and in revised form, April 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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As in the case of other forms of tolerance, oral tolerance is mediated at
the T cell level (6,
7). Three mechanisms are well
documented for oral tolerance induction: (a) Ag-specific T cell
clonal deletion (8,
9), (b) functional
unresponsiveness of T cells to Ags (anergy)
(1013),
and (c) active suppression mediated by regulatory T cells that
produce immunosuppressive cytokines, such as transforming growth factor-
and interleukin (IL)-10
(1417).
However, the precise molecular mechanisms underlying the induction of oral
tolerance remain unclear.
Previous studies demonstrated that Ag-specific T cell hyporesponsiveness was caused by defects in intracellular signaling events from the T cell receptor (TCR) and co-stimulatory molecules, such as incomplete protein phosphorylation (1821) and impaired calcium/nuclear factor of activated T cells (NFAT) signaling (22). Furthermore, genetic experiments, such as DNA array methods, have recently been performed to analyze hyporesponsive T cells entirely from multidirectional points (2325). However, to date, there are no detailed studies dealing with tolerant T cells examining total specific protein expression levels.
In the postgenomic era, proteome analysis is expected to be the bridge between the genomic sequence and protein characteristics underlying cellular behavior, since translated proteins can be posttranslationally modified (2628). The development of two-dimensional electrophoresis (2-DE) provides the high resolution of complex protein mixtures with high reproducibility. In addition, the combination of 2-DE, modern mass spectrometry (MS) technology, and rapidly accumulating genomic sequence data permits accurate and speedy identification of cellular proteins. With these advances, proteome analysis can be utilized in the research of intracellular protein changes in cells following a certain stimulus.
We have previously reported that long term feeding of dietary Ag to ovalbumin (OVA)-specific TCR transgenic mice (OVA23-3 mice) induced oral tolerance of peripheral T cells (29). In this experimental model, orally tolerant T cells showed impaired calcium/NFAT signaling following TCR-mediated activation, despite normal activation of the mitogen-activated kinase pathway. In this study, we used oral tolerance-induced OVA23-3 mice to investigate the expression of intracellular proteins using 2-DE to analyze the specific characteristics of in vivo orally tolerized splenic CD4 T cells compared with unsensitized CD4 T cells.
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EXPERIMENTAL PROCEDURES |
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OVA-specific Oral Tolerance Induction in OVA23-3 Mice68-week-old OVA23-3 mice were orally administered with dietary Ag containing 20% hen egg white (egg white diet; Funabashi Farm, Funabashi, Japan) or a control diet (commercially available CE-2; Clea Inc.), for 2830 days.
Antibodies (Abs) and ReagentsFor purification of CD4 T
cells and flow cytometric analyses, FITC- or allophycocyanin-conjugated
anti-mouse CD4 monoclonal Ab (mAb) (H129.19), biotinylated anti-mouse
CD45R/B220 mAb (RA3-6B2), and phycoerythrin-conjugated anti-mouse TCR
chain mAb (H57-597) were all purchased from BD PharMingen (San Diego, CA).
Anti-mouse major histocompatibility complex class II mAb (M5/114.15.2) and
anti-mouse CD11c mAb (N418) were purified from ascites and biotinylated in our
laboratory according to standard techniques. Propidium iodide (PI) was
purchased from Sigma. For the enrichment of CD4-positive cells, anti-mouse CD4
microbeads (Militenyi Biotec, Bergisch Gladbach, Germany) were used. For
immunoprecipitation and Western blotting experiments, anti-X-linked inhibitor
of apoptosis (XIAP) mAb (BD Transduction Laboratories, Lexington, KY),
anti-Grb2-related adaptor downstream of Shc (GADS) polyclonal Ab (pAb)
(Upstate Biotechnology, Inc., Lake Placid, NY), anti-phospholipase C-
1
(PLC-
1) mAb mixture (Upstate Biotechnology), anti-linker for activation
of T cells (LAT) pAb (Upstate Biotechnology), anti-Src homology 2
domain-containing leukocyte protein of 76 kDa (SLP-76) pAb (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA), anti-TCR-
chain mAb (Zymed
Laboratories Inc., South San Francisco, CA), horseradish peroxidase-conjugated
anti-phosphotyrosine mAb (RC20; BD Transduction Laboratories), horseradish
peroxidase-conjugated anti-rabbit IgG, and anti-mouse IgG (New England
Biolabs, Boston, MA) were used.
