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Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains. The tyrosine kinase ZAP70 subsequently associates with
the phosphorylated -chains, and activation of ZAP70 leads to
downstream signaling events (3). Further downstream, through a
mechanism that is not fully defined in T cells, is the activation of
the Ras pathway leading to mitogen-activated protein
(MAP)3 kinase activation
(2).
The study of APL has contributed to our understanding of T cell
activation (4, 5). Th cells engaging APL have altered TCR -chain
tyrosine phosphorylation and altered association with, and activation
of, ZAP70 (6, 7). Engagement of APL leads to altered signaling in
CD8+ cells in the absence of esterase release and cytolysis
(8). A hierarchy of cellular responses has been demonstrated in Th
cells depending on whether the stimulus is an agonist, partial agonist,
or antagonist (9). The different cellular responses following APL
engagement indicate that the events of T cell activation can be
uncoupled from one another and that TCR/CD3 complex engagement does not
always lead to complete activation.
It has been suggested that a threshold of TCR occupancy is necessary for sustained signaling and subsequent T cell activation (10, 11). The formation of intracellular signaling complexes could be an obligatory consequence of TCR occupancy that is necessary for full cellular activation, as suggested by Wange et al. (12). Given this information, it seemed probable that the method of T cell stimulation may influence the activation outcome and may provide insight into the requirements for full T cell activation. We tested the functional differences in stimulating CTL clones with Ab to the TCR/CD3 complex in immobilized and soluble cross-linked form. Our data directly demonstrate that the induction of tyrosine phosphorylation can be uncoupled from a functional response. Furthermore, we present evidence supporting the need for sustained signaling through the TCR/CD3 complex to generate downstream signaling responses and T cell activation as measured by CTL degranulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine CD8+ CTL clones, clone 11 and clone AB.1 (H-2d anti-H-2b), have been described previously (13). The clones are stimulated weekly with irradiated spleen cells from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) and used 4 to 7 days later.
Abs and reagents
The hybridoma producing 145-2C11 (anti-CD3
) was obtained
from the American Type Culture Collection (ATCC, Manassas, VA), and
PY-72 (anti-phosphotyrosine) was provided by Dr. B. Sefton (The
Salk Institute, La Jolla, CA). They were grown in Protein-Free
Hybridoma Medium-II (Life Technologies, Burlington, ON), and the Abs
were purified by ammonium sulfate precipitation followed by either
protein A or protein G chromatography, if required. Anti-MAP kinase
(Erk 1) and anti-p56lck mAbs were purchased
from Transduction Laboratories (Lexington, KY). Phospho-specific MAP
kinase Ab was purchased from New England Biolabs (Beverly, MA). Goat
anti-hamster, rabbit anti-hamster, and goat
anti-mouseHRP Abs were purchased from Jackson
Immunoresearch Laboratories (West Grove, PA). PMA and A23187 were
purchased from Sigma Chemical Company (Mississauga, ON) and Calbiochem
(San Diego, CA), respectively. The MAP kinase kinase (MEK) inhibitor PD
98059 was purchased from Calbiochem.
Protein immobilization
Ninety-six well, flat-bottom plastic microtiter plates (Becton Dickinson, Oxnard, CA) were coated with 145-2C11 at the indicated concentration overnight at 4°C. Wells were then washed twice with PBS, blocked with 2% BSA in PBS for 30 min at 37°C, and then washed twice with PBS.
Degranulation assay
Degranulation was measured by the release of serine esterase (13). Clone AB.1 or clone 11 cells were washed three times in RPMI 1640 with 2% newborn calf serum. Cells (1.5 x 105) in 150 µl of 2% FCS in RPMI 1640 were added to each well of a microtiter plate. For the soluble cross-linked Ab stimulation, cells at 107 cells per ml were incubated with various concentrations of 145-2C11 for 30 min on ice, washed, and resuspended in RPMI 1640 supplemented with 2% FCS. The cells were added directly to the wells immediately after addition of either 1.0 or 10.0 µg/ml goat anti-hamster Ab. Cells were pretreated with PD 98059 for 30 min at 37°C before addition of the cells to the Ab-coated wells. For assays employing PMA (125 ng/ml) and/or A23187 (50 µM), reagents were added to cells immediately after adding the cross-linking Ab. After 4 to 5 h at 37°C, 25 µl of supernatants were tested for benzyloxycarbonil-L-lysine thiobenzyl ester (BLT)-esterase activity (13). Results were read at 405 nm using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA). All samples were done in triplicate or quadruplicate, and the SD is shown.
