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Pfizer Global Research and Development, Fresnes, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in Th2 than in Th1 cells after TCR engagement. Taken
together, these results constituted evidence that early events in the
TCR signaling cascades are distinct in human Th1 and Th2
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Imbalance between Th1 and Th2 cell-mediated functions has become a paradigm often referred to as an explanation for immune-related disorders (6). Initially, Th1 and Th2 subsets were defined by the distinct profiles of cytokine they produce (7). Several surface markers were also shown to be preferentially expressed by Th1 and Th2 cells (8), such as signaling lymphocyte activation molecule (9) and chemoattractant receptor-homologous molecule expressed on Th2 cells (10), respectively. Recently, the transcription factors T-bet and GATA-3 (11) and the Tec family tyrosine kinases Txk (12) and Itk (13) were reported to control the differentiation of Th subsets. Even though progress has been made in deciphering the mechanisms of differentiation, little is known concerning the signaling events in already differentiated cells.
Identification of molecules specifically involved in TCR-mediated signaling in human Th1 or Th2 subsets would be helpful to define new therapeutic targets implicated in immunopathological processes. In the mouse model, TCR engagement, mimicked by anti-CD3 Ab cross-linking, was found to elicit distinct TCR-associated signaling pathways in Th1 and Th2 clones. In particular, calcium fluxes were reduced in Th2 compared with Th1 cells (14). Earlier TCR-mediated activation signaling events were also shown to be distinct in Th1 and Th2 clones. Namely, the activity of the tyrosine kinases Fyn and ZAP-70 was reduced in Th2 lymphocytes (15). While activation events distinguishing Th1 and Th2 cells have been documented in the mouse, to our knowledge no such differences in the human system have been reported to date. Indeed, it was proposed that TCR signaling pathways are identical in human Th1 and Th2 cells. This idea was suggested by the observations that TCR engagement, mimicked by anti-CD3 Ab cross-linking, resulted in similar protein tyrosine phosphorylation profiles in both Th1 and Th2 cells (16). Thus, observations made on Th cells in the murine and human systems are apparently at variance. One explanation is that murine and human Th1 and Th2 cells represent Th subsets with distinct features in these two species (17).
The aim of this work was to study the requirements for T cell
activation and TCR-mediated signaling events distinguishing human Th1
and Th2 cells. The results presented document that human Th1 and Th2
cells have distinct activation characteristics when engaged with
immobilized peptide/DR1 complexes or anti-CD3 mAb. A biochemical
analysis of TCR signaling events induced by anti-CD3 Ab revealed
fainter tyrosine phosphorylation profiles and a weaker CD3/ZAP-70
association in human Th2 than in Th1 cells. Distinct proximal events of
the TCR signaling cascades between Th1 and Th2 cells might be due to
distinct profiles of phosphatases found in Th1 and Th2 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were obtained by Ficoll-Paque (Amersham Pharmacia Biotech, Orsay, France) density gradient centrifugation of heparinized blood from healthy donors. Measles virus fusion protein-derived peptide 254–268 (F254)-specific, HLA-DRB1*0101-restricted Th1 and Th2 clones were isolated from PBMC (1.5 x 105 cells/200 µl) cultured for 1 wk in the presence of 100 ng/ml of F254 peptide. The cultures were performed under Th1- and Th2-polarizing conditions as previously described (18). Briefly, for differentiation of Th1 cells, cultures were supplemented with 2 ng/ml IL-12 (BD Biosciences, San Jose, CA) and 125 ng/ml anti-IL-4 mAb (clone 8D4-8; BD Biosciences). For differentiation of Th2 cells, cultures were supplemented with 7 ng/ml IL-4 (R&D Systems, Minneapolis, MN) and 2 µg/ml anti-IL-12 mAb (clone C8.6; BD Biosciences). Isolation and maintenance of the Th clones were previously reported in detail (19). The three Th1 (F254.7.2, SDMV3.13, and Th1 no. 4) and three Th2 (FR6.6, SDMV3.5, and Th2 no. 1) clones used in this study were isolated from independent cultures, and from two donors.
