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From the
Superantigens are able to stimulate T lymphocyte populations
expressing T cell antigen receptors (TCR) belonging to particular
V
Superantigens have the capacity to strongly activate oligoclonal
populations of T lymphocytes expressing antigen receptors homologous
within their
T cell antigen
receptor (TCR)
Although both
conventional T cell antigens and superantigens drive T cells to
proliferate and secrete lymphokines, several characteristics
distinguish superantigen from conventional antigen T cell recognition.
(i) Superantigen stimulation, although usually requiring the presence
of major histocompatibility complex class II
(MHC
Among the known superantigens,
S. aureus enterotoxins are the best characterized structurally
and functionally. These toxins are structurally related
(11) .
Mutational analysis revealed one TCR binding site and one or two MHC
binding sites located on separate regions of the toxin
(1) .
S. aureus enterotoxins generally bind to MHC
Although
MHC
The cellular mechanisms modulating T cell receptor
levels upon interaction with superantigens have not been defined yet.
The aim of our study was to elucidate how the interaction with
bacterial toxin superantigens may regulate the molecular dynamics of
surface T cell receptors. In order to avoid interference from other
molecular interactions provided by superantigen-presenting cells, we
made use of cells from the human tumor T cell line, Jurkat, which do
not express MHC
The samples were examined under a confocal
microscope (Leica) attached to a diaplan microscope (Leitz) equipped
with a double laser, argon-krypton. Serial optical sections were
recorded at 0.5 µm intervals with a 63
We first estimated the half-life of surface
TCR
To analyze whether SEB could augment T cell receptor
endocytosis, internalization of TCR
The mechanisms involved in the modulation of T cell receptor
expression upon antigen or superantigen recognition are not well
defined. Different cellular processes that regulate steady state levels
of surface molecules may be affected such as synthesis and secretion of
new polypeptides, internalization, recycling back to the plasma
membrane, and degradation.
Here we show that SEB superantigen
induced a rapid down-modulation of TCR
Although an increase in receptor internalization may account for the
rapid TCR down-regulation observed, the recycling and/or degradation
pathways might also be affected. Some of our observations suggest that
accumulated receptors ended up being degraded. Thus, after 6 h of
treatment with SEB, accumulation of TCR
Our experiments suggest
that inhibition of the secretory pathway is not responsible for the
rapid SEB-induced TCR down-regulation, since the half-life of surface
TCR
In line with previous
results
(19) , we observed a biological effect of SEB on
V
We further investigated whether other
enterotoxin superantigens could down-regulate their specific TCR in a
similar fashion to SEB. To this end, Jurkat cells expressing V
Down-regulation of signaling
receptors upon ligand interaction may alter the biology of a particular
receptor system by removing the receptor from the cell surface. This
may diminish further responsiveness of the cells simply by the loss of
available receptors or by uncoupling them from the signal transduction
machinery. Receptor internalization through the receptor-mediated
endocytosis pathway is a very efficient cellular mechanism to rapidly
reduce the number of surface receptors
(31) .
Cellular
unresponsiveness and concomitant down-regulation of T cell receptor
and/or co-receptors has been observed in some experimental models of
tolerance in vitro(19, 26, 35) or
in vivo(20, 36) , suggesting that receptor
down-modulation may be of functional significance for cellular
inactivation. However, anergy is most likely the result of a more
complex molecular regulation and not solely the result of modification
in the expression of membrane receptors. Thus, in other experimental
systems, T cells rendered tolerant to antigen or superantigen, in
vitro(21) or in vivo(22) , displayed
normal levels of TCR and CD4. To explain the different experimental
observations, Arnold et al.(37) proposed a model where
multiple levels of tolerance may exist. This may involve
down-regulation of TCR and/or CD4 or CD8 coreceptors as well as other
intracellular mechanisms and would condition the capacity of T cells to
be reactivated. Tolerant T cells would still be susceptible to further
tolerogenic signals, driving them to a deeper state of
tolerance
(37) .
T cell receptor and/or CD4 or CD8 coreceptor
down-regulation has been observed upon physiological stimulation of T
cell clones with appropriately presented antigen. In this case,
down-modulation of T cell receptors may contribute to arrest the
activation process and to create the transient period of
unresponsiveness that follows cell
stimulation
(38, 39, 40, 41, 42, 43) .
