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CUTTING EDGE |
*
Beirne B. Carter Center for Immunology Research, and
Departments of Pathology and Microbiology, University of Virginia, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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A number of studies suggest that lipid rafts promote the activation of T cells. Many key TCR-associated signaling molecules, such as the Src family kinases Lck and Fyn, are constitutively associated with lipid rafts (3). These kinases, in concert with other raft-associated molecules, transduce intracellular signals after TCR ligation that lead to tyrosine phosphorylation, Ca2+ flux, and ultimately T cell proliferation (4). Further evidence supporting a role for lipid rafts in regulating T cell activation comes from studies showing that lipid rafts accumulate at the site of TCR engagement, that T cell priming induces plasma membrane lipid raft formation, and that disruption of these microdomains abrogates essential T cell-specific signaling cascades (5, 6, 7).
We recently reported that CD8+ T cells directed
to a subdominant epitope of the influenza hemagglutinin
(HA)3 protein activate
in response to Ag and express normal levels of TCR but lack effector
activity and fail to bind specific MHC class I tetramers
(8). On further stimulation with specific Ag, these
activated CD8+ T cells become cytolytic, produce
IFN-
, and transition from a tetramer-negative (nonbinding) to a
tetramer-positive (binding) phenotype, without any change in the level
of cell surface TCR. At the time, we speculated that the display or
arrangement of TCR on the surface of these activated T cells might
influence the efficiency of binding of TCR by tetramer, possibly as a
result of an interaction or association of TCR with plasma membrane
lipid rafts (8).
In this report, we examine the role of lipid rafts and lipid raft integrity on the strength of the interaction of MHC class I tetramers with TCR on a functional, activated CD8+ T cell population. Using several methods to disorder lipid rafts, we show that disruption of low density microdomains leads to a loss of tetramer binding by the CD8+ T cells without affecting expression of TCR or other cell surface molecules. Furthermore, we demonstrate that the TCR on activated CD8+ T cells is displayed on the cell surface in a nonuniform, asymmetrical pattern and that disruption of lipid rafts leads to a change in TCR display from an asymmetrical to diffuse distribution. Thus, the results of this analysis provide evidence that TCR may interact with (is associated with) lipid rafts before TCR engagement and that lipid raft organization influences cell surface TCR array.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Methyl-
-cyclodextrin (MCD; Sigma, St. Louis, MO) was
dissolved in water (0.5 M) and diluted in DMEM containing 50 mM HEPES
buffer. Filipin (Sigma) was dissolved in absolute ethanol (5 mg/ml) and
prepared in PBS at the indicated concentrations. Simvastatin
(Calbiochem, La Jolla, CA) was prepared as described
(9).
Fluorescently labeled anti-TCR (H57-597), anti-CD3
(145-2C11), anti-CD11a (2D7), anti-CD25 (RD4), and
anti-Ly6C (AL-21) Abs were purchased from PharMingen (San Diego,
CA). FITC-conjugated cholera toxin subunit (CTx; Sigma) was used to
label lipid raft ganglioside GM1. MHC class I
tetramers were prepared as described (10) using a plasmid
DNA encoding the extracellular domain of the
H-2Kd heavy chain and human
2-microglobulin provided by J. D. Altman
(Emory University, Atlanta, GA). Synthetic influenza HA and
nucleocapsid protein (NP) peptides were synthesized at the Biomolecular
Core Facility at the University of Virginia Medical Center. An optimal
tetramer concentration of 3.5 µg/ml was used for cell surface
staining.
Disruption of lipid rafts
To selectively remove or intercalate cholesterol in lipid rafts,
cells were washed in PBS, incubated in serum-free DMEM containing
MCD for 30 min at 37°C or PBS containing filipin for 15 min at
room temperature, and then washed extensively in PBS and stained for
flow cytometry at 4°C using staining and washing solutions containing
0.05% sodium azide. For cholesterol synthesis inhibition, cells were
washed thoroughly in cholesterol-free OptiMEM medium (Life
Technologies, Gaithersburg, MD) and then plated in OptiMEM containing
human recombinant IL-2 and L-glutamine in the presence or
absence of 5 µg/ml simvastatin. Cells were incubated for 12–48 h at
37°C and then stained for flow cytometric analysis. To prepare cells
for confocal microscopic analysis, untreated or MCD-treated D4 clone
was labeled with CTx, Abs, or MHC tetramer at 4°C in buffer
containing 0.05% sodium azide, fixed with 2% paraformaldehyde, and
cytospun onto glass slides.