Preparation of Splenic CD4 T LymphocytesPreparation of
splenic CD4 T cells of OVA23-3 mice was performed using an Anti-FITC MultiSort
kit (Militenyi Biotec) as follows. Splenocytes were labeled with anti-mouse
CD4 mAb conjugated with FITC, biotinylated anti-mouse major histocompatibility
complex class II mAb, biotinylated anti-mouse CD11c mAb, and biotinylated
anti-mouse CD45R/B220 mAb. After washing and secondary labeling with anti-FITC
microbeads, cells were subjected to positive enrichment via the magnetic
column. After release of the anti-FITC microbeads, cells were relabeled with
streptavidin-conjugated microbeads for negative selection. Isolated cells were
both CD4- and TCR chain-positive and at greater than 98% purity, as
assessed by flow cytometric analyses, and were regarded as CD4 T
lymphocytes.
Proliferation Assay and Cytokine Assay with Enzyme-linked Immunosorbent
Assay1 x 105 purified CD4 T cells were cultured
with 0, 0.1, or 1.0 mg/ml OVA in flat bottom 96-well plates and 4 x
105 mitomycin C-treated BALB/c splenocytes as APCs in RPMI 1640
medium containing 5% heat-inactivated fetal calf serum (Sigma). For cytokine
assays, supernatant samples were collected at 24 h for IL-2 and 48 h for IL-4,
interferon (IFN)-, and IL-10. For the proliferation assays, cultures
were pulsed with [3H]thymidine (1 µCi/well, ICN Pharmaceuticals,
Costa Mesa, CA) after 48 h of incubation and harvested 24 h later. Cells were
collected on glass fiber, and the incorporated radioactivity was measured by
scintillation counting. IL-2, IL-4, and IFN-
cytokine production was
measured by enzyme-linked immunosorbent assay using paired Abs (BD
PharMingen), as described previously
(31). IL-10 production was
measured using the OptEIATM mouse IL-10 set (BD PharMingen) according to
the manufacturer's instructions.
2-DE and Silver StainingPurified CD4 T lymphocytes were lysed in 7 M urea, 2 M thiourea, 4% CHAPS, 20 mM dithiothreitol, 2.5 µg/ml DNase I, 2.5 µg/ml RNase, protease inhibitor mixture (Sigma), followed by sonication. Samples were then centrifuged at 20,000 x g for 1 h, and the supernatants were used as the whole cell lysates. The first dimension of 2-DE was performed with linear pH 4.55.5 and 5.56.7/18-cm Immobiline DryStrips (Amersham Biosciences). DryStrips were rehydrated overnight at room temperature in 8 M urea, 30 mM dithiothreitol, 2% CHAPS, 2% IPG buffer (Amersham Biosciences) containing 150250 µg of proteins. The first dimension electrophoresis was run using the following conditions: 100 V for 3 h, 300 V for 1 h, 500 V for 2 h, 700 V for 1 h, 1,000 V for 1 h, 1,500 V for 1 h, 2,000 V for 1 h, 2,500 V for 1 h, and 3,000 V for 2024 h. After the first run, Immobiline DryStrips were equilibrated according to the manufacturer's recommendations and set on the 12.0% polyacrylamide gels for the second dimensional separation. After 2-DE, gels were silver-stained as previously described (32). Stained gels were imaged with Gel dock (Bio-Rad), and spot intensity was measured using ImageGauge software (Fuji Photo Film, Kanagawa, Japan). Normalized spot intensities were calculated from more than 10 spots of each gel.
Protein Identification by MALDI-TOF MSIn-gel tryptic
digestions of excised protein spots were performed with modified protocols as
previously described (33).