The experiments that we report here shed further light on the
mechanism of T cell receptor down-regulation induced by bacterial toxin
superantigens. The understanding of the effect of these bacterial
products at the cellular level may help to clarify the strategies that
these organisms use to elude the immune response of the host. Moreover,
it may provide us with the means to modulate the immune system in novel
ways.
We thank Raymond Hellio for help and advice with
confocal microscopy and David Ojcius for critical reading of the
manuscript.
Volume 270,
Number 21,
Issue of May 26, pp. 12839-12845, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
families. Moreover, the presence of these superantigens may
induce long term unresponsiveness (anergy) of these sensitive cells.
Some bacterial toxins are potent superantigens. We have analyzed in
vitro the capacity of some Staphylococcus aureus enterotoxin superantigens to modulate T cell antigen receptor
expression and the cellular mechanisms involved. Staphylococcus enterotoxin B (SEB) induced rapid down-regulation of surface T
cell antigen receptors in V3-expressing T lymphocytes, as assessed
by flow cytometry. This phenomenon was a consequence of the direct
interaction between the toxin and the TCR since it was observed in the
absence of cells expressing major histocompatibility complex class II
molecules. The cellular mechanism involved in SEB-induced
down-regulation of TCR was further investigated. Immunofluorescence and
confocal microscopy experiments showed that toxin B induced
intracellular accumulation of TCRCD3 in endocytic vesicles.
Moreover, SEB induced an increase in T cell receptor endocytosis as
measured using radiolabeled Fab fragments of an anti-CD3 monoclonal
antibody. Taken together, our observations indicate that
Staphylococcus enterotoxin B superantigen induced changes in
the dynamics of surface T cell receptors, which resulted in the fast
reduction of membrane receptor numbers.
chain variable regions (V families). Certain
strains of Staphylococcus or Streptococcus produce
exotoxins that are potent superantigens. Similar to endogenous murine
retroviral superantigens, the presence of bacterial superantigens may
shape the T cell repertoire by deletion or inactivation (anergy) of
reactive clones. The alteration in immune system homeostasis occurring
in infections caused by superantigen-producing bacteria may be the
basis for some of the clinical manifestations observed and could play a
role in disease pathogenesis
(1, 2) .
()
expressed on the majority of
peripheral T lymphocytes is a complex composed of the hypervariable
- heterodimer noncovalently linked to the monomorphic
CD3,
,
and
-
or
-
chains
(TCR
CD3). The
and chains contain immunoglobulin-like
variable regions responsible for antigen and superantigen
interactions
(3) , whereas the CD3 and the chains play a
role in signal transduction
(4) . Surface expression of the
TCR
CD3 complex requires association of all of their polypeptides.
Individual chains or partial complexes are retained and degraded before
reaching the plasma membrane
(5, 6) .
)-presenting cells, is not restricted to one
MHC
allele. (ii) Processing of superantigens into
antigenic peptides is not necessary. (iii) Superantigens are specific
for TCR-V
domains, with no particular TCR-V domains being
required. (iv) Residues involved in superantigen interactions with
MHC or TCR are different from those involved in
conventional antigen
recognition
(1, 7, 8, 9, 10) . It
has been proposed therefore that superantigens stimulate T cells by
bridging MHC
molecules on antigen presenting cells with
TCR-V
domains on T lymphocytes.
molecules with a much better affinity than to the TCR. Therefore,
it was suggested that toxin superantigens might bind to MHC
molecules before being able to interact with the
TCR
(12, 13, 14, 15, 16, 17) .
Recently, it has been reported that the formation of the ternary
complex, MHC-toxin-TCR, may stabilize the interactions of each of the
binary complexes
(18) .
-presenting cells are usually required
for superantigen recognition by T cells, some toxins may be able to
interact directly with the TCR in the absence of MHC
molecules. Thus, Hewitt et al. (19) reported that the
Staphylococcus enterotoxin B (SEB) could stimulate
V
3 human T cells in the absence of MHC
molecules, driving them to a state of anergy
(19) .
