Download Calcium Imaging and Electron Microscopy by Response to Antigen

Differential Roles of Lck and Itk in T Cell
Response to Antigen Recognition Revealed by
Calcium Imaging and Electron Microscopy
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of June 15, 2017.
Emmanuel Donnadieu, Valérie Lang, Georges Bismuth,
Wilfried Ellmeier, Oreste Acuto, Frédérique Michel and
Alain Trautmann
J Immunol 2001; 166:5540-5549; ;
doi: 10.4049/jimmunol.166.9.5540
http://www.jimmunol.org/content/166/9/5540
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Copyright © 2001 by The American Association of
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Supplementary
Material
Differential Roles of Lck and Itk in T Cell Response to
Antigen Recognition Revealed by Calcium Imaging and
Electron Microscopy1
Emmanuel Donnadieu,* Valérie Lang,2* Georges Bismuth,* Wilfried Ellmeier,† Oreste Acuto,‡
Frédérique Michel,3‡ and Alain Trautmann3,4*
O
ne of the earliest events occurring during TCR signaling
is the activation of different protein tyrosine kinases
(PTKs).5 Thus, it has been shown that Src kinases (Fyn
and Lck) are involved in the TCR-induced phosphorylation of the
different CD3 chains on their immunoreceptor-based activation
motif. This allows the SH2-dependent recruitment and the activation of ZAP-70, which leads, in turn, to the phosphorylation of key
downstream signaling molecules, such as linker for activation of T
cells (LAT) and SH2 domain-containing leukocyte protein of 76
kDa (SLP)-76 (1–3). The Tec family kinases also participate in
signal transduction pathways downstream of the TCR (for review,
see Refs. 4 and 5). In T cells, at least three members of this family,
Itk, Rlk/Txk, and Tec, are expressed. Genetic studies have underlined the importance of Itk in T cell function, as T cells from
Itk-deficient mice show impaired proliferation and Th2 differentiation (6, 7). Analysis of signaling events reveal that Itk⫺/⫺ and
*Laboratoire d’Immuno-Pharmacologie, Centre National de la Recherche Scientifique, Paris, France; †Institute of Immunology at Vienna International Research Cooperation Center, University of Vienna, Vienna, Austria; and ‡Molecular Immunology Unit, Department of Immunology, Institut Pasteur, Paris, France
Received for publication August 8, 2000. Accepted for publication January 17, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from Centre National de la Recherche Scientifique and by Ligue Nationale Centre le Cancer.
2
Current address: Division of Cellular Immunology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London, U.K. NW7 1AA.
3
F.M. and A.T. contributed equally to this work.
4
Address correspondence and reprint requests to Dr. Alain Trautmann, Laboratoire
d’Immuno-Pharmacologie, Centre National de la Recherche Scientifique UPR 415,
Institut Cochin de Génetique Moléculaire, 22 rue Méchain, 75014 Paris, France. Email address: [email protected]
Abbreviations used in this paper: PTK, protein tyrosine kinase; PLC-␥1, phospholipase C-␥1; KD, kinase-defective. LAT, linker for activation of T cells; SLP-76, SH2
domain-containing leukocyte protein of 76 kDa.
5
Copyright © 2001 by The American Association of Immunologists
Itk⫺/⫺ Rlk⫺/⫺ T cells have markedly decreased phospholipase
C-␥1 (PLC-␥1) tyrosine phosphorylation, inositol trisphosphate
production and Ca2⫹ mobilization, while the overall pattern of
immediate tyrosine phosphorylation appears to be normal (8, 9).
Thus, Itk in T cells is reminiscent of Btk, another Tec kinase,
which controls the Ag-dependent Ca2⫹ response in B cells (10).
Recent data suggest that Itk, through its interaction with the scaffolding membrane protein LAT and the adaptor protein SLP-76, is
involved in an amplification step that is necessary for a full activation of PLC-␥ and, therefore, of the Ca2⫹ response (11–13).
Most of these conclusions have been drawn from analysis of the
tyrosine phosphorylations induced by anti-CD3 Abs, which have
been very informative, but which fail to take into account cell-cell
interactions and, in particular, the spatial constraints imposed by
Ag recognition.
The importance of these spatial constraints has been underlined
in a number of recent reports. Thus, we and others have shown the
importance of the cytoskeleton alterations that can be detected together with and even before the Ca2⫹ response induced in T cells
by APC (Refs. 14 –16; for a review, see Ref. 3). Disruption of the
active cytoskeleton leads to inhibition of the Ca2⫹ response and
abrogation of T cell activation (16, 17).
