Primary Human CD4+ T Cells Have Diverse
Levels of Membrane Lipid Order That
Correlate with Their Function
This information is current as
of June 16, 2017.
Laura Miguel, Dylan M. Owen, Chrissie Lim, Christian
Liebig, Jamie Evans, Anthony I. Magee and Elizabeth C.
Jury
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J Immunol 2011; 186:3505-3516; Prepublished online 9
February 2011;
doi: 10.4049/jimmunol.1002980
http://www.jimmunol.org/content/186/6/3505
The Journal of Immunology
Primary Human CD4+ T Cells Have Diverse Levels of
Membrane Lipid Order That Correlate with Their Function
Laura Miguel,* Dylan M. Owen,†,1 Chrissie Lim,* Christian Liebig,†,2 Jamie Evans,*
Anthony I. Magee,† and Elizabeth C. Jury*
C
urrent evidence supports an important role for lipid
microdomains (lipid rafts) in the formation of the immunological synapse (IS) between T lymphocytes and
APC; this process involves the segregation and reorganization of
membrane lipids and proteins and is dependent on the actin cytoskeleton (1, 2). Although the functional outcome of T cell/APC
interactions depends on the nature of IS formation (3), the importance of membrane microdomains in the regulation of IS development, cell differentiation, and function of primary human
T cells is not fully understood.
*Division of Medicine, Centre for Rheumatology Research, University College London, London W1P 4JF, United Kingdom; and †Section of Molecular Medicine, National Heart & Lung Institute, Imperial College London, South Kensington, London
SW7 2AZ, United Kingdom
1
Current address: Centre for Vascular Research, University of New South Wales,
Sydney, Australia.
2
Current address: Hertie-Institute for Clinical Investigation, Cell Biology and Neurological Disease, Tübingen, Germany.
Received for publication September 3, 2010. Accepted for publication January 12,
2011.
This work was supported by an Arthritis Research UK Career Development award
to E.C.J. (18106) and a University College London Hospital Clinical Research and
Development Committee project grant (GCT/2008/EJ). A.I.M. is supported by Medical Research Council Grant G0700771.
Address correspondence and reprint requests to Dr. Elizabeth Jury, Centre for Rheumatology Research, University College London, Windeyer Building, 46 Cleveland
Street, London W1P 4JF, United Kingdom. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ANE, di-4-ANEPPDHQ; CTB, cholera toxin subunit B; GP, generalized polarization; IRM, interference reflection microscopy; IS,
immune synapse; 7KC, 7-ketocholesterol; PKC, protein kinase C; pY, phosphotyrosine; RA, rheumatoid arthritis; rh, recombinant human; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B; SLE, systemic lupus erythematosus;
SMAC, supramolecular activation complex; SS, Sjörgren’s syndrome; TIRF, total
internal reflection fluorescence.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002980
The organization of plasma membrane sphingolipids and cholesterol into microdomains with relative liquid-order compared
with the surrounding disordered membrane is the basis of the lipid
raft hypothesis in mammalian cells (4). Some cell surface proteins
preferentially associate with ordered lipid microdomains whereas
others are excluded and diffuse freely in the more disordered
membrane (5). A main issue when considering lipid microdomains
is their visualization (6) since they are dynamic and of a size too
small to resolve using conventional microscopy (7). Original
results based on the resistance of ordered lipid microdomains to
solubilization with nonionic detergents (so called detergent-resistant membranes) (8) and cross-linking cell surface domains with
multivalent probes such as cholera toxin subunit B (CTB) revealed that capping of lipid microdomains at the IS following
TCR stimulation facilitates coordination, localization, and function of proteins residing proximal to the TCR (4, 5, 9). However,
there is debate about whether these methods reliably identify ordered lipid microdomains as they exist in living cells (9, 10).
A new approach to their analysis has been to observe ordered and
disordered membranes in live T cells, using fluorescent membrane
probes such as LAURDAN and di-4-ANEPPDHQ (ANE). ANE
partitions into both liquid-ordered (raft) and liquid-disordered
(nonraft) membranes and senses the environmental difference
between the two regions. It is water-soluble yet binds to lipid
membranes with high affinity and is therefore easily loaded into
membranes (11). The incorporation of ANE into hydrophobic
(more ordered) and hydrophilic (less ordered) membranes influences its interaction with aqueous solution and its subsequent
fluorescent emission spectra (11). When ANE is excited in the
blue spectral region with single-photon excitation it exhibits a 60nm spectral blue shift between the disordered and ordered lipid
phases (11). The degree of membrane order can be calculated
from the dye’s emission properties and expressed as a generalized
polarization (GP) value (a normalized intensity ratio of two different spectral channels) (12). This approach has provided evi-
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Membrane lipid microdomains (lipid rafts) play an important role in T cell function by forming areas of high lipid order that
facilitate activation. However, their role in regulating T cell differentiation and function remains controversial. In this study,
by applying a new approach involving microscopy and flow cytometry, we characterize membrane lipid order in ex vivo primary
human CD4+ T cells. We reveal that differential membrane lipid order dictates the response to TCR stimulation. T cells with high
membrane order formed stable immune synapses and proliferated robustly, intermediate order cells had reduced proliferative
ability accompanied by unstable immune synapse formation, whereas low order T cells were profoundly unresponsive to TCR
activation. We also observed that T cells from patients with autoimmune rheumatic disease had expanded intermediate order
populations compared with healthy volunteers. This may be important in dictating the nature of the immune response since most
IFN-g+CD4+ T cells were confined within intermediate membrane order populations, whereas IL-4+CD4+ T cells were contained
within the high order populations. Importantly, we were able to alter T cell function by pharmacologically manipulating membrane order. Thus, the results presented from this study identify that ex vivo CD4+ T cells sustain a gradient of plasma membrane
lipid order that influences their function in terms of proliferation and cytokine production. This could represent a new mechanism
to control T cell functional plasticity, raising the possibility that therapeutic targeting of membrane lipid order could direct
altered immune cell activation in pathology. The Journal of Immunology, 2011, 186: 3505–3516.