Although the molecular mechanism responsible for anergy remained to be
elucidated, it was suggested that T cell receptor down-regulation might
contribute to reduce the sensitivity to new stimulations. Consistent
with this proposal, treatment of cells with SEB reduced TCR levels in a
dose-dependent manner. Moreover, the capacity of the cells to increase
cytosolic free Ca
concentration in response to a
subsequent exposure to optimal concentrations of SEB was proportional
to TCR levels, suggesting that down-regulation might be of functional
significance
(19) . Studies in vivo also support the
idea that the level of T cell receptor may be of importance in the
mechanism of both T cell activation and anergy
(20) . However,
other intracellular mechanisms also seem to be involved, since in some
experimental models, cell anergy can be induced in vitro (21)
or in vivo(22) , without substantial TCR
down-regulation.
molecules. These cells had been
transfected with genes coding for the
and chains of the
influenza hemagglutinin-specific T cell receptor HA1.7
(V1.2-V3.1) and are able respond to peptide antigen or to SEB
by secreting lymphokines. Moreover, incubation of these cells with SEB
in the absence of MHC accessory cells had
been shown to induce anergy
(19) . Here we show that
Staphylococcus enterotoxin B superantigen induces a rapid,
specific, and dose-dependent down-modulation of surface T cell
receptors in these cells. Using confocal microscopy and biochemical
approaches, we demonstrated that SEB provokes changes in the
intracellular traffic of TCR. Cells treated with SEB show an increased
receptor internalization rate and accumulation of TCR in endocytic
vesicles.
Materials
Reagents
Staphylococcal enterotoxins SEA, SEB,
SED, SEE, and TSST1 were obtained from Toxin Technology, Inc. (Madison,
WI) or Sigma. Human transferrin (Sigma) was loaded with iron and
coupled to lissamine rhodamine (Eastman Kodak Co.) as described
previously
(23) . Monoclonal antibodies anti-CD2 (TS2.18,
IgG), anti-CD3 (OKT3, IgG), anti-CD4 (OKT4,
IgG
), and anti-CD45 (GAP8.3, IgG
) were
obtained from the American Type Culture Collection (Rockville, MD).
Anti-CD3 (UCHT1, IgG
) was a kind gift of Dr. P. Beverley
(Imperial Cancer Research Fund, London), and anti-CD5 (B36.1,
IgG) was a kind gift of Dr B. Perussia (Jefferson Cancer
Inst., Philadelphia, PA). The anti-V
3 mAb (JOVI-3,
IgG) has been described previously
(24) .
Fluorescein-conjugated goat anti-mouse Ig (Becton-Dickinson, San Jose,
CA) was used for flow cytometry and fluorescein-conjugated sheep
anti-mouse Ig (Amersham Corp.) was used for confocal analysis.
Cell lines
J77Cl20 (TCR V1.2 V8,
CD2, CD3
, CD4
,
CD5
, CD45
,
MHC
, MHC
)
is a subclone of the cell line Jurkat. CH7Cl7 (TCR V
1.2 V3.1,
CD2, CD3
, CD4
,
CD5
, CD45
,
MHC
, MHC
)
is a Jurkat transfectant expressing the
and chains of a
hemagglutinin-specific TCR HA1.7, described previously
(19) . All
cells were grown in RPMI 1640 medium supplemented with 10%
decomplemented fetal calf serum (FCS), 10 mM HEPES, pH 7.2, 2
mML-glutamine, 1 µg/ml penicillin, and 1
µg/ml streptomycin. CH7Cl7 cells were cultured in the presence of
400 µg/ml hygromycin and 4 µg/ml puromycin.
Methods
Preparation and
The OKT3 mouse monoclonal antibody
was purified from ascites by Protein A affinity chromatography
(Bio-Rad). Monovalent Fab fragments were prepared as follows. The OKT3
antibody was digested for 18 h by immobilized papain (Pierce Chemical
Co.) at 37 °C following manufacturer's instructions and
further purified through a protein A-Sepharose chromatography column.
The purity of monovalent Fab fragments was confirmed by
SDS-polyacrylamide gel electrophoresis. Fab fragments were radiolabeled
with I-Labeling of
Anti-CD3 Fab Fragments
I (Amersham, Corp.) by the chloramine T method to a
specific activity of 35 µCi/µg. For labeling, three successive
additions of chloramine T were performed within 5 min, at room
temperature, to a final concentration of 25 µg/ml. The reaction was
stopped after another 5 min, and labeled ligand was separated from free
I by passage through an Exocellulose GF-5 column (Pierce
Chemical Co.).