In addition, major redistribution of cell surface molecules is
observed when T lymphocytes recognize an Ag on APCs. Analysis
of fixed T cell/APC conjugates reveals that cell surface molecules
segregate into distinct regions or clusters, referred to as supramolecular activation clusters, with the TCR/peptide-MHC accumulating in a central region surrounded by a peripheral ring of adhesion molecules (18). Recently, through the use of live T cells and
lipid bilayer in which fluorescently labeled molecules have been
incorporated, the dynamic nature of these redistributions has been
underlined (19). These reorganizations appear to be required for
full T cell activation. Hence, immunological synapse formation
correlates with full T cell activation, and any processes known to
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Ag recognition triggered at the interface between a T cell and an APC is conditioned by cell-cell adhesion and cytoskeletal
remodeling. The role played in these phenomena by Lck and Itk, two protein tyrosine kinases essential for T cell signaling, was
examined. Early T cell responses (membrane ruffling, Ca2ⴙ response, APC-T cell adhesion) were monitored in T cells overexpressing kinase-defective (KD) Lck and Itk mutants by combining fluorescence imaging and electron microscopy. Neither Lck nor
Itk appears to be involved in the Ag-independent formation of a small and labile contact interface between T cells and APCs. By
contrast, the Ag-induced Ca2ⴙ response in a cell population is similarly blunted in both KD transfectants. However, the underlying
mechanisms are strikingly different for the two kinases. The major effect of Lck-KD is to reduce the probability of giving rise to
quasi-normal Ca2ⴙ responses, whereas overexpression of Itk-KD results in a tuning down of all single-cell Ca2ⴙ responses. In
addition, Lck, but not Itk, is required for the formation of a stable T/APC conjugate and for T cell polarization after Ag
stimulation. Overall, our results lead to a clear distinction between Lck and Itk. Lck plays an ignition role, controlling all the
downstream events tested here, whereas Itk amplifies the Ca2ⴙ response, but is dispensable for APC-induced adhesive and
morphological responses. The Journal of Immunology, 2001, 166: 5540 –5549.
The Journal of Immunology
ondary Ab. After two washes in PBS-saponin and three in PBS-BSA, cells
were subjected to flow cytometry (FACScan; Becton Dickinson, San
Jose, CA).
For intracellular detection of Lck by fluorescence microscopy, cells
plated on microgrid coverslips were washed twice in PBS and fixed at
room temperature with 500 ␮l of a 3% paraformaldehyde PBS solution for
10 min. The fixed cells were washed in PBS and incubated for 20 min in
PBS containing 0.1 M glycine, then permeabilized with 0.1% Triton X-100
in PBS for 10 min at room temperature and blocked with PBS containing
0.2% BSA for 20 min. The anti-Lck mAb (clone 3A5; Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1 ␮g/ml in blocking solution for
30 min at room temperature. Then cells were washed three times and incubated with a 100-fold dilution of FITC-labeled goat anti-mouse Ab. After washing, the coverslip was mounted on a slide using ProLong antifade
reagent (Molecular Probes, Eugene, OR). Immunofluorescence and transmission light images were acquired with Image Pro Plus (Media Cybernetics, Carlsbad, CA) on a Leica microscope equipped with a Sensys 400
cooled CCD camera (Photometrics, Huntington Beach, CA). Quantitative
analysis of Lck fluorescence staining was performed with NIH Image on
regions of interest defined around individual cells.
PTK expression was measured by Western blot as previously described
(23). In brief, cells were rapidly pelleted and lysed for 20 min on ice in 1%
Nonidet P-40 lysis buffer containing 20 mM Tris-HCl (pH 7.5), 140 mM
NaCl, and 1 mM EDTA in the presence of proteases inhibitors. Proteins in
postnuclear supernatant were then separated on a 8% SDS-polyacrylamide
gels and blotted onto nitrocellulose. Itk was detected with the Abs used for
intracellular detection. For Lck, immunoblotting was performed with Abs
from Upstate Biotechnology. Enhanced chemiluminescence was used to
reveal specific PTKs.
Single cell Ca2⫹ video imaging
Materials and Methods
Cells
T8.1 is a murine T cell hybridoma expressing a chimeric human-mouse
TCR specific for a tetanus toxin peptide (tt830 – 843) restricted by HLADRB1*1102. The DR-expressing murine fibroblasts L625.7 used as APC
also express endogenous B7-1 and ICAM-2 (data not shown). This T/APC
system has been previously characterized and used to study biochemical
events triggered by Ag recognition (20, 21). T8.1 was maintained in
DMEM with 10% FCS, 2 mM L-glutamine, and antibiotics (complete medium) supplemented with 400 nM methotrexate, 1 mg/ml G418, and 50
␮M 2-ME. L625.7 cells were cultured in complete DMEM with 250 ␮g/ml
G418. Stable transfectants expressing PTKs-KD were grown in the presence of 1 ␮g/ml puromycin. Stable transfectants overexpressing Lck-KD
have been previously used and characterized (21).
Plasmid construct
The murine Itk cDNA in pBS was mutated at the ATP binding site (R390F)
and subcloned in the eukaryotic expression vector, pSR␣-puro, a derivative
of pcDL-Sr␣296 in which a puromycin resistance cassette had been inserted (22). The mutant was referred to as Itk-KD.
Cell transfections
Stable transfectants expressing different amounts of Itk-KD were obtained
as previously described (21). T8.1 cells (1 ⫻ 107 in DMEM and 20% FCS)
were electroporated in a Gene Pulse cuvette (Bio-Rad, Hercules, CA) with
30 ␮g of plasmid at 960 ␮F and 250 V. After 48 h in normal medium, cells
were placed in 96-well tissue culture plates in DMEM containing methotrexate (400 nM), G418 (1 mg/ml), and puromycin (1 ␮g/ml). Puromycinresistant transfectants were selected for expression of the T cell markers
CD3, CD4, CD28, and CD45 similar to that of parental T8.1 cells.