3506
MEMBRANE LIPID ORDER CORRELATES WITH T CELL FUNCTION
Materials and Methods
Cell isolation
PBMC from 40 healthy donors were separated on Ficoll-Hypaque (Pharmacia Biotech). PBMC from 40 healthy donors (mean age, 34.7 y; 10 male,
30 female) and 10 patients with active systemic lupus erythematosus
(SLE), 10 patients with rheumatoid arthritis (RA), and 5 patients with
Sjörgren’s syndrome (SS) attending the Rheumatology Clinic at University
College Hospital were included in this study after receiving informed
consent. The study was approved by the local ethics committee. Purified
CD4+ lymphocytes were obtained by negative selection using magnetic
beads (Miltenyi Biotec) or by cell sorting using FACSAria (BD Biosciences).
Abs and reagents
Abs for flow cytometry included allophycocyanin-CD4, Pacific Blue-CD4,
allophycocyanin-CD3, FITC-CD8, PE-Cy5-CD25, allophycocyanin-CD45RA,
allophycocyanin-H7-CD27, allophycocyanin-CD69, allophycocyanin-CD25,
allophycocyanin-Cy7-IFN-g, Pacific Blue-IL-10, allophycocyanin-IL-4,
FITC-IL-2, PE-Ki67, allophycocyanin-T-bet, allophycocyanin-pSTAT6,
allophycocyanin-annexin V, and fluorochrome-conjugated isotype controls
(all from BD Biosciences). Abs for confocal microscopy included antiCD3 (OKT3) and anti-phosphotyrosine (pY) (4G10) with secondary antimouse IgG2a-Alexa Fluor 488 and anti-mouse IgG2b-Alexa Fluor 555
from Invitrogen. For functional experiments and for coating coverslips,
purified Abs to CD3 (HIT3a), CD28 (CD28.2), or IgG isoype controls
from BD Biosciences were used. For generation of polarized Th1/Th2
populations, anti–IL-4, anti–IL-12, anti–IFN-g (eBioscience), recombinant human (rh)IL-12p70, rhIL-2, and rhIL-4 (R&D Systems) were used.
The membrane order probe di-4-ANEPPDHQ, CTB-biotin, and PP2
were obtained from Invitrogen. DAPI was obtained from Sigma-Aldrich
(St. Louis, MO).
Flow cytometry
PBMC were stained for protein surface markers before washing and
resuspending with 4 mM ANE in PBS for up to 30 min at 37˚C. For each
population the geometric mean fluorescence intensity for wavelengths at
570 nm (FL2 channel) and 630 nm (FL3 channel) were used to make the
GP calculation (see Equation 1 below). Staining for annexin V was performed according to manufacturer’s instructions (BD Biosciences) following ANE labeling. Cells were analyzed without fixing using a BD LSR
II flow cytometer (BD Biosciences) and FlowJo software (Tree Star).
Staining for intracellular cytokines, Ki67, T-bet, and pSTAT6 was performed on FACS-sorted populations after fixation/permeabilization with
either saponin- or methanol-based buffer according to the manufacturer’s
instructions (eBioscience). Cell viability was assessed by annexin V
staining according to the manufacturer’s instructions and DAPI exclusion
by flow cytometry. For the flow cytometry-based conjugation assay, autologous APC were isolated from PBMC by removal of CD3+ cells using
magnetic columns (Milteni Biotec) and labeled with CellTracker Blue
CMHC (Invitrogen) following the manufacturer’s protocol before loading
with superantigen (1 mg/ml staphylococcal enterotoxin E, 2 mg/ml staphylococcal enterotoxin A [SEA]/staphylococcal enterotoxin B [SEB]) for 1 h at
37˚C. FACS-sorted low, intermediate, and high order CD4+ T cells were
labeled with CellTracker Green CMFDA (Invitrogen). APC and T cells were
mixed in a ratio of 1:1, briefly centrifuged for 1 min at 100 3 g to form
conjugates, and then incubated in complete RPMI 1640 medium (100 ml) at
37˚C for 5 min. Thereafter, T cell/APC mixtures were fixed in PBS containing 1% paraformaldehyde before analysis. The relative proportion of
orange, blue, and orange/blue events in each sample was determined. For
manipulation of membrane order, 15 mg/ml 7-ketocholesterol (7KC) and
cholesterol (Avanti Lipids, Alabaster, AL) in ethanol were combined in a
cholesterol-7KC ratio of 1:2. During 30 min, these were then added to a
solution of 50 mg/ml methyl-b-cyclodextrin (Sigma-Aldrich) in PBS at 80˚C
to a final sterol concentration of 1.5 mg/ml. Then, 15 ml/ml lipid solution
was added to cell medium containing 1 3 106 cells at 37˚C for 30 min before
labeling with ANE and analysis by flow cytometry and microscopy.
Differentiation of Th1 and Th2 phenotypes
FACS for CD4+CD82CD252CD45RA+ T cells were stimulated with antiCD3 and anti-CD28 (2 mg/ml each) and cultured in RPMI 1640, 10% (v/v)
FCS, penicillin/streptomycin, and 2 mM L-glutamine. To generate differentiated Th cell responses, Th1 cultures were supplemented with rhIL12p70 (10 ng/ml) and anti–IL-4 (10 mg/ml), and Th2 cultures were supplemented with anti–IL-12 (10 mg/ml), rhIL-4 (100 U/ml), and anti–IFN-g
(10 mg/ml). Cultures were supplemented with rhIL-2 (100 U/ml) on day 4
after the activation of cultures; on day 6 supernatants and cells were recovered. Th1/Th2 differentiation was confirmed by cytokine analysis of
cell supernatants.
