By
From the Basel Institute for Immunology, CH-4005, Basel, Switzerland
T cell activation by specific antigen results in a rapid and long-lasting downregulation of triggered T cell receptors (TCRs). In this work, we investigated the fate of downregulated TCR-
CD3- complexes. T cells stimulated by peptide-pulsed antigen-presenting cells (APCs) undergo an antigen dose-dependent decrease of the total cellular content of TCR-
, CD3-
, and
chains, as detected by FACS® analysis on fixed and permeabilized T-APC conjugates and by
Western blot analysis on cell lysates. The time course of CD3-
chain consumption overlaps
with that of TCR downregulation, indicating that internalized TCR-CD3 complexes are
promptly degraded. Inhibitors of lysosomal function (bafilomycin A1, folimycin) markedly reduced
chain degradation, leading to the accumulation of
chain in large Lamp1+ vesicles.
These results indicate that in T cell-APC conjugates, triggered TCRs are rapidly removed
from the cell surface and are degraded in the lysosomal compartment.
Tlymphocytes are activated by the engagement of their
clonally expressed TCR with peptide-MHC complexes displayed on the APC surface. The antigen receptor
of T cells is a multimeric protein complex composed of the
clonotypic Upon conjugation with APCs, T cells undergo a sustained
[Ca2+]i increase (3, 4) that results from the serial engagement and triggering of many TCRs by a small number of
peptide-MHC complexes (5). A key feature of T cell antigen recognition is that the process of TCR/peptide-MHC
interaction is self-limited by the downregulation of triggered TCR complexes (5, 6). Indeed, downregulation of
triggered TCRs leads to extinction of sustained signaling in
T-APC conjugates and affects T cell responsiveness to further antigenic stimulation (6).
The mechanisms that lead to downregulation of triggered TCRs are presently unknown and, in particular, it is
not clear whether these TCRs are indeed degraded or simply internalized. In this study we investigated the fate of
triggered TCR-CD3 components in human T cells interacting with peptide-pulsed APCs. We report that stimulation by the specific antigen results in rapid and profound
loss of TCR- T Cell Clones and APCs.
Two DRBI*1104-restricted T cell clones
(KS140 and KS70) specific for the tetanus toxin peptide (TT830-
843; QYIKANSKFIGITE) were used. Autologous Epstein-Barr virus (EBV)-B cells were used as APCs (4).
Intracellular Staining for CD3- TCR Downregulation, IFN- T cells conjugated with
peptide-pulsed APCs undergo a rapid downregulation of
triggered TCR-CD3 complexes that reaches a plateau in
1-2 h (5). To investigate whether downregulated receptors accumulate inside of T cells or are degraded, we measured
the surface levels and the total cellular content of different
TCR-CD3 components by FACS® analysis. As shown in
Fig. 1, A-B, in T cells conjugated with peptide-pulsed
APCs the surface level of CD3-
To investigate whether the reduced staining for CD3-
The above results demonstrate that TCR triggering by
specific antigen results in a net decrease in the cellular content of TCR and CD3- A wellknown mechanism for the inactivation of hormone and
growth factor receptors is their internalization upon ligand
binding followed by their degradation in lysosomes (7). To
find whether triggered TCRs are degraded via a similar
mechanism we stimulated T cells with peptide-pulsed APCs
in the presence of bafilomycin A1 or folimycin, drugs that
affect lysosomal function by increasing lysosomal pH (8, 9).
These drugs do not interfere with TCR downregulation,
sustained [Ca2+]i increase, and IFN-
To identify better the site of TCR-CD3
complex degradation, we investigated by confocal microscopy the fate of triggered TCRs. T cells were conjugated with
peptide-pulsed or unpulsed APCs and after 2 h the conjugates were fixed, permeabilized, and stained with anti- In unstimulated T cells most of the
In specific T-APC conjugates TCRs are downregulated in
an antigen dose- and time-dependent fashion (5). The reduction of TCR levels plays an important role in extinguishing
the signaling process and reduces T cell responsiveness to
antigenic stimulation (6). So far, TCR downregulation has
been defined only as a long-lasting disappearance of TCRs
from the cell surface, whereas their fate has not been investigated. In this work, we demonstrate that triggered
TCR-CD3- Whereas CD3 and TCR are tightly associated, the The mechanisms involved in TCR downregulation by
specific antigen are presently unknown. It has been shown
that phosphorylation of Ser 126 of the CD3- As are many other surface receptors, TCR-CD3- What could be the physiological relevance of antigeninduced TCR degradation? It is interesting to note that the
time kinetics of receptor degradation overlaps with the time
kinetics of sustained [Ca2+]i increase and TCR downregulation (Fig. 2; references 5, 6). This indicates that serial
TCR triggering and degradation are ongoing phenomena.