Briefly, destained gels were reduced and alkylated, followed by tryptic
digestion (Promega, Madison, WI). Digested peptides were eluted with 5% formic
acid, 50% acetonitrile. Peptides were concentrated and desalted using
ZipTipC18 (Millipore Corp., Bedford, MA) according to the
manufacturer's protocol. Purified peptides were mixed with 10 mg/ml
-cyano-4-hydroxycinnamic acid, 0.2% aqueous trifluoroacetic acid,
acetonitrile (1:1) as a matrix. The mass spectra were recorded by Voyager-DE
STR (Applied Biosystems, Foster City, CA). Internal calibration was performed
using autoproteolytic trypsin peptide fragments of 842.50 and 2211.1046 Da,
and specific peaks were applied to the MS-fit program (available on the World
Wide Web at
prospector.ucsf.edu/ucsfhtml4.0/msfit.htm).
Proteins were identified with 50 ppm accuracy and a minimum of four matching
peptides and 25% peptide coverage of total amino acids.
Stimulation of CD4 T Lymphocytes with AbsPurified CD4 T cells were stimulated with co-cross-linking of TCR and CD4 molecules with Abs for the indicated times as previously described in Ref. 29.
Immunoprecipitation and Western BlottingFor Western blots of the pro form and active form of caspase-3, harvested cells were lysed in CHAPS cell extract buffer (50 mM Pipes/KOH, pH 6.5, 0.1% CHAPS, 2 mM EDTA, 5 mM dithiothreitol, protease inhibitor), and detections were performed using an apoptosis sampler kit (Cell Signaling Technology, Beverly, MA) according to the manufacturer's instructions. Harvested cells were lysed in 0.5% Nonidet P-40 lysis buffer for immunoprecipitation of stimulated CD4 T cells and in PLC lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, protease inhibitor) for Western blot of whole cell lysate. Western blots of phosphotyrosine were performed as described in Ref. 29. Other Western blots were performed as follows. Immunoblots were blocked with 5% skim milk in TBS-Tween for 1 h, followed by probing with primary antibody for1hat room temperature or overnight at 4 °C. After washing with TBS-Tween three times, immunoblots were incubated with secondary antibody for 1 h. Immunoblots were then washed with TBS-Tween three times, and detections were performed with ECL (Amersham Biosciences).
Early Apoptotic and Dead Cell Detection by Fluorescence-activated Cell SortingSplenocytes were enriched for CD4-positive cells with anti-CD4 microbeads and magnetic separation columns, according to the manufacturer's recommended protocol. Cells were then labeled with FITC-conjugated annexin V (Roche Applied Science), PI, and APC-conjugated anti-mouse CD4 mAb according to the manufacturer's instructions. Their staining profiles were analyzed by fluorescence-activated cell sorting LSR and CellQuest software (BD Biosciences, Mountain View, CA).
DNA Fragmentation AssayTriplicate 4 x 106 purified T cells were subjected to a DNA fragmentation assay with diphenylamine reagent as previously described in Ref. 34.
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RESULTS |
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In Vivo Protein Expression Analysis of Orally Tolerant CD4 T Lymphocytes with 2-DETo characterize protein expression levels of orally tolerant CD4 T cells, we compared these cells with unsensitized CD4 T cells obtained from control diet-fed OVA23-3 mice by 2-DE. We used whole cell lysates to represent the in vivo state of cellular proteins and to make an accurate comparative study. We also utilized narrow range immobilized pH gradient gels for the first dimension electrophoresis to obtain high resolution separation. Fig. 2A shows silver-stained two-dimensional separated gels from both control and orally tolerant CD4 T cells. The arrowheads show the up-regulated protein spots when the two gels were compared. We also used luminescent staining, which is known to deliver a wide quantitation range (35), but could not produce the same sensitivity as observed using silver staining (data not shown). In relation to the detected spots, similar results were obtained when comparing spot intensities between gels. We detected 42 changed spots between pH 4.5 and 6.7, of which 26 were increased and 16 decreased for orally tolerant CD4 T cells. Of the differentially expressed spots, we identified 35 by peptide mass fingerprinting. Table I shows the peptide mass fingerprint results, which contain experimental and theoretical pI and molecular weights, NCBI protein accession number, the number of matching peptides, and the amino acid coverage of the obtained peptides. The -fold induction of the protein spots (orally tolerant versus control CD4 T cells) are also shown. There were some unidentified spots that had no matching proteins in the data base to date, although we could identify specific digested peptide peaks, or in some cases, the amount of protein expressed was too little to be precisely identified by this method.