T cell lines were established by culture of PBMC at 106 cells/ml for 2 wk with 10 ng/ml toxic shock syndrome toxin superantigen (Sigma, Saint Quentin Fallavier, France) in the presence of cytokines and mAb for differentiation into Th1 and Th2 cells, as described for the T cell clones.
Th1 and Th2 polarization was determined by ELISA. Cells producing
IFN-
, but not IL-5, were considered Th1, and cells producing IL-5,
but not IFN-, were considered Th2. Since differentiation of Th2
cells was conducted in medium containing IL-4, IL-5, rather than IL-4,
was determined to establish Th phenotypes.
T cells were cultured in RPMI medium (Life Technologies, Cergy Pontoise, France) supplemented with glutamine, sodium pyruvate, nonessential amino acids, kanamycin, 5% decomplemented AB human serum, and 100 U/ml IL-2 (Tebu, Le Perray en Yvelines, France). Th1 and Th2 cells were simultaneously restimulated for expansion, and were used at least 15 days after restimulation.
The HLA-DR1-transfected HeLa cell line was cultured in RPMI medium supplemented with glutamine, sodium pyruvate, nonessential amino acids, kanamycin, 10% decomplemented FCS, and 1 mg/ml geneticin (Life Technologies) (20).
Antibodies and chemicals
For T cell stimulation, anti-human CD3 mAb TR66 (mouse IgG1)
was a gift from Dr. Lanzavecchia (Biomedical Research Institute,
Bellinzona, Switzerland). Anti-human CD28 mAb (clone CD28.2, mouse
IgG1) and goat anti-mouse IgG Ab were obtained from Tebu. For
Western blotting analyses, anti-phosphotyrosine (4G10) and
anti-Lck (clone 3A5) mAb were purchased from Euromedex
(Souffelweyersheim, France); anti-p38 (clone A-12), anti-CD3
(clone 6B10.2), anti-ZAP-70 (clone LR), anti-Src homology 2
containing protein tyrosine phosphatase
(SHP)3-1 (clone
C-19), and anti-SHP-2 (clone C-18) mAb were obtained from Tebu.
Anti-CD45 (clone BRA-55) mAb was obtained from Sigma. HRP-labeled goat
anti-mouse IgG and goat anti-rabbit IgG Ab were from Amersham
Pharmacia Biotech (Uppsala, Sweden). Phenylarsine oxide and okadaic
acid were purchased from France Biochem (Meudon, France). For flow
cytometric analysis, anti-CD25 (clone M-A251) and isotype-matched
IgG1 (clone A112-2) mAb were obtained from BD Biosciences.
Preparation of soluble F254 peptide-loaded HLA-DR1 protein
DR1 protein was purified by affinity chromatography on anti-DR mAb L.243-coupled Sepharose as previously described (21). For peptide loading, DR1 was incubated for 2 days at 37°C at pH 5 in the presence of a 60-fold molar excess of synthetic F254 peptide (sequence 254–268 of the measles virus fusion protein). Synthesis and purification of the F254 peptide were previously reported (20). DR samples were then dialyzed against 0.1% sodium deoxycholate in 20 mM Tris, pH 8.0. Papain was activated by incubation for 10 min at 37°C in 1 mM EDTA, 1 mM DTT, and 20 mM Tris, pH 8.0. Digestion was performed by incubation of 7 vol DR1 protein and 3 vol activated papain for 1 h at 37°C. The reaction was terminated by the addition of 1 vol of a protease inhibitor mixture (Mini Complete; Roche Diagnostics, Mannheim, Germany). DR1 material was subsequently chromatographed over a Superose 12 column (Amersham Pharmacia Biotech) equilibrated in PBS. DR material was analyzed by SDS-PAGE on 12% gels, followed by Coomassie and silver staining (Accurate Chemical & Scientific, Westbury, NY).
