From the Department of Microbiology and Immunology
and
Department of Pediatric Cardiovascular Surgery, The Heart
Institute of Japan, Tokyo Women's Medical University, Tokyo 162-8666, Japan and the ¶ Program in Molecular Immunology, Institute of
Molecular Medicine and Genetics, Medical College of Georgia,
Augusta, Georgia 30912-2600
Received for publication, February 8, 2001
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ABSTRACT |
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Human thymic
CD1a Human thymic CD1a Signal transduction via TCR stimulation involves at least two pathways:
(i) activation of phospholipase C- In the present study, we addressed the question of why human thymic
CD1a Antibodies--
Antibodies (Abs) I2C3 (anti-HLA-DR/DP) and Nu
Ts/c (anti-CD8) have been described previously (13). The following Abs
were available commercially: a hybridoma cell line, OKT6 (anti-CD1a, American Type Culture Collection, Manassas, VA); FITC-conjugated SK3
(anti-CD4), phycoerythrin-conjugated SK1 (anti-CD8) and FITC- or
phycoerythrin-conjugated SK7 (anti-CD3, Becton Dickinson, Mountain View, CA); RD-1 conjugated I2 (anti-DR, Coulter Immunology, Hialeah, FL); biotin-conjugated E22E7.2 (anti-V Preparation of APB CD4+ and Thymic
CD1a Preparation of Antigen-presenting Cells--
DR+ L
cells (8124) were used as antigen-presenting cells (APC). Before use as
APC, DR+ L cells were irradiated at 30 gray with an
x-ray irradiator and treated with 50 µg/ml mitomycin C for 30 min at
37 °C. APC were added to each well of microplates, 1 × 105 cells/well for 48-well plates or 1 × 106 cells/well for 6-well plates, and allowed to adhere
overnight at 37 °C.
Preparation of TSST-1-induced APB and Thymic T-cell
Blasts--
TSST-1-induced T-cell blasts were obtained as described
previously (3). Briefly, APB CD4+ and thymic
CD1a Assay for Production of IL-2--
For the assay of IL-2, T-cell
blasts (5 × 105 cells/well) were stimulated with 10 ng/ml TSST-1 on an APC monolayer for various periods in 0.5-ml volumes
in 48-well culture plates (Corning Glass). IL-2 activity in the
culture supernatants was determined using IL-2-dependent
CTLL-2 cells as reported previously (14). Data are presented as
units/ml IL-2.
Flow Cytometric Analysis--
For expression of CD4
versus CD8, CD3 versus V Immunoprecipitation and Immunoblotting--
T-cell blasts
(2 × 107 cells/well) were restimulated with 2 µg/ml
TSST-1 at 37 °C in 1-ml volumes in 6-well culture plates containing
tightly adherent APC. At different time points, cultures were
terminated by adding 5 ml of cold PBS, and T-cell blasts were collected
immediately. After washing with cold PBS, the T-cell blasts were lysed
in cold TNE buffer (containing 10 mM Tris-HCl (pH 7.4), 1%
Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, 1 mM Na3VO4, 10 µg/ml aprotinin and
300 µg/ml phenylmethylsulfonyl fluoride). Cell lysates were
pre-cleared with Protein A/G Plus-agarose (Oncogene Science Inc.,
Uniondale, NY) and then incubated with Protein A/G Plus-agarose coupled
to the respective antibody for 2 h at 4 °C. The
immunoprecipitates were subjected to 10% SDS-polyacrylamide gel
electrophoresis under reducing conditions, transferred to a
polyvinylidene difluoride membrane, and immunoblotted with the indicated Ab followed by horseradish peroxidase-conjugated protein G
(Zymed Laboratories Inc. Laboratories, San Francisco,
CA) as the second Ab. The immunoblots were developed with the ECL
system (Amersham Pharmacia Biotech, Buckinghamshire, UK).
In Vitro Kinase Assay--
T-cell blasts were solubilized in TNE
buffer as described for immunoprecipitation, and the supernatants were
subjected to immunoprecipitation for 2 h with an anti-Lck Ab.