Detection of PTK expression
PTK expression was measured by flow cytometry, fluorescence microscopy, and Western blot. For intracellular detection by flow cytometry, T8.1
cells and KD transfectants were washed and resuspended in 0.5% PBSBSA (106 cells in 100 ␮l). Cells were fixed at room temperature by adding
500 ␮l of a 4% paraformaldehyde PBS-BSA solution for 20 min, washed
with PBS-BSA, and resuspended for 30 min in PBS containing 0.1% saponin and specific Abs or control isotypes. Monoclonal anti-Lck and antiItk were obtained from Transduction Laboratory (Lexington, KY) and Upstate Biotechnology (Lake Placid, NY), respectively. Cells were washed
three times in PBS-BSA-saponin and stained with a FITC-conjugated sec-
Twelve to 20 h before the experiment L625.7 cells (0.4 ⫻ 106) were plated
on glass coverslip-mounted petri dishes. Fibroblasts were pulsed with 5
␮g/ml of tt830 – 843 peptide unless otherwise stated. For successive measurements of Ca2⫹ and Lck levels in the same cells, fibroblasts were plated
on 175-␮m CELLocate microgrid coverslips (Eppendorf, Hamburg,
Germany).
T cells (5 ⫻ 105) were incubated for 20 min at 37°C with 1 ␮M fura2/AM (Molecular Probes). Cells were then washed and added to fibroblasts. Measurements of the intracellular calcium concentration were performed at 37°C in mammalian saline buffer (140 mM NaCl, 5 mM KCl, 10
mM HEPES (pH 7.3), 1 mM CaCl2, and 1 mM MgCl2) with a Diaphot 300
microscope (Nikon, Melville, NY) and an IMSTAR imaging system as
described previously (14). Averaged Ca2⫹ responses were calculated either
for all cells or for responding cells, i.e., cells showing an amplitude of Ca2⫹
increase of at least 80 nM. When several Ca2⫹ traces were averaged, the
rising phases of the traces were first synchronized so that the time course
of the average was not “filtered” by the asynchrony of the different responses. Transmission light images acquired to visualize the APCs interacting with the T cells were taken every 10 s in turn with fluorescence
images.
Adhesion assay
Ag-independent and -dependent adhesion between murine T cells hybridoma and L625.7 cells was quantified as follows. Fibroblasts were cultured
overnight on petri dishes mounted with glass coverslips, in the presence of
0, 0.5, or 5 ␮g/ml of peptide. Fura-2-loaded T8.1 cells were left to adhere
to fibroblasts at 37°C in 2 ml of medium. After 30 min of interaction,
fluorescent images of several microscopic fields with at least 50 T cells
were acquired. Standardized washes were then applied to cells. To this end,
the medium was rapidly removed, and 2 ml of prewarmed solution was
added either at the center of the dish for strong washes or at the periphery
of the dish for mild washes. Two rounds of washes were preformed for
each condition. The number of adherent T cells was counted from processed fluorescent images before and after washes. The percentage of remaining cells was calculated from two or three independent experiments.
Scanning electron microscopy
Twelve to 20 h before the experiment L625.7 cells (0.4 ⫻ 106) were plated
on glass coverslip-mounted petri dishes. Fibroblasts were pulsed with 5
␮g/ml of tt830 – 843 peptide unless otherwise stated. T8.1 cells and transfectants were allowed to adhere to L625.7 for 5 and 20 min at 37°C in mammalian saline buffer. Cells were fixed at room temperature with 2.5% prewarmed glutaraldehyde for 30 min. After washing, the samples were
dehydrated in five successive and graded ethanol baths (from 25 to 100%),
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prevent T cell activation such as antagonist peptides, fail to trigger
the redistribution of surface molecules (19).
The intracellular signaling events required for these earliest responses are to a large extent undetermined. In a previous work we
have begun to characterize the signaling pathway linking Ag recognition to T cell membrane ruffling and Ca2⫹ increase in murine
naive T lymphocytes during their interaction with B cells (16).
Using a pharmacologic approach, our study highlighted the major
role played by PTKs because Ag-induced cytoskeletal responses
were entirely blocked by PTKs inhibitors.
Here we have analyzed more precisely the role played by two
PTKs, Lck and Itk, in the earliest steps of Ag recognition. To this
end, we have used a dominant-negative approach based on overexpression of catalytically inactive mutants of these PTKs in a
murine T cell hybridoma. The effects of Lck and Itk kinase-defective (KD) mutants in early Ag-induced T cell responses (conjugate
formation, membrane ruffling, Ca2⫹ response, T-APC adhesion)
were then monitored by combining video imaging, electron microscopy, and adhesion measurements. Our results show that none
of these PTKs is involved in Ag-independent adhesion, and that
both contribute by distinct mechanisms to a full-blown Ca2⫹ response. In addition, Lck-KD and Itk-KD differ in that only Lck,
not Itk, is strictly required for the control of Ag-driven morphological and adhesive responses.
5541
5542
DIFFERENTIAL ROLES OF Lck AND Itk IN T CELL RESPONSE TO Ag RECOGNITION
dried by the critical point method using liquid CO2, coated with gold by
sputtering, and observed with a scanning electron microscope (JSM.840.A,
JEOL, Peabody, MA).
Quantitation of shape changes
The percentage of polarized T cells able to form a lamellipodium during
their interaction with peptide-pulsed L625.7 cells was scored from transmission light images. At least 30 cells were analyzed from each separate
experiment. T/APC contact length was quantitated using NIH Image software. The largest visible diameter of the T cell protrusion at the contact
interface between the two cells was measured from scanning electron microscope images.
Statistics
Data are expressed as mean ⫾ SD, and the significance of differences
between two series of results was assessed using Student’s t test.