Flow cytometric analysis
TCR engagement was performed using plastic-bound F254 peptide/DR1 complexes or anti-CD3 mAb TR66. Immobilization of TCR ligands was obtained by a 4- to 6-h incubation in PBS at 4°C in wells of microtiter plates. Alternatively, soluble anti-CD3 mAb, followed by the addition of goat anti-mouse IgG Ab, were used at the indicated concentrations. In experiments using APC for stimulation, T cells (105) were combined with irradiated (5000 rad) DR1-transfected HeLa cells (2 x 105) in the presence of graded concentrations of synthetic F254 peptide. Direct immunofluorescence determination was performed 24 h later. PE-labeled anti-CD25 mAb and isotype-matched control were incubated with 3 x 106 cells/ml for 1 h at 4°C in PBS, 0.1% sodium azide, and 1% FCS, then washed twice in PBS. Mean fluorescence was measured by flow cytometry on an EPICS XL cytometer (Coulter, Miami, FL).
Cell stimulation and lysis
Cells were washed twice, resuspended at 12 x 106 cells/ml in prewarmed RPMI and incubated in a water bath at 37°C for 15 min. Stimulation was then performed on 6 x 106 cells/point by addition of soluble TR66 mAb, in the presence or the absence of anti-CD28 mAb, followed by addition of goat anti-mouse IgG Ab and incubation at 37°C for the indicated periods of time. Stimulation was stopped by centrifugation, followed by solubilization of the cell pellets for 45 min on ice in 100 µl lysis buffer (1% Igepal (Sigma) in 20 mM Tris (pH 7.5) and 140 mM NaCl, supplemented with 1 mM EDTA, 1 mM sodium orthovanadate, and a mixture of protease inhibitors (Mini Complete; Roche Diagnostics)) used according to the manufacturer’s instructions. Each condition of stimulation was simultaneously performed on Th1 and Th2 cells. After removal of nuclei and cellular debris by centrifugation, the protein concentration was determined (bicinchoninic acid; Pierce, Interchim, Asnières, France), and constant amounts of protein sample were diluted in Laemmli sample buffer (Bio-Rad, Marne la Coquette, France) with 5% 2-ME and boiled for 3 min before SDS-PAGE analysis.
Lipid raft fractionation
Cell lysates (15 x 106 cells/point) were prepared as described above in 1 ml lysis buffer, except that 1% Triton X-100 was used as a detergent. The samples were centrifuged through a sucrose step gradient as described by Xavier et al. (22). Briefly, following 30-min incubation on ice, the lysates were gently mixed with 1 ml 85% sucrose in lysis buffer. The samples were placed in the bottom of a tube, overlaid by 6 ml 35% sucrose, followed by 4 ml 5% sucrose in lysis buffer, and centrifuged in a SW40 Beckman rotor (Fullerton, CA) for 16 h at 180,000 x g and 4°C without stopping. The gradients were separated into 11 1.2-ml fractions by collection from the bottom of the tube. Each fraction was precipitated by addition of 0.2 ml 100% TCA in water, and the pellet was washed twice with acetone. The pellets were resuspended in Laemmli sample buffer plus 5% 2-ME and boiled for 3 min before SDS-PAGE analysis. One-fifth of each fraction was loaded on 12% SDS-PAGE gels.
Immunoprecipitations
Cell lysates (6 x 106 cells/point in 100 µl lysis buffer) were prepared as described for lipid raft fractionation. Following incubation for 2 h on ice with the indicated mAb, immunoprecipitated material was recovered with 20 µl protein G bound on agarose beads (France Biochem) for 1 h at 4°C under continuous agitation. After three washings in lysis buffer, immune complexes were eluted by boiling for 3 min in Laemmli sample buffer and 5% 2-ME before SDS-PAGE analysis.
