By
From the * Department of Microbiology and Immunology, McGill University, Montreal, Quebec,
Canada H3A 2B4; the Department of Immunology, University of Toronto, and The Arthritis and
Immune Disorder Research Center, Toronto, Ontario, Canada M5G 2M9; the § Department of
Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33101; the
McGill Cancer Centre and the Departments of Biochemistry, Medicine, and Oncology, McGill
University, Montreal, Quebec, Canada H3G 1Y6; and the ¶ Howard Hughes Medical Institute,
University of Chicago, Chicago, Illinois 60637
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Abstract |
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T cell activation and clonal expansion is the result of the coordinated functions of the receptors
for antigen and interleukin (IL)-2. The protein tyrosine kinase p56lck is critical for the generation of signals emanating from the T cell antigen receptor (TCR) and has also been demonstrated to play a role in IL-2 receptor signaling. We demonstrate that an IL-2-dependent, antigen-specific CD4+ T cell clone is not responsive to anti-TCR induced growth when propagated in IL-2, but remains responsive to both antigen and CD3-specific monoclonal antibody. Survival of this IL-2-dependent clone in the absence of IL-2 was supported by overexpression of exogenous Bcl-xL. Culture of this clonal variant in the absence of IL-2 rendered it
susceptible to anti-TCR-induced signaling, and correlated with the presence of kinase-active
Lck associated with the plasma membrane. The same phenotype is observed in primary, resting
CD4+ T cells. Furthermore, the presence of kinase active Lck associated with the plasma membrane correlates with the presence of ZAP 70-pp21
complexes in both primary T cells and T
cell clones in circumstances of responsive anti-TCR signaling. The results presented demonstrate that IL-2 signal transduction results in the functional uncoupling of the TCR complex
through altering the subcellular distribution of kinase-active Lck.
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Introduction |
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T cell activation and subsequent growth is dependent
upon the coordinated expression and function of the
/
-TCR and the IL-2 receptor (1). Expression of the
high affinity receptor for IL-2, as well as the de novo transcription and translation of IL-2, is induced upon T cell antigen recognition (7). The control of T cell growth in
these circumstances is regulated through the autocrine production and use of IL-2, which are interrelated processes in
that the loss of high levels of membrane IL-2R
chain expression and the production of IL-2 are coordinated (11). IL-2 has also been shown to predispose T cells to apoptosis
upon subsequent interaction with antigen (16). Furthermore, the exposure of T cell clones to exogenous IL-2 can
result in their inability to respond to antigen (17). Thus,
IL-2 is essential in supporting T cell clonal expansion, and
is also involved in a feedback regulatory mechanism limiting cellular expansion and/or function.
The JAK/STAT pathway is central in the transduction
of IL-2R signaling (18). However, a role for src family
tyrosine kinases has also been demonstrated. Specifically,
stimulation of human T cell clones with exogenous IL-2
results in the activation of p56lck, which is independent of
its association with either CD4 or CD8 (24, 25). Lck has
also been shown to associate with the chain of the IL-2
receptor (25), which is critical for IL-2-mediated Lck activation (26).
We have developed a T cell clonal system that provides
further insight into the mechanisms underlying the coordinated function of the TCR and the IL-2R. The antigen receptor signaling phenotypes of CD4+ and CD4 variants
of an IL-2-dependent, ovalbumin-specific T cell clone have been described previously (27). Specifically, although both variants respond to antigen and mAbs specific for
CD3
, only the CD4
variant responds to mAbs specific
for TCRC
or V
. Expression of exogenous wild-type
CD4, but not mutated CD4 unable to bind Lck, in CD4
variants restored their TCR-CD3 signaling phenotype to
that of cells expressing endogenous CD4. Thus, the refractoriness of clones expressing wild-type CD4 to TCR-specific mAbs correlates with the sequestration of cellular Lck
by CD4 (27).
The role of Lck in T cell antigen receptor signaling is well established. Biochemical (28), and genetic (39) analyses demonstrate that its presence and activity are required for the most proximal signaling events initiating through the TCR. Thus, it is plausible that the sequestration of Lck by CD4 in the above described clonal system disables the transmission of the most proximal signals emanating from the TCR.