T cell functional assays
T cells sorted for high, intermediate, and low membrane order were
stimulated with either plate-bound anti-CD3 (2 mg/ml) and anti-CD28 (2
mg/ml) Abs, Human T-Activator CD3/CD28 beads (Dynabeads; Invitrogen), or superantigen-coated (SEA/SEB) autologous APC. Three-day
culture supernatants were analyzed for cytokine production by Cytokine
Bead Array (BD Biosciences). Intracellular staining for IFN-g, IL-4, IL-2,
and IL-10 was carried out after incubation with 50 ng/ml PMA, 250 ng/ml
ionomycin, and 2 mM monensin (Golgi-Plug; BD Biosciences) for 4 h
before fixation/permeabilization. For proliferation, cells were stimulated
with anti-CD3/CD28 (2 mg/ml each) for 3 d then pulsed with [3H]thymidine for 16 h, followed by harvesting and scintillation analysis.
Interference reflection microscopy
Glass coverslip cell culture chambers (Intracel, London, U.K.) were coated
with Abs to CD3/CD28 or isotype control (10 mg/ml) in PBS for a minimum of 2 h. Sorted high, intermediate, and low order cells were layered
onto Ab-coated coverslips at 37˚C. Cells were imaged as they attached to
the coverslip surface using a Zeiss confocal microscope and 488 nm argonion laser excitation and a 363 oil objective. Reflected and transmitted light
images were obtained at 30-s intervals. Images were merged using ImageJ
software to reveal areas of cell attachment.
T cell/APC conjugates
Sorted high, intermediate, and low order cells were cocultured with
superantigen (1 mg/ml staphylococcal enterotoxin E, 2 mg/ml SEB/SEA)loaded Raji B cells for 10 min at 37˚C. Conjugates were attached to polyL-lysine–coated coverslips and fixed with 2% paraformaldehyde before
permeabilization with 0.2% Triton X-100 for 10 min and blocking with 5%
BSA/PBS for 2 h. Cells were stained with anti-CD3 and anti-pY Abs
followed by appropriate secondary Abs before mounting onto glass slides
using anti-fade mountant (Invitrogen). Conjugates were imaged using 488
nm argon-ion, 633 nm helium, and 400 nm ultraviolet laser excitation and
a 363 oil immersion lens using a Zeiss LSM-510 inverted laser-scanning
confocal microscope. Approximately 10 conjugates were imaged from
each sample, and 0.5-mm z-slices were acquired through each conjugate.
The Pearson correlation coefficient of anti-CD3 and anti-pY staining at the
IS for each z-slice in the conjugates was measured using Zeiss LSM
software. Three-dimensional reconstructed images were obtained from
deconvoluted images using Volocity software.
Total internal reflection fluorescence (TIRF) microscopy was performed
on a custom-built microscope with excitation at 473 nm and a 360, 1.45NA
oil-immersion TIRF objective. Fluorescence was collected on an electronmultiplying CCD camera (iXon; Andor, Belfast, U.K.) in the range 500–
593 and 600–680 nm using a two-channel imager (Dual-View; Optical
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dence confirming the importance of ordered lipid microdomains
in IS formation and T cell function (6, 13–16).
Using the lipid probe ANE to identify lipid microdomains in ex
vivo human CD4+ T cells, we reveal an array of plasma membrane
lipid order, ranging from low, intermediate, to high order. We
demonstrate that this gradient of membrane lipid order dictates
the outcome of CD4+ T cell responses to activation. Upon TCR
stimulation, high order T cells formed stable IS and proliferated
robustly, intermediate order cells had reduced proliferative ability
accompanied by unstable IS formation, but low order T cells were
profoundly unresponsive. Pharmacologically reducing T cell
membrane order from high to intermediate induced unstable IS
formation and reduced proliferation. Furthermore, most IFN-g+
CD4+ T cells were characterized by intermediate order, whereas
IL-4+CD4+ T cells were within the high order population. Interestingly, patients with autoimmunity had increased intermediate
order T cell populations accompanied by reduced proliferation
and increased production of IFN-g compared with healthy controls. Therefore, the results presented in this study identify a new
mechanism to control T cell functional plasticity, raising the possibility that therapeutic targeting of membrane lipid order could
direct altered immune cell activation in pathology.
The Journal of Immunology
3507
Insights). Data were processed using custom software (LabVIEW; National Instruments, Austin, TX). The sample was maintained at 37˚C using
a stage and objective heater. Quantification of image data was performed
by measuring average GP values (a normalized intensity ratio of two
different spectral channels: green [ordered] and red [disordered]) where
high GP (increased green fluorescence compared with red) represents high
membrane lipid order (12):
GP ¼
I500 2 570 2 I620 2 750
:
I500 2 570 þ I620 2 750
ð1Þ
Confocal microscopy
Sorted high, intermediate, and low order cells were labeled with 4 mM ANE
for 30 min and applied to coverslip chambers at 37˚C. Attachment of cells
was imaged at 5-min intervals up to 30 min from addition of cells to the
chamber, using single-photon excitation confocal microscopy on an
inverted laser-scanning confocal microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany; or Zeiss LSM-510) with a 363, 1.25NA oilimmersion objective lens. Excitation was at 488 nm from an argon-ion
laser. Fluorescence detection was in the wavelength bands 500–580 and
620–750 nm using internal photomultiplier tubes. GP values were calculated from the two wavelength channels according to Equation 1, where I
is the intensity of emission. The z-slices were acquired every 0.5 mm.
All values are expressed as mean 6 SEM. We performed analysis of
significance in Prism (GraphPad Software) by the Mann–Whitney U test,
Student t test, or paired t test as appropriate.
Results
Distinct CD4+ T cell subsets identified based on plasma
membrane lipid order
To investigate the role of membrane lipids in ex vivo human CD4+
T cells, we used a new approach; that is, cells were labeled with
the lipid probe ANE and analyzed by confocal microscopy. The
degree of membrane order was calculated and expressed as a GP
value where high GP represents high membrane lipid order (Fig.