Immunoblotting
Equal amounts of protein, as determined by Bradford assay (Bio-Rad), were run on 12% SDS-PAGE gels and then electrotransferred onto nitrocellulose (Bio-Rad). The membranes were saturated in 20 mM Tris (pH 7.4) and 150 mM NaCl (TBS) containing 3% gelatin (Bio-Rad). Immunoblotting was performed using the indicated Ab, followed by HRP-labeled goat anti-mouse Ab or goat anti-rabbit IgG Ab in TBS, 0.1% Tween 20, and 1% gelatin. Revelation was performed using ECL (Amersham Pharmacia Biotech). Stripping was performed by incubation of nitrocellulose for 30 min at 50°C in 62.5 mM Tris (pH 6.7), 100 mM 2-ME, and 2% SDS, followed by four washings in TBS containing 0.1% Tween 20.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the TCR-mediated activation requirements of Th1 and Th2
lymphocytes under defined experimental conditions closely mimicking the
physiological situation, soluble peptide/MHC class II complexes were
chosen as a stimulus of choice. Rather than using a classical strategy
based on the expression of soluble recombinant MHC class II
heterodimers, an alternative approach was followed, which consisted of
the biochemical manipulation of naturally expressed DR1 protein.
Following purification by affinity chromatography from EBV-B cells, DR1
protein was loaded with synthetic F254 peptide by incubation under
optimal conditions using a large molar excess of peptide. Then, a
method was established for the production of soluble DR1 material
devoid of detergent. Samples of DR1 protein were treated with
increasing concentrations of preactivated papain, and the extent of
digestion was evaluated by analysis of Coomassie-stained SDS-PAGE gels.
By increasing the papain concentration, a concomitant fraction of
polypeptide corresponding to the DR1 
- and 
-chains showed
increased mobility, indicating that these conditions resulted in
limited digestion of the DR1 protein. Treatment with 40 ng/ml papain
led to a complete shift in mobility of both DR1 chains. When higher
concentrations of papain were used, the DR1 material was progressively
more degraded, up to a point where digestion was complete (Fig. 1
A). Based on this initial
experiment, a large sample of DR1 (300 µg) was treated with 40 ng/ml
papain and fractionated by size exclusion chromatography. Material was
eluted as an isolated peak with an apparent m.w. similar to that of
BSA, which was expected to correspond to solubilized DR1, without the
transmembrane and cytoplasmic domains (Fig. 1B). This
assumption was further supported by the analysis of material from this
peak on silver-stained SDS-PAGE gels. Two bands of lower m.w. than the
undigested DR1 - and -chains were resolved (Fig. 1C).
In addition, this chromatography was expected to produce material
devoid of papain, Ag peptide, and detergent. Therefore, this procedure
resulted in the isolation of soluble, peptide-loaded DR1 protein
suitable for cellular assays.
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Isolated F254 peptide/DR1 complexes offered the opportunity to
compare activation of Th1 and Th2 cells following engagement of the TCR
by their physiological ligands in the absence of the contribution of
accessory and adhesion molecules. As assessed by the percentage of CD25
up-regulation relative to CD25 expression by nonstimulated cells,
activation in three F254-specific, DR1-restricted Th1 clones was
elicited by at least 10 times less immobilized F254 peptide/DR1
complexes than in three Th2 clones exhibiting the same Ag and
restriction specificity (Fig. 2A). To facilitate comparison
between clones, since the magnitude of CD25 up-regulation was higher in
Th2 than in Th1 cells, results were expressed as the percentage of CD25
up-regulation (23). Biochemical analyses of signaling
events triggered by immobilized F254 peptide/DR1 complexes were
foreseen as technically difficult. For that reason we evaluated whether
the use of anti-CD3 mAb, a more suitable reagent for the purpose of
signal transduction studies, resulted in similar activation profiles
distinguishing Th1 and Th2 cells. Similar to the previous situation,
100-fold lower amounts of immobilized anti-CD3 mAb induced CD25
up-regulation by Th1 than by Th2 cells (Fig. 2B). Likewise,
stimulation with soluble anti-CD3 mAb cross-linked with
anti-mouse IgG Ab resulted in a 10-fold more sensitive
dose-response in Th1 than in Th2 cells (Fig. 2B). However,
it could not be formally excluded that a fraction of soluble Abs might
be adsorbed on the plastic wells and contributed to T cell activation.
