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Correlate with Their FunctionLevels of Membrane Lipid Order That

T Cells Have Diverse+Primary Human CD4

JuryLiebig, Jamie Evans, Anthony I. Magee and Elizabeth C. Laura Miguel, Dylan M. Owen, Chrissie Lim, Christian

http://www.jimmunol.org/content/186/6/3505doi: 10.4049/jimmunol.1002980February 2011;

2011; 186:3505-3516; Prepublished online 9J Immunol 

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The Journal of Immunology

Primary Human CD4+ T Cells Have Diverse Levels ofMembrane 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*

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 mem-

brane 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.

Current evidence supports an important role for lipidmicrodomains (lipid rafts) in the formation of the im-munological synapse (IS) between T lymphocytes and

APC; this process involves the segregation and reorganization ofmembrane lipids and proteins and is dependent on the actin cy-toskeleton (1, 2). Although the functional outcome of T cell/APCinteractions depends on the nature of IS formation (3), the im-portance of membrane microdomains in the regulation of IS de-velopment, cell differentiation, and function of primary humanT cells is not fully understood.

The organization of plasma membrane sphingolipids and cho-lesterol into microdomains with relative liquid-order comparedwith the surrounding disordered membrane is the basis of the lipidraft hypothesis in mammalian cells (4). Some cell surface proteinspreferentially associate with ordered lipid microdomains whereasothers are excluded and diffuse freely in the more disorderedmembrane (5). A main issue when considering lipid microdomainsis their visualization (6) since they are dynamic and of a size toosmall to resolve using conventional microscopy (7). Originalresults based on the resistance of ordered lipid microdomains tosolubilization with nonionic detergents (so called detergent-resis-tant membranes) (8) and cross-linking cell surface domains withmultivalent probes such as cholera toxin subunit B (CTB) re-vealed that capping of lipid microdomains at the IS followingTCR stimulation facilitates coordination, localization, and func-tion of proteins residing proximal to the TCR (4, 5, 9). However,there is debate about whether these methods reliably identify or-dered 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 membraneprobes such as LAURDAN and di-4-ANEPPDHQ (ANE). ANEpartitions into both liquid-ordered (raft) and liquid-disordered(nonraft) membranes and senses the environmental differencebetween the two regions. It is water-soluble yet binds to lipidmembranes with high affinity and is therefore easily loaded intomembranes (11). The incorporation of ANE into hydrophobic(more ordered) and hydrophilic (less ordered) membranes influ-ences its interaction with aqueous solution and its subsequentfluorescent emission spectra (11). When ANE is excited in theblue spectral region with single-photon excitation it exhibits a 60-nm spectral blue shift between the disordered and ordered lipidphases (11). The degree of membrane order can be calculatedfrom the dye’s emission properties and expressed as a generalizedpolarization (GP) value (a normalized intensity ratio of two dif-ferent spectral channels) (12). This approach has provided evi-

*Division of Medicine, Centre for Rheumatology Research, University College Lon-don, London W1P 4JF, United Kingdom; and †Section of Molecular Medicine, Na-tional Heart & Lung Institute, Imperial College London, South Kensington, LondonSW7 2AZ, United Kingdom

1Current address: Centre for Vascular Research, University of New South Wales,Sydney, Australia.

2Current address: Hertie-Institute for Clinical Investigation, Cell Biology and Neu-rological Disease, Tubingen, Germany.

Received for publication September 3, 2010. Accepted for publication January 12,2011.

This work was supported by an Arthritis Research UK Career Development awardto E.C.J. (18106) and a University College London Hospital Clinical Research andDevelopment Committee project grant (GCT/2008/EJ). A.I.M. is supported by Med-ical Research Council Grant G0700771.

Address correspondence and reprint requests to Dr. Elizabeth Jury, Centre for Rheu-matology Research, University College London, Windeyer Building, 46 ClevelandStreet, London W1P 4JF, United Kingdom. E-mail address: e.jury@ucl.ac.uk

The online version of this article contains supplemental material.