1A) (12). We observed heterogeneous membrane order in ex vivo
negatively purified CD4+ T cells from healthy volunteers. This
was depicted by a wide spectrum of intensity of ANE staining,
revealing cells with relative low (green/blue in the GP image),
intermediate (orange/green in the GP image), and high (red in the
GP image) plasma membrane lipid order. To confirm these findings and to examine a larger number of cells from many individuals, a method was developed combining ANE labeling with
multiparameter flow cytometry (Fig. 1B). Again, three populations
were defined with relative low (1.4 6 1.3% SD), intermediate
(8.28 6 5.1% SD), and high (90.47 6 5.7% SD) plasma membrane order (Fig. 1B, 1C, 1D). Intermediate and high order cells
were viable by DAPI exclusion and annexin V staining (Fig. 1E).
Low order cells had comparatively reduced viability with ∼50%
being annexin V positive, indicating that many were preapoptotic.
Furthermore, when FACS-sorted low, intermediate, and high order
T cells were cultured overnight and relabeled with ANE, they
maintained their order (Fig. 1F).
The ability of flow cytometry and ANE labeling to distinguish
between plasma compared with intracellular membranes (which are
largely low order) (13, 17) was verified by assessing ANE labeling
of CD4+ T cells over time. ANE was rapidly incorporated into
cellular membranes; plasma membrane order was observed at
early time points and remained unchanged over time as observed
in the representative merged intensity images of intermediate and
high order cells (Fig. 1G). The accompanying RGB profiles represent ANE emission spectra detected in 500–570 (green) and
620–750nm (red) channels obtained from equatorial cross-sections
of the intermediate and high order cells. They reveal that ANE was
rapidly incorporated into the plasma and intracellular membranes
of the intermediate (Fig. 1G) and low order cells (data not shown),
Differential membrane order is associated with distinct
patterns of IS formation
Because recent work has shown that high lipid order is important
for stable IS formation (14, 16), the effect of differential global
plasma membrane order on IS development was examined by
interference reflection microscopy (IRM). FACS-sorted low, intermediate, and high order T cells were applied to coverslips
coated with anti-CD3/CD28 or isotype control and visualized for
up to 10 min. Low order cells demonstrated limited and transient
attachments compared with partial interactions by intermediate
order cells (Fig. 2A). In contrast, high order cells exhibited robust
and symmetrical attachments (Fig. 2A). These results were confirmed by confocal microscopy (Supplemental Fig. 1A). Quantitation of imaging data verified that high order cells formed a more
substantial contact surface area with the coverslip compared with
intermediate and low order cells (Fig. 2B). All three populations
possessed similar expression levels of CD3, excluding the possibility that differences in CD3 expression might explain the altered
patterns of synapse formation (Supplemental Fig. 1B).
Given that increased membrane order at the IS facilitates T cell
activation (14, 15, 18), a more accurate assessment of lipid order
at the cell/coverslip interface in FACS-sorted intermediate and
high order populations was made using TIRF microscopy (the
more transient nature of low order cell interactions made it difficult for them to be visualized using this technique). Representative
GP/TIRF images (Fig. 2C) depict areas of highest membrane order in red and lowest order in blue/green. High order cells had the
highest average order at the IS compared with intermediate order
cells (Fig. 2D). The apparent differential nature of IS formation in
the three populations was also tested in a more physiological
T cell/APC system. FACS-sorted low, intermediate, and high order
cells were cocultured with superantigen-loaded Raji B cells, and
T cell/APC conjugates were stained with anti-CD3 (green) and
anti-pY (red) and analyzed by confocal microscopy. Representative images are shown in Fig. 2E. CD3 was patched at the IS in
both intermediate and high order cells (Fig. 2E, 2H) accompanied
by pY accumulation (Fig. 2E, 2G, 2I). CD3 did not accumulate at
the interface between low order T cells and APC (Fig. 2E, 2F,
2H), and no pY accumulation was seen (Fig. 2F, 2G, 2I). Although
both the intermediate and high order cells formed functional
synapses in terms of pY accumulation, differences were seen in
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Statistics
corresponding with the preference for ANE to incorporate into
disordered membranes (13). In contrast, ANE labeling was confined mainly to the plasma membrane in the high order cells, with
intracellular membranes becoming labeled only after 17 min (Fig.
1G, arrow). These results were confirmed by flow cytometry (Fig.
1H), showing that even after 1 min after ANE staining the three
distinct populations were revealed and did not change significantly
over time. Finally, we related the relative level of plasma membrane lipid order to the expression of lipid microdomain-associated
lipids, cholesterol, GM1, and GM3. Intermediate and high order
cells had increased expression of cholesterol compared with low
order cells (assessed by filipin binding), and a significant positive
correlation was seen between global lipid order and membrane
levels of cholesterol (Fig. 1I, upper panels). Alternatively, GM1
(measured by CTB binding) was upregulated in the intermediate
and low order cells, and a significant negative correlation was seen
between membrane order and CTB binding (Fig. 1I, middle panels). Interestingly, glycosphingolipid GM3 was not differentially
expressed between the subsets (Fig 1I, lower panels). These results
reveal differences between measurement of global membrane lipid
order and the traditional markers for assessing membrane lipid
microdomains.