Immunofluorescence and Flow Cytometry
Cells at
10
cells/ml were incubated in growth medium containing
toxins at the concentrations indicated for various times at 37 °C.
Cells were then washed in cold phosphate-buffered saline (PBS), 10
mM phosphate buffer, pH 7.3, 150 mM NaCl, containing
1% FCS (1% FCS-PBS), and cell surface expression of TCR or accessory
molecules was assessed by indirect immunofluorescence. Cells were
stained for 45 min at 4 °C using saturating concentrations of the
appropriate murine monoclonal antibodies, washed twice in cold 1%
FCS-PBS, and incubated for an additional 45 min with
fluorescein-conjugated goat anti-mouse Ig (1/20, Becton Dickinson, San
Jose, CA). Cells were washed twice and resuspended in 1% FCS-PBS. Five
thousand viable cells, identified by their ability to exclude propidium
iodide, were analyzed immediately after labeling on a FACScan flow
cytometer (Becton Dickinson, San Jose, CA). The mean fluorescence
intensity was obtained from the recorded data, and the results were
expressed as the percentage of fluorescence intensity of cells
incubated without toxins.
Immunofluorescence and Confocal
Microscopy
Exponentially growing cells were preincubated at
10 cells/ml in serum-free RPMI 1640 medium supplemented
with 20 mM HEPES buffer pH 7.2 and 1 mg/ml bovine serum
albumin (BSA) for 30 min at 37 °C in order to deplete transferrin
from cells. SEB at 10 µg/ml and/or 600 nM
rhodamine-transferrin were then added, and cells were incubated for 3 h
at 37 °C. Cells were then washed twice in cold PBS containing 1
mg/ml BSA (BSA-PBS) and fixed for 30 min at 4 °C in PBS containing
3.7% paraformaldehyde and 30 mM sucrose. After quenching
formaldehyde for 10 min in 50 mM NH
Cl-PBS, the
cells were washed once in BSA-PBS and permeabilized for 15 min at 37
°C in BSA-PBS containing 0.05% saponin. Subsequent steps were
performed at room temperature in permeabilizing buffer. Cells were
incubated with anti-CD3 antibody (UCHT1) at 1:500 ascites dilution
for 45 min. After two washes, the presence of antibodies was revealed
by incubating the cells for 45 min with fluorescein
isothiocyanate-coupled sheep anti-murine Ig antibody (1/50, Amersham
Corp.). After three washes in permeabilizing buffer and one wash in
PBS, the cells were mounted on microscope slides in 25 mg/ml Dabco
(1,4-diazalbicyclo[2.2.2]octane, Sigma), 100 mg/ml Mowiol
(Calbiochem, La Jolla, CA), 25% (v/v) glycerol, 100 mM
Tris-HCl, pH 8.5.
lens. Photographs
were taken on Kodak Ektachrome 100 ASA. No immunofluorescence staining
was ever observed when second antibodies were used without the first
antibody or with an irrelevant first antibody.
Endocytosis of Radiolabeled Anti-CD3
Cells, 3 Fab
Fragments
10
/point, were
preincubated at 37 °C for 30 min in 100 µl of growth medium.
The I-labeled Fab fragment of anti-CD3 OKT3 mAb was then
added to a final concentration of 40 nM, either alone or
together with SEB (final concentration, 10 µg/ml). At the end of
the incubation times, cells were chilled in 2 ml of RPMI medium
containing 20 mM HEPES buffer pH 7.2 and 1 mg/ml BSA (wash
medium). Cells were washed twice at 4 °C to remove unbound ligand.
They were then subjected to two successive acid pH treatments. For each
treatment, cell pellets were resuspended in 300 µl of acid medium
(RPMI 1640 medium, 25 mM sodium acetate, brought to pH 2.8
with HCl) for 2.5 min and then neutralized with 0.8 ml of RPMI 1640
medium brought to pH 9 with NaOH. This treatment removed
surface-associated
I-Fab fragments with an efficiency of
85-90%. Results are expressed as internalized
I-Fab
anti-CD3. Data were corrected taking into account the efficiency of the
acid wash, and the percentage of internalized receptors was calculated
as described previously
(25) .