Abbreviations used in this article: ANE, di-4-ANEPPDHQ; CTB, cholera toxin sub-unit B; GP, generalized polarization; IRM, interference reflection microscopy; IS,immune synapse; 7KC, 7-ketocholesterol; PKC, protein kinase C; pY, phosphotyr-osine; RA, rheumatoid arthritis; rh, recombinant human; SEA, staphylococcal en-terotoxin A; SEB, staphylococcal enterotoxin B; SLE, systemic lupus erythematosus;SMAC, supramolecular activation complex; SS, Sjorgren’s syndrome; TIRF, totalinternal reflection fluorescence.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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dence confirming the importance of ordered lipid microdomainsin 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 membranelipid order, ranging from low, intermediate, to high order. Wedemonstrate that this gradient of membrane lipid order dictatesthe outcome of CD4+ T cell responses to activation. Upon TCRstimulation, high order T cells formed stable IS and proliferatedrobustly, intermediate order cells had reduced proliferative abilityaccompanied by unstable IS formation, but low order T cells wereprofoundly unresponsive. Pharmacologically reducing T cellmembrane order from high to intermediate induced unstable ISformation and reduced proliferation. Furthermore, most IFN-g+

CD4+ T cells were characterized by intermediate order, whereasIL-4+CD4+ T cells were within the high order population. In-terestingly, patients with autoimmunity had increased intermediateorder T cell populations accompanied by reduced proliferationand increased production of IFN-g compared with healthy con-trols. Therefore, the results presented in this study identify a newmechanism to control T cell functional plasticity, raising the pos-sibility that therapeutic targeting of membrane lipid order coulddirect altered immune cell activation in pathology.

Materials and MethodsCell isolation

PBMC from 40 healthy donors were separated on Ficoll-Hypaque (Phar-macia 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 withSjorgren’s syndrome (SS) attending the Rheumatology Clinic at UniversityCollege Hospital were included in this study after receiving informedconsent. The study was approved by the local ethics committee. PurifiedCD4+ lymphocytes were obtained by negative selection using magneticbeads (Miltenyi Biotec) or by cell sorting using FACSAria (BD Bio-sciences).

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 anti-CD3 (OKT3) and anti-phosphotyrosine (pY) (4G10) with secondary anti-mouse IgG2a-Alexa Fluor 488 and anti-mouse IgG2b-Alexa Fluor 555from Invitrogen. For functional experiments and for coating coverslips,purified Abs to CD3 (HIT3a), CD28 (CD28.2), or IgG isoype controlsfrom BD Biosciences were used. For generation of polarized Th1/Th2populations, anti–IL-4, anti–IL-12, anti–IFN-g (eBioscience), recombi-nant human (rh)IL-12p70, rhIL-2, and rhIL-4 (R&D Systems) were used.The membrane order probe di-4-ANEPPDHQ, CTB-biotin, and PP2were obtained from Invitrogen. DAPI was obtained from Sigma-Aldrich(St. Louis, MO).

Flow cytometry

PBMC were stained for protein surface markers before washing andresuspending with 4 mM ANE in PBS for up to 30 min at 37˚C. For eachpopulation the geometric mean fluorescence intensity for wavelengths at570 nm (FL2 channel) and 630 nm (FL3 channel) were used to make theGP calculation (see Equation 1 below). Staining for annexin V was per-formed according to manufacturer’s instructions (BD Biosciences) fol-lowing ANE labeling. Cells were analyzed without fixing using a BD LSRII flow cytometer (BD Biosciences) and FlowJo software (Tree Star).Staining for intracellular cytokines, Ki67, T-bet, and pSTAT6 was per-formed on FACS-sorted populations after fixation/permeabilization witheither saponin- or methanol-based buffer according to the manufacturer’sinstructions (eBioscience). Cell viability was assessed by annexin Vstaining according to the manufacturer’s instructions and DAPI exclusionby flow cytometry. For the flow cytometry-based conjugation assay, au-tologous APC were isolated from PBMC by removal of CD3+ cells using