However, the above observations in the CD4+ T cell
clones do not correspond well with TCR-CD3 signaling
phenotypes observed in primary CD4+ T cells. Specifically,
the latter do respond to mAb specific to TCRC, albeit
less robustly than they respond to mAb specific for CD3
(47). An obvious difference within this comparison is
the state of activation of the cells involved. Primary CD4+
lymph node T cells are in the majority resting, with >95%
in the G0 phase of the cell cycle. In contrast, the CD4+ T
cell clones are maintained in exogenous IL-2 before assay, and are in the majority, cycling. Although a significant proportion of these cycling cells contain a diploid content of
DNA, these cells cannot be equated with noncycling cells
in G0. Since IL-2 has been shown to affect the physiology
of Lck (24, 25), coupled with the obligate role of Lck in
TCR signaling, the observed TCR signaling phenotype in
CD4+ IL-2-dependent clones may reflect an IL-2-mediated physical and/or functional redistribution of cellular Lck.
This study was undertaken to characterize the molecular
basis of IL-2-mediated perturbation of TCR signaling. We
demonstrate that exogenous IL-2 disables anti-TCR-, but
not anti-CD3-induced T cell growth, and that the permissive anti-TCR signaling phenotype correlates with the
presence of kinase-active Lck associated with the plasma
membrane. Primary CD4+ T cells, which are also responsive to anti-TCR-induced growth, are also shown to have
kinase-active Lck at the plasma membrane. Furthermore,
permissive anti-TCR signaling is shown to correlate with
the presence of ZAP-70-pp21 complexes in both primary
T cells and clones deprived of IL-2.
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Materials and Methods |
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Mice.
C57BL/6 male mice, 6-8 wk old, were purchased from Charles River Canada, Inc. (Laval, Quebec, Canada) or The Jackson Laboratory (Bar Harbor, ME), and maintained in a pathogen-free animal facility at the Ontario Cancer Institute (Toronto, Ontario, Canada).Antibodies.
Hamster mAbs H57.597 (anti-TCRCCell Preparation and Culture.
Clones were maintained at 0.5- 1.0 × 106 cells/ml in serum-free IMDM supplemented with 10 U/ml (1%) rIL-2 as previously described (27). Experiments involving rescue of responsive anti-TCR signaling in the presence of low concentrations of IL-2 required that CD4+ clones be antigen pulsed, which increased their sensitivity to IL-2. Specifically, clone 2.5 was cocultured with irradiated C57BL/6 spleen cells and 1-10 µg/ml of the ovalbumin-derived peptide143-157 for 48 h in the absence of rIL-2. Cultures were harvested and irradiated filler cells were removed by centrifugation through Lympholyte-M (Cedarlane Labs. Ltd., Hornby, Ontario, Canada). Viable T cell blasts were washed and seededCell Fractionation.
Preparation of membranes and cytosol used procedures previously described in detail (57). In brief, cells were pelleted and resuspended in ice-cold calcium- and magnesium-free PBS in chilled borosilicate tubes (Fisher Scientific Co., Pittsburgh, PA). After two more washes in ice-cold PBS, the cells were resuspended to a final volume not exceeding 0.5 ml at 2 × 107 cells/ml in ice-cold hypotonic extraction buffer containing 25 mM Hepes, pH 7.0, 1 mM MgCl2, 1 mM EGTA, and 100 µg/ml each of leupeptin and aprotinin. Cells were incubated on ice for 40 min, then subjected to 20 "shears" per tube (avoiding foaming) using a 30-gauge needle on a 1-ml syringe barrel. Sheared material was examined microscopically to verify that no intact cells remained and nuclear material was removed by centrifugation. The volume of the final supernatant was measured, transferred to Eppendorf tubes, and centrifuged at 12,000 g for 10 min. The pellet from this spin contains the heavy membrane fraction (HMF) and the supernatant contains the cytosol. The cytosol fraction was centrifuged at 100,000 g for 1 h at 4°C, the supernatant of which was resolubilized in 0.1% Triton X-100 for 45 min with gentle agitation before immunoprecipitation. The HMF was washed once in 200 µl of ice-cold extraction buffer containing 0.1% delipidated BSA (Boehringer Mannheim), and resuspended to 4 × 107 cell equivalents/ml in ice-cold extraction buffer containing 0.1% Triton X-100. This suspension was solubilized with gentle agitation and then centrifuged at 12,000 rpm for 10 s to remove detergent-insoluble material. The supernatant of this spin contains the detergent-soluble HMF. Biotinylation of cell surface proteins was achieved as follows. Cells were harvested and washed in ice-cold calcium- and magnesium-free PBS and resuspended to 107 cells/ml in borate buffer containing 10 mM anhydrous sodium borate and 150 mM NaCl, pH 8.8. Long chain biotin stock (Pierce Chemical Co., Rockville, IN) was dissolved in DMSO to a concentration of 10 mg/ml, and the appropriate volume was added to the cell suspension to achieve a final concentration of 50 µg/ml. Cells were incubated with biotin for 10 min at room temperature with frequent, gentle vortexing. The reaction was stopped by the addition of 10 µl/ml of 1 M NH4Cl. Cells were washed three times in room temperature PBS containing 10 mM Tris and 1mM EDTA and was examined microscopically for viability, which was typically >95%. An aliquot of cells was stained with PE-conjugated streptavidin (Southern Biotechnology Associates, Huntington, AL) to determine efficiency of cell biotinylation.Immunoprecipitation and Immunoblotting.