Results
To delineate the functional role of Lck and Itk in controlling
Ca2⫹, morphological, and adhesive responses induced in T cells
by APCs, we used a dominant-negative mutant approach in the
murine T cell hybridoma T8.1 (20). Stable transfectants expressing different amounts of KD mutants of Lck (21) and Itk
were used.
The level of expression of each kinase in two different clones
for each type of transfectants was compared with the level of
expression of the wild-type endogenous counterpart detected in
T8.1 cells (Fig. 1). Overexpression of PTKs-KD was evaluated
by Western blot and flow cytometry after intracellular staining.
Analysis of the intracellular staining allows measurement not
only of the average expression of the mutant PTK but also of
the variability of their expression in the cell population. This
distribution appeared homogeneous and unimodal in all cases.
As previously reported (21) the level of Lck detected by Western blot in T8.1 cells was very low. T8.1 cells also express a
barely detectable level of endogenous Itk. Surface expressions
FIGURE 1. PTK expression measured in KD transfectants and T8.1
cells. PTK expression was measured in two ways: intracellular staining (A)
and Western blots (B). Two different clones for each condition are
presented.
Morphological alterations and Ca2⫹ response induced by APCs
in T8.1 cells
The normal alterations in the morphology and the intracellular
Ca2⫹ concentration of T8.1 cells induced by Ag stimulation were
first visualized by scanning electron microscopy and fluorescence
imaging of fura-2-loaded T cells. T cells interactions were monitored using as APC, class II-transfected murine fibroblast, L625.7
cells (20). At the beginning of a typical 20-min experiment, T8.1
cells were allowed to settle on a monolayer of L625.7 cells that had
been previously pulsed, or not, with 5 ␮g/ml of tetanus toxin
(tt830 – 843) peptide for 12 h.
Fig. 2A and movie 16 show a sequence of images captured
every 18 s in one T8.1 cell interacting with peptide-pulsed
APCs. The first detectable event was the formation of a stable
T/APC conjugate in which the T cell contacted an APC through
fine cellular processes (marked by arrows). The T cell origin of
these processes is clearly visible on movie 1. After an average
delay of 207 ⫾ 87 s (n ⫽ 14 cells), this contact led to a large
and sustained Ca2⫹ increase. Three individual Ca2⫹ responses
elicited in T8.1 cells by L625.7 are shown in Fig. 2B. The
percentage of Ca2⫹-responding cells was then analyzed. Cells
were scored as responsive when the minimal Ca2⫹ increase was
⬎80 nM. Due to the high frequency of T8.1 cells presenting a
Ca2⫹ response (71 ⫾ 15% of the cells in seven separate experiments in which ⬎30 cells were analyzed/experiment), the average response of all cells (dotted line) is close to the average
response of responding cells (continuous line).
This Ca2⫹ response was entirely Ag specific. Indeed, in the
absence of Ag, no Ca2⫹ response occurred despite the formation of numerous T-APC conjugates. The small junction observed in the absence of Ag looked quite similar to the transient
one observed in the presence of Ag, just before the Ca2⫹ response started (not shown).
The detailed structure of the contact zone cannot be visualized
in transmitted light images, but can be further analyzed by scanning electron microscope (Fig. 2C). In the absence of Ag, T8.1
interacted with L625.7 cells by a bundle of filopodia forming a
structure with an average diameter at the contact interface of
2.03 ⫾ 0.63 ␮m (n ⫽ 7 cells; left panel of Fig. 2C). In the presence
of Ag, this initial phase was followed by marked morphological
changes, first characterized by the appearance of extended lamellipodia projected from the T cell body toward the APC. This cell
polarization, which “blossoms” in a few seconds (as judged from
video recordings), is illustrated in an electron micrograph shown in
the middle panel of Fig. 2C where a T cell had been in contact with
the APC for 5 min. At this stage, two distinct compartments can be
distinguished in the T cell: a small and shrunken cell body and a
spectacular lamellipodium. The average diameter of the lamellipodium measured at the cell-cell contact interface was 7.18 ⫾ 2.61
␮m (n ⫽ 8 cells). The cell body appears rounded and dented,
whereas the lamellipodium is smooth, with a prominent leading
edge. Note that the surfaces of both structures display very few
microvilli, which contrasts with the spherical unstimulated T cells,
which are covered with short microvilli. After a few minutes, T
cells revert to a more spherical shape, but an active membrane
6
The on-line version of this article contains supplemental material.
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Characteristics of stable transfectants overexpressing KD forms
of Lck and Itk
of CD3, CD4, CD28, and CD45 were similar in transfectants
and T8.1 cells (data not shown). IL-2 production in response to
Ag stimulation was inhibited in T8.1 cells expressing PTKsKD; the inhibition was positively correlated to the expression of
PTKs-KD (data not shown).
The Journal of Immunology
5543
FIGURE 3. Different effects of Lck-KD on Ag-induced T cells responses. A, Examples of three Lck-KD 3.4 transfectants added to L625.7 cells. Image
interval, 23 s. The arrow indicates the cell that displays near-normal cytoskeletal and Ca2⫹ responses. B, Three single-cell Ca2⫹ responses and the average
Ca2⫹ response measured only in responding cells (—) or in all cells (----). C, Scanning electron microscope analysis of conjugates between Lck-KD 3.4
transfectants and L625.7 cells. Bars ⫽ 1 ␮m.