magnetic columns (Milteni Biotec) and labeled with CellTracker BlueCMHC (Invitrogen) following the manufacturer’s protocol before loadingwith superantigen (1 mg/ml staphylococcal enterotoxin E, 2 mg/ml staphy-lococcal enterotoxin A [SEA]/staphylococcal enterotoxin B [SEB]) for 1 h at37˚C. FACS-sorted low, intermediate, and high order CD4+ T cells werelabeled with CellTracker Green CMFDA (Invitrogen). APC and T cells weremixed in a ratio of 1:1, briefly centrifuged for 1 min at 100 3 g to formconjugates, and then incubated in complete RPMI 1640 medium (100 ml) at37˚C for 5 min. Thereafter, T cell/APC mixtures were fixed in PBS con-taining 1% paraformaldehyde before analysis. The relative proportion oforange, blue, and orange/blue events in each sample was determined. Formanipulation of membrane order, 15 mg/ml 7-ketocholesterol (7KC) andcholesterol (Avanti Lipids, Alabaster, AL) in ethanol were combined in acholesterol-7KC ratio of 1:2. During 30 min, these were then added to asolution of 50 mg/ml methyl-b-cyclodextrin (Sigma-Aldrich) in PBS at 80˚Cto a final sterol concentration of 1.5 mg/ml. Then, 15 ml/ml lipid solutionwas added to cell medium containing 13 106 cells at 37˚C for 30 min beforelabeling with ANE and analysis by flow cytometry and microscopy.

Differentiation of Th1 and Th2 phenotypes

FACS for CD4+CD82CD252CD45RA+ T cells were stimulated with anti-CD3 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 differ-entiated Th cell responses, Th1 cultures were supplemented with rhIL-12p70 (10 ng/ml) and anti–IL-4 (10 mg/ml), and Th2 cultures were sup-plemented 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 4after the activation of cultures; on day 6 supernatants and cells were re-covered. Th1/Th2 differentiation was confirmed by cytokine analysis ofcell supernatants.

T cell functional assays

T cells sorted for high, intermediate, and low membrane order werestimulated with either plate-bound anti-CD3 (2 mg/ml) and anti-CD28 (2mg/ml) Abs, Human T-Activator CD3/CD28 beads (Dynabeads; Invi-trogen), or superantigen-coated (SEA/SEB) autologous APC. Three-dayculture supernatants were analyzed for cytokine production by CytokineBead 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/mlionomycin, and 2 mM monensin (Golgi-Plug; BD Biosciences) for 4 hbefore fixation/permeabilization. For proliferation, cells were stimulatedwith anti-CD3/CD28 (2 mg/ml each) for 3 d then pulsed with [3H]thy-midine for 16 h, followed by harvesting and scintillation analysis.

Interference reflection microscopy

Glass coverslip cell culture chambers (Intracel, London, U.K.) were coatedwith Abs to CD3/CD28 or isotype control (10 mg/ml) in PBS for a mini-mum of 2 h. Sorted high, intermediate, and low order cells were layeredonto Ab-coated coverslips at 37˚C. Cells were imaged as they attached tothe coverslip surface using a Zeiss confocal microscope and 488 nm argon-ion laser excitation and a363 oil objective. Reflected and transmitted lightimages were obtained at 30-s intervals. Images were merged using ImageJsoftware to reveal areas of cell attachment.

T cell/APC conjugates

Sorted high, intermediate, and low order cells were cocultured withsuperantigen (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 poly-L-lysine–coated coverslips and fixed with 2% paraformaldehyde beforepermeabilization 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 Absfollowed by appropriate secondary Abs before mounting onto glass slidesusing anti-fade mountant (Invitrogen). Conjugates were imaged using 488nm argon-ion, 633 nm helium, and 400 nm ultraviolet laser excitation anda 363 oil immersion lens using a Zeiss LSM-510 inverted laser-scanningconfocal microscope. Approximately 10 conjugates were imaged fromeach sample, and 0.5-mm z-slices were acquired through each conjugate.The Pearson correlation coefficient of anti-CD3 and anti-pY staining at theIS for each z-slice in the conjugates was measured using Zeiss LSMsoftware. Three-dimensional reconstructed images were obtained fromdeconvoluted images using Volocity software.