Precipitation of Lck, CD4, and Bcl-xL for immunoblotting was achieved by lysing either T cell clones or primary CD4+ lymph node T cells at 4 × 107 cells/ml in hypotonic TNE lysis buffer (50 mM Tris, pH 8.0, 20 mM EDTA, 100 µM oxidized sodium orthovanadate, 50 mM sodium fluoride, 20 µg/ml each of leupeptin and aprotinin [Boehringer Mannheim], 100 µg/ml Pefabloc [Boehringer Mannheim], 50 µg/ml L-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone hydrochloride [TLCK; Boehringer Mannheim], and 1% NP-40). Cells were lysed in prechilled Eppendorf tubes on ice for 40 min and centrifuged at 4°C at 12,000 g for 10 min. The supernatant was transferred to fresh Eppendorf tubes and maintained on ice until ready for use. Immunoblotting for Bcl-xL from whole cell lysates was achieved by adding 12.5 µl of lysate (0.5 × 106 cell equivalents) to 12.5 µl of 2× Laemmli sample buffer plus 2.5 µl ![]() |
Results |
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Both the CD4+ (2.5)
and CD4 (2.10) variants of the ovalbumin-specific T cell
clone were propagated in varying concentrations of rIL-2
for 1 wk. Viable cells were harvested and their capacity to
respond to anti-V
4 and antigen was assessed. As illustrated in Fig. 1, there is an inverse correlation between the
amount of exogenous IL-2 used to propagate the CD4+
variant and its capacity to subsequently respond to anti-V
4. This IL-2-dependent refractoriness to anti-TCR
stimulation is also observed using an mAb specific for
TCRC
(data not shown). The inability to respond to
anti-TCRV
or anti-TCRC
is not due to the compromised expression of the antigen receptor complex at the
plasma membrane. Direct assessment of the membrane levels of TCR and CD3 by immunofluorescence revealed no
differences in cells grown over the range of IL-2 concentrations used in this study (data not shown). The highest
concentration of IL-2 used in this experiment (1% = 10 U/ml) results in a 20-fold inhibition of the anti-TCR response of CD4+ variants. However, the antigen responses
of cells cultured in 10 vs. 1 U/ml of IL-2 were within a
factor of two of one another (Fig. 1). Thus, IL-2 is not predisposing cells to die upon subsequent ligation of the
TCR-CD3 complex as previously described (16). Rather,
it is profoundly affecting their capacity to respond to TCR-specific mAbs.
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The involvement of cellular Lck in this signaling phenotype is derived from the observation that CD4 clonal variants are not susceptible to this IL-2-mediated effect. Thus,
whether clone 2.10 was propagated in 0.1 or 1.0% IL-2,
the subsequent response to anti-TCR was comparable.
Shown in Fig. 1 is the anti-V
4 response of clone 2.10, which was propagated in 1.0% IL-2. To elucidate the role
of Lck, both membrane and cytosolic fractions of clones
2.5 and 2.10 were prepared and the content and enzymatic
activity of Lck contained within these fractions was assessed
quantitatively. This analysis was performed using cells
propagated in 1% IL-2, circumstances in which the CD4+
clone 2.5 does not respond to anti-TCR.
As illustrated in Fig. 2 A, quantitative precipitation of Lck from membrane and cytosolic fractions of clone 2.5 followed by immunoblotting revealed three pools, two of which were membrane associated, and the third of which was cytosolic. Phosphorimaging revealed that ~70% of cellular Lck in clone 2.5 is associated with the membrane fraction, and that within this fraction >85% is associated with CD4. A comparable membrane/cytosol distribution of Lck is observed in clone 2.10 despite the absence of CD4 (Fig. 2 A). Immune complex kinase assays were performed in parallel with Lck protein analyses within each of the isolated pools, from each of the clonal variants. As shown in Fig. 2 B, the cytosolic pools from both clonal variants contained kinase-active Lck, revealing a robust phosphorylation of the exogenous substrate, enolase, coupled with a weak autophosphorylation signal. Neither the CD4-associated nor the non-CD4-associated pools of membrane Lck of clone 2.5 exhibited detectable kinase activity (Fig. 2 B). In marked contrast, membrane-associated Lck derived from clone 2.10 was kinase active (Fig. 2 B).