Immunoprecipitates were washed with radioimmune precipitation buffer
(TNE buffer containing 0.1% sodium deoxycholate and 0.1% SDS)
followed by washing buffer containing 20 mM Tris-HCl (pH
7.4) and 0.5 M LiCl. Immunoprecipitates were incubated with
30 µl of kinase reaction buffer containing 40 mM HEPES
(pH 7.4), 3 mM MnCl2, 10% glycerol, 10 µM cold ATP, and 10 µCi of [ Precipitation of Tyrosine-phosphorylated Lck Carboxyl-terminal
Peptide--
The sequence of a tyrosine-phosphorylated Lck
carboxyl-terminal peptide (LckP peptide) containing the
carboxyl-terminal 11 amino acids of Lck,
Thr-Ala-Thr-Glu-Gly-Gln-Tyr-(PO3H2)-Gln-Pro-Gln-Pro, has been described by Sieh et al. (15). Biotin-conjugated
LckP peptide was synthesized by BEX (Tokyo, Japan). Precipitation of LckP peptide was done as described previously (15). Briefly, cell
lysates from T-cell blasts stimulated as described in
immunoprecipitation were incubated with 50 µg/ml biotinylated LckP
peptide for 2 h at 4 °C followed by incubation with
streptavidin-conjugated agarose beads (Sigma) for 2 h. The
precipitates were subjected to analysis by immunoblotting procedures as
described above.
Immunofluorescence and Confocal Microscopy--
For
immunofluorescence staining of CD45 and Lck, T-cell blasts were washed
with PBS and fixed for 30 min in 3.7% paraformaldehyde in PBS at room
temperature on glass slides pretreated with Cell-Tak (Collaborative
Biomedical Products, Bedford, MA). Glass slides were then incubated
with 0.1% Triton X-100 in PBS for 5 min to permeabilize the attached
T-cell blasts and blocked for 15 min with 1% skimmed milk. The glass
slides were incubated overnight at 4 °C with biotinylated anti-Lck
Ab and FITC-conjugated anti-CD45 Ab at the appropriate concentrations
followed by the incubation with Texas red-conjugated avidin as the
second Ab overnight at 4 °C. After extensive washing with PBS, the
glass slides were mounted using a glycerol solution containing
anti-fade reagent (Molecular Probes, Eugene, OR).
Confocal imaging was performed with a laser head (MRC-600, Bio-Rad).
Samples were excited at 488 and 568 nm, and fluorescein and Texas red
signals were detected through the K2 and K1 filter blocks,
respectively. Images were collected using the photon counting mode of
the COMOS program (Bio-Rad). In double-labeling experiments, bleed-through of the Texas red signal into the fluorescein channel was negligible.
Sucrose Gradient Centrifugation--
To obtain the raft membrane
fraction, T-cell blasts (7.5 × 107) were lysed with 1 ml of cold TNE buffer containing 10 mM Tris-HCl (pH 7.4),
1% TX-100, 0.15 M NaCl, 1 mM EDTA, 1 mM Na3VO4, 10 µg/ml aprotinin.
The lysates were centrifuged for 5 min at 1300 × g to
remove the nuclei and large cellular debris. For equilibrium centrifugation of the TX-100 lysates, the lysates was diluted with an
equal volume of 80% w/v sucrose in TNE buffer. Then 0.8 ml of
lysate-sucrose mixture was overlaid sequentially with 2 ml of 30%
sucrose and 1 ml of 4% sucrose prepared in TNE, and the mixture was
centrifuged at 200,000 × g for 16 h at 4 °C in an RPS 50-2 rotor (Hitachi, Tokyo, Japan). The gradient was
fractionated into 0.5-ml fractions from the top of the tube. The raft
and TX-100-soluble fractions were obtained in fractions 1-3 and 6-7, respectively.