We and others have shown that in T cells undergoing sustained signaling, treatments that terminate the process of TCR engagement result in the extinction of the signaling
process within a few minutes (4, 19). A rapid degradation
of triggered TCRs could be important to inactivate already
triggered receptors as soon as they have been internalized
while new receptors are interacting with the ligands. As a
consequence, any single TCR would be triggered by a single peptide-MHC complex only once and the length of
signaling would reflect the length of the serial receptor engagement. This mechanism could be at work to ensure a
strict control of the extent of T cell activation by a defined number of antigenic determinants.
heterodimer, the CD3
chains, and the
homodimer. Whereas the
heterodimer is responsible for specific recognition, the associated CD3 chains and
homodimer are necessary for signal transduction (1). The
assembly of this multichain receptor complex is highly regulated in T cells, because only correctly assembled receptors can reach the cell surface (2).
, CD3-
, and
chain due to degradation in
the lysosomal compartment.
and
Chain.
EBV-B cells were
pulsed for 2 h at 37°C with various concentrations of TT830-843
in RPMI, 5% FCS. During the last 10 min, 1 µM BCECF-AM
(2
,7-bis-(carboxyethyl)-5(6
)-carboxyfluorescin; Calbiochem, San
Diego, CA) was added and the cells were washed four times. T cells
were mixed with EBV-B cells at a 1:2 ratio in 200 µl RPMI, 5%
FCS in U-bottomed microplates, centrifuged 1 min at 1,500 rpm
to allow conjugate formation, and incubated at 37°C. In some experiments, T cells were pretreated for 1 h with 10 µg/ml cycloheximide and the drug was present throughout the assay. At different
times, the cells were resuspended, washed in PBS, 0.5 mM EDTA
and fixed for 10 min with 3% paraformaldehyde. The cells were
permeabilized for 10 min at room temperature with washing buffer
(Hepes-buffered PBS containing 0.1% saponin) and stained with
anti-CD3 (TR66; reference 4), anti-V
2 (Immunotech, Marseille,
France), or anti-
(Santa Cruz Biotechnology, Santa Cruz, CA) in
Hepes-buffered PBS containing 0.1% saponin and 2% BSA (or 5%
FCS), followed by a goat anti-mouse PE-labeled Ab (SBA, Birmingham, AL). The TCR, CD3, and
fluorescence were analyzed on a FACScan® (Becton Dickinson, Mountain View, CA).
EBV-B cells were gated out using both forward and side scatter
(FSC/SSC) parameters and green BCECF fluorescence. In some
experiments, T cells were conjugated with unstained peptidepulsed EBV-B cells; at different times of incubation at 37°C, the
cells were gently resuspended and laid on poly-L-lysine-coated
slides for 10 min at 37°C. The cells were fixed for 10 min with
3% paraformaldehyde, permeabilized for 10 min at room temperature with washing buffer, and stained with an anti-
(Santa
Cruz) in Hepes-buffered PBS containing 0.1% saponin and 4%
BSA, followed by FITC-labeled goat anti-mouse (SBA) and anti-
human Lamp-1 (provided by Dr. S. Carlsson, Umea University, St. Louis, MO) followed by Texas Red-labeled goat anti-rabbit antibody (SBA). The samples were mounted in 90% glycerol-PBS
containing 2.5% 1-4-diazabicyclo (2.2.2) octane (DABCO; Fluka
AG, Buchs, Switzerland). In some experiments, T cells were pretreated with 0.5 µM bafilomycin A1 (Calbiochem) or with the
vehicle of the drug only (DMSO 0.5%) for 1 h at 37°C before
conjugate formation. The samples were examined using a BioRad MRC 1024 confocal microscope (Bio-Rad, Richmond, CA).
Chain Detection by Western Blot.