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Caspase Activities in Orally Tolerant CD4 T Lymphocytes Up-regulated proteins in orally tolerant CD4 T cells included both caspase-1 and caspase-3. The caspases are a family of cysteine aspartic acid proteases that play a central role in the regulation of apoptosis (36). Activation of members of the caspase family requires their cleavage; for example, the pro form of caspase-3 (32 kDa) is cleaved into its active form (17 or 20 kDa) (37). Furthermore, the effector caspases are responsible for the cleavage of target proteins. In our results of 2-DE gels, some of the proteins identified with decreased expression levels in orally tolerant CD4 T cells have been reported to undergo caspase-dependent cleavage: i.e. STE20-like kinase MST-1, which promotes apoptosis after cleavage (38, 39), and GADS (40, 41). In addition, actin is cleaved into 40-, 31-, 29-, and 15-kDa polypeptides by caspase-1 and -3 (42, 43), and we found that the 31- and 29-kDa cleaved forms of actin were increased in orally tolerant CD4 T cells (Table I). These findings also suggest that active caspase-3 or -1 was up-regulated in orally tolerant CD4 T cells, although we only detected the pro form of caspase-3 or -1 up-regulation in 2-DE gels. Therefore, we next analyzed caspase-3 cleavage by Western blotting. As expected, the pro form and active form of caspase-3 were increased in orally tolerant CD4 T cells (Fig. 3A). These results suggest that caspases play an important role in orally tolerant CD4 T cells.
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Although the activation of caspases contributes to apoptosis, there are some reports that caspase-3 activation occurs in nonapoptotic T cells (4446). We therefore examined whether orally tolerant CD4 T cells underwent apoptosis. As shown in Fig. 3B, very few apoptotic (annexin V-positive) and necrotic (PI-positive) cells were identified in both control and orally tolerant CD4 T cells. In addition, purified CD4 T cells that were subjected to 2-DE and Western blots exhibited negligible levels of DNA fragmentation (Fig. 3C). These results indicated that orally tolerant CD4 T cells, which were nonapoptotic, up-regulated caspase activation and suggested the possibility that orally tolerant CD4 T cells augmented the expression of antiapoptotic proteins, such as caspase suppressors. One of the known endogenous caspase inhibitor protein families is the inhibitors of apoptosis (IAPs) protein family (47). XIAP is the most potent of the IAPs and is restricted to targeting caspase-9 and -7 and the active form of caspase-3 (47, 48). We therefore proposed that the up-regulation of XIAP in orally tolerant CD4 T cells may prevent the cells from undergoing apoptosis. To test this possibility, we performed Western blot analyses using an antibody against XIAP (Fig. 3D). As predicted, orally tolerant CD4 T cells showed increased XIAP protein levels when compared with control cells, suggesting that XIAP aids in maintaining the viability of orally tolerant CD4 T cells.
Caspases in TCR Signaling Impairment of Orally Tolerant CD4 T
LymphocytesWe previously reported that orally tolerant CD4 T cells
in this experimental system exhibited impairment in the NFAT/calcium signaling
pathway (29). Since the 2-DE
studies of orally tolerant CD4 T cells showed degradation of intact GADS,
which is involved in proximal TCR signaling
(49), we proposed that the
caspase-dependent cleavage of TCR signaling components contributed to
signaling impairment. To confirm whether GADS was cleaved by caspase-3,
Western blot analysis was performed. As predicted, the 26-kDa
caspase-3-dependent cleaved GADS
(40) was detected in orally
tolerant CD4 T cells (Fig.
4A). We then investigated GADS-associated molecules in
stimulated CD4 T cells by immunoprecipitation with anti-GADS pAb.
Fig. 4B shows the
tyrosine phosphorylation of GADS-associated molecules. The TCR-induced
calcium/NFAT pathway is critically initiated by SLP-76 and PLC-1.