3508
MEMBRANE LIPID ORDER CORRELATES WITH T CELL FUNCTION
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FIGURE 1. Identification of distinct CD4+ T cell subsets based on membrane lipid order. Negatively selected CD4+ T cells were labeled with ANE. A,
Confocal microscopy showing intensity and GP images, revealing cells with low (***), intermediate (**), and high (*) order. Scale bar, 10 mm. B, Flow
cytometry detecting ANE in FL2 (570 nm) and FL3 (630 nm) channels. Three cell populations were gated according to FL2 versus FL3 intensity;
a representative dot plot is shown. Cumulative data from 18 healthy volunteers showing (C) percentage CD4+ cells in each population and (D) corresponding GP value depicting membrane order. E, Cell viability determined by DAPI exclusion (left panel) and annexin V binding (right panel) in CD4+
T cells from six healthy donors. F, FACS-sorted ANE-labeled high, intermediate, and low order T cells from three healthy volunteers were cultured for 18 h,
relabeled with ANE, and GP was assessed by flow cytometry. G, ANE incorporation into plasma and intracellular membranes. ANE was added to CD4+
T cells and imaged under physiological conditions for 30 min. Representative merged intensity images (green, 500–570 nm and red channels, 620–750 nm)
from intermediate and high order cells. Arrows indicate intracellular membrane staining. Scale bars, 5 mm. Right panels show corresponding RGB profiles
(green and red channels) obtained in ImageJ software from equatorial cross-section through the cell. H, Negatively isolated CD4+ T cells were labeled with
ANE and analyzed by flow cytometry at 1, 7, and 30 min. Results from three healthy control samples showing number of cells (%) in low, intermediate, and
high populations. I, FACS-sorted high, intermediate, and low order T cells from 10 healthy individuals were labeled with either filipin, CTB, or anti-GM3 to
detect levels of surface lipids. Cumulative results are show in bar graphs. **p # 0.002, *p = 0.05 low versus high. Lipid expression was assessed in whole
The Journal of Immunology
3509
the pattern of CD3 arrangement; significantly fewer intermediate
cells formed synapses with distinct CD3 patching (Fig. 2H), and
most intermediate order T cells displayed a characteristic diffuse
pattern of CD3/pY colocalization at the IS (Fig. 2E, 2F). Thus,
these results suggest that in ex vivo T cell populations, heterogeneous membrane order may influence the nature and stability of
IS formation with APC.
Plasma membrane order is associated with distinct T cell
function
The dramatic differences in IS formation suggested that membrane
lipid order may influence T cell function following TCR stimulation. IS stability was correlated with the speed of T cell/APC conjugate formation. The three membrane order subsets were FACSsorted and interacted with superantigen-loaded APC and the
CD4+ T cell populations from eight healthy controls by correlating total CD4+ GP with either filipin or CTB binding or staining with anti-GM3 Abs. **r2=
0.0713, p = 0.009; *r2 = 0.728, p = 0.01.
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FIGURE 2. Differential IS formation in T cells with low, intermediate, and high membrane order. A, FACS-sorted high, intermediate, and low order
CD4+ T cells from six healthy donors interacted with anti–CD3/CD28- or isotype-coated coverslips and imaged by IRM; images were taken at 30-s
intervals for 10 min. Representative images of cell/coverslip attachment at 5 min (dark/black color), with unattached cell body outlined in intermediate and
low order cells. Scale bar, 5mm. B, Cell contact area (pixels) was measured; cumulative results from ∼50 cells/condition from six individuals are shown.
***p = 0.0001 IgG versus high, *p = 0.01 IgG versus intermediate. C, Cell/coverslip interaction analyzed by TIRF microscopy; representative GP images
from high and intermediate order cells 30 min after application to coverslips are shown. Red, high order; blue/green, low order. Scale bars, 5 mm. D,
Cumulative TIRF/GP values from ∼25 cells/condition in five individuals. *p = 0.01. E, FACS-sorted ANE-labeled high, intermediate, and low order CD4+
T cells from three individuals interacted with superantigen-loaded Raji B cells. Cells stained for anti-CD3 (green) and anti-pY (red) and imaged by confocal
microscopy (∼10 conjugates/sample) are shown. Representative conjugates show differential interference contrast and deconvoluted images for anti-CD3,
anti-pY, and merged intensity. Scale bars, 5 mm. F, Representative three-dimensional–rendered images from merged CD3/pY confocal z-stacks at the IS.
Scale bars, 1 mm G, Representative Pearson correlation coefficient (R) of red/green colocalization at the IS shown in E and F. H, Pattern of CD3 accumulation at IS for each conjugate; cumulative results showing percentage of cells with CD3 patched at IS. *p = 0.03 high versus intermediate and low order
cells. I, Cumulative data of red/green colocalization across each IS (∼10 conjugates/sample). **p # 0.006, high and intermediate versus low order cells.
3510
MEMBRANE LIPID ORDER CORRELATES WITH T CELL FUNCTION
kinetics of conjugate formation were observed by flow cytometry
(Fig. 3A, 3B). These experiments revealed that high order T cells
form conjugates more slowly than intermediate and low order
cells, although as shown above, the IS is more stable once it is
formed. Changes in IS formation and stability were associated
with differences in cell function. Assessment of low, intermediate,
and high membrane order T cells following 3 d coculture with
superantigen-loaded APC revealed that high order T cells displayed elevated levels of the proliferation marker Ki-67 (Fig.
3C), compared with intermediate and low order cells. Differential
proliferation between the three populations was confirmed by
thymidine incorporation assays following stimulation with antiCD3/CD28 (Fig. 3D), and it was found to be associated with reduced production of IL-2 in the intermediate order T cells (Fig.
3E, 3F). A more detailed assessment of cytokine production in
FACS-sorted high, intermediate, and low order T cells revealed
that TCR stimulation of intermediate order cells preferentially
induced INF-g and IL-6 production compared with high order
cells that produced more IL-4 and IL-10 (Fig. 3G). CD4+ T cells
characterized by low lipid order did not proliferate or produce IL2 in response to TCR stimulation; furthermore, they were more
prone to cell death following activation (Fig. 3H) but were rescued
by exogenous IL-2 or stimulation by PMA/ionomycin (Fig 3I),
suggesting an unresponsive or tolerized phenotype (19).
Phenotypic characterization of the three populations in terms
of memory and activation marker expression using a panel of surface markers (CD45RA, CD27, CD25, and CD69) and multiparameter flow cytometry is shown in Supplemental Fig. 2. The results
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FIGURE 3. Plasma membrane lipid order affects speed of T cell/APC conjugate formation potency of T cell proliferation and cytokine production.
