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Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-S126 and the CD3 leucine-based
internalization motif are required, constitutive TCR cycling required
neither PKC nor CD3-S126 but only the CD3
leucine-based motif. Having identified these requirements, we next
studied ligand-induced internalization in cells with abolished
constitutive TCR cycling. We found that ligand-induced TCR
internalization was not dependent on constitutive TCR internalization.
Likewise, constitutive internalization and recycling of the TCR were
independent of an intact ligand-induced internalization of the TCR. In
conclusion, ligand-induced TCR internalization and constitutive cycling
of the TCR represents two independent pathways regulated by different
mechanisms. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In resting T cells, the TCR constitutively cycles between the plasma
membrane and intracellular compartments (9, 10, 11, 12); 70–85%
of the TCR in the cycling pool is expressed on the cell surface and
15–30% is found inside the cell (9, 10, 11, 12). The
constitutive TCR internalization rate constant is 
0.012
min-1, meaning that 1.2% of
surface-expressed TCR is internalized per minute. Likewise, the
exocytic rate constant is 0.055 min-1, meaning
that 5.5% of the intracellular pool of recycling receptors is
transported to the plasma membrane per minute (12). The
TCR amino acid motifs involved in constitutive TCR cycling and how the
cycling is regulated still remain to be determined.
Activation of protein kinase C
(PKC)3 induces TCR
down-regulation from the cell surface by increasing the TCR
internalization rate constant 10 times without affecting the
exocytic rate constant (12). PKC-induced phosphorylation
of CD3-S126 most probably induces a
conformational change, which leads to exposure of the CD3 leucine
(L)-based receptor-sorting motif. This motif consists of the DxxxLL
sequence that binds the AP-2 at the plasma membrane (13, 14). AP-2, in turn, links the TCR to the clathrin-dependent
internalization machinery. Some studies have indicated that PKC plays a
role in the constitutive cycling of the TCR and that the PKC-induced
pathway is identical with the constitutive pathway (10, 15). However, in these studies cells were stimulated with
phorbol esters and the role of PKC in the constitutive cycling of the
TCR in unstimulated cells is still not known.
Finally, TCR surface down-regulation is seen after ligand stimulation. In several studies, TCR surface down-regulation is observed within minutes of ligand stimulation, implying that ligand stimulation increases the TCR internalization rate (7, 16, 17). However, a recent study showed that ligand-induced TCR down-regulation did not increase the TCR internalization rate and that the observed surface down-regulation was due to lack of recycling of ligated TCR (11). From this, it was suggested that the ligand-induced internalization pathway was in fact identical with the constitutive TCR internalization pathway and therefore absolutely dependent on a functional constitutive cycling of the TCR.
Thus, whether the TCR can be sorted via one or more different pathways is still a matter of debate. In this work, we focused on the mechanisms regulating the constitutive and the ligand-induced pathways. We speculated that if the ligand-induced internalization pathway is indeed identical with the constitutive pathway then a block in the ligand-induced pathway should parallel a block in the constitutive pathway. Given that the mechanisms regulating the constitutive cycling of the TCR were unknown, the aim of this study was first to identify these mechanisms and thereafter to examine whether the same mechanisms were involved in ligand-induced internalization of the TCR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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JGN and E3 TCR cell surface-negative variants of the human T