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Of note is that, based on the enolase signals observed,
the specific activity of cytosolic Lck derived from clone
2.10 appears higher than that of the analogous Lck pool derived from clone 2.5 (Fig. 2 B). Densitometric analysis of
these enolase signals, normalized to the content of Lck
within the respective precipitates, reveals that the signal derived from clone 2.10 is 2.3-fold higher than that derived
from clone 2.5. Thus, the differences in the kinase activities
observed in these clonal variants may not be restricted to
the plasma membrane-associated pools of Lck, exclusively. The comparison of these clonal variants suffers from what
may be more complicated affects mediated by CD4 on cellular Lck in general. Although the distinct subcellular distribution of Lck kinase activity in the CD4+ and CD4 clonal
variants described above could reflect the underlying differences in their susceptibility to anti-TCR-mediated growth, a potential caveat relates to the role of CD4 in altering the function of cellular Lck. A direct comparison of the subcellular distribution and enzymatic activity of Lck in anti-TCR permissive and nonpermissive CD4+ variants is required to obviate this concern. The low number of cells
rescued after culturing CD4+ clone 2.5 in concentrations
of exogenous IL-2 that supported subsequent responses to
anti-TCR (Fig. 1) precluded our analyses. Therefore, we
took advantage of the recent demonstration showing that
forced expression of exogenous Bcl-xL was able to maintain the viability of IL-2-dependent T cells after withdrawal of IL-2 (58).
Bcl-xL was cloned into the expression vector pSFFV-neo (55, 56) and electroporated into clone 2.5. G418 resistant cells were propagated and clones were derived from this expanded drug resistant population. The level of Bcl-xL expressed in clone 2.5.2, in comparison to that expressed by clone 2.5, is illustrated in Fig. 3 A. Quantitation of this immunoblotting analysis revealed that the transfected clone 2.5.2 expressed ~2.5-fold more Bcl-xL than the endogenous levels expressed by clone 2.5.
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Clone 2.5.2 was then cultured in the presence of irradiated syngeneic splenocytes in medium containing either 10 U/ml of rIL-2 or no IL-2. Viable cells harvested 24 h after
initiation of these cultures were assessed for cell cycle status
using propidium iodide (59). As shown in Fig. 3 B, of those
cells expressing 2n DNA content, roughly one-third of
the cells derived from IL-2-containing cultures were in the
S and G2/M phases of cycle, compared with 2% of the cells
that were harvested from cultures containing no IL-2. Of note
is that levels of expression of TCRC
, CD3
, and CD4, as
assessed by immunofluorescence analysis of clone 2.5.2, were unaffected by exposure to IL-2 (data not shown).
The question followed whether clone 2.5.2 cultured in
the absence of exogenous IL-2 was now responsive to anti-TCR mediated growth. As depicted in Fig. 3 C, this was
indeed the case. Thus, once "rested" for 24 h in the absence of exogenous IL-2, clone 2.5.2 responded as robustly
to anti-TCR as it did to anti-CD3 (Fig. 3 C). In contrast,
and as for clone 2.5, clone 2.5.2 was unresponsive to anti-TCR if derived from cultures containing 10 U/ml rIL-2.
However, consistent with the phenomenon of IL-2-mediated functional uncoupling of TCR and CD3 signaling,
these cells responded robustly to anti-CD3
(Fig. 3 C).
The availability of anti-TCR responsive and nonresponsive CD4+ clonal variants enabled a direct comparison of the cellular distribution and activity of Lck in these two circumstances. Towards this end, CD4+ clone 2.5.2 was propagated in 10 U/ml IL-2, or rested in the absence of exogenous IL-2. Viable cells were rescued from each of the two culture conditions, followed by the preparation of membrane and cytosol fractions. To control the cell fractionation technique used, cells derived from the two conditions were biotinylated and CD4 was used as a membrane marker. As illustrated in Fig. 4, immunoprecipitation from "membrane" but not "cytosolic" fractions with anti-CD4 revealed the appropriate signal. Sequential precipitation with anti-CD4, from biotinylated membrane fractions of both resting and IL-2- propagated 2.5.2 was followed by sequential precipitation with anti-Lck. Precipitates were fractionated by SDS-PAGE, immunoblotted and developed with enzyme-linked streptavidin.
As illustrated in Fig. 4, the CD4 signal was cleared by anti-CD4 and not detectable in subsequent anti-Lck precipitates. Stripping the immunoblot followed by probing with anti-Lck revealed that CD4 associated Lck had been cleared, and that membrane Lck not associated with CD4 was subsequently rescued, quantitatively, with anti-Lck (Fig. 4). The cytosolic fractions from the two 2.5.2 populations were sequentially precipitated with anti-CD4, followed by anti-Lck, as described above for the membrane fractions. As shown in Fig. 4, developing the immunoblot with enzyme-linked streptavidin did not reveal a CD4 signal, consistent with this fraction being devoid of material derived from the plasma membrane. However, non-CD4-associated cytoplasmic Lck was evident (Fig. 4).