TSST-1-induced Human Thymic CD4+ T-cell Blasts Are in
an Anergic State--
A summary of the surface phenotypes of
TSST-1-induced thymic and APB CD4+ T-cell blasts
used in this study is presented in Table
I (under "Immunologic
phenotypes"). Both type of cells show the phenotypes of
TSST-1-reactive T cells. The percentage of TCR V Reduced Tyrosine Phosphorylation of the TCR Activation of Lck in Thymic CD4+ T-cell Blasts Is
Impaired--
Because up-regulation of the tyrosine kinase activity of
Lck plays a critical role in T-cell activation through TCR (19), the
kinase activity of the Lck present in TSST-1-induced thymic and APB
CD4+ T-cell blasts was studied next. Lck immunoprecipitates
were prepared from restimulated T-cell blasts and were subjected to an
in vitro kinase assay in which autophosphorylation of Lck
was measured. In three independent experiments, a 2-14-fold increase
in autophosphorylation was observed in the APB CD4+ T-cell
blasts after restimulation with TSST-1 (Fig. 1B). In
contrast, no elevation in autophosphorylation was observed in the
thymic CD4+ T-cell blasts (Fig. 1B). These
results indicate that the Lck kinase activity of APB CD4+
T-cell blasts increases markedly upon restimulation with TSST-1, whereas no such increase is induced in thymic CD4+ T-cell blasts.
Tyrosine kinase activity of src family kinases is regulated by
phosphorylation and dephosphorylation of two tyrosine residues (Tyr-394
and Tyr-505 in Lck) (20-22). To determine why thymic CD4+
T-cell blasts restimulated with TSST-1 showed only weak Lck kinase activity, the extent of Lck tyrosine phosphorylation was compared in
thymic and APB CD4+ T-cell blasts. Lck was
immunoprecipitated from cell lysates of these T-cell blasts after
restimulation with TSST-1 on an APC monolayer, and the extent of
tyrosine phosphorylation was analyzed by Western blotting with an
anti-phosphotyrosine Ab. Lck was highly tyrosine-phosphorylated in both
thymic and APB CD4+ T-cell blasts when no restimulation was
carried out. After restimulation, the extent of tyrosine
phosphorylation was markedly reduced within 5 min in APB
CD4+ T-cell blasts, whereas almost no change was observed
in thymic CD4+ T-cell blasts (Fig. 1C, upper
panels). Lck expression of was similar in both types of
T-cell blasts (Fig. 1C, lower panels). Similar findings
were observed 20 min after restimulation (data not shown).
Up-regulation of the Lck kinase activity is associated with
phosphorylation of Tyr-394 (the autophosphorylation site) and dephosphorylation of the carboxyl-terminal tyrosine (Tyr-505) (20-22).
The results shown in Fig. 1, B and C, suggests
that TSST-1 restimulation induced dephosphorylation of phosphorylated
Tyr-505 in APB CD4+ T-cell blasts but not in thymic
CD4+ T-cell blasts. Phosphorylated Tyr-505 of Lck has been
shown to bind to its own Src homology-2 domain, and it is postulated
that this binding can cause the molecule to fold and thereby block kinase activity (23, 24). When phosphorylated Tyr-505 is
dephosphorylated, Lck "opens out" and recovers the capacity to
interact with a tyrosine-phosphorylated Lck carboxyl-terminal peptide
(LckP peptide) (15).
To determine whether the Tyr-505 residue of Lck is the site at which
dephosphorylation induced by restimulation with TSST-1 occurs in APB
CD4+ T-cell blasts, binding of dephosphorylated Lck to LckP
peptide was examined. Cell lysates prepared from T-cell blasts
restimulated with TSST-1 on an APC monolayer were incubated with
biotinylated LckP peptide. The precipitates were isolated with
avidin-conjugated agarose beads and analyzed by Western blotting using
an anti-Lck Ab. As shown in Fig. 1D (upper
panels), the extent of precipitated Lck was minimal in the absence
of TSST-1 restimulation, but restimulation in APB CD4+
T-cell blasts significantly increased the binding of Lck. In contrast,
the LckP peptide-bound Lck did not increase after restimulation and rather showed a slight decrease in thymic CD4+ T-cell
blasts. No difference in expression of the Lck was detected between the
two T-cell populations (Fig. 1D, lower panels). These results show that the dephosphorylated Tyr-505 of Lck in TSST-1-induced APB CD4+ T-cell blasts is in the "open" configuration,
whereas Lck of thymic blasts is in a "closed" form.