SEB Superantigen Induces a Rapid Down-regulation of
TCR
To study in detail the effect of
SEB superantigen on TCRCD3 Surface Expression
CD3 surface expression, we analyzed the
dose-response and the kinetics of down-regulation. To this end,
V
3-expressing Jurkat cells were treated with various doses of
toxin for 18 h, and TCRCD3 surface expression was measured by
immunofluorescence and flow cytometry using the anti-CD3
mAb OKT3.
As shown in Fig. 1A, SEB induced a dose-dependent
down-regulation of the surface TCR
CD3 complex. The S. aureus enterotoxins A and D, also able to stimulate V
3-expressing T
lymphocytes
(26) , displayed only minor or undetectable effects.
Other S. aureus enterotoxin (SE) superantigens, such as SEE or
the toxic shock syndrome toxin 1 (TSST1), which are specific for other
V families
(11) , did not have any effect on TCRCD3
expression in V
3-expressing cells. The kinetics of down-regulation
are shown in Fig. 1B. The addition of SEB induced a
rapid decrease in TCRCD3 surface expression. Receptor numbers
decayed rapidly during the initial 30 min, falling to
50% of the
initial levels (Fig. 1B) and then falling more slowly to
30-40%, and they remained stable for the time that the toxin was
present (up to 72 h tested) (data not shown). Only a transient minor
effect was obtained with SED (Fig. 1B), whereas other
enterotoxins tested, such as SEA, SEE, or TSST1, did not induce this
effect.
Figure 1:
Staphylococcus enterotoxin B
induces the rapid down-regulation of TCRCD3 independently of MHC
class II molecules. Jurkat transfectants expressing V
3 TCR (CH7C17
cells) were incubated for 18 h with various concentrations of
Staphylococcus enterotoxin superantigens (panelA) or with various toxins at 10 µg/ml for different
times (panelB). Surface TCRCD3 levels were
analyzed by immunofluorescence and flow cytometry using saturating
concentrations of the anti-CD3 mAb OKT3. Mean fluorescence intensity
was measured at each point. Results are given as percentage of control
cells (incubated in medium alone). Each point represents the average
± S.D. (n = 5).
, SEB;
, SED;
, SEE; , SEA;
, TSST1.
The T cell antigen receptor complex is functionally and
physically linked to other surface molecules such as CD2, CD4, CD5, and
CD45. These molecules cooperate with the TCR during antigen recognition
and participate in signal transduction
(27, 28) . In
order to analyze whether T cell receptor down-regulation induced by
toxin superantigens could affect the expression of any of these
``accessory'' molecules, we treated V3-expressing Jurkat
cells with appropriate doses of SEB, and we followed TCRCD3
surface expression together with that of CD2, CD5, or CD45 by flow
cytometry. The CD4 molecule was not tested since it is very weakly
expressed in these cells. As shown in Fig. 2, only TCR
CD3
expression was affected, whereas surface levels of CD2, CD5, or CD45
remained unchanged. No significant changes in CD2, CD5, or CD45
expression were observed upon 16-h treatment at various concentrations
of SEB (data not shown). As expected, changes in TCR
CD3
expression followed either by an anti-CD3
(OKT3) or an
anti-V
3 (JOVI-3) mAb led to identical results.
Figure 2:
Staphylococcus enterotoxin B
induces down-regulation of TCRCD3 but did not change the
expression of accessory molecules. Jurkat transfectants expressing
V
3 TCR (CH7C17 cells) were incubated for various times with SEB at
10 µg/ml. Cells were then washed and stained for immunofluorescence
and flow cytometry using mAbs OKT3 anti-CD3 (), JOVI-3
anti-TCRV
3 (), TS2.18 anti-CD2 (), B36.1 anti-CD5
(
), or GAP8.3 anti-CD45 (
). Mean fluorescence intensity
was measured at each point. Results are given as percentage of control
cells (incubated in medium alone). Each point represents the average
± S.D. (n = 3) for CD3 and TCR, and a
representative experiment is shown for the other
molecules.