This analysis revealed that the cellular distribution of Lck was not significantly altered in cycling and noncycling populations of clone 2.5.2. Thus, as observed in clone 2.5 (Fig. 2), the majority of cellular Lck was associated with the plasma membrane fraction, and of the two pools comprising this fraction, the majority of Lck was found to be associated with CD4.
In marked contrast to the unaltered cellular distribution of Lck, its enzymatic activity was profoundly affected by exogenous IL-2 (Fig. 5). Immune complex kinase assays were performed on membrane and cytosolic fractions of clone 2.5.2 cultured in the presence and absence of exogenous IL-2 as described above. As illustrated in Fig. 5, the cytosolic fractions of 2.5.2 cultured in either condition contained kinase-active Lck. As shown, the enolase signal is easily detectable, and although the position of autophosphorylated Lck is indicated, this signal is barely detectable. Thus, if kinase-active Lck is required for responsiveness to anti-TCR stimulation, cytosolic Lck is probably not playing a role.
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Assessment of the kinase activities contained within the two pools of membrane-associated Lck from clone 2.5.2 cultured in the presence and absence of exogenous IL-2 revealed striking differences (Fig. 5). Clone 2.5.2 cultured in the absence of IL-2 and responsive to anti-TCR-induced growth (Fig. 3 C) contained membrane-associated Lck that was kinase active. Both Lck associated with CD4 and that which was not gave detectable enolase signals, and for the cytosolic Lck described above, autophosphorylation signals were virtually undetectable in precipitates from the number of cell equivalents assayed (Fig. 5). In marked contrast, kinase activity within the analogous pools of membrane-associated Lck derived from clone 2.5.2 cultured in 10 U/ml IL-2 was undectable. Thus, as concluded from the results obtained with clones 2.5 and 2.10 (Fig. 2), the presence of membrane-associated, kinase-active Lck correlates with a permissive anti-TCR signaling phenotype.
CD4-Associated Lck in Clone 2.5.2 Propagated in IL-2 can be Acutely Activated.There is an obligate requirement for
Lck in the generation of the most proximal signals emanating from the TCR complex. The observation that clone
2.5.2 propagated in IL-2 cannot respond to TCRV-specific mAb raises the question of how the involvement of
Lck is manifest in response to antigen. An obvious difference between anti-TCR and antigen-induced T cell activation is the latter's capacity to recruit CD4, and thus the
CD4-associated pool of Lck, to the activation complex.
However, as shown in Fig. 5, the CD4-associated pool of
Lck is kinase inactive in clone 2.5.2 propagated in IL-2.
The question is whether the activity of this pool may be altered during the T cell response to antigen. Although it has
not been reported that antigen-mediated coaggregation of
CD4 and TCR-CD3 results in the activation of CD4-associated Lck, it has been demonstrated that mAb-mediated
aggregation of CD4 does (50, 60). Therefore, we determined whether mAb-mediated aggregation of CD4 resulted in an alteration in the phosphotyrosyl content and
kinase activity of associated Lck derived from clone 2.5.2 propagated in IL-2.
As illustrated in Fig. 6, this was indeed the case. Specifically, and corresponding with the results shown in Fig. 5, when CD4 is not ligated, the associated pool of Lck is not kinase active (Fig. 6, middle), nor does it contain detectable levels of phosphotyrosine (Fig. 6, top). However, upon mAb-mediated aggregation of CD4, both the phosphotyrosyl content and the kinase activity of the pool of associated Lck is elevated (Fig. 6). Thus, the IL-2-mediated downregulation of the activity of the CD4-associated pool of Lck in clone 2.5.2 propagated in IL-2 is not irreversible. Furthermore, these results are consistent with the capacity of antigen, but not anti-TCR, to recruit and use the CD4-associated pool of Lck.
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To determine whether the observations described above did or did not reflect the idiosyncratic behavior of a T cell clone, we assessed the distribution and enzymatic activity of cellular Lck in primary CD4+ T cells. However, although the partitioning of membrane and cytosolic fractions was as efficient as that observed with the T cell clones, we were unable to detect kinase activity associated with Lck derived from any of the fractions. Cells were therefore lysed using standard technique and immune complex kinase assays were performed with anti-CD4 and anti-Lck precipitates from total cell lysate.