cell line Jurkat that synthesizes no CD3 were produced in our
laboratory (18). JGN- and E3-WT cells express wild-type
(WT) CD3, JGN- and E3-L131/132A cells express CD3 with leucine
131 and 132 to alanine mutations, and JGN- and
E3-S126V cells express CD3 with serine 126 to
alanine mutation (19). J76 is a WT clone of Jurkat
(20). J.CaM1.6, a p56lck-deficient
variant of the Jurkat clone E6-1 (21), J45.01 (J45), a
CD45-deficient variant of E6-1 (22), and the Burkitt’s
lymphoma cell line Raji were from American Type Culture Collection
(Manassas, VA). J.CaM1.6 and J45 cells were transfected as previously
described with p56lck and CD45, respectively
(17). DO11.10 is a mouse T cell hybridoma
(23). In some experiments, CD3-L131/132A or WT was
cloned into pEGFP-N1 (Clontech, Palo Alto, CA). These constructs were
subsequently transfected into JGN cells (JGN-L131/132A-GFP and
JGN-WT-GFP) as previously described (19). Cells were
cultured in RPMI 1640 supplemented with penicillin (2 x
105 U/L; Leo Pharmaceutical Products, Ballerup,
Denmark), streptomycin (50 mg/L; Merck, Darmstadt, Germany), and 10%
(v/v) FCS (Life Technologies, Paisley, U.K.) at 37°C in 5%
CO2. PE -conjugated and unconjugated UCHT1
anti-human CD3
mAb were obtained from Dakopatts (Glostrup,
Denmark). PE-conjugated and unconjugated anti-murine CD3
(145-2C11) and anti-murine Ti-V
8 (F23.1) mAbs were obtained from
BD PharMingen (San Diego, CA). The anti-TCR mAb F101.01 was
produced in our laboratory (24). Cy5-conjugated
F(ab)2 fragments of donkey anti-mouse IgG H +
L and PE-conjugated F(ab)2 fragments of goat
anti-mouse IgG H + L were obtained from Jackson ImmunoResearch
(West Grove, PA). The phorbol ester phorbol 12,13-dibutyrate
(PDB) and the protein synthesis inhibitor cycloheximide were from Sigma
(St. Louis, MO). Superantigen staphylococcal enterotoxin E (SEE) was
from Toxin Technology (Sarasota, FL), and brefeldin A (BFA) was from
Boehringer Mannheim (Mannheim, Germany).
Confocal microscopy
Cells were washed in PBS and fixed for 10 min with 1% paraformaldehyde-PBS at room temperature. The cells were permeabilized for 10 min at room temperature with washing buffer (HEPES-buffered PBS containing 0.1% saponin) and incubated with primary Abs at room temperature for 10 min. Cells were washed three times in washing buffer and stained with Cy5-conjugated secondary Abs at room temperature for 10 min. Confocal microscopy was performed on a Zeiss LSM510 connected to a Zeiss Axiovert 100 M microscope (Carl Zeiss, Jena, Germany). Green-fluorescent protein (GFP) and Cy5 fluorescence were detected using band pass filter BP 505–550 and long pass filter LP 650, respectively.
Recycling assay, TCR down-regulation, and phosphotyrosine blots
TCR recycling was analyzed by determining the size of the
intracellular pool of recycling receptors as follows. First, all TCR
expressed at the cell surface was blocked by incubating the cells with
saturating amount of unconjugated anti-CD3 or anti-TCR mAb
(1 µg/ml) at 4°C for 30 min. After extensive washing at 4°C, the
cells were transferred to a 37°C water bath for the indicated time to
allow TCR cycling. The cells were subsequently transferred to 4°C,
stained with PE-conjugated mAb of the same type as used for TCR
blockade, and analyzed using a FACSCalibur flow cytometer (BD
Biosciences, Mountain View, CA). The recycling pool of TCR was
calculated as the percentage of TCR expressed at the cell surface using
the equation ((H - C)/T) x
100%, where H is the mean fluorescence intensity (MFI) of
cells incubated with unconjugated mAb at 4°C followed by incubation
at 37°C and staining with PE-conjugated mAb, C is the MFI
of cells incubated with unconjugated mAb followed by incubation at
4°C and staining with PE-conjugated mAb, and T is the MFI
of cells directly stained by incubation with PE-conjugated mAb. The
initial slope of the curves reflects the exocytic rate constant.