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FIGURE 2. Ag-induced cytoskeletal and Ca2⫹ responses in wild-type T8.1 cells. A, Example of one T8.1-L625.7 cell interaction. Image interval, 18 s.
The arrows indicate the small contact interface observed before the Ca2⫹ increase. B, Three single-cell Ca2⫹ responses and the average Ca2⫹ response
measured only in responding cells (—) or in all cells (----). C, Scanning electron microscope analysis of conjugates between T8.1 cells and L625.7 cells.
Bars ⫽ 1 ␮m.
5544
DIFFERENTIAL ROLES OF Lck AND Itk IN T CELL RESPONSE TO Ag RECOGNITION
ruffling persists, essentially restricted to the contact interface between the two cells. One example of a T8.1 cell in contact with
L625.7 cells for 20 min is illustrated in the right panel of Fig. 2C.
A typical sequence of events occurring in a T8.1 cell in interaction
with an Ag-pulsed APC, captured with the imaging setup, is shown
in movie 2.
Effect of Lck-KD on Ag-induced Ca2⫹ responses and
morphological alterations
Effect of Itk-KD on Ag-induced Ca2⫹ responses and
morphological alterations
Fig. 5A and movie 4 illustrate typical Ca2⫹ and morphological
responses of two Itk-KD 2.5 transfectants interacting with Agpulsed APCs. In one cell marked by the arrow a weak Ca2⫹ increase was observed despite extensive cell shape changes (open
arrow). The other cell remained unresponsive during the course of
the experiment.
The percentage of Itk-KD transfectants (clones 2.5 and 3.11)
presenting a Ca2⫹ response was modestly reduced relative to
that of control T8.1 cells. At 5 ␮g/ml of Ag, 49 ⫾ 4% (n ⫽ 3
experiments in which ⬎30 cells were scored/experiment) of
Itk-KD 2.5 cells showed a Ca2⫹ response (compared with 71 ⫾
15% for T8.1 cells; p ⬍ 0.05). Similar results were obtained
with Itk-KD 3.11 transfectants expressing a higher amount of
mutated Itk (see below).
If it did not markedly affect the percentage of responding cells,
KD-Itk had profound effects on the characteristics of the Ag-induced Ca2⫹ response. These responses were not only smaller, but
also briefer, than their control counterparts. Examples of such
Ca2⫹ responses, measured in single Itk-KD 2.5 cells, are shown in
Fig. 5B.
In contrast to these strongly impaired Ca2⫹ responses, the
changes in T cell shape induced by Ag-loaded APCs were not
markedly altered. When L625.7 cells were pulsed with 5 ␮g/ml
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The specific Ca2⫹ and morphological responses elicited in
Lck-KD transfectants were examined next. Fig. 3A and movie 3
show an example of three individual Lck-KD 3.4 cells that have
been added to peptide-pulsed APCs. In two of the three T cells
shown, no Ca2⫹ increase or shape changes were detected despite
the existence of a contact with the APC. However, one T cell
(marked by an arrow in Fig. 3A) displayed marked cytoskeletal and
Ca2⫹ responses.
The percentage of Lck transfectants that presented a clear contact with the APC and showed an increase in Ca2⫹ ⬎80 nM was
then analyzed. In the presence of APCs loaded with 5 ␮g/ml of Ag,
the percentage of Lck-KD 3.4 transfectants showing a Ca2⫹ response (32 ⫾ 14%; n ⫽ 4 experiments in which ⬎30 cells were
scored/experiment) was markedly reduced compared with that of
control T8.1 cells (71%; p ⬍ 0.003). When the APCs were loaded
with 0.5 ␮g/ml peptide, the difference between Lck-KD cells and
control cells was even more marked (see below). Similar results
were obtained with another Lck-KD transfectant expressing less
mutated Lck.
Remarkably, in the cells expressing Lck-KD that did show a
Ca2⫹ response, the amplitude of this Ca2⫹ increase was only
slightly smaller than the control response detected in T8.1 cells (in
the two Lck-KD transfectants, 8% smaller, on the average, than the
response measured in T8.1 cells). Examples of individual Ca2⫹
responses elicited in Lck-KD 3.4 transfectants by L625.7 cells are
shown in Fig. 3B. Note that the rate of rise of the Ca2⫹ increase in
Lck-KD cells was significantly slower than that in wild-type cells
and that two distinct phases could be distinguished, as if the Ca2⫹
response hesitated to start. Thus, the global Ca2⫹ response elicited
in a cell population was smaller in Lck-KD transfectants essentially because the percentage of responding cells was reduced. As
a result, this global Ca2⫹ response differed from the average response of responding cells (Fig. 3B), contrary to what had been
observed in T8.1 cells.
We next assessed the influence of Lck on the morphological
modifications induced in T8.1 cells by APCs. Scanning electron
microscope experiments showed that the small protrusion formed
by the Lck-KD 3.4 (Fig. 3C, left panel) transfectant toward unpulsed APCs was very similar to that observed in control T8.1
cells. The diameter of the small protrusion measured in Lck-KD
3.4 cells (1.52 ⫾ 0.94 ␮m; n ⫽ 6 cells) was comparable to that
measured in T8.1 cells (2.03 ⫾ 0.63 ␮m; n ⫽ 7 cells). By contrast,
the percentage of Lck-KD 3.4 transfectant able to form a conspicuous lamellipodium in the presence of Ag (34 ⫾ 12%; n ⫽ 5
experiments in which ⬎30 cells were scored/experiment) was very
much reduced compared with that in parental T8.1 cells (73 ⫾
16%; n ⫽ 9; p ⬍ 0.0003). Similarly, the diameter of the T/APC
contact zone for Lck-KD 3.4 cells (4.24 ⫾ 2.86; n ⫽ 13) was
significantly smaller than that of parental T8.1 cells (7.18 ⫾ 2.61
␮m; n ⫽ 8; p ⫽ 0.029). The middle and right panels of Fig. 3C
illustrate the morphology of Lck-KD 3.4 transfectants after 5 min
of interaction with peptide-pulsed APC. A majority of cells did not
change their shape (middle panel), whereas a few cells displayed
large, well-shaped lamellipodia (right panel).