Total internal reflection fluorescence (TIRF) microscopy was performedon a custom-built microscope with excitation at 473 nm and a360, 1.45NAoil-immersion TIRF objective. Fluorescence was collected on an electron-multiplying 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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Insights). Data were processed using custom software (LabVIEW; Na-tional Instruments, Austin, TX). The sample was maintained at 37˚C usinga stage and objective heater. Quantification of image data was performedby measuring average GP values (a normalized intensity ratio of twodifferent spectral channels: green [ordered] and red [disordered]) wherehigh GP (increased green fluorescence compared with red) represents highmembrane lipid order (12):

GP ¼ I5002 570 2 I6202 750

I5002 570 þ I6202 750: ð1Þ

Confocal microscopy

Sorted high, intermediate, and low order cells were labeled with 4 mMANEfor 30 min and applied to coverslip chambers at 37˚C. Attachment of cellswas imaged at 5-min intervals up to 30 min from addition of cells to thechamber, using single-photon excitation confocal microscopy on aninverted laser-scanning confocal microscope (TCS SP2; Leica Micro-systems, Wetzlar, Germany; or Zeiss LSM-510) with a 363, 1.25NA oil-immersion objective lens. Excitation was at 488 nm from an argon-ionlaser. Fluorescence detection was in the wavelength bands 500–580 and620–750 nm using internal photomultiplier tubes. GP values were calcu-lated from the two wavelength channels according to Equation 1, where Iis the intensity of emission. The z-slices were acquired every 0.5 mm.

Statistics

All values are expressed as mean 6 SEM. We performed analysis ofsignificance in Prism (GraphPad Software) by the Mann–Whitney U test,Student t test, or paired t test as appropriate.

ResultsDistinct CD4+ T cell subsets identified based on plasmamembrane 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 withthe lipid probe ANE and analyzed by confocal microscopy. Thedegree of membrane order was calculated and expressed as a GPvalue where high GP represents high membrane lipid order (Fig.1A) (12). We observed heterogeneous membrane order in ex vivonegatively purified CD4+ T cells from healthy volunteers. Thiswas 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 theGP image) plasma membrane lipid order. To confirm these find-ings and to examine a larger number of cells from many indi-viduals, a method was developed combining ANE labeling withmultiparameter flow cytometry (Fig. 1B). Again, three populationswere 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 mem-brane order (Fig. 1B, 1C, 1D). Intermediate and high order cellswere 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 orderT cells were cultured overnight and relabeled with ANE, theymaintained their order (Fig. 1F).The ability of flow cytometry and ANE labeling to distinguish

between plasma compared with intracellular membranes (which arelargely low order) (13, 17) was verified by assessing ANE labelingof CD4+ T cells over time. ANE was rapidly incorporated intocellular membranes; plasma membrane order was observed atearly time points and remained unchanged over time as observedin the representative merged intensity images of intermediate andhigh order cells (Fig. 1G). The accompanying RGB profiles rep-resent ANE emission spectra detected in 500–570 (green) and620–750nm (red) channels obtained from equatorial cross-sectionsof the intermediate and high order cells. They reveal that ANE wasrapidly incorporated into the plasma and intracellular membranesof the intermediate (Fig. 1G) and low order cells (data not shown),

corresponding with the preference for ANE to incorporate intodisordered membranes (13). In contrast, ANE labeling was con-fined mainly to the plasma membrane in the high order cells, withintracellular 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 threedistinct populations were revealed and did not change significantlyover time. Finally, we related the relative level of plasma mem-brane lipid order to the expression of lipid microdomain-associatedlipids, cholesterol, GM1, and GM3. Intermediate and high ordercells had increased expression of cholesterol compared with loworder cells (assessed by filipin binding), and a significant positivecorrelation was seen between global lipid order and membranelevels of cholesterol (Fig. 1I, upper panels). Alternatively, GM1(measured by CTB binding) was upregulated in the intermediateand low order cells, and a significant negative correlation was seenbetween membrane order and CTB binding (Fig. 1I, middle pan-els). Interestingly, glycosphingolipid GM3 was not differentiallyexpressed between the subsets (Fig 1I, lower panels). These resultsreveal differences between measurement of global membrane lipidorder and the traditional markers for assessing membrane lipidmicrodomains.