For TCR down-regulation, cells were adjusted to 1 x
106 cells per ml of medium and incubated at
37°C with various concentrations of PDB, the anti-TCR mAb
F101.01, or MHC class II-positive Raji cells pulsed for 2 h with
different concentrations of SEE. At the indicated time, cells were
transferred to ice-cold PBS containing 2% FCS and 0.1%
NaN3 and washed twice. The cells were stained
directly with PE-conjugated anti-CD3 and analyzed by flow
cytometry. In experiments using the anti-TCR mAb F101.01 for TCR
down-regulation, TCR expression was measured by staining the cells with
saturating amounts of F101.01 followed by PE-conjugated goat
anti-mouse Ig. MFI was recorded and used in the calculation of
percentage anti-CD3/TCR binding: (MFI of treated cells/MFI of
untreated cells) x 100%. Phosphotyrosine blots were performed as
previously described (15).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-based motif is required for constitutive TCR cycling
It has been shown that the CD3 L-based motif is required for
PKC-induced internalization of the TCR (19). However, it
is not known whether the CD3 L-based motif is also required for
constitutive TCR internalization and recycling. To examine this we used
two CD3-deficient cell lines (JGN and E3) transfected with wild-type
CD3 (CD3-WT) or CD3 with a nonfunctional L-based motif due to
a CD3-Leu131/132 to alanine mutation (CD3-L131/132A). Three
different methods were used to study the constitutive cycling of the
TCR in these cells. 1) Blocking exocytosis while leaving endocytosis
intact with the fungal metabolite BFA should lead to a decrease in
surface-expressed TCR in cells with functional cycling of the TCR.
Accordingly, a decrease in TCR surface expression was observed in JGN-
or E3-WT cells treated with BFA (Fig. 1
, A and B). In contrast, BFA treatment did not
decrease TCR surface expression in JGN- and E3-L131/132A cells. This
indicated that constitutive TCR internalization was abolished in these
cells (Fig. 1, A and B). 2) To analyze TCR
exocytosis (recycling), cells were incubated with the unconjugated
anti-CD3 mAb UCHT1 at 4°C to block cell surface expressed TCR.
After extensive washing the cells were incubated at 37°C for
different time periods and subsequently analyzed by FACS using
PE-conjugated UCHT1. This procedure allowed us to specifically label
and analyze only newly expressed TCR. The results showed an almost
complete lack of TCR recycling in the mutant T cells JGN- and
E3-L131/132A (Fig. 1, C and D). As expected,
treatment of control cells with BFA inhibited TCR recycling (Fig. 1C). 3) As a final approach, WT and L131/1132 cells were
analyzed by confocal microscopy. According to the previous experiment
demonstrating an almost complete lack of an intracellular pool of
recycling TCR, it could be speculated that intracellular TCR staining
would be reduced in L131/132A cells compared with WT cells. JGN cells
(and E3, data not shown) transfected with CD3-WT stained positive
for CD3 at the cell surface and in intracellular vesicles (Fig. 2A). The same staining pattern
was observed for PBL (Fig. 2B). In contrast, although
JGN-L131/132A showed a clear surface staining, a significant decrease
in intracellular staining was observed (Fig. 2C). Likewise,
T cells expressing the CD3-WT chain linked to GFP (JGN-WT-GFP)
showed a significantly higher staining of intracellular vesicles
compared with JGN-L131/132A-GFP cells (Fig. 2, D and
E). The reduced occupancy of TCR in intracellular vesicles
supported that constitutive TCR cycling in JGN- and E3-L131/132A cells
was abolished.
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L-based
motif is required for constitutive internalization and recycling of
the TCR. The vast majority of newly expressed TCR represents recycling TCR
In the analyses used in this study, we measured exocytosis of
unlabeled TCR. These TCR could come either from the recycling pool of
TCR, from newly synthesized TCR, or from a combination of the two. The
half-life of fully assembled TCR has been determined to be 15 h
(25, 26). This means that 0.08%
(ln2/t1/2) of the TCR at the cell surface
is exchanged with newly synthesized TCR per minute. At the same time,
1.2% of the TCR at the cell surface is exchanged with recycling TCR
per minute. Thus, in theory the vast majority ((1.2/(1.2 + 0.08)
x 100% = 94%) of newly expressed TCR should come from the recycling
pool. To address this question experimentally, cells were preincubated
with or without the protein synthesis inhibitor cycloheximide and then
analyzed in the recycling assay. As seen in Fig. 3A, cycloheximide did not
affect the appearance of newly expressed TCR in agreement with the
theoretical considerations. Similar results were obtained for
cycloheximide-treated JGN- and E3-WT cells (data not shown).