Next we examined why the Ca2⫹ response in Lck-KD transfectants seems to be almost all or none. One hypothesis is that the
amount of dead kinase expressed in a given transfectant cell controls the ignition of the response, and that in the lowest Lck-KD
expressors, partial inhibition of the endogenous Lck is not sufficient to inhibit the triggering of the signal. This hypothesis was
tested by examining whether there is a correlation between the
expression of Lck-KD measured at the single-cell level and the
existence of an APC-induced Ca2⫹ response in the very same
cells. Abs against Lck recognize both endogenous and Lck-KD.
However, the staining of endogenous Lck in wild-type cells was so
faint (data not shown) that all significant staining in transfectants
can be attributed to Lck-KD.
Lck-KD 3.4 transfectants were allowed to settle on a monolayer
of peptide (5 ␮g/ml)-pulsed L625.7 cells. The Ca2⫹ level in individual cells was monitored during the first 10 min of T/APC interaction. Cells were then fixed, permeabilized, and stained with an
anti-Lck mAb followed by a FITC-labeled goat anti-mouse Ab.
Fig. 4A shows the Ca2⫹ level and staining of Lck in three individuals Lck-KD 3.4 cells interacting with peptide-pulsed APC. It
is clear in this example that Ca2⫹ responses are observed in the
two cells showing the lowest level of Lck (i.e., essentially
Lck-KD).
The amount of Lck was then quantified by measuring the
fluorescence intensity on regions of interest defined around individual cells. Fig. 4B shows the distribution of Lck in Ca2⫹responding and nonresponding individual cells. The Ca2⫹-responding cells expressed 27.3 ⫾ 7.6 fluorescence intensity units
(n ⫽ 45 cells), whereas the nonresponding cells expressed
32.6 ⫾ 11.9 (n ⫽ 36). This difference in the amount of Lck was
statistically different ( p ⬍ 0.02). In fact, this was an underestimation of the true difference, because a fraction of the least
adhering cells may have been lost during the washes because
strong expression of Lck-KD leads to reduced APC/T cell adhesion (see below). These data suggest a correlation between
the amount of dead kinase expressed in individual cells and the
inhibition of the Ca2⫹ response.
The Journal of Immunology
5545
FIGURE 5. Overexpression of Itk-KD mutant inhibits the Ag-induced Ca2⫹ response, but not cytoskeletal alterations. A, Examples of two Itk-KD 2.5
transfectants added to L625.7 cells. Image interval, 40 s. The black arrow points to the cell displaying a weak Ca2⫹ increase despite extensive morphological
changes. The white arrows point to the lamellipodium, which was too thin to give rise to a detectable fluorescent signal. B, Three single-cell Ca2⫹ responses
and the average Ca2⫹ response measured only in responding cells (—) or in all cells (---). B, Scanning electron microscope analysis of conjugates between
Itk-KD 2.5 transfectants and L625.7 cells. Bars ⫽ 1 ␮m.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 4. Correlation between the amount of Lck-KD and the inhibition of the Ca2⫹ response. A, Measurement of the Ca2⫹ level and the amount of
Lck in the same cells. Upper panel, Ca2⫹ level in three Lck-KD 3.4 cells in interaction with L625.7 cells for 5 min (left) and the corresponding transmission
light image (right). Lower panel, Amount of Lck in the same three cells revealed by immunofluorescence (left) and the corresponding transmission light
image (right). After 10 min of interaction, cells were fixed, permeabilized, and stained with an anti-Lck mAb followed by FITC-conjugated goat anti-mouse
IgG Ab. The fluorescence level was coded with an 8-bit gray scale under nonsaturating conditions. B, Distribution of Lck-KD level in Ca2⫹ responding
and nonresponding cells. The amount of Lck was determined by measuring the fluorescent intensity of every Ca2⫹-responding and nonresponding individual
cells. Each dot represents one cell. The mean for each group is indicated by a white dot. Values were pooled from three independent experiments. The
mean ⫾ SD for each group were calculated and compared using unpaired t test.
5546
DIFFERENTIAL ROLES OF Lck AND Itk IN T CELL RESPONSE TO Ag RECOGNITION
of Ag, a majority (53 ⫾ 15%; n ⫽ 5 experiments in which ⬎30
cells were scored/experiment) of Itk-KD 2.5 cells formed a conspicuous lamellipodium, i.e., slightly less than in T8.1 cells
(73 ⫾ 16%; p ⬍ 0.03). The contact length measured in Itk-KD
2.5 interacting with APCs (6.05 ⫾ 2.37; n ⫽ 11) was not statistically different from that measured in T8.1 cells interacting
with APCs (7.18 ⫾ 2.61 ␮m; n ⫽ 8). Examples of shape
changes at 5 and 20 min after the initial contact are illustrated
in Fig. 5C (middle and right panels).