As illustrated in Fig. 7 A, sequential precipitation from
lysate derived from primary CD4+ lymph node T cells
with anti-CD4 followed by anti-Lck revealed that the majority of cellular Lck is associated with CD4. The remaining Lck would be partitioned between the membrane and
cytosol. Immune complex kinase assays were performed on
both total cellular Lck and on CD4-associated Lck to ensure that we were assessing the activity of some membrane-associated Lck. As shown in Fig. 7 B, both anti-Lck and
anti-CD4 precipitates mediated robust enolase phosphorylation. An Lck autophosphorylation signal was not observed in anti-CD4 precipitates, and only marginally in
anti-Lck precipitates (Fig. 7 B). Anti-TCRC precipitates
derived from the same lysate were kinase negative (Fig. 7
B). These results support the conclusion that membrane
Lck is kinase active in primary, resting CD4+ T cells.
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One of the roles of Lck in TCR signaling is to mediate
the phosphorylation of the chain (33, 34, 36, 60), thus
enabling the recruitment and subsequent activation of
ZAP-70 (32, 33, 35). Given that kinase-active Lck is
present in resting primary T cells and T cell clones, it was
of interest to determine the phosphorylation status of the
chain and whether it was associated with ZAP-70. Towards this end, three populations of T cells were analyzed:
clone 2.5.2 cultured in the presence of IL-2; clone 2.5.2 cultured in the absence of IL-2; and primary CD4+ lymph
node T cells. Anti-ZAP precipitates were isolated from lysates of each of these three populations, resolved on SDS-PAGE, and immunoblotted with phosphotyrosine-specific
mAb 4G10 (52) with
chain-specific mAb, G3 (53), and
anti-ZAP-70 (51). The top panel in Fig. 7 C depicts the
pp21 signal that coprecipitated with anti-ZAP-70 in each
of the three populations, revealed with phosphotyrosine-specific mAb. The middle panel of Fig. 7 C illustrates that
the pp21 signals were also revealed with the
chain-specific mAb. The bottom panel of Fig. 7 C demonstrates that
equivalent amounts of ZAP-70 were present in precipitates
from each of the three populations.
The results presented in Fig. 7 C indicate that the chain is at least partially phosphorylated in primary resting
T cells and in clonal populations that exhibit a permissive
anti-TCR signaling phenotype. Notably, pp21
is present
at much reduced levels in clonal populations derived from
cultures containing IL-2, which are nonpermissive to anti-TCR. Furthermore, this pp21
is constitutively associated
with ZAP-70 in resting primary T cells, as previously demonstrated (34), and in resting T cell clones. Thus, both primary resting T cells, and resting clones exhibit a basal level of pp21
, at least some of which is constitutively associated with ZAP-70. This phenotype correlates with both the
presence of kinase-active Lck at the plasma membrane and
responsiveness to anti-TCR-mediated growth.
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Discussion |
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To gain insight into the mechanism through which IL-2
can affect TCR-CD3 function, we have analyzed the signaling capacity of elements of the antigen receptor complex in a CD4+, IL-2 dependent clone. We demonstrate
that IL-2, in a dose dependent fashion, inhibits the capacity
of TCR, but not CD3 specific mAbs to induce T cell proliferation. The forced expression of exogenous Bcl-xL enabled the withdrawal of exogenous IL-2 from clone 2.5.2, and the rescue of cells, >98% of which were in G0 phase of
cycle. This resting clonal population is responsive to anti-TCR. However, responsiveness to anti-TCR is not strictly
a cell cycle dependent phenomenon. Clone 2.5 propagated
in low concentrations of exogenous IL-2 (1 and 3 U/ml),
do contain a significant proportion of cycling cells, ranging
from 5 to 20%, and yet are responsive to anti-TCR. Thus,
IL-2 appears to function in two domains, one in which it supports T cell growth without disabling anti-TCR signaling, and another in which IL-2 in excess prohibits the induction of the most membrane proximal signaling events
induced by TCRV- and TCRC
-specific mAbs (27).
Responsiveness to TCR-specific mAbs in clone 2.5.2 and primary CD4+ T cells correlates with the presence of detectable kinase active Lck at the plasma membrane, and this compartment of Lck is modulated in response to exogenous IL-2. Specifically, IL-2-mediated refractoriness to anti-TCR in clone 2.5.2 correlates with the absence of detectable kinase-active, membrane-associated Lck. In contrast, the cytosolic pool of Lck derived from clone 2.5.2 was kinase active whether or not the cells were responsive to anti-TCR. Thus, kinase-active cytosolic Lck is probably not involved in anti-TCR-mediated T cell growth. Consistent with this tenet is previous work demonstrating that forced expression of exogenous constitutively active Lck results in a hypersensitive TCR signaling phenotype (29) only when Lck can tether to the inner leaflet of the plasma membrane (30). These results support the conclusion that plasma membrane-associated and cytosolic pools of Lck support distinct cellular functions (61), and the results presented here demonstrate that IL-2 may induce a redistribution of activity within these pools.