Lck and CD45 Are Physically Uncoulpled in Thymic CD4+
T-cell Blasts--
Because the Tyr-505 site of Lck is a potential
substrate for the protein tyrosine phosphatase CD45 (25), CD45 may play
a major role in the dephosphorylation of Lck in APB CD4+
T-cell blasts after restimlulation with TSST-1. To determine how Lck
dephosphorylation is suppressed in thymic T-cell blasts, the
subcellular localization of CD45 and Lck was determined by confocal
microscopy. T-cell blasts restimulated with TSST-1 on an APC monolayer
were separated from the monolayer and stained with FITC-conjugated
anti-CD45 Ab and biotinylated anti-Lck Ab followed by Texas
red-conjugated avidin. If Lck (red) and CD45 (green) were present in
close proximity, the appearance of yellow dots would be observed. As
shown in Fig. 2, Lck was expressed in
both the membrane and cytoplamic areas of unstimulated thymic and APB
CD4+ T-cell blasts, whereas CD45 was expressed mainly in
the membrane. No colocalization of Lck and CD45 was observed in these
cells. After restimulation with TSST-1, the intensity of the yellow
color increased in the membrane of the APB CD4+ T-cell
blasts, predominantly on one side of the blast, indicating that Lck and
CD45 had become colocalized in the plasma membrane region after
restimulation with TSST-1. In contrast, no colocalization of these two
molecules was induced by restimulation of thymic CD4+
T-cell blasts. Although the distributions of Lck and CD45 are comparable with that of APB CD4+ T-cell blasts, double
staining with anti-CD45 and anti-Lck clearly shows these molecules are
present in discrete areas on the cell membrane.
Loss of Lck Localization into the Membrane Raft in Thymic T-cell
Blasts--
Recent studies have shown that sphingolipid- and
cholesterol-rich plasma membrane microdomains, termed membrane rafts,
play an important role in TCR signal transduction. Raft aggregation promotes recruitment of signaling proteins such as Lck but excludes the
tyrosine phosphatase CD45 (26, 27). Our data from confocal microscopy
suggests that the physical interaction between CD45 and Lck in thymic
T-cell blasts is much less than in APB T-cell blasts.
To test whether membrane rafts play a role in the process of Lck
activation, we next determined the localization of Lck and CD45 inside
and outside of the raft fraction. Cell lysates prepared from T-cell
blasts restimulated with TSST-1 on an APC monolayer were lysed in a
buffer containing nonionic detergent, and the lysates were fractionated
by sucrose gradient centrifugation. As shown in Fig.
3A, Lck was mostly present
outside of rafts in unstimulated T-cell blasts. After restimulation
with TSST-1, a significant increase of Lck in rafts was observed in APB
CD4+ T-cell blasts. In contrast, no increase of Lck in
membrane rafts was induced by restimulation of thymic CD4+
T-cell blasts. This increase of Lck in the raft was more striking after
a 70-min restimulation of APB T-cell blasts. Even at that point, no
increase was observed in thymic T-cell blasts. Immunoprecipitation of
Lck from the same fractionated lysates confirmed these results (Fig.
3A). In contrast to this accumulation of Lck in the raft, CD45 was present outside of the raft fraction throughout the
restimulation in both types of T-cell blasts as previously reported
(Fig. 3C).
Human thymic CD1a The activity of CD45 is regulated by several mechanisms including
isoform specific regulation (28). In this study, the level of CD45RO
expression was similar in both thymic and APB CD4+ T-cell
blasts (Table I), ruling out the possibility that isoform differences
caused the impairment of CD45 function in thymic CD4+
T-cell blasts. Instead, our results show a remarkable difference in the
physical localization of CD45 and Lck after restimulation with TSST-1
in thymic CD4+ T-cell blasts. An analysis of the
subcellular localization of CD45 and Lck in thymic and APB
CD4+ T-cell blasts after restimulation with TSST-1 showed
that Lck and CD45 were physically associated in the membrane region of APB CD4+ T-cell blasts but not thymic CD4+
T-cell blasts (Fig. 2).
Membrane-associated molecules are known to localize to a specifically
designated area composed of discrete lipid microdomains (26, 27). Some
but not all of the Lck localizes in sphingolipid-cholesterol rafts,
whereas no association of CD45 with these rafts has been observed (27,
29). Several studies have shown that a synaptic structure is formed by
dynamic relocalization of various molecules at the site of the
interaction between T cells and APC (30-32). The central zone of the
synaptic structure formed between T cells and APC is rich in TCR, CD4,
and CD28, whereas CD45 remains on the outside (31, 32). This discrete
localization of CD45 and Lck indicates that there is a dynamic process
involved in the interaction between these two molecules. A potential
model is that repartitioning of Lck from the outside to the inside of
sphingolipid-cholesterol rafts is regulated by interaction with CD45.