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B, the
theoretical curve for appearance of unlabeled TCR at the cell surface
reflected the experimentally obtained curves.
Finally, another important point to address was whether mAb-induced TCR
ligation, signaling, and internalization affected the appearance of
newly expressed TCR as measured by the recycling assay. To study this
point, we took advantage of the existence of both activating and
nonactivating mAb against the TCR expressed by the mouse T cell
hybridoma DO11.10. Thus, triggering the TCR with the activating
anti-CD3 mAb 145-2C11 induced both TCR internalization and
tyrosine phosphorylation of intracellular substrates (Fig. 3, C and D). In contrast, incubation of the cells
with the anti-V8 mAb F23.1 induces neither TCR internalization
nor tyrosine phosphorylation (Fig. 3, C and D).
However, both mAb produced very similar results in the recycling assay,
strongly indicating that mAb-induced TCR internalization and signaling
did not affect the appearance of newly expressed TCR (Fig. 3E).
Constitutive TCR cycling is independent of CD3-S126
and PKC activity
The results presented thus far showed that the CD3 L-based
motif is required for constitutive TCR internalization and recycling.
Because PKC-induced TCR internalization in addition to the CD3
L-based motif is absolutely dependent on
CD3-S126, we next analyzed the role of
CD3-S126 in constitutive TCR cycling. We used
JGN and E3 cells expressing a CD3 chain with a
CD3-S126 to valine mutation (CD3-S126V). In
these transfectants PKC-induced internalization is completely inhibited
(Fig. 4A and Ref.
17). Surprisingly, BFA treatment produced the same degree
of TCR surface down-regulation as observed for JGN- and E3-WT cells
(Fig. 4B). If constitutive internalization was indeed intact
in JGN- and E3-S126V cells, it would be expected that constitutive
recycling should also be intact in these cells. Accordingly, analyses
of TCR recycling showed no significant difference between WT and S126V
cells (Fig. 4C). Taken together, these experiments showed
that constitutive TCR internalization and recycling is independent of
CD3-S126.
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-S126 is a substrate for PKC (27, 28). If constitutive internalization were independent of PKC,
this would explain why CD3-S126 was not
required. We therefore examined the role of PKC in constitutive cycling
of the TCR. Cells were treated with BAPTA-AM and EGTA that bind
intracellular and extracellular Ca2+. This
treatment completely inhibited PKC-induced TCR internalization (Fig. 5A). Thereafter, we used two
approaches: 1) TCR recycling after treatment with BAPTA-AM-EGTA for 30
min at 37°C was directly analyzed as described above. These
experiments showed that recycling of the TCR was unaffected by the lack
of PKC activity (Fig. 5B). 2) Cells were pretreated with
BAPTA-AM-EGTA and subsequently treated with BFA to block spontaneous
exocytosis. Despite the lack of PKC activity, BFA treatment produced
the same degree of TCR surface down-regulation as observed for
untreated cells (Fig. 5C). Thus, these experiments
demonstrated that constitutive internalization and recycling of the TCR
do not require CD3-S126 and PKC activity.