Western blotting
CTL clones were harvested and washed in D-PBS (Life Technologies). Cells (1.5 x 105) in 50 µl were stimulated as for the degranulation assay but in serum-free D-PBS. Cells were incubated at 37°C for the indicated times. Cells were lysed by the addition of 40 µl 2x Laemmli reducing sample buffer and boiled for 2 min. Whole cell lysates were loaded on either 7.5% standard (37.5:1 acrylamide:Bis) or 8.5% low Bis (56.6:1 acrylamide:Bis) SDS-PAGE, electrophoresed, and the proteins transferred to Immobilon P (Millipore Corporation, Bedford, MA) overnight at 75 mA. For the MAP kinase mobility shift assays, cell lysates were subjected to SDS-PAGE on a 15% low Bis gel (175:1 acrylamide:Bis). Immunoblotting was done using anti-phosphotyrosine (PY-72), anti-p56lck, or anti-MAP kinase and rabbit anti-mouseHRP Ab. Bound Ab was detected by Enhanced Chemiluminescence (NEN, Life Science Products, Boston, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As we have previously demonstrated (14), immobilized Ab to CD3
(145-2C11) or the TCR (H57-597; data not shown) stimulates a
degranulation response by CTL clone AB.1 (Fig. 1
). Cells were plated on microtiter wells
coated with 2C11 at various concentrations, and the serine esterase
release was assayed to measure degranulation. In contrast, when we
performed the same assay using cross-linked Abs, we observed that
soluble cross-linked Ab to either CD3 (Fig. 1) or the TCR (data not
shown) was unable to stimulate a degranulation response. Cells were
incubated with soluble 2C11 at various concentrations for 30 min on
ice. Cells were either used directly or cross-linked with secondary
goat anti-hamster Ab at either 1.0 or 10.0 µg/ml. Degranulation
significantly above that of background was not observed at a range of
both primary and secondary Ab concentrations (Fig. 1, and data not
shown); no cross-linking conditions were found that could stimulate
degranulation above the background control of BSA alone. Ice
pretreatment had no effect on the degranulation in response to
immobilized anti-CD3 stimulation (data not shown).
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Since degranulation is a downstream event following TCR engagement
and it is not triggered upon soluble Ab cross-linking of the TCR, we
examined a more membrane-proximal event. We initially examined the
tyrosine phosphorylation of total cellular proteins following both
stimulation conditions. This is the earliest event detected thus far
following TCR engagement, and a number of studies have shown that
cross-linked Ab stimulates tyrosine phosphorylation. When CTL clones
are stimulated with soluble cross-linked Ab to CD3, there is an early
and transient tyrosine phosphorylation of a number of proteins (Fig. 2). Phosphorylation of the majority of
proteins occurs by 5 min and begins to diminish by 20 min. Immobilized
anti-CD3-stimulated CTL clones have similar levels of protein
tyrosine phosphorylation at 10 min compared with soluble cross-linked
Ab stimulation, and it is sustained until at least 60 min. Both
stimulation conditions yield similar patterns of phosphorylation, with
apparently similar proteins becoming phosphorylated to the same extent.
The major difference appears to be in the duration of the tyrosine
phosphorylation. Even considering an initial lag time for cells to
contact the immobilized Ab, the duration of tyrosine phosphorylation is
about 15 min for soluble cross-linked stimulation, as compared with 50
min for immobilized conditions. The same results are seen with
anti-TCR (H57–597) stimulation by soluble cross-linked and
immobilized conditions, respectively (data not shown).
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p56lck undergoes a mobility shift only with immobilized Ab stimulation
TCR/CD3 complex stimulation leads to the recruitment and
activation of p56lck. The enzymatic activity of
p56lck plays a pivotal role in the downstream
tyrosine phosphorylation events that result in T cell activation (15, 16). It has also been shown that p56lck
undergoes a mobility shift to p60lck following
anti-CD3 stimulation (17, 18, 19). This shift may be due to increased
serine phosphorylation of p56lck (17, 18), the
function of which is not clearly understood. To determine whether these
stimulation conditions lead to this migratory shift, CTL clones were
stimulated with either soluble cross-linked or immobilized
anti-CD3. Total cell lysates were separated by SDS-PAGE using 8.5%
low Bis gels, which have higher resolution in the 40- to 60-kDa range,
and probed with a mAb to p56lck. The resulting
immunoblot (Fig. 3) indicates that,
following soluble cross-linked anti-CD3 stimulation, there is a
dominant form of p56lck (arrow 1) and a minor
higher m.w. form (arrow 2). p56lck from CTLs
stimulated with immobilized anti-CD3 undergo a more extensive
mobility shift to the higher m.w. form (arrow 2). Also, two higher m.w.
p56lck forms (arrows 3 and 4) are present at 30
min following immobilized Ab stimulation, and they are sustained for at
least 60 min.