In this view, Lck, which resided outside of the raft, gets
dephosphorylated after stimulation and localizes in the raft. With
thymic T-cell blasts, the interaction between CD45 and Lck is blocked,
and thus this translocation into the raft is blocked (Fig.
4, MODEL 1). Alternatively, although CD45 is not within the raft, it only interacts with Lck within
the raft. In thymic T-cell blasts, Lck localization into rafts is
blocked, and as a result, its interaction with CD45 is impaired (Fig.
4, MODEL 2). We are currently testing these hypotheses.
CD4+ T cells in the final stage of
thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome toxin-1 (TSST-1). Thymic CD4+ T-cell
blasts, established by stimulating human thymic
CD1a
CD4+ T cells with TSST-1 in
vitro, produce a low level of interleukin-2 after
restimulation with TSST-1, whereas TSST-1-induced adult peripheral
blood (APB) CD4+ T-cell blasts produce high levels of
interleukin-2. The extent of tyrosine phosphorylation of the T-cell
receptor
chain induced after restimulation with TSST-1 was
2-4-fold higher in APB CD4+ T-cell blasts than in thymic
CD4+ T-cell blasts. The tyrosine kinase activity of Lck was
low in both thymic and APB CD4+ T-cell blasts before
restimulation with TSST-1. After restimulation, the Lck kinase activity
increased in APB CD4+ T-cell blasts but not in thymic
CD4+ T-cell blasts. Surprisingly, Lck was highly
tyrosine-phosphorylated in both thymic and APB CD4+ T-cell
blasts before restimulation with TSST-1. After restimulation, it was
markedly dephosphorylated in APB CD4+ T-cell blasts but not
in thymic CD4+ T-cell blasts. Lck from APB CD4+
T-cell blasts bound the peptide containing the phosphotyrosine at the
negative regulatory site of Lck-505 indicating that the site of
dephosphorylation in TSST-1-activated T-cell blasts is Tyr-505.
Confocal microscopy demonstrated that colocalization of Lck and CD45
was induced after restimulation with TSST-1 in APB CD4+
T-cell blasts but not in thymic CD4+ T-cell blasts.
Further, remarkable accumulation of Lck in the membrane raft was
observed in restimulated APB CD4+ T-cell blasts but not in
thymic CD4+ T-cell blasts. These data indicate that
interaction between Lck and CD45 is suppressed physically in thymic
CD4+ T-cell blasts and plays a critical role in sustaining
an anergic state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CD4+ T cells in the
final stage of maturation (1) and cord blood CD4+ T cells
are susceptible to anergy induction by in vitro stimulation with a superantigen, toxic shock syndrome toxin-1
(TSST-1).1 Thymic and cord
blood CD4+ T-cell blasts, prepared by stimulating human
thymic CD1a
CD4+ and cord blood
CD4+ T cells with TSST-1, exhibited low IL-2, IL-4, and
interferon-
production and reduced proliferation after restimulation
with TSST-1, whereas adult peripheral blood (APB) CD4+
T-cell blasts prepared in the same way exhibited high responses (2, 3).
These characteristics indicate that cord blood and thymic
CD4+ T cells are still functionally immature and that a
post-thymic maturation process occurs in human peripheral blood T
cells. To understand why these cells respond differently, it is
essential to analyze the signal transduction pathway via T-cell
receptor stimulation in anergic TSST-1-induced thymic CD4+
T-cell blasts.
1 and (ii) activation of the
Ras-MAPK (mitogen-activated protein kinase) pathway. Both pathways
require the activation of Src family kinases such as Lck and Fyn.