Nevertheless, in both conditions CD25 up-regulation was triggered by
less anti-CD3 mAb in Th1 than in Th2 cells. These results indicated
that TCR engagement resulted in distinct signaling events in Th1 and
Th2 cells regardless of the form of the anti-CD3 mAb, immobilized
or soluble and cross-linked. Similar results were obtained with the
other Th1 and Th2 clones used in this study (data not shown).
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Remarkably, when Th1 and Th2 cells were triggered with F254
peptide-pulsed DR1-transfected HeLa cells, the two Th subsets did not
exhibit marked differences in their sensitivities. The most sensitive
clones were the Th2 FR6.6 and Th1 6396 F254.7.2 clones. The four other
Th1 and Th2 clones showed similar Ag dose responses (Fig. 3). Stimulation by F254 peptide-pulsed
HeLa cells resulted in profiles of cytokine production that paralleled
the patterns of CD25 up-regulation (data not shown). Since HeLa cells
are nonprofessional APC that are largely devoid of costimulatory
capacity, it indicated that the different thresholds of activation
observed with purified DR1 material were probably not due to distinct
costimulation requirements. Rather, these results suggested that Th2
cells might rely on a higher level of TCR aggregation for activation
than is the case for Th1 cells.
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The distinct stimulation thresholds of Th1 and Th2 cells obtained
with immobilized TCR ligands prompted us to evaluate whether TCR
signaling pathways differed in these two types of cells. Profiles of
tyrosine phosphorylation exhibited by Th1 and Th2 cells were analyzed
by Western blotting as a first step aimed at assessing multiple
signaling events following TCR engagement. Strikingly, stimulation with
soluble and cross-linked anti-CD3 mAb led to overall fainter
phosphotyrosine profiles in Th2 than in Th1 cells. In parallel, p38
mitogen-activated protein kinase was Western blotted to determine that
equal amounts of protein were loaded in each lane (Fig. 4A). Similarly, faint
phosphotyrosine profiles were observed before and after TCR engagement
in three other Th2 clones compared with three other Th1 clones (data
not shown). Suboptimal TCR engagement also resulted in fainter
phosphotyrosine profiles in Th2 than in Th1 polyclonal lines,
indicating that following comparable levels of TCR ligation, a more
sustained cascade of signaling events was generated in Th1 than in Th2
cells (Fig. 4B). Tyrosine phosphorylation profiles were also
fainter in Th2 than in Th1 cells regardless of the time following TCR
engagement. Maximal intensity in both Th1 and Th2 cells was observed at
150 s after TCR ligation, followed by decay of the phosphotyrosine
signal (Fig. 4C). This finding was observed for three Th1
and three Th2 clones (data not shown). Together these data established
that anti-CD3 mAb-mediated TCR engagement resulted in a lower
phosphotyrosine content in Th2 than in Th1 cells. These differences
could not be explained by distinct optimal intensities of stimulation
or by different response kinetics. Rather, they indicated that Th2
cells were inherently less capable than Th1 cells of anti-CD3 mAb
TCR-induced tyrosine phosphorylation.
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It is well documented that signaling pathways emanating from the
TCR and CD3 complex alone are not sufficient for T cell activation.