We further
investigated whether other toxins able to activate cells expressing T
cell receptors belonging to other V families could also induce
TCRCD3 down-regulation. Thus, Jurkat cells expressing V
8 TCR
were tested against a panel of toxins. As shown in Fig. 3, none
of the toxins tested, SEA, SEB, SED, SEE, or TSST1, were able to
down-regulate TCRCD3 at any of the concentrations used. The lack
of effect of these toxins on T cell receptor expression was not due to
general unresponsiveness of these cells, since nanomolar concentrations
of SED or SEE could induce IL2 secretion, provided that
superantigen-presenting cells expressing MHC
molecules
were present (data not shown). Moreover, SEE, and to a lesser extent
SED, induced TCR
CD3 down-regulation of V
8-expressing Jurkat
cells when MHC superantigen presenting
cells were present in the assay.
()
This suggests
that SED and SEE, when used in soluble form, have lower affinities for
their corresponding T cell receptor than that displayed by SEB for
V3.
Figure 3:
Staphylococcus enterotoxin
superantigens do not induce down-regulation of TCRCD3 in
V
8-expressing Jurkat cells. J77cl20 cells were incubated for 18 h
with various concentrations of toxins. Surface TCRCD3 levels were
analyzed by immunofluorescence and flow cytometry using saturating
concentrations of the anti-CD3 mAb OKT3. Mean fluorescence intensity
was measured at each point. Results are given as percentage of control
cells (incubated in medium alone). Each point represents the average
± S.D. (n = 3).
, SEB;
, SED;
, SEE; , SEA;
, TSST1.
SEB Superantigen Induces Accumulation of TCR
Steady state display of surface
receptors is the result of a dynamic equilibrium maintained by the
membrane expression of newly synthesized molecules, internalization,
recycling to the cell surface, and degradation. In order to
characterize the mechanism that modulates TCRCD3
Complexes in Endocytic Vesicles
CD3 expression in
the presence of SEB, we looked for changes in any of the components of
this equilibrium.
CD3 by measuring the decay of receptor surface expression in
the presence of cycloheximide to block protein synthesis. The half-life
of surface TCR
CD3 in Jurkat cells was estimated to be longer than
10 h (data not shown). The fact that SEB-induced down-regulation was
rapid, reaching a plateau at 30 min, indicated that changes in
endocytosis or recycling might account for this phenomenon rather than
inhibition of the secretory pathway. To address this question, we
analyzed the effect of SEB on subcellular localization of TCR
CD3
complexes by confocal microscopy. Thus, V
3-expressing Jurkat
transfectants were treated with SEB for various times and then fixed,
permeabilized, and stained using anti-CD3 mAbs and fluorescent second
antibodies. The staining of CD3 in untreated cells is shown in
Fig. 4A. The immunofluorescence pattern observed within
the cytoplasm resembles that of the endoplasmic reticulum (ER). The
black area corresponds to the nucleus. Due to the brightness
of the ER, membrane staining is not clearly appreciable. Strong ER
labeling was expected, since CD3 is synthesized in excess and
assembled with other TCR
CD3 subunits in the ER. Unassembled
single polypeptides or partial complexes are retained in the ER and
eventually degraded before reaching the cell
surface
(5, 6) . Cells treated with SEB showed a
different pattern of immunofluorescence, since their TCR
CD3
accumulated in intracellular vesicles (Fig. 4B).
Vesicular staining was maximal at 2-3 h, disappearing afterwards.
No patching or capping of receptors was observed even at shorter times
of treatment. After 6 h of continuous incubation with SEB, no clear
differences were observed between the immunofluorescence patterns
displayed by treated and untreated cells. Accumulated TCR
CD3 was
not reexpressed on the cell surface, since expression quantitated by
flow cytometry remained at
30-40% of control levels for up to
72 h (not shown).
Figure 4:
SEB induces accumulation of TCRCD3
in intracellular vesicles. Jurkat transfectants expressing V
3 TCR
(CH7C17 cells) were incubated for 3 h at 37 °C in medium alone
(panelA) or in the presence of 10 µg/ml SEB
(panelB). Cells were then fixed, permeabilized, and
stained for immunofluorescence using UCHT1 anti-CD3 mAb and
fluorescein-coupled second antibodies. A Z series of optical sections
was performed at 0.5-µm increments. The image shows a projection of
four medial optical cuts of a representative cell. The color scale used
ranges from red (weak staining) to yellow (bright
staining). Bar, 10 µm.