To overcome the effects of cholesterol synthesis inhibition, cells were
cultured in Optimem containing simvastatin (5 µg/ml) and 10% FBS,
1% bovine cholesterol-enriched low density lipoprotein (LDL; Sigma),
or 0.5% squalene (Sigma) for 18 h and then analyzed for cell
surface marker expression by flow cytometry. To reverse the effects of
cholesterol depletion, MCD-treated cells were incubated at 37°C
for 3 h in serum-free medium containing 1% LDL-cholesterol, 400
µg/ml water-soluble cholesterol (Sigma), or 0.2 mM MCD-cholesterol
complexes (11).
Bulk cultures and cell lines
BALB/cAnNTac mice (Taconic Farms, Germantown, NY) were primed i.p. with 5 x 106 PFU (25 HA U) of infectious A/Japan/305/57. After >3 wk, immune splenocytes were stimulated in vitro with HA204 peptide-pulsed (10-9 M) splenocytes as described (8). Cells were harvested 5–6 days post in vitro stimulation. CD8+ T cell clones D4 and 14-13 are stimulated biweekly with the HA210–219 and NP147–155 epitopes of A/Japan virus, respectively.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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CD
and analyzed for cell surface TCR expression and
HA210 tetramer binding. Treatment with MCD
over a range of concentrations did not alter cell surface TCR
expression (Fig. 1
), a finding consistent
with previous evidence that disruption of lipid rafts on T cells has
little or no effect on cell surface TCR levels (13). By
contrast, MCD treatment substantially decreased the efficiency of
tetramer binding by the T cells in a dose-dependent manner (Fig. 1).
Disruption of lipid rafts had little effect on the level of the T cell
surface molecules CD11a (Fig. 1), CD3, CD8
, CD8, or CD25 (data
not shown), but did result in a modest (2-fold) decrease in the
staining intensity of the GPI-linked, raft-associated Ly6C molecule
(data not shown), suggesting that treatment with MCD may
preferentially affect the detection of certain lipid raft-associated
molecules. In addition, the preferential effect of MCD treatment on
TCR binding to tetramer was not dependent on active energy metabolism
in the T cells, given that D4 cells treated with MCD in the presence
or absence of sodium azide showed a comparable selective decrease in
tetramer staining (data not shown). This finding suggests that the loss
of tetramer binding by TCR after MCD treatment likely represents a
direct effect of lipid raft disruption rather than an energy-dependent
signaling event associated with MCD treatment.
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CD (data not shown).
MCD disrupts the integrity of lipid rafts by extracting cholesterol
from plasma membranes (15). If, as the above results
suggest, the efficiency of TCR binding to tetramer is influenced by the
integrity of plasma membrane lipid rafts, then other methods to disrupt
lipid raft organization on the CD8+ T cell
membrane should likewise affect the interaction of TCR with tetramer.
To evaluate this possibility, we examined the effect of filipin or the
inhibitor of cholesterol synthesis, simvastatin, on TCR expression and
tetramer binding by D4 cells. Unlike MCD, filipin disrupts lipid
microdomains by binding to and intercalating cholesterol-rich lipid
rafts (16). As Fig. 2 shows,
tetramer binding on filipin-treated D4 T cells decreases in a
dose-dependent manner, whereas TCR levels remain constant or increase
slightly when cells are treated with a high dose of the drug.
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). Treatment of the
cells with simvastatin for longer times (24–48 h) led to a more
dramatic selective reduction in tetramer binding (data not shown), but
the inhibition of tetramer-TCR interaction was associated with a
significant decrease in the viability of D4 cells (>60–70% cell
loss), presumably because of serum and/or sterol deprivation.