Although mature T cells are highly resistant to anergy induction, they
are rendered anergic when stimulated through TCR in the absence of
costimulatory signals such as the CD28 molecule (4). Anergic T cells
display various alterations in the two above mentioned pathways,
including failure of ZAP-70, Ras, ERK (extracellular
signal-related kinase), JNK (c-Jun NH2-terminal kinase), or
AP-1 activation after restimulation with antigens (5-9). They also
display constitutively elevated concentrations of intracellular free
calcium and inositolphosphate, as well as increased tyrosine
phosphorylation of phospholipase C-
1, Fyn, and Cbl (10-12). It is
not yet clear whether these defects are involved in the anergic state
induced by TSST-1 in human thymic CD4+ T cells.
CD4+ T cells are induced into an anergic
state after stimulation with TSST-1 by comparing signal transduction in
anergic TSST-1-induced thymic CD4+ T-cell blasts with that
in highly responsive TSST-1-induced APB CD4+ T-cell blasts.
The results strongly suggest that the absence of an interaction between
Lck and CD45 (which possesses phosphatase activity) is responsible for
maintaining the anergic state of human thymic
CD1a
CD4+ T cells induced by TSST-1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2) and FITC-conjugated UCHL.1
(anti-CD45RO, Immunotech, Marseille, France); FITC-conjugated anti-CD28 (Immunotech, Marseille, France); FITC-conjugated avidin (Vector Laboratories, Burlingame, CA); 4G10 (anti-phosphotyrosine, Upstate Biotechnology Inc., Lake Placid, NY); 6B10.2 (anti-TCR
and
anti-CD45 (M-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA); and
FITC-conjugated anti-CD45 and Texas red-conjugated avidin (PharMingen,
San Diego, CA). The anti-Lck Ab was kindly provided by Dr. Y. Koga
(Tokai University, Japan).
CD4+ T Cells--
The procedures used
for preparing human lymphoid cells have been described previously (2).
Briefly, APB mononuclear cells were isolated from the peripheral blood
of healthy adult donors by Ficoll-Conray density gradient
centrifugation. Whole APB T cells were obtained by the sheep red blood
cell rosette method. After obtaining written informed consent,
single-cell suspensions of thymocytes were obtained from thymus
fragments dissected from donors, ranging in age from 3 to 24 years,
during corrective cardiac surgery. To obtain APB CD4+ T
cells, whole APB T cells were treated with Abs Nu Ts/c and I2C3. To
obtain thymic CD1a
CD4+ T cells, thymocytes
were first treated with peanut agglutinin, and then non-aggregated T
cells were treated with a combination of Abs Nu Ts/c, I2C3, and OKT6.
After washing, these Ab-treated cells were mixed with anti-mouse IgG
coupled to magnetic beads (Dynabeads, Dynal, Oslo, Norway), and the
mixtures were kept on ice for 30 min. Unbound cells were separated with
a magnet and suspended in RPMI 1640 supplemented with 100 µg/ml
streptomycin, 100 units/ml penicillin, 10% fetal calf serum, and
5 × 10
5 M
2-mercaptoethanol. The preparations of APB CD4+ and thymic
CD1a
CD4+ T cells contained <5%
CD8+ T cells, 5% CD8+ T cells, and 1%
CD1a+ T cells, respectively.
CD4+ T cells were stimulated with 10 ng/ml TSST-1 (Toxin Technology, Inc., Sarasota, FL) on an APC monolayer
for 3 days. Recovered cells were subjected to Percoll (density = 1.068) centrifugation. The large lymphoblasts obtained at the interface
of the culture medium and Percoll were expanded by incubation with 100 units/ml recombinant IL-2 (Takeda Chemical Industries, Kyoto,
Japan) for 4 days. Recovered cells were subjected to Percoll
centrifugation. T-cell blasts were obtained at the interface between
densities 1.050 and 1.068.
2, CD3
versus CD28, CD3 versus CD45RO, and CD3
versus HLA-DR in the T-cell preparations, T cells were
stained with several combinations of the appropriate phycoerythrin-,
RD1-, and FITC-conjugated Abs and examined by two-color flow cytometric
analysis using an EPICS CS flow cytometer (Coulter Electronics,
Hialeah, FL) as described previously (3). All procedures for cell
staining were conducted on ice.