PLC-
1 activity is regulated by tyrosine phosphorylation
(50) and association with LAT
(51) and SLP-76
(52). We found that
GADS-associated SLP-76 and GADS-LAT-SLP-76-associated PLC-
1
(53) were decreased in both
phosphorylation and association in orally tolerant CD4 T cells. Most notably,
the association between GADS and SLP-76 was dramatically disrupted. In
addition, the phosphorylation of TCR-
chain and LAT was reduced in
orally tolerant CD4 T cells. We have previously described the reduced
phosphorylation of TCR-
chain and PLC-
1 independent of their
abilities to associate with GADS
(29), so we further analyzed
the phosphorylation of SLP-76 and LAT. As shown in
Fig. 4C, the
phosphorylation of SLP-76 and LAT was reduced in orally tolerant CD4 T cells.
The protein levels of LAT and PLC-
1 were not altered in both
populations (data not shown), whereas SLP-76 was slightly decreased in orally
tolerant CD4 T cells (Fig.
4D). It has also been implied that SLP-76 undergoes
caspase-dependent cleavage
(40), so we investigated
caspase-dependent cleavage of SLP-76. As shown in
Fig. 4D, SLP-76 was
cleaved in orally tolerant CD4 T cells. These results suggest that the
caspase-dependent cleavage of GADS and SLP-76 inhibit the associations that
are formed by these molecules, thus contributing to TCR signaling impairment
in orally tolerant CD4 T cells.
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DISCUSSION |
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In this experimental model of oral tolerance induced by long term feeding of dietary Ag, splenic CD4 T cells exhibited a lack of proliferative ability and reduced cytokine production (Fig. 1, A and B). These hyporesponsive CD4 T cells therefore resembled previously described anergic T cells. Since immunosuppressive cytokine production was not detected in this model (Fig. 1C) (29), the induction of oral tolerance under these conditions is not considered to be caused by active suppression but rather by clonal deletion or anergy. Recently, the suppressive function of CD25+CD4 T cells has been demonstrated in oral tolerance (17, 54). The CD4 T cells from orally tolerant mice in our system contained CD25+CD4 T cells (29); however, the depletion of these cells did not affect the response of CD4 T cells from the egg white diet-fed mice (29). Thus, the reduced response of CD4 T cells after Ag feeding was not mediated by CD25+CD4 T cells in our system, although these CD25+CD4 T cells themselves are in a hyporesponsive state (17, 54). The observation that we could not find any evidence of active suppression in our system is consistent with previous studies examining Ag dose-dependent oral tolerance (55, 56); low dose Ag preferentially induces tolerance mediated by regulatory T cells, whereas high dose Ag deletes or anergizes CD4 T cells (8, 11, 31, 5759). Our two-dimensional gel electrophoresis studies (Fig. 2, Table I) reflected the in vivo state directly and revealed that caspase-dependent protein cleavages were frequently occurring in orally tolerant CD4 T cells. Although these findings suggest that orally tolerant CD4 T cells underwent clonal deletion by apoptosis, most of them were nonapoptotic when freshly isolated (Fig. 3, B and C). Our model thus explains the link between clonal anergy and clonal deletion. Previous studies suggest that caspase activation is involved in the early steps of nonapoptotic T cell activation (46). In cases when caspase activation does not result in apoptosis, antiapoptotic factors, such as IAPs, may rescue cells from apoptosis (60). We observed an increase of XIAP in orally tolerant CD4 T cells (Fig. 3D). These T cells may have acquired resistance to apoptosis and therefore survived deletion, most likely by activation-induced cell death, by expressing antiapoptotic factors, which result in an anergic-like state. Indeed, the number of splenic CD4 T cells in transgenic mice decreased significantly in the first 3 weeks of egg white diet feeding; however, the decrease from 3 weeks onward was slight, which suggests that extensive deletion occurs mainly in the early stages of Ag feeding (data not shown). Consequently, orally tolerant CD4 T cells appear to maintain the subtle balance between their survival and apoptosis by adjusting pro- and antiapoptotic proteins.