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Recently it was reported that the Ca2+ flux in T cells
is more sustained following stimulation with surface-attached ligands
than soluble ligands (20). One possibility is that the soluble
cross-linking conditions do not stimulate a sustained Ca2+
flux, and we have shown that Ca2+ is required for
degranulation by this CTL clone (21). If this is the case, then we
should be able to restore degranulation in the CTL clones with a
Ca2+ ionophore. Adding the Ca2+ ionophore
A23187 and the phorbol ester PMA together triggers CTL degranulation
whereas either alone does not (Fig. 4).
Treatment of cells with A23187 along with soluble cross-linked Ab does
not induce degranulation in the CTL clones. Addition of PMA following
soluble cross-linking Ab stimulation also does not induce degranulation
as compared with the control treatment with both A23187 and PMA. This
would indicate that the defect in CTL degranulation is not simply a
lack of increased intracellular Ca2+ or protein kinase C
(PKC) activation but suggests that both pathways are insufficiently
triggered by cross-linked Abs.
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Since both stimulation conditions induce tyrosine phosphorylation
in the CTL clones, whereas degranulation is not triggered with soluble
cross-linked Ab, we examined another downstream effector outcome
following TCR complex stimulation. We examined the consensus MAP kinase
activation pathway. Activation of MAP kinase is required for CTL
degranulation since PD 98059, an inhibitor of this pathway, decreases
the serine esterase release response (Fig. 5). Previous data in fibroblasts
indicated that MAP kinase activation was dependent on cell anchorage
and that greater MAP kinase activation occurred in cells adhering to
fibronectin than those in suspension (22). It is possible that similar
criteria are necessary for T cells even though T cells are not
considered adherent. CTL clones were stimulated as described above, and
whole cell lysates were run on a low-Bis 15% SDS-PAGE and
immunoblotted for MAP kinase (Fig. 6).
MAP kinase undergoes decreased mobility due to phosphorylation, which
can be used to assess the extent of activation of the enzyme when
low-Bis gels are employed. Following soluble cross-linked Ab
stimulation, there is transient and partial activation of MAP kinase
for about a 5-min duration as indicated by the mobility shift.
Following immobilized Ab stimulation, the mobility shift is evident at
20 min, and some shifted material is present even at 60 min.
Furthermore, the extent of MAP kinase activation is significantly
greater after stimulation with immobilized Abs. These results were
confirmed using a phospho-specific MAP kinase Ab that recognizes only
catalytically activated p42 and p44 MAP kinases (Fig. 6, bottom
panel). Again, there is only very transient and limited MAP
kinase activation after induction with cross-linked Ab whereas
extensive and prolonged MAP kinase activation is induced with
immobilized Ab. Thus, for sustained MAP kinase activation, an
immobilized stimulus is required.
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Upon incubation with soluble cross-linked Ab, there will be a
uniform distribution of Ab on the cell surface with most receptors
engaged as compared with the more polarized engagement with immobilized
Ab. It is plausible that the tyrosine phosphorylation induced with
immobilized anti-CD3 requires an intact cytoskeleton, possibly for
surface receptor redistribution, to be sustained. We hypothesized that
inhibiting cytoskeleton assembly with cytochalasin E treatment would
alter the tyrosine phosphorylation differently in the cells stimulated
in the two different ways. CTL clones were treated with cytochalasin E
before stimulation at 37°C for various times, and tyrosine
phosphorylation of total cellular proteins was examined. Figure 7 shows that cells stimulated with
immobilized 2C11 had a near complete inhibition of protein tyrosine
phosphorylation in the presence of cytochalasin E (right) as
compared with the normal pattern of tyrosine phosphorylation
(left). In contrast, cells stimulated with soluble
cross-linked 2C11 undergo the same tyrosine phosphorylation events in
the absence (left) and presence (right) of
cytochalasin E (Fig. 7). Consequently, in contrast to the immobilized
stimulus, the soluble cross-linked Ab-induced phosphorylation is not
inhibited by cytoskeleton disruption.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Valitutti et al. (10, 11) have shown that the effector responses elicited from CTLs can be uncoupled from one another, and this is attributed to different levels of TCR occupancy. Glickstein et al. demonstrated that anti-CD3-stimulated apoptosis and cytokine secretion by a Th hybridoma can be uncoupled from each other by using immobilized vs soluble Ab (23). It has also been suggested that T cells have the ability to count the number of TCRs triggered with MHC/peptide (24), and, in the absence of threshold TCR engagement, there is no cell activation. Although our data support that a minimum TCR engagement at the cell surface is necessary for T cell activation, our system suggests that there is more to activating T cells than simple receptor occupancy, since stimulation with soluble cross-linked Ab will result in essentially all the TCR complexes being engaged. Consistent with our results, it has been shown that prolonged TCR/CD3 engagement is needed for a sustained Ca2+ flux, which is required for T cell activation (25). We propose that, under our immobilized Ab conditions, there is a prolonged and sufficient engagement of the TCR complex to allow sustained tyrosine phosphorylation and MAP kinase activation, p56lck mobility shift, and degranulation, none of which is achieved with cross-linked Ab.