FACS-sorted high, intermediate, and low order CD4+ T cells from four individuals were labeled with CellTracker Orange and cocultured with superantigenloaded APC labeled with blue CellTracker for 5 min. A, Representative dot plots showing rate of T cell/APC conjugate formation (%) and (B) cumulative
data for the three populations. *p = 0.015. C, Representative dot plot showing T cell expression of proliferation marker Ki-67 following 72 h coculture with
superantigen-loaded APC. D, FACS-sorted high, intermediate, and low order CD4+ T cells from eight individuals were cultured with or without anti-CD3/
CD28 for 72 h. Cell proliferation was determined by thymidine incorporation. **p = 0.007 high versus intermediate order. E, Representative dot plot
showing IL-2 production in T cells following 72 h coculture with superantigen-loaded APC. F, Cumulative results from four experiments showing IL-2
production in high, intermediate, and low order cells.*p = 0.05. G, Combined results from eight individuals comparing the percentage difference in cytokine
production by intermediate order cells compared with high order cells. *p $ 0.05 IL-4 and IL-10 compared with both INF-g and IL-6 ,**p = 0.007 IL-2
compared with INF-g. H, FACS-sorted cells cultured with or without TCR stimulation for 24 h and analyzed for cell viability. I, Viability of low order cells
cultured in the presence of IL-2 and PMA. *p = 0.02.
The Journal of Immunology
Table I.
3511
Characteristics of high, intermediate, and low order CD4+ T cells
Membrane Order
High
Intermediate
Low
Area of T cell/APC contact
Speed of T cell/APC conjugate formation
Accumulation of CD3/pY at T cell/APC IS
Proliferation
IL-2 production
IL-4/IFN-g production
Apoptosis
Associated membrane lipids
Cholesterol
GM1
GM3
++
+
+++/+++
+++
+++
+++/+
2
++
+++
++/+++
++
++
+/+++
2
+
+++
2/2
2
ND
ND
+
+++
+
++
+++
++
++
++
+++
++
+++, strong; ++, intermediate; +, weak; ND, not detected.
FIGURE 4. Expansion of intermediate order
T cell population in patients with autoimmune
rheumatic disease. A, Membrane order was
assessed in ex vivo T cells from healthy volunteers (n = 10) and patients with active SLE
(n = 10), RA (n = 10) and SS (n = 5). Representative dot plots and (B) cumulative data are
shown. ***p # 0.0008. CD4+ T cells isolated
from healthy controls and patients with SLE,
RA, and SS were assessed for strength of proliferation by thymidine incorporation (C) and
IFN-g production by intracellular staining (D).
by the addition of protein tyrosine kinase inhibitor PP2 (Supplemental Fig. 3C), indicating that signaling via TCR-associated
kinases mediates an effect on membrane order.
The results described above suggest that membrane lipid order
could represent a phenotypic marker that reflects the functional
capability of T cells with a gradient ranging from T cells with high
membrane order with a naive phenotype, which proliferate robustly
and produce IL-2 and IL-4, to intermediate order cells that have
a memory phenotype, proliferate weakly, but produce IFN-g, and
finally to low membrane order T cells that have an activated
phenotype, are unresponsive to TCR stimulation, and are more
prone to apoptosis (Table I).
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revealed that high order cells were predominantly naive/
nonactivated cells whereas intermediate order cells had a more
activated/effector memory phenotype (Supplemental Fig. 2C–E).
Analysis of the low order population revealed that a high percentage were CD45RA+, but they also revealed a more activated
phenotype expressing high levels of CD69 and CD25 (Supplemental Fig. 2E), further supporting their “anergic-like” profile
(20). In support of the phenotyping profiles, in vitro TCR triggering using soluble anti-CD3 at a range of concentrations together
with costimulation with anti-CD28 (1 mg/ml) reduced global
membrane order and led to an increase in the intermediate and low
populations (Supplemental Fig. 3A, 3B). This effect was inhibited
3512
MEMBRANE LIPID ORDER CORRELATES WITH T CELL FUNCTION
Differential membrane order in patients with autoimmune
rheumatic disease
To relate CD4+ T cell membrane order to in vivo function in
humans, we investigated autoimmune T cells, characterized by
chronic activation, isolated from patients with RA, SLE, and SS,
compared with healthy volunteers. The representative dot plots
and cumulative data (Fig. 4A, 4B) show that patients with active
lupus and RA have a significant expansion of the intermediate
order population compared with healthy volunteers. Interestingly,
those patients with expanded intermediate order populations had
lower proliferation (Fig. 4C) and increased production of INF-g in
response to in vitro TCR stimulation (Fig. 4D) compared with
healthy controls. These results imply that membrane order could
play an important role in determining the response of T cells to
TCR stimulation in vivo.
Th cell phenotypes are associated with specific plasma
membrane lipid order
FIGURE 5. Th1 and Th2 cells are
associated with different relative
levels of plasma membrane lipid
order. A, Naive T cells (CD4+CD82
CD45RA+CD252) were sorted from
four individuals and cultured under
Th1- or Th2-polarizing conditions.
B, Representative dot plot showing
polarized Th1 and Th2 cells labeled
with ANE. C, Cumulative results
from five experiments showing percentage of cells with intermediate and
high order membranes, *p # 0.05
Th1 versus Th2. To confirm phenotype differentiation, cells and supernatants from 6 d polarizing cultures
were analyzed for production of
IFN-g and expression of T-bet (D) or
production of IL-4 and expression
of pSTAT6 (E). Results shown are
from four individual experiments.
*p # 0.05.
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Previous reports examining polarized Th cell populations revealed
differences in IS formation between Th1 and Th2 phenotypes (21,
22). We reasoned that the distinct patterns of IS formation and
alterations in proliferation and cytokine production seen in ex vivo
intermediate and high order CD4+ populations could be connected
with different function in human Th cell subsets.
To examine this possibility we cultured FACS-sorted naive
T cells under Th1- or Th2-polarizing conditions (Fig. 5A) and then
labeled the differentiated cells with ANE to evaluate their plasma
membrane order. The vast majority of naive T cells (CD4+
CD45RA+CD252) had relatively high membrane order (Fig. 5B).