If cholesterol depletion and the associated lipid raft disruption
result in the decreased efficiency of tetramer binding by TCR, the
reincorporation of cholesterol into the plasma membrane should reverse
the effect of these cholesterol-active agents. To test this
possibility, D4 cells treated with MCD were incubated for 3 h
in serum-free medium containing soluble complexes of MCD and
cholesterol, which have been reported to facilitate the incorporation
of exogenous cholesterol into membranes (18). As Fig. 3 shows, treatment of
cholesterol-depleted D4 cells with MCD-cholesterol complexes
resulted in a partial restoration of tetramer binding by the cells and
also reversed the modest decrease in TCR staining intensity exhibited
by the T cells after lipid raft disruption. These results are
consistent with a recent study showing that the addition of cholesterol
to a transgenic T cell line increased specific MHC dimer binding
(19).
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). We
also attempted to reverse the effects of MCD and simvastatin on
tetramer-TCR interaction by adding squalene, a partially water-soluble
intermediate in the cholesterol-biosynthetic pathway (20),
water-soluble cholesterol, or purified cholesterol-enriched LDL to the
treated cells, but these strategies were unsuccessful (data not shown).
The reasons for this lack of reversal are not clear but may be due in
one instance to the amphiphilic properties of sterols (squalene) in
aqueous solution and in the other to the inefficient uptake of these
isolated lipoprotein-cholesterol complexes by the cells through the
endocytic pathway (15).
The above observations suggest that TCR are displayed on the activated
T cell in an array favorable for efficient TCR interaction with soluble
MHC class I tetramers and that the arrangement of TCR on the cell
surface is influenced by the integrity of plasma membrane lipid rafts.
To examine the display of lipid rafts and TCR on intact D4 T cells, we
used confocal microscopy using CTx to identify the ganglioside
GM1, which is enriched in and preferentially
associated with lipid rafts, and anti-TCR -chain Ab to label
TCR. To evaluate the impact of lipid raft disruption on TCR
arrangement, untreated and MCD-treated cells were examined. For this
analysis (as well as in the experiments described above), staining of
lipid rafts and TCR was conducted at 4°C in the presence of sodium
azide to block any energy-dependent signaling events associated with
CTx or anti-TCR Ab binding to the cells.
On untreated D4 T cells harvested at least 7 days after in vitro
stimulation with specific Ag, GM1 was distributed uniformly
throughout the plasma membrane in a diffuse, ring-like manner, typical
of the lipid raft distribution seen on other cell populations
(5). There were several areas of more intense
GM1 staining, suggesting some asymmetry in the
array of lipid rafts on the plasma membrane surface (Fig. 4). By contrast, we repeatedly observed
that TCR was not uniformly distributed on the cell surface but rather
was primarily asymmetrically localized to distinct regions of the
plasma membrane (Fig. 4). An identical TCR staining pattern was
observed when fluorochrome-labeled HA210 tetramer
was used to label this cell surface marker (data not shown).
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).
Importantly, aggregation of lipid rafts also resulted in the
colocalization of TCR to the raft cap (Fig. 4).
Treatment of the T cells with MCD had a minimal effect on the
pattern of CTx staining (Fig. 4). Treated cells exhibited a relatively
uniform ring-like staining pattern with notably fewer areas of focal
increased GM1 labeling than observed on untreated
cells. The effect of MCD on TCR arrangement was more dramatic, with
TCR now exhibiting a much more diffuse, ring-like distribution (Fig. 4). Importantly, cholesterol depletion by MCD inhibited both the
formation of lipid raft caps after CTx cross-linking (Fig. 4) and TCR
aggregation after cross-linking of cell surface bound anti-TCR Ab
(data not shown). In keeping with the above observations (Fig. 1),
MCD treatment did not result in a decrease in
GM1 content on the plasma membrane, as determined
by the intensity of CTx staining in companion flow cytometric analyses
(data not shown), which further supports the view that lipid raft
disruption results in a redistribution, rather than a loss, of cell
surface molecules.