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Recent studies have indicated that the ligand-induced TCR
internalization pathway is identical with the constitutive TCR
internalization pathway (11). JGN- and E3-L131/132A cells
exhibited a block in constitutive TCR internalization and recycling,
which allowed us directly to study whether ligand-induced TCR
internalization was dependent on intact constitutive TCR
internalization. WT and L131/132A cells were stimulated with different
concentrations of the anti-TCR mAb F101.01 or incubated with APC
pulsed with different concentrations of the superantigen SEE. Efficient
ligand-induced TCR internalization was observed for all cell lines
independently of the CD3 L-based motif, although E3-WT cells
responded to somewhat lower concentrations of ligand than JGN-WT and
JGN- and E3-L131/132A cells (Fig. 6, A and B). Furthermore, stimulating for different
time periods with a fixed amount of anti-TCR mAb or APC pulsed with
a fixed amount of SEE did not reveal any significant differences
between WT and L131/132A cells (Fig. 6, C and D).
These experiments demonstrated that ligand-induced internalization of
the TCR is functional in cells with abolished constitutive TCR
cycling.
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Because constitutive TCR cycling was not required for
ligand-induced TCR internalization, we next examined whether a
functional ligand-induced TCR internalization was required for TCR
cycling. Given that lack of tyrosine kinase
p56lck or phosphatase CD45 significantly reduces
ligand-induced TCR internalization, we analyzed TCR cycling in J.CaM1.6
cells deficient of p56lck (21, 29)
and J45 cells deficient of CD45 (22). As expected,
anti-TCR mAb or superantigen-induced TCR internalization in J45 and
J.CaM1.6 cells was significantly reduced and could be restored by
transfection with CD45 and p56lck, respectively
(Fig. 7A and data not shown).
Importantly, TCR recycling was similar in cells deficient of CD45 or
p56lck when compared with J45 cells transfected
with CD45 or J.CaM1.6 transfected with p56lck,
respectively (Fig. 7, B and C). Likewise, lack of
p56lck or CD45 did not significantly affect TCR
surface down-regulation after BFA treatment (Fig. 7, D and
E). This indicated that constitutive TCR cycling does not
require a functional ligand-induced TCR internalization pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although constitutive TCR cycling has been observed in several studies
(9, 10, 15), the mechanisms controlling this process have
remained unknown. In the present study, we showed that constitutive
internalization and recycling of the TCR is dependent on the CD3
L-based motif. Mutation of the CD3 L-based motif abrogated the
internalization step in the constitutive cycling of the TCR and thus
impaired TCR cycling. Previous studies have suggested that a basic PKC
activity resulting in low rate phosphorylation of
CD3-S126 might be required for constitutive
TCR internalization (10, 15). Surprisingly, we found that
constitutive TCR cycling did not require PKC activity or
CD3-S126.
Thus, although treatment of cells with BAPTA-AM-EGTA completely
inhibited PKC-induced TCR internalization, constitutive TCR cycling was
unaffected by this treatment. In addition to PKC activity,
BAPTA-AM-EGTA treatment most likely inhibited other calcium-dependent
activities in the cell; however, this did not affect constitutive TCR
cycling. The independence of constitutive TCR cycling for PKC activity
was further substantiated by the independence of constitutive TCR
cycling for the PKC TCR substrate CD3-S126. It
could be suggested that the CD3 L-based motif is to some degree
exposed in nonligated, nonphosphorylated TCR and thus partially
available for interaction with the AP-2 components of the cellular
endocytic machinery. PKC-mediated phosphorylation of
CD3-S126 probably induces a conformational
change that fully exposes the CD3 L-based motif leading to an
increased association with the AP-2 complex and consequently to an
increase in the TCR internalization rate. In conclusion, we found that
constitutive cycling of the TCR was dependent on the CD3 L-based
motif but independent on CD3-S126 and PKC
activation.