However, after 6-d differentiation, naive CD4+ T cells cultured
under Th1-polarizing conditions assume an intermediate order
phenotype (lower GP) (Figs. 5B, 5C, 6A). Successful Th1 differentiation was confirmed by the production of high levels of IFN-g
and expression of the transcription factor T-bet (Fig. 5D). In
contrast, CD4+ T cells cultured under Th2-polarizing conditions
retained a relative high membrane order (higher GP) (Figs. 5B,
5C, 6A), produced high levels of IL-4, and exhibited increased
levels of phosphorylated STAT6, an established Th2 marker (23)
(Fig. 5E).
The link between membrane order and Th cell function was
strengthened when IS formation and proliferation were assessed
as described previously. Th1 cells with intermediate lipid order
formed less stable IS compared with Th2 cells with higher lipid
order (Fig. 6A), thus confirming the aforementioned findings in
Fig. 2A. Furthermore, Th1 cells formed T cell/APC conjugates
more quickly (Fig. 6C, 6D) but had reduced proliferation (Fig. 6E)
compared with the Th2 cells, again corroborating the results described previously (Fig. 3A–D).
The Journal of Immunology
3513
Discussion
FIGURE 6. IS formation and proliferation in Th1- and Th2-differentiated cells mirror the characteristics of intermediate and high membrane
order populations. Naive T cells were sorted from four individuals and
cultured under Th1- or Th2-polarizing conditions. A, Th1- and Th2-differentiated cells were analyzed by IRM. Representative images show the
pattern of cell/coverslip attachment with representative GP image showing
membrane order. Scale bars, 5 mm. B, Cell contact area (pixels) from the
IRM images; a representative experiment of four is shown. ***p = 0.001.
Th1- and Th2-differentiated cells and superantigen-loaded APC were labeled with CellTracker and cocultured for 5 min. Representative dot plots
show (C) T cell/APC conjugates and (D) cumulative data from four
individuals. *p # 0.05. E, Th1- and Th2-differentiated cells were cocultured with superantigen-loaded APC for 48 h before either staining for Ki67 expression (left panel) or assessing for thymidine incorporation (right
panel). Results are expressed as percentage change in proliferation from
Th1 to Th2 populations. *p = 0.03
Manipulation of membrane order modulates T cell function
The results described so far suggest that the ability of T cells to
respond to TCR stimulation might be associated with mechanisms
that control membrane order. Therefore, to further understand the
relationship between lipid order and T cell function we used the
oxysterol 7KC, which has been shown to reduce lipid order at the
T cell/APC IS and inhibit T cell proliferation and IL-2 production
(14, 24). First, we confirmed this finding in primary T cells; CD4+
T cells cultured with 7KC show a rapid reduction in membrane
order as shown by reduced GP values (Supplemental Fig. 4A) and
an increase in the number of intermediate and low order cells
(Supplemental Fig. 4B). TCR-induced proliferation (Supplemental
Fig. 4C) and IL-2 production (data not shown) were also inhibited
as described previously (14). Having established that 7KC could
One of the determining properties of lipid raft microdomains is that
they form areas of lipid order in biological membranes that help to
compartmentalize signaling molecules (18, 25). Lipid order affects
membrane fluidity; relative disordered membranes support free/
random movement of protein molecules within the membrane,
whereas movement of molecules within more ordered membrane
may be restricted (26). However, controversy has surrounded the
role, and very existence, of these regions mainly because they
have been difficult to visualize by microscopy (9, 10). The use of
membrane probes such as ANE and LAURDAN (12, 27) has gone
some way to resolving some of these difficulties, and the importance of membrane order in the formation of stable IS in both
T cell lines and in primary T cells has been established (13, 14,
16). In this study, by labeling human CD4+ T cells with ANE, we
go further and reveal that ex vivo T cells are heterogeneous in
terms of global plasma membrane lipid order, and three distinct
populations can be discerned. Importantly, we found that not only
can the overall level of T cell plasma membrane lipid order predict
the stability and pattern of IS formation, but it can also predict
T cell function in terms of proliferative ability and profile of cytokine production. Moreover, we were able to manipulate T cell
function by changing membrane lipid order. These results offer
a possible mechanism by which T cells could control functional
plasticity and raise the possibility that therapeutic targeting of
membrane lipid order could direct immune cell activation, helping
to correct abnormal immune responses in conditions such as autoimmunity.
High lipid order at the IS has been shown by several groups,
mainly in Jurkat T cell lines. Combining imaging of the IS with
LAURDAN labeling has shown that, at the T cell/APC interface,
lipid order is high compared with low order in the surrounding
membrane (14, 15). These results corroborate the wealth of data
associating lipid microdomains with T cell activation (1, 2, 4, 18,
28, 29). Additionally, the results indicate that global membrane
lipid order, not just order at the IS, plays a role in shaping the
interaction between a T cell and APC. This could provide an
additional mechanism influencing how T cells relate with other
cells in their immediate environment and may ultimately determine T cell differentiation and function. High order T cells
formed IS with a large surface area that resembled the classical
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reduce global membrane order in primary T cells, we tested its
ability to influence T cell function in FACS-sorted ex vivo CD4+
intermediate and high order populations. Cells were treated with
7KC or left untreated for 30 min and IS formation was assessed by
IRM as before. 7KC treatment of intermediate order T cells did
not influence their membrane order or their ability to form
attachments to Ab-coated coverslips (Fig. 7A, 7B). However, when
high order T cells were treated with 7KC, their membrane order
was reduced (shown in the GP images in Fig. 7A), accompanied
by a significant reduction in the area of cell/coverslip contact (Fig.
7A, 7B). Despite this, 7KC treatment did not significantly influence the number of cells within each population interacting
with anti–CD3/CD28-coated coverslips (Supplemental Fig. 4D).
Interestingly, by reducing membrane order in high order T cells
(using 7KC) we were able to induce a phenotype similar to the
intermediate order population; namely, proliferation was inhibited
(Fig. 7C) and production of IFN-g was increased (Fig. 7D, 7E),
although no significant differences in IL-4 production were seen
(data not shown). 7KC did not influence cytokine production by
intermediate order T cells. Taken together, these results provide
evidence that changes in membrane order could influence T cell
plasticity in terms of cytokine production and proliferation.