-32P]ATP
(PerkinElmer Life Sciences) per sample. The reaction was allowed
to proceed for 10 min at 30 °C; the samples were then centrifuged,
and the supernatants were separated by reducing 8% SDS-polyacrylamide
gel electrophoresis. After drying, the radioactivity bound to the gels
was analyzed with a Molecular Imager System FX (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2+ T
cells, which are the major TSST-1-reactive human T-cell fraction (16),
was around 80% in both the thymic and APB T cell preparations. The
intensity of TCR expression was equivalent in both preparations (data
not shown). Most of the V
2-negative T cells among the thymic and APB
T-cell blasts were likely to be other TSST-1-reactive fractions such as
V
4+ T cells (17). The percentage of CD28+ T
cells increased from around 70% before to around 90% after blast
formation in both the thymic and APB T cell preparations, and the
percentage of CD45RO+ T cells increased to a similar extent
among both types of T-cell blasts. It should be noted that the
percentage of HLA DR+ T cells was 3-fold lower among the
thymic CD4+ T-cell blasts than among the APB
CD4+ T-cell blasts. The APB and thymic CD4+
T-cell blasts were then restimulated with TSST-1 (10 ng/ml) on an APC
monolayer and examined for IL-2 production (Table I, under "IL-2
production"). IL-2 production was much lower in the thymic CD4+ T-cell blasts than in the APB CD4+ T-cell
blasts; the amount of IL-2 produced by the former was at most 10% of
that produced by the latter. However, both the thymic and APB
CD4+ T-cell blasts exhibited substantial IL-2 production
upon stimulation with a combination of PMA and ionomycin (data not
shown). These results indicate that thymic CD4+ T-cell
blasts are in an anergic state following exposure to TSST-1.
Immunologic phenotypes of thymic and APB CD4+ T cells before
and after blast formation and TSST-1-induced IL-2 production of thymic
and APB CD4+ T-cell blasts
Chain in Thymic
CD4+ T-cell Blasts--
When T cells are activated by
antigens, tyrosine phosphorylation of the TCR
chain is induced (18).
The extent of tyrosine phosphorylation of the TCR
chain was
therefore compared between TSST-1-induced thymic and APB
CD4+ T-cell blasts. T-cell blasts were restimulated with
TSST-1 (2 µg/ml) on an APC monolayer and cell lysates were prepared.
The
chain was immunoprecipitated and the extent of tyrosine
phosphorylation was analyzed by Western blotting with an
anti-phosphotyrosine Ab. As shown in the upper part of Fig.
1A, tyrosine phosphorylation of the
chain was minimal in the absence of TSST-1 restimulation but
showed significant increase after restimulation in both the thymic and
APB T-cell blasts. The extent of phosphorylation was 2- to 4-fold
higher in APB CD4+ T-cell blasts than in thymic
CD4+ T-cell blasts. No difference in expression of the
chain was detected between the two T-cell populations (lower
part of Fig. 1A).
View larger version (28K):
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Fig. 1.
Lck in thymic CD4+ T-cell blasts
is not functionally linked with TCR stimulation and stays
constitutively tyrosine-phosphorylated at the negative regulator site
Tyr-505. Thymic and APB CD4+ T-cell blasts were
prepared as described in Table I and then restimulated with 2 µg/ml
TSST-1 or unstimulated (Med) on an APC monolayer for the
indicated times. A, the CD4+ T-cell blasts
(1.2 × 107 cells/sample) were then lysed and
immunoprecipitated (IP) with anti-TCR Ab. The
immunoprecipitates were blotted with anti-phosphotyrosine Ab (top
panels) or anti-TCR
Ab (bottom panels).
B, Lck was immunoprecipitated from the CD4+
T-cell blasts (2 × 107 cells/sample). The
incorporated 32P count was compared between stimulated
(TSST-1) and unstimulated (Med) samples, and the
ratio of kinase activity presented is that from three independent
autophosphorylation experiments. C, the CD4+
T-cell blasts (1.5 × 107 cells/sample) were lysed and
immunoprecipitated with anti-Lck Ab. The immunoprecipitates were
blotted with anti-phosphotyrosine Ab (upper panels) or
anti-Lck Ab (lower panels). D, the
CD4+ T-cell blasts (1.2 × 108
cells/sample) were lysed and mixed with the biotinylated LckP peptide.
The peptide complex was precipitated with avidin-agarose beads. The
precipitates and the total cell lysates were subjected to Western blot
analysis with anti-Lck Ab.