Comparison between Ag-induced Ca2⫹ responses in Lck-KD
and Itk-KD transfectants
As mentioned above and shown in more detail in Fig. 6A, the
percentage of cells showing a Ca2⫹ response in the presence of
Ag-pulsed APCs was more profoundly affected by the inhibition of
Lck than by that of Itk. The difference between the two KD PTKs
was most obvious at a nonsaturating Ag concentration (0.5 ␮g/ml).
Fig. 6B illustrates the fact that if the average Ca2⫹ response in
the whole cell population is significantly reduced in both Itk-KD
and Lck-KD transfectants, the average response in responding
cells is mostly affected in Itk-KD transfectants. This conclusion
holds true for two clones that express different levels of the two
KD PTKs. This clearly points to distinct mechanisms underlying
Lck- and Itk-dependent inhibition of the Ca2⫹ response.
Effects of Lck-KD and Itk-KD on Ag-induced T/APC adhesion
Because full T cell activation requires a long-lasting APC-T cell
interaction (24), it is important to evaluate not only the initiation of
FIGURE 7. Ag-independent and -dependent adhesion measured in LckKD, Itk-KD, and T8.1 cells. A, In the absence of Ag, the percentage of
remaining cells was scored after mild washes. The control represents nonspecific binding of T cells to HeLa cells. B, In the presence of Ag, the
percentage of remaining cells was scored after strong washes.
TCR signaling, but how it can be sustained by a prolonged T-APC
interaction. The impact of Lck and Itk-KD was thus evaluated on
the adhesion of T cells to L625.7 cells pulsed, or not, with the
peptide.
T8.1 cells were left to adhere to L625.7 cells for 30 min, a
duration allowing maximum adhesion (25). At this time cells were
counted before and after standardized washes. By using washes of
two different intensities, either mild or strong, we were able to
distinguish among three levels of adhesion: nonspecific, light, and
strong.
A monolayer of HeLa cells was used as a negative control for
the evaluation of nonspecific binding. Thus, 16 ⫾ 2.8% of T8.1
cells were still in contact with HeLa cells after mild washes (and
zero after strong washes). In contrast, when T cells were left to
adhere to unpulsed L625.7 cells, 64 ⫾ 19% of the T cells were still
in contact with L cells after mild washes. Thereafter, strong
washes removed all these T cells. Thus, significant light adhesion
was measured between T8.1 cells and APC-expressing MHC class
II, B7-1, and ICAM-2 in the absence of Ag (Fig. 7A). The Agindependent adhesion to L625.7 cells, measured after mild washes
was not statistically different between T8.1 cells and Lck and
Itk-KD transfectants (Fig. 7A).
Ag-dependent adhesion was then quantified (Fig. 7B). After
strong washes, 40% of T8.1 cells still adhered to L625.7 cells that
had been pulsed with 0.5 or 5 ␮g/ml of peptide. Overexpression of
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FIGURE 6. Effects of Lck and Itk-KD mutants on the characteristics of
the Ag-induced Ca2⫹ response. A, Percentage of Ca2⫹-responding T8.1
cells and transfectants as a function of the Ag concentration. Only cells
presenting an increase in Ca2⫹ ⬎80 nM and a clear contact with L625.7
were taken into account. B, Average Ca2⫹ response of T8.1, Lck-KD, and
Itk-KD cells measured either in all cells or only in responding cells.
The Journal of Immunology
KD forms of Lck induced a significant reduction of Ag-induced T
cell adhesion. For this PTK, the most severe inhibition was observed with the highest expressor of Lck-KD (clone 3.4). This
result is in sharp contrast with what was observed with Itk-KD
transfectants (clones 2.5 and 3.11), which adhered to L625.7 cells
nearly as well as T8.1 cells.
Taken together these data show that none of the KD mutants
affected the basal light Ag-independent adhesion. In addition, KD
forms of Lck had severe effects on the stability of the conjugates,
while Itk-KD did not affect this stability.
Discussion
main difference does not reside so much in the affinities but, rather,
in the relative abundance of the ligands for CD2 and CD28 compared with the very small number of specific MHC-peptide complexes sufficient to trigger a response (34, 36, 37). We have shown
here that this Ag-independent adhesion is weak, but measurable,
and that it is not affected by the inhibition of activity of Lck and
Itk. Additional experiments indicate that KD forms of Fyn and
ZAP-70 are unable to inhibit this Ag-independent adhesion (data
not shown). In transmitted light images, one can simply ascertain
that an Ag-independent contact exists, as judged namely by the
sudden immobilization of the T cell, which emits poorly resolved
cellular processes. The characteristics of this tiny contact zone
were revealed by scanning electron microscopy: a bundle of filopodia (which might have been former microvilli) forming a small
protrusion ⬍1 ␮m in diameter. It is tempting to draw a parallel
between the initial T/APC interaction and the contact between a
leukocyte and an endothelial cell even though adhesion molecules
are probably different in both cases. Initial contact between a leukocyte and an endothelial cell is made by adhesion molecules (Lselectin, ␣4 integrin) preferentially localized at the tip of leukocyte
microvilli (38, 39). Indeed, microvilli are particularly well suited
to promote the initial T/APC interaction. Beside the presence of
adhesion receptors, microvilli are enriched in actin bundles and
cytoskeletal proteins such as ezrin, which link adhesion molecules
to actin (40, 41). The functional importance of microvilli in the
context of T/APC Ag-independent interaction deserves further
investigation.