Our study extends those first describing the capacity of IL-2 to alter Lck function (24, 25), with some notable differences. The latter studies demonstrated an IL-2-mediated transient activation of cellular Lck. Mitogen-activated T cells were propagated in IL-2, then starved of IL-2 before assessing the effects of subsequent IL-2 exposure on Lck activity. Neither the kinase activity of Lck in the initial resting T cell population, nor the subcellular distribution of the kinase active Lck induced by IL-2, was assessed. In contrast, we have observed an IL-2-mediated downregulation of Lck kinase activity, specifically of that Lck associated with the plasma membrane. The high basal level of cytosolic Lck kinase activity observed in both resting and cycling T cell clones may have precluded our detection of an IL-2- mediated increase in Lck activity in the cytosolic pool. However, this basal level of Lck kinase activity appears to be physiological, as it is observed in primary resting T cells.
No differences in the subcellular distribution of Lck protein could be correlated with IL-2-induced alterations in the cellular distribution of Lck kinase activity. It is possible that, once activated by IL-2, Lck is dislodged from the plasma membrane to the cytosol, and replaced by kinase-inactive Lck. This Lck would in turn become activated and redistribute to the cytosol under continuing IL-2-mediated pressure. Thus, we posit that use of Lck in IL-2 receptor signaling (26) results in the redistribution of kinase-active protein, and thus impairs subsequent anti-TCR-mediated responses.
According to this paradigm, IL-2-mediated inhibition of
anti-TCR-induced growth would be ameliorated in the
absence of membrane CD4 expression. Anti-TCR and antigen-mediated T cell activation would not engage the
CD4-associated pool of Lck in similar fashions. mAb-mediated T cell activation may predominantly engage that pool of membrane-associated Lck that is not bound to CD4. In
the absence of membrane CD4 expression, the available
pool of membrane-associated Lck required for anti-TCR
signaling would be larger and therefore proportionally less
depleted by IL-2. This would explain the anti-TCR responsiveness of clone 2.10, the CD4 clonal variant of clone
2.5. Thus, when cultured in concentrations of IL-2 that inhibit anti-TCR-induced growth of the CD4+ clone 2.5, clone 2.10 is still responsive to anti-TCR-induced growth
(Fig. 1 and reference 27). Furthermore, kinase-active Lck
associated with the plasma membrane is observed in clone
2.10 (Fig. 2). The prediction follows that increasing the
concentration of IL-2 used to propagate clone 2.10 should
ultimately result in the inhibition of anti-TCR-induced
growth, and indeed antigen-induced growth. Anti-TCR-
mediated growth of clone 2.10 propagated in 50-100 U/ml
IL-2 is inhibited, as is the antigen response of both clones
2.5 and 2.10 propagated in these conditions (data not
shown). Similar IL-2-mediated inhibition of the antigen
response of CD4+ T cell clones have been reported by
other investigators (17). It is possible that these high concentrations of exogenous IL-2 have predisposed cells to antigen receptor-induced apoptosis (16).
The observed differences in the sensitivity of anti-TCR and antigen induced responses to exogenous IL-2 may reflect the differential involvement of the CD4 associated compartment of membrane Lck in these two modes of activation. Thus, doses of IL-2 that virtually ablate the anti-TCR responses of clones 2.5 and 2.5.2 do not impair their responses to antigen (Figs. 1 and 3). Membrane-associated Lck is nonetheless kinase inactive in these cells before antigen stimulation (Figs. 2 and 5). As previously described for clone 2.5, antibody-mediated coaggregation of CD4 with TCR does result in robust responses, but only if CD4 is associated with Lck (27). Thus, as with coaggregation of mAbs, antigen-mediated juxtaposition of CD4-Lck with the TCR-CD3 complex (62) obviates the requirement for preexisting membrane-associated kinase-active Lck associated with anti-TCR responsiveness in resting clones and primary T cells.