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Fig. 2.
Colocalization of CD45 and Lck is
induced by TSST-1 stimulation on APB CD4+ T-cell blasts but
not in thymic CD4+ T-cell blasts. Thymic and APB
CD4+ T-cell blasts were prepared as described in Table I
and then restimulated with 2 µg/ml TSST-1 or unstimulated
(Medium) on an APC monolayer for 10 min. The T-cell blasts
were fixed, permeabilized, and double-stained with anti-Lck
Ab (red) and anti-CD45 Ab (green). The
upper two rows show the fluorescence from each antibody, and
the third row shows the combined fluorescence. Bright
field pictures are shown in the bottom row.
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Fig. 3.
Redistribution of Lck between inside and
outside raft fraction is induced by TSST-1 stimulation on APB
CD4+ T-cell blasts but not in thymic CD4+
T-cell blasts. Thymic and APB CD4+ T-cell blasts were
prepared as described in Table I and then restimulated with 2 µg/ml
TSST-1 or unstimulated (Medium) on an APC monolayer
for indicated time. The CD4+ T-cell blasts (3 × 107 cells/sample) were then lysed in a buffer containing
nonionic detergent, and the raft was fractionated by sucrose gradient
centrifugation. The gradient was fractionated into 0.5-ml fractions
from the top of the tube. A, each fraction was blotted with
anti-Lck Ab. B, each fraction was immunoprecipitated with
anti-Lck Ab. The precipitates were blotted with anti-Lck Ab.
C, each fraction was blotted with anti-CD45 Ab.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CD4+ T cells are highly
susceptible to anergy induction by superantigens, whereas APB
CD4+ T cells are resistant, as shown in a previous study
(2) and in Table I. T cells are rendered anergic when they are
stimulated through TCR in the absence of costimulatory signals such as
the CD28 molecule (4). However, the absence of a costimulatory signal
is not responsible for the high susceptibility of thymic CD1a
CD4+ T cells to TSST-1-induced anergy,
because the percentage of CD28+ T cells among unstimulated
thymic and APB CD4+ T cells was similar. We did not find
any differences in surface phenotypes to explain the differences in
susceptibility to anergy induction between unstimulated thymic and APB
CD4+ T cells (Table I). In the present study, we examined
signal transduction in thymic and APB CD4+ T-cell blasts
before and after restimulation with TSST-1. The results strongly
suggest the presence of a novel regulatory mechanism that controls an
interaction between Lck and CD45.
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Fig. 4.
Models for the regulatory mechanism of the
Lck activity: interaction with CD45 and localization to the raft.
Model 1 predicts that Lck interacts with CD45 prior to accumulation in
the raft, whereas model 2 predicts that Lck interacts with CD45 after
it localizes within the raft. The data presented here shows that
interaction between CD45 and Lck is suppressed. This inhibition could
take place outside (MODEL 1) or inside (MODEL 2)
of the raft. In both cases, dephosphorylated Lck (Lck*)
accumulates within the raft.
Taken together, these results suggest the presence of a novel
regulatory mechanism that controls localization of CD45 and Lck in
activated T cells. Most importantly, this regulation may play a
critical role in the maintenance of the anergic state of human thymic
CD1aCD4+ T cells induced by superantigens.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Takamitsu Fujimaki (University of Teikyo, Japan) for helpful discussion during the experiments, Dr. Pandlakis Koni (Medical College of Georgia) for critical reading of the manuscript, and to Dr. Yasuhiro Koga (Tokai University, Kanagawa, Japan) for providing the anti-Lck antibody.
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FOOTNOTES |
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* This work was supported in part by Grants-in-aid 08670320 and 11670276 from the Ministry of Education, Science, Culture and Sports of Japan and by a grant from the Itoe Okamoto Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel.: 81-3-3353-8111; Fax: 81-3-5269-7411; E-mail: wakae@research.twmu.ac.jp.
Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M101072200
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ABBREVIATIONS |
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The abbreviations used are: TSST-1, toxic shock syndrome toxin-1; IL, interleukin; APB, adult peripheral blood; TCR, T-cell receptor; Ab, antibody; FITC, fluorescein isothiocyanate; APC, antigen-presenting cell; PBS, phosphate-buffered saline.
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