Simultaneous engagement of costimulatory molecules, in particular CD28,
is required to achieve full activation of T cells. Therefore, it was
evaluated whether simultaneous engagement of CD3 and CD28 increased
tyrosine phosphorylation in Th2 cells to levels matching those attained
in Th1 cells. First, immunofluorescence staining established that
expression levels of CD28 were similar in Th1 and Th2 clones (data not
shown). Then, it was found that CD3 and CD28 coengagement with mAb did
not augment the intensity of the overall phosphotyrosine profiles in
Th2 cells to levels attained in Th1 cells. A slight increase in the
phosphorylation of a 38-kDa protein, which might correspond to p38
mitogen-activated protein kinase, was nevertheless noted (Fig. 5). Similar results were found with three
pairs of Th1 and Th2 clones (data not shown). Since strong TCR
engagement as well as ligation of the costimulatory molecule CD28
failed to induce tyrosine phosphorylation levels in Th2 cells matching
those observed in Th1 cells, it was next questioned whether these
differences might be due to a greater ability of Th2 cells to
dephosphorylate tyrosine residues.
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An intrinsic low kinase activity in Th2 compared with Th1 cells
might explain the faint intensity of the phosphotyrosine profiles after
TCR engagement in Th2 cells. Alternatively, the phosphatase activity
might be higher in Th2 than Th1 cells. As a result, tyrosine
phosphorylation would be more inhibited in Th2 than in Th1 cells. To
discriminate between these two hypotheses, Th1 and Th2 cells were
stimulated with anti-CD3 mAb in the presence or the absence of
phosphatase inhibitors. Remarkably, the tyrosine phosphorylation
profiles in Th2 cells treated with the tyrosine phosphatase inhibitor
phenylarsine oxide were as intense as those in Th1 cells similarly
treated, while the serine and threonine phosphatase inhibitor okadaic
acid failed to restore intense tyrosine phosphorylation profiles in Th2
cells after TCR engagement (Fig. 6A). Analysis of three Th1 and
three Th2 clones gave the same results (data not shown). This
circumstantial evidence indicated that Th2 cells are endowed with as
high a tyrosine phosphorylation capacity as Th1 cells. It therefore
indirectly suggested that distinct phosphatases of higher activity or
expressed at higher levels made Th2 cells less reactive than Th1 cells
to TCR-mediated stimulation. To evaluate whether higher overall
expression levels of phosphatases in Th2 than in Th1 cells might
explain the distinct profiles of tyrosine phosphorylation in these two
cell types, the relative amounts of CD45, SHP-1, and SHP-2 in Th1 and
Th2 cells were assessed by Western blotting. Expression levels of these
three tyrosine phosphatases were similar in Th1 and Th2 cells before
and after TCR engagement (Fig. 6B). The absence of
differences in total phosphatase expression levels between Th1 and Th2
cells suggested that the fainter phosphotyrosine profiles in Th2 than
in Th1 cells might be alternatively explained by distinct enzymatic
activities of phosphatases involved in the TCR signaling pathway or by
distinct subcellular localization of phosphatases in the two Th
subsets. The latter possibility was assessed by evaluating whether
tyrosine phosphatases were excluded from lipid rafts to a comparable
extent in Th1 and in Th2 cells before TCR engagement (22).
Th cell lysates were separated by sucrose gradient centrifugation, and
the presence of phosphatases in the fractions was probed by Western
blotting. CD45, SHP-1, and SHP-2 proteins were not detected in low
density fractions, while the tyrosine kinase Lck was found in the low
density material of Th1 and Th2 cells, indicating that membranes
originating from lipid rafts concentrated in these fractions (Fig. 6C). Collectively, these results indicated that, for the
three abundant phosphatases analyzed and previously shown to be
implicated in TCR-mediated signaling pathways, the fainter
phosphotyrosine profiles in Th2 than in Th1 cells cannot be explained
by distinct expression levels before and after TCR engagement or
subcellular localization before TCR engagement.