By identification of some of the mechanisms required for constitutive
TCR cycling, we had the opportunity to examine ligand-induced TCR
internalization in cells with abrogated constitutive TCR cycling. We
found that ligand-induced internalization of the TCR was intact in two
independently derived cells with abolished constitutive TCR cycling due
to a disrupted CD3 L-based motif. This was observed by using both
anti-TCR mAb and superantigen as ligands and is in agreement with
previous studies, which showed that ligand-induced internalization of
the TCR is functional despite of a mutated or truncated CD3 L-based
motif (17, 34, 37). E3-WT cells were slightly more
sensitive for ligand-induced TCR internalization than JGN-WT and JGN-
and E3-L131/132A cells. This could indicate that cross-talk between
ligand-induced and PKC/CD3-dependent TCR internalization might take
place in these cells. However, from the present studies, we could
conclude that ligand-induced TCR internalization is functional in cells
with abolished constitutive TCR internalization and cycling.
We also found that the opposite was a fact, namely that constitutive TCR internalization was intact in cells with severely impaired ligand-induced TCR internalization. This strongly indicated that constitutive TCR cycling and ligand-induced TCR internalization represent two different pathways. Our results are in contrast to a recent work in which ligand-induced TCR internalization was suggested to be absolutely dependent on the constitutive TCR internalization pathway and where these two types of TCR internalization actually were proposed to be identical (11).
The physiological role of constitutive TCR cycling is unknown. It has
been suggested that constitutive cycling of the TCR might serve as a
quality check of the receptor as only fully functional and assembled
TCR can proceed through the entire cycling event. Receptors that are
not properly assembled may have lost the ability to mask the CD3
L-based motif and consequently are sorted to the lysosomes
(38). Such a task would require a motif that could
function both as an internalization signal as well as a lysosomal
sorting signal, which is in fact the case of the CD3 L-based motif
(26, 39, 40). Another role for constitutive cycling might
be to ensure a constant internal store of TCR. By generating an
internal store of TCR that can be rerouted to the site of stimulation,
constitutive TCR cycling may play a role in T cell activation. However,
even though several studies have shown that TCR are recruited to the
site of stimulation (41, 42, 43, 44, 45, 46), it is not known to which
degree these TCR originate from internal stores. Further clarification
of these issues awaits the analyses of the CD3 dileucine-mutated
knock in mice (C. Menné, M. C. Haks, A.M. Kruisbeek, and C.
Geisler, manuscript in preparation).
In conclusion, in this study we showed that constitutive TCR cycling is
not a requirement for ligand-induced TCR internalization and vice
versa. Furthermore, constitutive TCR internalization is dependent on
the CD3 L-based motif but independent of
CD3-S126 and PKC activity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carsten Geisler or Dr. Jes Dietrich, Institute of Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, Building 18.3, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark. E-mail address: cgtcr{at}biobase.dk
3 Abbreviations used in this paper: PKC, protein kinase C; PDB, phorbol 12,13-dibutyrate; SEE, staphylococcal enterotoxin E; BFA, brefeldin A; MFI, mean fluorescence intensity; L-based, leucine-based; WT, wild type; GFP, green-fluorescent protein.
Received for publication November 13, 2001. Accepted for publication March 29, 2002.
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References |
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production. Eur. J. Immunol. 31:2487.[Medline]
DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro. J. Cell Biol. 138:271. influences T cell responsiveness and controls T cell receptor cycling. J. Biol. Chem. 273:24232.
complexes after antigen stimulation. J. Exp. Med. 185:1859. chain: consequences for T cell antigen receptor assembly, processing, and expression. J. Immunol. 148:2437.[Abstract]
contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor. EMBO J. 13:2156.[Medline]
chain with the 
-T cell receptor/CD3 complex. J. Immunol. 145:1761.[Abstract]
chain is phosphorylated at serine 126 in response to T lymphocyte activation. J. Biol. Chem. 262:10918. subunit of the TCR contains endocytosis signals. J. Immunol. 163:25. complex, recruits intracellular signaling proteins to the plasma membrane. J. Exp. Med. 188:561. chain. J. Exp. Med. 193:1269.
or CD3 cytoplasmic domains, necessary in response to anti-CD3 antibody. Int. Immunol. 11:1731. allows stable expression of receptors containing the CD3 leucine-based receptor-sorting motif. J. Biol. Chem. 273:26281.This article has been cited by other articles:
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