Consistent with these notions is the demonstration in this study that mAb-mediated aggregation of CD4 on clone 2.5.2 propagated in IL-2 results in the induction of kinase activity within the CD4-associated pool of Lck (Fig. 6). This result supports the conclusion that the capacity of antigen, but not anti-TCR, to induce the growth of clone 2.5.2 propagated in these conditions reflects the differential recruitment of the CD4-associated pool of Lck to the antigen receptor complex. Furthermore, the inactivity of "membrane" Lck in clone 2.5.2 propagated in IL-2 correlates with undetectable levels of phosphotyrosyl content, suggesting that phosphorylation of Y394, demonstrated by others to predicate Lck activation, is a probable basis for this IL-2-mediated Lck phenotype. Thus, IL-2-mediated downregulation of the basal kinase activity of membrane-associated Lck in clone 2.5.2 is not irreversible, and can presumably be rescued by antigen-mediated coaggregation of CD4 with the TCR-CD3 complex.
The functional uncoupling of mAb-mediated TCR and
CD3 signaling observed in this study is not unprecedented.
The first demonstration involved the characterization of
the differential capacity of anti-TCRC and anti-CD3
to
induce calcium flux in thymocytes and lymph node T cells
(63). Subsequent studies extended this signaling phenotype
to include mature primary T cells (47). The central role
of Lck in this uncoupling phenotype is supported by results
obtained using Lck-deficient T cells. Studies using Lck
variants of the T cell line, Jurkat, demonstrated that signaling mediated by Ti-specific mAb was profoundly reduced
in comparison to that mediated by CD3
-specific mAb
(42). Furthermore, mAb-mediated signaling through the
TCR in primary T cells from Lck-deficient animals is ablated, whereas that through CD3
is only partially affected
(43). The results presented in this study demonstrate that
IL-2, through altering the subcellular distribution of kinase-active Lck, mediates the same uncoupling phenotype.
Taken together, the results support the conclusion that
mAb-mediated signaling through TCR and CD3 has differential requirements for Lck.
Although the role of Lck in generating proximal signals
emanating from the TCR has been established, there is a
paucity of information correlating its cellular location with
its delivery of function. A plausible function of the kinase-active, membrane-associated Lck described in this study is
the maintenance of a basal level of TCR- phosphorylation. In vitro observations have demonstrated that the
chain can be a substrate for Lck (35, 36). In this study we
demonstrate that the presence of kinase-active Lck at the
plasma membrane correlates with the presence of pp21
and its constitutive association with ZAP-70. This result
supports and extends the recently published observation
that thymocytes isolated from mice containing a targeted
disruption of the Lck locus do not contain pp21
, whereas
those thymocytes from Lck-sufficient animals do (36). The
pp21
observed in this study represents a hypophosphorylated form of the molecule. As previously described (64), it
is accompanied by a pp23 form, induced upon aggregation of the antigen receptor. Furthermore, immune complex kinase assays indicate that the ZAP-70 in pp21
-ZAP 70 complexes is inactive and is activated only upon aggregation of the antigen receptor complex (34). Thus, resting T
cells are poised to respond through TCR, and the basal
configuration of the antigen receptor complex supporting
this phenotype is mediated at least in part through the presence of kinase-active, membrane-associated Lck.
Recent results provide a possible mechanism through
which the basal level of Lck kinase activity reported here
could be induced and maintained in primary resting T cells,
and by extension in resting T cell clones cultured in the
presence of syngeneic irradiated splenocytes. The survival
of mature CD4+ and CD8+ naive T cells is profoundly
compromised in the absence of MHC class II and MHC
class I expression, respectively, in the periphery (65, 66).
Thus, in MHC null mice, the half-life of mature T cells of
either lineage is markedly reduced. These results support the conclusion that continuous, and probably low affinity,
interaction of TCR with self-MHC molecules in the absence of nominal peptide is providing critical survival signals. The basal configuration of the TCR-CD3 complex
described in this study, comprised of pp21 and its complexes with ZAP-70, could reflect the biochemical consequences of these interactions on the activity of Lck.
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Footnotes |
---|
Address correspondence to Michael Julius, Arthritis and Immune Disorder Research Centre, Suite 700, 620 University Ave., c/o 610 University Ave., Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-6549; Fax: 416-946-6589; E-mail: michael.julius{at}utoronto.ca
Received for publication 17 February 1998 and in revised form 30 July 1998.
L. Haughn was supported by a fellowship from the Cancer Research Society, Inc. B. Leung was supported by a Medical Research Council studentship. A. Veillette is the recipient of an MRC Scientist Award. This work was supported by grants from the Medical Research Council of Canada, the National Cancer Institute, and the National Institutes of Health.We thank C. Cantin and Denis Bouchard and the flow cytometry unit at the Ontario Cancer Institute; Mina Marmor for help in the isolation of primary CD4+ lymph node T cells; and Philippe Poussier and Robert Rottapel for helpful discussion and critical review of the manuscript.
Abbreviations used in this paper HMF, heavy membrane fraction; HRP, horseradish peroxidase.
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