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in Th2 than in Th1
cells
Beside the overall fainter intensity of tyrosine phosphorylation
profiles in Th2 than in Th1 cells, no individual bands were found to
exhibit a markedly different ratio of intensity between Th1 and Th2
cells. This observation suggested that the Th1 and Th2 TCR signaling
cascades differ at an early step. This possibility prompted us to
assess whether one of the earliest signaling events, the association
between CD3 and ZAP-70, was different in Th1 and Th2 cells. CD3
material was immunoprecipitated from both Th1 and Th2 cells before and
after TCR engagement. The presence of coimmunoprecipitated ZAP-70
protein was then evaluated by Western blotting (Fig. 7). Following TCR engagement,
phosphorylated ZAP-70 was associated with CD3 in activated Th1
cells. By contrast, in Th2 cells no ZAP-70 protein was detected in the
material immunoprecipitated by anti-CD3 mAb. In a control
experiment using anti-TNP mAb instead of anti-CD3 mAb, no
ZAP-70 was detected by Western blotting. Similar results were observed
in three Th1 and three Th2 clones (data not shown). A lower association
between CD3 and ZAP-70 in Th2 cells compared with Th1 cells by
controlling the TCR signaling cascades might at least partly determine
the overall fainter phosphorylation profiles in Th2 than in Th1
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ZAP-70 in Th2 than in Th1 cells. MHC class II protein preparations isolated from B cells were biochemically manipulated to obtain soluble, largely monomeric, Ag peptide/MHC class II complexes. The absence of detergent in this material made it suitable for cellular assays. In addition, this procedure offered the clear advantage that it did not require the development of expression vectors for each allelic form of MHC class II molecules. Since EBV-transformed B cells express high levels of DR protein, soluble peptide/DR complexes can be readily obtained in large quantities (300 µg DR protein/l EBV-B cell culture following this procedure). In contrast with MHC class II protein produced by recombinant techniques in insect cells or bacteria, MHC class II molecules isolated from B cells are not empty, but are occupied by cell- and medium-derived material. For that reason, it is expected that complete loading with Ag peptide cannot be attained, since, depending on the MHC class II alleles, occupancy with exogenously added peptides varies between <5% and up to 20% (24).
This study provided evidence that activation of Th1 cells was elicited at lower densities of immobilized peptide/MHC class II complexes, or anti-CD3 mAb than that for Th2 cells. In contrast, Th2 cells were as sensitive as Th1 cells upon TCR engagement with peptide/MHC class II complexes displayed by transfection at the surface of nonprofessional APC. These findings could be rationalized along two lines of reasoning. First, immobilized peptide/MHC class II complexes and anti-CD3 mAb cannot be clustered by T cells, while peptide/MHC class II molecules expressed on APC are laterally mobile and can be capped at the area of contact with T cells. Our observations might therefore suggest that Th2 cells require a greater degree of TCR clustering for activation than is the case for Th1 cells. Second, APC can provide Th2 cells with accessory signals needed for activation. DR1-transfected HeLa cells were used as a model of nonprofessional APC. It was assumed that these cells were largely devoid of secondary signaling molecules. In particular, they lack the expression of CD80, CD86, and CD40 molecules (25). In addition, it was found that CD3 and CD28 coengagement failed to increase tyrosine phosphorylation in Th2 cells to levels reached by Th1 cells. These results indicated that the lower activation ability of Th2 than of Th1 cells was intrinsic to the TCR signaling cascades, rather than due to the absence of CD28 costimulatory signals. Together, our results favored the idea that activation of Th2 cells might require more robust TCR-mediated signals than is the case for Th1 cells. Interestingly, it was recently shown in the murine system that TCR complexes of Th1 cells are efficiently recruited into rafts at the area of contact with peptide/MHC class II complexes, while such marked TCR partitioning does not occur in Th2 cells (26). Our results might suggest that these differences in plasma membrane dynamics are also found between human Th1 and Th2 cells.