EBV-B cells were pulsed
with various concentrations of TT830-843. 5 × 105 T cells were
pretreated for 1 h with 10 µg/ml cycloheximide mixed with 106
EBV-B cells in 500 µl RPMI 5% FCS in U-bottomed tubes,
centrifuged to allow conjugate formation, and incubated at 37°C
for 2 h in the presence of cycloheximide. In some experiments,
T cells were pretreated with 1 µM bafilomycin A1 or with 1 µM
folimycin (Calbiochem) or with the vehicle of the drugs only
(DMSO 1%) for 1 h at 37°C before conjugate formation. The
drugs were present in the culture throughout the assay. At different timepoints, the cells were mixed with PBS, lysed in either
ice-cold RIPA buffer containing 1 mM NaVO4 or in prewarmed
Laemmli buffer, sonicated, and boiled. After separation on 12.5%
SDS-PAGE and transfer to nitrocellulose, membranes were blocked
1 h at room temperature with blocking buffer (5% nonfat dry
milk, 0.05% Tween-20 in Tris-buffered saline) and incubated for
1 h with 1 µg/ml anti-
(Santa Cruz) in blocking buffer. After
washing, the membranes were incubated for 1 h with HRP-labeled
goat anti-mouse Ig (SBA) in blocking buffer. Filters were developed using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL). Densitometric analysis was performed using a Computing Densitometer 300A (Molecular
Dynamics, Sunnyvale, CA).
Production, and [Ca2+] Measurement.
TCR downregulation, IFN-
production, and [Ca2+];
were measured as previously described (4, 5). In some experiments, T cells were pretreated with 1 µM bafilomycin A1 or with
1 µM folimycin for 1 h at 37°C before conjugate formation and
the drugs were kept in the culture through all the assay. Control
cultures were done in the presence of vehicle only (1% DMSO).
Reduced Content of TCR-, CD3-
, and
Chains in T
Cells after Antigen Stimulation.
and the total content of
CD3-
and
chains decreased in a parallel fashion with increasing doses of antigen. Blocking of protein synthesis did
not affect the extent of CD3-
and
chain decrease, demonstrating that disappearance of these components was not
due to a decreased synthesis (Fig. 1 C ). Similar results were
observed when levels of CD3-
and TCR-
were measured in a different T cell clone (Fig. 1 D).
Fig. 1.
Parallel decrease in surface expression and total cellular content of TCR-, CD3-
and
chains in T cells as a function of antigenic
stimulation. T cells (clone KS140) were conjugated at 37°C with APCs
pulsed with various doses of peptide. After 2 h the cells were either fixed
and stained with anti-CD3-
or anti-V
2, or fixed, permeabilized, and
stained with anti-CD3-
, anti-V
2, or anti
. (A) Staining for total CD3-
(left) and
(right) in T cells conjugated with unpulsed or peptide-pulsed
APCs. (B and C ) Levels of surface CD3-
(
), total CD3-
(
) and total
(
) chains as a function of antigen concentration. In C, T cells were pretreated for 1 h with cycloheximide and the drug was present throughout the assay. (D) Levels of surface and total V
2 and CD3-
in T cell
clone KS70 stimulated with 100 nM (diagonal stripes) or 10 µM (empty)
peptide expressed as percent of the staining of unstimulated cells (closed).
[View Larger Version of this Image (32K GIF file)]
and
chain could be due to the localization of triggered receptors in cellular compartments not accessible to antibodies,
we measured
chain expression by Western blot. As shown
in Fig. 2 A, the lysates of T-APC conjugates showed a
dose-dependent decrease in the content of
chain. These
results are comparable to those observed by FACS® analysis
(Fig. 1). In addition, the time kinetics of
chain decrease
parallels that of surface TCR downregulation (5, 6) reaching a plateau in about 2 h (Fig. 2 B).
Fig. 2.
Dose response and kinetics of antigen-induced chain loss.
T cells were conjugated in the presence of cycloheximide with APCs
pulsed with various peptide concentrations. At the indicated time points
the cells were lysed and the amount of
chain was measured by Western
blot. (A)
chain loss as a function of antigen concentration. (B) Time
course of
chain loss in T cells conjugated with APC pulsed with 10 µM
peptide.
[View Larger Version of this Image (27K GIF file)]
and
molecules. This indicates
that downregulated TCR-CD3-
complexes are promptly
degraded.
in Activated T Cells.
production (Fig. 3),
indicating that they are not toxic for the T cell biological
response. However, bafilomycin A1 and folimycin dramatically inhibited
chain degradation as detected by Western
blot analysis on total cell lysates (Fig. 4). The inhibition of
chain degradation by bafilomycin A1 and folimycin was
also observed when protein synthesis was blocked by pretreating T cells with cycloheximide (Fig. 4). This result indicates that the effect of bafilomycin A1 and folimycin is
not due to the accumulation of newly synthesized unassembled components, but actually results from a block of
the degradation of triggered receptors.
Fig. 3.