To analyze whether TCRCD3 complexes observed
in intracellular vesicles were endocytosed receptors, we simultaneously
labeled the endocytic compartment using transferrin coupled to
rhodamine, and we followed both fluorescent labels by dual
immunofluorescence and confocal microscopy. The intracellular endocytic
pathway of transferrin and its receptor has been extensively studied in
numerous cell types. Transferrin accompanies its receptor through the
recycling pathway
(29, 30) and thus defines early and
recycling endocytic organelles. Cells were incubated in the presence or
absence of SEB in a medium containing rhodamine-transferrin, and then
CD3 was localized on permeabilized cells as described above using a
fluorescein-coupled second antibody. The results are shown in
Fig. 5
. Untreated cells displayed an immunofluorescence pattern
corresponding to CD3
, which accumulated mainly in the endoplasmic
reticulum (green). In addition, vesicles containing rhodamine
transferrin (red) were readily observed in the same cells. As
described above, SEB-treated cells displayed CD3
that accumulated
in the ER as well as in intracellular vesicles (green). In the
computer-generated composite image, the areas of colocalization of both
fluorochromes appear as yellow. Most of the intracellular
vesicles containing CD3 were stained in yellow. This indicates
that, upon SEB interaction, TCR
CD3 accumulated in organelles
containing transferrin, i.e. in the early endocytic and
recycling compartment. This suggests that SEB may provoke changes in
TCR
CD3 endocytosis and/or recycling.
Figure 5:
SEB induces accumulation of TCRCD3
in endocytic vesicles. Jurkat transfectants expressing V
3 TCR
(CH7C17 cells) were incubated for 3 h in medium containing 600
nM rhodamine transferrin in the absence (A-C)
or presence (D-F) of 10 µg/ml SEB. Cells were then
fixed, permeabilized, and stained by immunofluorescence using UCHT1
anti-CD3 mAb and fluorescein-coupled second antibodies. A Z series of
optical sections was performed at 0.5-µm increments. Measurements
of fluorescein and rhodamine emissions were acquired simultaneously.
The image shows a medial optical cut of a representative cell. A and D, fluorescein CD3-labeling; B and
E, rhodamine-transferrin-labeling; C and F,
combined images. Areas of colocalization appear yellow in the
computer-generated composite image. Bar, 10
µm.
SEB Treatment Increases the Internalization of
TCR
Interaction of ligands with cell surface
receptors may induce receptor internalization through the well
described pathway of receptor-mediated endocytosis
(31) . The
fate of a receptor along its intracellular route can be followed by
means of a radiolabeled ligand. Binding of toxin superantigens to TCR
is very difficult to measure due to their low affinity (see
``Discussion''). Therefore, we used radiolabeled mAbs to
follow the TCRCD3 Complex
CD3 complex. In order to avoid interference of
antibodies with SEB binding to TCR, we used mAbs directed to epitopes
on the CD3
chain, rather than antibodies against TCR
or
chains. Moreover, to prevent the influence of antibody
cross-linking on receptor endocytosis
(32) , we used purified Fab
fragments.
CD3 was followed using
I-Fab fragments of the anti-CD3
mAb OKT3, either
alone or in the presence of 10 µg/ml SEB. As shown in Fig. 6,
untreated cells rapidly internalized
I-Fab fragments,
reaching a plateau at 3 min. This shows, in agreement with previously
reported data
(32, 33) , that TCR
CD3 is
constitutively endocytosed and recycled. 18% of the total surface
receptors were found inside the cells at 7 min. When cells were
incubated in the presence of SEB,
I-Fab-OKT3 was
internalized more efficiently than in control cells (Fig. 6). The
receptors internalized at 7 min represent 32% of total surface
receptors. These data indicate that TCR
CD3 internalization was
augmented in the presence of SEB.
Figure 6:
SEB
increases TCRCD3 internalization. Jurkat transfectants expressing
V
3 TCR (CH7C17 cells) were incubated in growth medium at 37
°C. Then, I-Fab fragments of the anti-CD3 mAb OKT3
were added alone (opencircles) or together with SEB
(closedcircles) to a final concentration of 40
nM for
I-Fab and 10 µg/ml for SEB. Aliquots
of cells were taken at each time point, and the amount of
I-Fab internalized was determined as described under
``Methods.'' The figure shows a representative experiment out
of three independent experiments carried out. At 7 min, the amount of
I-Fab fragments internalized represented 18% of cell
associated ligand in control cells and 32% in SEB-treated
cells.