TCR engagement leads to down-regulation of TCR surface expression by
internalization (24, 26). Engagement of the TCR complex on T cells with
soluble cross-linked 2C11, using the same conditions that were used for
this study, leads to TCR down-regulation (27), but this is apparently
insufficient for activation, as indicated by the lack of degranulation
(Fig. 1
). Recent studies have demonstrated that TCR down-regulation can
occur in the absence of T cell activation and that TCR down-regulation
does not necessarily lead to subsequent T cell activation (28, 29).
Taken together, these data indicate that TCR down-regulation and T cell
activation can be uncoupled.
Intracellular signaling events that lead to T cell activation require multimeric complexes to form between signaling molecules and the cytoskeleton (12). Wulfing et al. suggest that the nature of the stimulus will dictate the assembly of intracellular molecules and hence the outcome of stimulation (30). How sustained the TCR engagement is will be important to the formation of such complexes and the subsequent signaling events. Shaw and Dustin (31) also suggest that T cell activation is attained only after the correct signaling complexes have formed and that this requires a given amount of time. The time is needed to provide a stimulus of threshold strength and duration. We propose that this threshold is not achieved with soluble cross-linked Ab, and the absence of these events may result in the lack of effector functions such as degranulation. The necessary strength and/or duration of stimulus is achieved only with the immobilized stimulus. We speculate that the p56lck mobility shift that occurs only following immobilized stimulation may contribute to the formation of such protein complexes, possibly through protein-protein interactions as a result of serine phosphorylation (18).
Previous evidence has shown that the cytoskeleton is necessary for T
cell effector functions (32) and that this may be required to sustain
the intracellular signals (10). We found that an intact cytoskeleton is
required for the sustained tyrosine phosphorylation induced by
immobilized Ab and that this is not a requirement for the transient
phosphorylation induced by soluble cross-linked Ab (Fig. 7). We predict
that the cytoskeleton is required for cell spreading on the Ab-coated
surface and engagement of sufficient receptors for threshold
stimulation. As already discussed, however, TCR occupancy alone is not
sufficient for T cell activation, and we conclude that the cytoskeleton
is required for the subsequent sustained phosphorylation. The
cytoskeleton, either directly or indirectly, is likely needed for the
assembly of signaling molecules into multimeric complexes necessary for
activation and subsequent effector functions such as degranulation.
Our data show that MAP kinase is activated only transiently with cross-linked Ab but, with immobilized Ab MAP kinase, is activated to a greater extent, and the activation is sustained. Based on a number of studies in neuronal cells, Marshall has proposed that the duration of MAP kinase activation leads to functionally different responses (33). Although our systems differ, our data are in agreement with this model, in that a longer duration of MAP kinase activation correlates with a degranulation response in CTL.
Our results suggest that T cell occupancy is not sufficient to stimulate activation. Since we used high affinity Abs to stimulate activation and the Ab was not limiting in our system, we can assume that essentially all of the receptors were "occupied," yet no degranulation response was observed. On the other hand, if the Abs were immobilized, which does not necessarily change the extent of TCR occupancy, a degranulation response was observed. The striking difference between these two stimulation conditions is that the duration of a number of signals that we measured was significantly longer when immobilized Abs were used. Taken together, these results suggest that the duration of the response, which likely involves cytoskeletal rearrangements, dictates whether a response ensues. This observation can have significant implications for vaccine design since triggering the TCR is not sufficient to guarantee a response, but the duration of the response must also be sustained.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hanne L. Ostergaard, Department of Medical Microbiology and Immunology, 141 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7. E-mail address:
3 Abbreviations used in this paper: MAP, mitogen-activated protein; APL, altered peptide ligands; Bis, N,N'-Methylene-bis-acrylamide.
Received for publication March 12, 1998. Accepted for publication May 12, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and lack of ZAP70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
chain and displays a unique activation-dependent phosphorylation pattern. J. Biol. Chem. 271:20705.This article has been cited by other articles:
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