Interestingly, Ag-independent interaction with L625.7 cells
never triggered Ca2⫹ responses in T8.1 cells, contrary to what has
been reported when T cells interact with dendritic cells (34, 42). It
is conceivable that the dendritic cell-induced signal requires a molecule that is uniquely expressed on dendritic cells, such as DCSIGN (43). Alternatively, the specific ability of dendritic cells to
induce an Ag-independent signal may result from their high expression level of costimulatory molecules (35).
Ag-induced Ca2⫹ responses are obviously conditioned by the
adhesion events just discussed. However, some additional, specific
features of Ag-induced Ca2⫹ responses deserve further examination. The inhibition of Lck and Itk leads to an apparently similar
reduction of the Ag-induced Ca2⫹ responses averaged over a number of T cells. However, the single-cell analysis performed in the
present work allowed us to make a clear distinction between the
two modes of inhibition. The inhibition of Lck resulted essentially
in a reduced probability of triggering a Ca2⫹ response, but in the
few cells that did respond, the amplitude of the Ca2⫹ response was
90% that of the control (even though the kinetics of the response
were slowed). This trend toward an all-or-none inhibition of the
Ca2⫹ response is suggestive of a threshold effect; when a sufficiently small fraction of Lck is inactivated, the signaling process
remains unaltered, and when enough Lck is inactivated, the signaling cascade is completely blocked. Although in the two clones
used the expression of the dead kinases was unimodal, as judged
by its measurement by flow cytometry, we have shown that the
difference between the low and high expressors contributes to allow the lowest dead kinase expressors to escape from the
inhibition.
The situation was different when Itk was inhibited. In this
case, a Ca2⫹ response could still be triggered in a large fraction
of the T cells (70% of the control), but the amplitude of these
responses was strongly reduced (by ⬎50%). In this case, there
was no all-or-none effect but, rather, a global tuning of the
whole response.
These results suggest that in Ag-induced Ca2⫹ signaling Lck
plays an ignition role, while Itk plays an amplification role. This
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In the present work by combining Ca2⫹ imaging, electron microscopy, and cell-cell adhesion measurements, we have analyzed the
functional importance of Lck and Itk in Ag recognition by T cells.
To compare the roles of the two PTKs in the same T/APC system,
KD forms of these PTKs were stably overexpressed in a murine T
cell hybridoma. With this type of approach, one cannot exclude
that overexpression of one PTK-KD may result in the inhibition of
a group of PTKs (for instance, overexpression of Itk-KD may inhibit not only Itk but also other members of the same family, such
as Rlk or Tec). However, this should not affect our main conclusion concerning the differential regulation exerted by kinases typified by Lck and Itk, during T-APC interaction.
Lck and Itk have been shown to play an important role in T cell
development and TCR signaling (for a review, see Ref. 26). However, many questions remain unanswered, in particular the participation of Lck and Itk in the signaling pathways leading to alterations of the T cell cytoskeleton. This last point is of importance,
because actin remodeling is thought to be essential for T cell activation (3, 16, 17). In a previous study using anti-CD3-coated
beads and variants of Jurkat T cells defective in Lck and ZAP-70,
Lowin-Kropf et al. pointed to the participation of these two kinases
in actin remodeling and reorientation of the microtubule-organizing center (27). However, anti-CD3 stimulation does not necessarily mimic all aspects of APC stimulation. By using a more
physiological setting, we have examined in more detail the APCinduced changes in T cell shape. They begin with the rapid formation of a large lamellipodium protruding from the T cell body
toward the APC. This allows the T cell to translocate most of its
cytoplasmic and plasma membrane material to the contact interface. Indeed, a redistribution of both cytoplasmic vesicles and cell
surface molecules toward the contact zone has been described (for
a review, see Ref. 28). In addition, the large lamellipodia protruded
toward the APC increases the contact zone size, which is thought
to stabilize the contact between T cells and APCs. We have shown
that these events are dependent on Lck, but not on Itk. Recent data
have provided insight into the molecular chain linking the TCR to
actin. A multimolecular complex including SLP-76, SLP-76-associated protein (Fyb/SLAP), Nck, Vav, and Wiskott-Aldrich syndrome protein (WASP) is suggested to play an important role in
these processes (29 –33). Based on our results, one could hypothesize that Lck, but not Itk, should be involved in the formation of
this complex.
Another important question that has been addressed here is that
of the APC-T cell adhesion. We and others have previously shown
that T-APC adhesion can take place in the absence of Ag and
presumably constitutes a first checkpoint in Ag recognition (34,
35). Even when Ag is present at the APC surface, it is likely that
the initial T-APC interaction occurs in an Ag-independent way,
because the avidity of a number of costimulatory molecules, such
as CD2 and CD28, for their respective ligands is likely to be much
larger than that of TCR for cognate MHC-peptide complexes. The
5547
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DIFFERENTIAL ROLES OF Lck AND Itk IN T CELL RESPONSE TO Ag RECOGNITION
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
Acknowledgments
28.
We thank Clotilde Randriamampita, Jérôme Delon, and Patrick Revy for
their comments on the manuscript.
29.
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triggering of a Ca2⫹ response is clearly dependent upon the
amount of KD-PTK expressed in the clone. By contrast, once
the response is initiated, it becomes almost insensitive to the
amount of KD-PTK, as the amplitude of the Ca2⫹ signal is
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