Bafilomycin A1 and folimycin do not interfere with T cell
activation induced by specific antigen. T cells pretreated for 1 h with bafilomycin A1 (), folimycin (
), or vehicle only (
) were conjugated
with APCs pulsed with various peptide concentrations and the drugs were
left in culture throughout the assay. (A) CD3 downregulation after 4 h;
(B) IFN-
production after 4 h; (C ) [Ca2+]i increase in T cells conjugated
with unpulsed APC (a and d ) with APC pulsed with 25 nM (b and e), or
10 µM peptide (c and f ). T cells were either pretreated with bafilomycin
A1 (d, e, f ) or with the vehicle only (a, b, c).
[View Larger Version of this Image (40K GIF file)]
Fig. 4.
Antigen-induced degradation of chain is prevented by bafilomycin A1 and folimycin. T cells were pretreated for 1 h with bafilomycin A1 or folimycin in the presence or absence of cycloheximide. The
cells were conjugated with APCs pulsed with 10 µM peptide and total
content of
chain was determined after 2 h by Western blot. The drugs
were present throughout the assay. (A) Western blot. (B) Densitometric
analysis of the bands obtained in the absence (closed ) or in the presence
(empty) of cycloheximide.
[View Larger Version of this Image (27K GIF file)]
to
the Lysosomes.
chain antibodies.
chain is associated
with the plasma membrane and this staining pattern is not
affected by preincubation with bafilomycin A1 (Fig. 5, A,
B, E ). In T cells that had been conjugated for 2 h with
peptide-pulsed APCs, the staining was much weaker and
did not show the ring-shaped pattern of
chain surface expression (Fig. 5 C ). Strikingly, when conjugates were
formed in the presence of bafilomycin A1 a dramatic accumulation of
chain was observed in intracellular vesicles
(Fig. 5 D). In these vesicles, most of the internalized
chain colocalized with the lysosomal marker LAMP-1 (Fig.
5 F). These results demonstrate that upon TCR/peptide-
MHC interaction
chains are promptly removed from the
cell surface and degraded in the lysosomes.
Fig. 5.
Triggered CD3- is targeted to the lysosomes for degradation. T cells were conjugated with either unpulsed APCs (a, b, e) or peptide-pulsed
(10 µM) APCs (c, d, f ). T cells were treated with bafilomycin A1 1 h before conjugate formation (b, d, e, f ) or with vehicle only (a and c) the drug was
left in culture throughout the assay. After 2 h the conjugates were fixed, permeabilized, and stained with anti-
(green) and anti-Lamp-1 (red ).
[View Larger Version of this Image (83K GIF file)]
complexes are degraded in the lysosomes after antigenic stimulation. This conclusion is based on measurements of the total cellular content of TCR-
, CD3-
,
and
chains that are quantitatively lost in an antigen dependent fashion and on the effect of drugs that block lysosomal degradation.
chain, which plays a dominant role in TCR-mediated signal
transduction, is associated to the complex in a loose fashion.
Indeed, whereas
is required for assembly and surface expression of the TCR (2, 10), it has been proposed that it
may be exchanged on mature receptors (11). Our data
demonstrate that in the process of antigen-induced downregulation TCR-CD3 and
chains are irreversibly linked.
Recently, Cai et al. (12) showed that in mouse resting T
cells TCR downregulation is due to internalization rather
than shedding, but degradation of the TCR-CD3 complexes has not been investigated.
chain is both
necessary and sufficient for TCR downregulation induced
by pharmacological stimulation of PKC (13). However, the
mechanisms of TCR downregulation induced by PMA or
specific ligand are fundamentally different, because mutations of CD3-
that abolish PMA-induced downregulation
do not affect ligand-induced downregulation (Salio, M., S. Valitutti, and A. Lanzavecchia, manuscript in preparation).
complexes are constitutively internalized and recycled to the
plasma membrane (14, 15). Our results suggest that triggering by cognate ligand leads to the failure of TCRs to recycle back to the surface due to their targeting to lysosomes.
Among possible signals for targeting triggered TCR complexes to lysosomes, it is interesting to consider ubiquitination (7, 16). Indeed, ubiquitination of TCR-CD3 subunits
(17) and especially
chains (18), have been demonstrated following TCR cross-linking.
Address correspondence to Salvatore Valitutti, Institute of Biochemistry, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.
Received for publication 10 March 1997.
The Basel Institute for Immunology was founded and is supported by Hoffman-La Roche Ltd. Co., Basel, Switzerland.We thank M. Dessing for help in [Ca2+]i measurements; M. Dessing and S. Meyer for image processing; K. Campbell, K. Karjalainen and J. Pieters for discussion and critical reading of the manuscript.
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