With regard to TCR signaling impairment of orally tolerant CD4 T cells, we have previously reported that the calcium/NFAT pathway is abnormal in these cells despite normal activation of the mitogen-activated kinase pathway (29). In this report, we showed intact GADS degradation and cleavage in orally tolerant CD4 T cells (Table I, Fig. 2, and Fig. 4A), whereas GADS mRNA levels are not significantly altered (data not shown). GADS is a hematopoietic adaptor protein and plays an important role in proximal TCR signaling by LAT and the SLP-76 (49, 6164) and is shown to undergo caspase cleavage. Our results showed the appropriate molecular weight of the predicted GADS cleavage product in orally tolerant CD4 T cells (Fig. 4A). We also detected in the 2-DE comparative study another hematopoietic-restricted scaffold protein, Grb2-related adaptor protein (Grap) (65), which belongs to the Grb2 family and regulates TCR signaling as does GADS (64, 66) but contains no caspase-cleaved site (40). In contrast to GADS, Grap was expressed at the same level in both populations of CD4 T cells (data not shown). These results strongly suggest that degradation of intact GADS and SLP-76 molecules is caused by selective caspase-dependent protein cleavage. GADS is known to undergo caspase cleavage at amino acid sequences between its Src homology 2 domain, which associates with LAT, and C-terminal Src homology 3 domain, which associates with SLP-76 (40, 41). It has been reported that GADS caspase-3-cleaved products also have binding capacity to these molecules and perturb TCR signaling by SLP-76 and LAT, leading to impairment of NFAT activation (40, 41). Furthermore, SLP-76 cleavage products may also perturb TCR signaling. Thus, it is possible that GADS and SLP-76 cleavage products may inhibit TCR signaling in orally tolerant CD4 T cells.
Yankee et al. (41)
demonstrated that FAS signaling leads to GADS cleavage, which subsequently
leads to impairment in the GADS·SLP-76 complex. Our results clearly
showed that the formation of the TCR signaling complex involving GADS was
defective in in vivo induced nonapoptotic orally tolerant CD4 T
cells, suggesting that caspase-dependent cleavage of GADS and SLP-76 is one of
the causes (Fig. 4). These
caspase-dependent defects in the assembly of TCR signaling molecules may cause
the reduced phosphorylation of LAT, SLP-76, and PLC-1. However, it is
also possible that the abilities of these molecules to undergo phosphorylation
are altered independent of GADS association in orally tolerant CD4 T cells by
other mechanisms (Fig.
4C). In any case, we demonstrated that PLC-
1
activity is down-regulated in orally tolerant CD4 T cells because of reduced
phosphorylation and incomplete association with the
LAT·SLP-76·GADS complex (Fig.
4B). PLC-
1 controls intracellular calcium
elevation after TCR engagement
(67), leading to NFAT
activation (68). Our data
strongly suggest that the caspase-dependent defects in the proximal TCR
signaling complex contribute to the impairment of the calcium/NFAT pathway in
orally tolerant CD4 T cells.
Our findings indicate that orally tolerant CD4 T cells up-regulate caspase activation and show a selective decrease in the levels of caspase-targeted proteins and that antiapoptotic factors were concomitantly up-regulated. Furthermore, the cleavage of key TCR signaling molecules by caspases appears to result in defects in the TCR signaling. Conclusively, our results suggest that orally tolerant CD4 T cells undergo caspase activation to maintain their unique tolerant characteristics.
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FOOTNOTES |
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Present address: Dept. of Immunology, National Institute of Infectious
Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
¶ To whom correspondence should be addressed. Tel.: 81-3-5841-5137; Fax: 81-3-5841-8029; E-mail: ahachi{at}mail.ecc.u-tokyo.ac.jp.
1 The abbreviations used are: Ag, antigen; IL, interleukin; NFAT, nuclear
factor of activated T cells; OVA, ovalbumin; TCR, T cell receptor; 2-DE,
two-dimensional electrophoresis; MS, mass spectrometry; Ab, antibody; mAb,
monoclonal antibody; pAb, polyclonal antibody; PI, propidium iodide; XIAP,
X-linked inhibitor of apoptosis; GADS, Grb2-related adaptor downstream of Shc;
SLP-76, Src homology 2 domain-containing leukocyte protein of 76 kDa; LAT,
linker for activation of T cells; PLC-1, phospholipase C-
1; APC,
antigen-presenting cell; IFN-
, interferon-
; IAP, inhibitor of
apoptosis; FITC, fluorescein isothiocyanate; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Pipes,
1,4-piperazinediethanesulfonic acid; MALDI-TOF, matrix-assisted laser
desorption/ionization time-of-flight.
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ACKNOWLEDGMENTS |
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REFERENCES |
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