The results reported here suggest that the arrangement of TCR on the
surface of activated CD8+ T cells is influenced
by the organization and integrity of lipid rafts in the plasma
membrane. The difference between the TCR-tetramer interaction and the
TCR-anti-TCR Ab interaction, in their respective sensitivity to
lipid raft disruption, may be related to the relatively low intrinsic
affinity of the interaction between TCR and individual peptide-MHC
class I complexes (21). If so, then the TCR are likely to
be normally clustered or arrayed on the cell surface in a manner that
facilitates the multivalent interaction between the low affinity TCR
and its tetramer ligand, and redistribution of cell surface TCR after
lipid raft disruption may adversely affect tetramer binding while not
affecting the binding of Ab to TCR. Because CD8 can influence the
binding of particular MHC tetramers to TCR (22) and
because the chain of this coreceptor partitions to lipid rafts
(23), it is possible that the decrease in tetramer binding
after lipid raft disruption may also reflect a change in the
interaction of cell surface TCR with CD8, which in turn could alter the
strength of tetramer binding.
Observations from a number of laboratories indicate that TCR is not associated with low density, detergent-insoluble lipid domains before Ag contact (3, 24), but only colocalizes transitorily with lipid rafts after Ag encounter (13). The finding of extensive colocalization of TCR with lipid rafts on intact cells reported here and recently by Janes et al. (5) suggests the possibility of a baseline steady state association of TCR with lipid rafts and that such interactions at the plasma membrane may not always withstand physical membrane isolation procedures (1, 5). Furthermore, the possible requirement for TCR to be recruited to lipid rafts to engage critical raft-associated signaling intermediates necessary for T cell activation do not exclude TCR-lipid raft interactions before TCR engagement if there is heterogeneity among lipid rafts in their molecular composition (1).
The observations described herein showing that lipid raft integrity affects the display of cell surface TCR provide a possible explanation for our recent finding that virus-specific CD8+ T cells undergoing incomplete activation in response to virus infection fail to bind specific MHC class I tetramers (8); that is, the display of TCR on the surface of these partially activated CD8+ T cells is unfavorable for efficient tetramer binding. A study by Fahmy et al. (19) supports this claim by demonstrating that TCR on activating T lymphocytes redistributes to an arrangement that greatly enhances the binding of specific MHC dimers and that the modulation of plasma membrane cholesterol content likewise influences the efficiency of TCR binding to MHC dimers. Furthermore, recent studies demonstrating that binding of MHC class II tetramers to CD4+ T cells depends on the activation state of the cell (25) and that recruitment of BCR to lipid rafts after receptor cross-linking is dependent on the state of B cell maturation (26) also suggest that the arrangement of Ag receptors on lymphocytes, through their association with lipid rafts, may be regulated during cell differentiation and activation. The finding that T cell activation induces the rapid synthesis and recruitment of lipid raft ganglioside GM1 to the plasma membrane also suggests that low density microdomains may regulate T cell responsiveness (6). Our results (8) showing that activated CD8+ T cells can exhibit Ag-specific effector activity, i.e., in vitro cytolysis or cytokine secretion, without staining with specific tetramer may also reflect lipid raft-mediated control of TCR arrangement. Additional studies will determine the extent of the impact of these TCR-lipid raft interactions on T lymphocyte activation and function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas J. Braciale, Beirne B. Carter Center for Immunology Research, University of Virginia, Box 801386, MR4 Building, HSC Box 4012, Charlottesville, VA 22906. E-mail address: tjb2r{at}virginia.edu
3 Abbreviations used in this paper: HA, hemagglutinin; MCD, methyl--cyclodextrin; CTx, cholera toxin subunit; NP, nucleocapsid protein; LDL, low density lipoprotein.
Received for publication March 13, 2001. Accepted for publication April 16, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-cyclodextrins and liposomes as water-soluble carriers for cholesterol incorporation into membranes and its evaluation by a microenzymatic fluorescence assay and membrane fluidity-sensitive dyes. Anal. Biochem. 258:277.[Medline]
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