3514
MEMBRANE LIPID ORDER CORRELATES WITH T CELL FUNCTION
bulls-eye pattern of IS formation, with peripheral supramolecular
activation complex (SMAC) and distal SMAC regions of attachment, whereas intermediate order cells did not form distinct distal
SMAC regions, had a smaller area of attachment, and interactions
were more rapid. Low order cells did not form a distinct IS pattern
of attachment visible by IRM, although they did form conjugates
with APC (3). It is possible that differences in global membrane
order could discriminate between cells that form long-lived stable
IS that last several hours and the more dynamic and transient
contacts lasting a few minutes, also known as kinapses (30). These
differences could also account for the striking variation in proliferative ability between T cells with low, intermediate, and high
lipid order since stable IS formation is required for full T cell
activation (31), although further work is needed to confirm this
proposal.
The different patterns of IS formation in the three lipid order
populations were characterized by a distinctive arrangement of
CD3 and accumulation of tyrosine phosphorylated proteins at the
T cell/APC interface and subsequently by altered T cell function.
These differences most likely reflect altered strength and organization of TCR-associated signaling events at the IS (31). Stability
of IS formation in naive, effector, and regulatory T cell subsets is
partly determined by differential accumulation of signaling proteins, including protein kinase C (PKC)-u and Wiskott–Aldrich
syndrome protein, that influence actin polymerization in the peripheral and distal SMAC regions of the synapse (3, 32). Differ-
ential membrane lipid order in T cells could regulate these (and
other) key signaling events by controlling the clustering of
membrane proteins at the IS, thereby shaping subsequent T cell
function. It is known, for example, that accumulation of PKC-u at
the IS is essential for Th2 cell development and production of
IL-4 (33), and that altered localization of key signaling molecules
at the IS, including PKC-u and CTLA-4, controls regulatory
T cell function (34, 35). It remains to be determined whether
molecules such as PKC-u preferentially accumulate at the IS in
the high order cells, thereby contributing to more robust proliferation. The unresponsive nature of low order cells, together
with their inability to partition CD3 and the accompanying low
levels of protein tyrosine phosphorylation at the IS, is suggestive
of a tolerized or terminally differentiated phenotype (19, 36).
In vivo-tolerized cells are able to form conjugates with APC but
are unable to mobilize TCR, PKC-u, or lipid raft clustering at the
immune synapse and do not induce tyrosine phosphorylation of
signaling proteins (37). It seems likely therefore that low plasma
membrane order could be a hallmark of tolerized T cells. These
cells were also more prone to apoptosis. It is plausible that differential membrane order could influence multiple T cell intracellular signaling pathways, including cell survival and apoptosis
(38), as indicated by recent work showing that T cell apoptosis is
associated with differential localization of PKC family proteins
with membrane lipid microdomains (39). It remains to be seen
whether lipid order plays a role in such mechanisms, and detailed
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FIGURE 7. Manipulation of membrane order
modifies T cell function. FACS-sorted CD4 + intermediate and high order cells from three healthy
volunteers were cultured for 30 min either without
sterols or with 7KC. A, Cells were labeled with ANE
and analyzed by IRM as described in Fig. 2A. Representative IRM and corresponding GP images. GP
panels: red, high order; green/blue, low order. Scale
bars, 5 mm. B, Cumulative results from three experiments showing the contact area (pixels) of each cell
from the IRM images. *p = 0.02 high order no treatment versus 7KC. C, Proliferation in untreated and
7KC-treated intermediate and high order T cells
assessed by thymidine incorporation. Cytokine production assessed by intracellular staining for IFN-g in
intermediate and high order T cells treated with and
without 7KC. Representative dot plots (D) and cumulative data from five experiments are shown (E). **p =
0.001 high versus high7KC, p = 0.008 high versus intermediate.
The Journal of Immunology
membrane order is associated with higher levels of membrane
cholesterol as might be expected since cholesterol gives structure
to the membrane by allowing lipids to become tightly packed (50,
51). However, CTB binding to GM1 was associated with the less
ordered populations, suggesting that membrane lipid order does
not reflect lipid microdomains as they are described in the literature. More detailed analysis of membrane lipid content using more
accurate methods to determine lipid species will help improve our
understanding of the role played by membrane lipids in T cell
activation and function.
The relevance of T cell membrane order was assessed in a range
of patients with autoimmune rheumatic disease. Patients with lupus
and RA are characterized by chronic immune cell activation. We
reveal that these patients have increased numbers of T cells with
intermediate membrane order, which was associated with reduced
proliferation and increased production of proinflammatory cytokine IFN-g. Lupus T cells are characterized by altered expression
of raft-associated lipids, defective raft-associated signaling, and
altered patterns of IS formation when compared with T cells from
healthy volunteers (52, 53). The enrichment of the intermediate
order population may reflect the altered lipid composition of T cell
membranes in patients with lupus. Whether this is a result of
chronic activation by endogenous Ags is unclear. A more detailed
examination of lipids in T cell membranes from patients with
autoimmune disease is underway.
To conclude, we show in this study that membrane lipid order
is heterogeneous in primary T cells and associated with distinct
Th1-like, Th2-like, and tolerogenic characteristics. However, how
plasma membrane lipid order relates to the previously described
lipid raft microdomains remains unclear. Artificial manipulation of
lipid order influenced IS formation, which in turn may influence the
ability of membrane proteins to cluster at the IS and shape T cell
function. This raises the possibility that therapeutic targeting of
molecules controlling membrane lipid order could regulate immune activation and help control abnormal immune responses in
conditions such as autoimmunity (29).
Acknowledgments
We thank the Facility for Imaging by Light Microscopy at Imperial College.
We also thank Claudia Mauri, Panagiotis Kabouridis, and David Isenberg
for discussions, input, and critical reading of the manuscript.
Disclosures
The authors have no financial conflicts of interest.
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