The differential reactivity of human Th1 and Th2 cells observed in this
study could be explained by distinct features in the proximal events of
the TCR signaling machinery, as revealed by a weaker association
between CD3 and ZAP-70 in human Th2 than in Th1 cells. In the mouse
model a looser interaction between CD3 and ZAP-70 in Th2 than in Th1
cells was similarly reported by Tamura et al. (15). These
authors suggested that the lower activities of Fyn and ZAP-70 observed
in Th2 cells could explain this weaker association. As postulated in
the mouse system, the weaker association between CD3 and ZAP-70 in
human Th2 cells than in Th1 cells might be due to a weaker activity of
Fyn and ZAP-70 kinases. Interestingly, the activities of Fyn and of
ZAP-70 kinases were reduced in human Th cells activated by altered
peptide ligands promoting Th2-like phenotypes (27). In
contrast, a previous study showed that in differentiated human Th cells
the two subsets do not differ in the overall tyrosine phosphorylation
profiles, particularly in tyrosine phosphorylation of ZAP-70
(16). Different procedures for the isolation of human Th1
and Th2 cells and the resulting distinct phenotypes of these cells
might explain the apparent discrepancies between these studies.
Tyrosine phosphatases have been shown to play a key role in the early
steps of TCR signaling (28). In particular, CD45 was shown
to activate Lck, a protein kinase involved in the first steps of the
TCR signaling cascades (29, 30). To identify which
phosphatases might be responsible for the faint phosphotyrosine
profiles in Th2 cells, expression levels of CD45, SHP-1, and SHP-2 were
assessed in Th1 and Th2 cells. Comparable amounts of these three
phosphatases were detected in lysates of Th1 and Th2 cells. Moreover,
CD45, SHP-1, and SHP-2 could not be detected in lipid rafts of both Th
subtypes before TCR engagement. These observations indicated that
before TCR engagement, neither the overall expression level nor the
subcellular localization of these phosphatases could account for the
reduced phosphotyrosine profiles in Th2 compared with Th1 cells.
Interestingly, it was recently shown that CD45 was recruited in lipid
rafts after TCR engagement in mouse Th1, but not Th2, cells
(26). It may be postulated that CD45 recruitment also
occurs in human Th1, but not Th2, cells after TCR engagement. In Th2
cells the absence of clustering of TCR and CD45 in membrane domains
involved in signal transduction might result in the failure of
efficiently initiating the TCR signaling cascades. Nevertheless, it
cannot be excluded that other phosphatases could be involved in the
distinct TCR signaling events found in Th1 and Th2 cells. It was
suggested that phosphatases other than CD45, SHP-1, and SHP-2 are
present in the T cell plasma membrane (31). Moreover,
distinct phosphatase expression patterns were reported in Th1 and Th2
subsets. While CD45
was detected in murine Th1, but not in Th2,
clones (32), Fas-associated phosphatase 1 (FAP-1) mRNA was
expressed at high levels in Th2, but not in Th1, cells
(33). It was suggested that FAP-1, a membrane tyrosine
phosphatase, is involved in resistance to activation-induced cell death
in Th2 cells, suggesting a role for FAP-1 in TCR signaling
cascades.
Identification and characterization of molecules such as phosphatases that differentially control early steps of TCR signaling cascades in Th1 and Th2 cells might provide new avenues for the pharmacological manipulation of dysregulated Th cell functions. Future work would aim at evaluating whether interfering with early steps of the TCR signaling cascades distinguishing Th1 and Th2 cells might result in a beneficial impact on various immune-related pathological manifestations, such as autoimmune diseases and allergic reactions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stéphane Demotz at the current address: Dictagene, 4 chemin de la Vulliette, CH-1000 Lausanne 25, Switzerland. E-mail address: stephane.demotz{at}dictagene.ch
3 Abbreviations used in this paper: SHP Src homology 2 containing protein tyrosine phosphatase; FAP-1, Fas-associated phosphatase 1.
Received for publication December 18, 2001. Accepted for publication June 6, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in human T lymphocytes. J. Exp. Med. 190:1147. dimers released from complexes with invariant chain fail to stimulate alloreactive T cell clones. Eur. J. Immunol. 23:2100.[Medline]
mRNA in murine Th1 but not Th2 clones. Immunobiology 201:506.[Medline]
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