CD3 surface expression due,
at least in part, to increased receptor internalization. This is
indicated by the following observations. First, we observed an
accumulation of TCR
CD3 complexes in intracellular vesicles that
colocalized with internalized transferrin. Since transferrin is
endocytosed and recycled together with its
receptor
(29, 30) , it is a marker widely used for early
endocytic and recycling organelles. Accumulation of TCR
CD3 in
these organelles suggests that SEB induces changes in internalization
and/or recycling. Second, SEB treatment augmented the efficiency of
internalization of the TCR
CD3 complex as measured using
radiolabeled Fab fragments of anti-CD3 mAb, thus providing further
evidence for a role of endocytosis in T cell receptor down-regulation.
CD3 complexes in
intracellular vesicles was no longer detected by confocal microscopy.
In addition, surface levels of the receptor did not recover; on the
contrary, they continued diminishing during 18 h and then remained low
for the time that the toxin was present (up to 72 h tested). Additional
experiments are in progress to formally prove degradation. Furthermore,
from our data we cannot rule out the possibility that TCR
CD3 that
accumulated in intracellular vesicles might be further dispersed in
smaller undetectable subcellular structures.
CD3 was estimated to be longer than 10 h, indicating that the
TCR
CD3 complex is secreted at low rates. It is therefore unlikely
that the secretory pathway plays a direct role in the rapid regulation
of receptor numbers. However, an effect at the level of gene
transcription, protein synthesis, and/or secretion may occur at longer
times of incubation with the toxin.
3-transfected Jurkat cells in the complete absence of MHC molecules. This indicates that the toxin can interact directly
with the TCR. In agreement with this, Seth et al. (18)
recently showed that SEB could form complexes in vitro with a
soluble form of the V
3 TCR HA1.7. If the interaction between SEB
and the TCR were strong enough to form stable complexes, one might
expect SEB to be endocytosed with its TCR. To address this point, we
labeled SEB with I and carried out ligand internalization
experiments. We were unable to detect significant levels of
internalized toxin. Moreover, we could not detect SEB inside the cells
by means of immunofluorescence using an anti-SEB antiserum. It is worth
noting that
I-SEB was able to induce down-regulation of
surface TCR
CD3 with the same efficiency as unlabeled toxin,
showing that radiolabeled toxin was biologically active. Our findings
indicate that SEB, although capable of interacting with the TCR to
induce a biological response, does not bind stably enough to be
endocytosed together with the receptor. The rapid dissociation and slow
association kinetics reported for the interaction between SEB and
V
3TCR could explain this fact
(18) . Therefore, in this
case, TCR internalization does not require stable association with the
ligand. It is tempting to speculate that, upon (even unstable)
interaction between SEB and TCR, a signal is transduced to the cell
that in turn triggers receptor down-regulation. Post-translational
modifications (i.e. phosphorylation) of CD3 chains that occur
upon T cell activation might be responsible for changes in the
intracellular traffic
(32, 33, 34) . Moreover,
our experiments indicate that low affinity ligands can induce receptor
endocytosis without being involved in further intracellular trafficking
of the receptor. This may be of physiological relevance for T cells,
since the natural ligand for T cell receptors, the peptide antigen/MHC
complex, is anchored on the surface of antigen-presenting cells.
Interaction between these two structures could provoke TCR
internalization, keeping the independence of each receptor on the
surface of its own cell.
8
TCR were tested against a panel of toxins. Previous experiments showed
that these cells responded to SED and SEE by secreting IL2, provided
that accessory cells expressing MHC molecules were present
(not shown). Our data showed that none of the toxins tested, including
SED and SEE, induced TCR down-regulation in the absence of
MHC
molecules. This is most likely due to
differences in affinity of these toxins for the TCR, since the addition
of MHC
accessory cells made SEE and SED
able to induce TCR
CD3 down-regulation on V
8 Jurkat
cells. Previous binding of toxins to MHC molecules on accessory cells may facilitate binding to TCR and
increase the stability of the interaction. This may improve the
intracellular signal required to induce down-regulation. Other
intercellular interactions provided by other surface molecules may also
play an important role in this process.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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