Differential membrane order is associated with distinctpatterns of IS formation

Because recent work has shown that high lipid order is importantfor stable IS formation (14, 16), the effect of differential globalplasma membrane order on IS development was examined byinterference reflection microscopy (IRM). FACS-sorted low, in-termediate, and high order T cells were applied to coverslipscoated with anti-CD3/CD28 or isotype control and visualized forup to 10 min. Low order cells demonstrated limited and transientattachments compared with partial interactions by intermediateorder cells (Fig. 2A). In contrast, high order cells exhibited robustand symmetrical attachments (Fig. 2A). These results were con-firmed by confocal microscopy (Supplemental Fig. 1A). Quanti-tation of imaging data verified that high order cells formed a moresubstantial contact surface area with the coverslip compared withintermediate and low order cells (Fig. 2B). All three populationspossessed similar expression levels of CD3, excluding the possi-bility that differences in CD3 expression might explain the alteredpatterns 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 orderat the cell/coverslip interface in FACS-sorted intermediate andhigh order populations was made using TIRF microscopy (themore transient nature of low order cell interactions made it diffi-cult for them to be visualized using this technique). RepresentativeGP/TIRF images (Fig. 2C) depict areas of highest membrane or-der in red and lowest order in blue/green. High order cells had thehighest average order at the IS compared with intermediate ordercells (Fig. 2D). The apparent differential nature of IS formation inthe three populations was also tested in a more physiologicalT cell/APC system. FACS-sorted low, intermediate, and high ordercells were cocultured with superantigen-loaded Raji B cells, andT cell/APC conjugates were stained with anti-CD3 (green) andanti-pY (red) and analyzed by confocal microscopy. Representa-tive images are shown in Fig. 2E. CD3 was patched at the IS inboth intermediate and high order cells (Fig. 2E, 2H) accompaniedby pY accumulation (Fig. 2E, 2G, 2I). CD3 did not accumulate atthe interface between low order T cells and APC (Fig. 2E, 2F,2H), and no pYaccumulation was seen (Fig. 2F, 2G, 2I). Althoughboth the intermediate and high order cells formed functionalsynapses in terms of pY accumulation, differences were seen in

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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) corre-

sponding 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

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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, hetero-

geneous membrane order may influence the nature and stability of

IS formation with APC.

Plasma membrane order is associated with distinct T cellfunction

The dramatic differences in IS formation suggested that membranelipid order may influence T cell function following TCR stimu-lation. IS stability was correlated with the speed of T cell/APC con-jugate formation. The three membrane order subsets were FACS-sorted 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.

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 accu-

mulation 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.

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kinetics of conjugate formation were observed by flow cytometry(Fig. 3A, 3B). These experiments revealed that high order T cellsform conjugates more slowly than intermediate and low ordercells, although as shown above, the IS is more stable once it isformed. Changes in IS formation and stability were associatedwith differences in cell function. Assessment of low, intermediate,and high membrane order T cells following 3 d coculture withsuperantigen-loaded APC revealed that high order T cells dis-played elevated levels of the proliferation marker Ki-67 (Fig.3C), compared with intermediate and low order cells. Differentialproliferation between the three populations was confirmed bythymidine incorporation assays following stimulation with anti-CD3/CD28 (Fig. 3D), and it was found to be associated with re-duced production of IL-2 in the intermediate order T cells (Fig.

3E, 3F). A more detailed assessment of cytokine production inFACS-sorted high, intermediate, and low order T cells revealedthat TCR stimulation of intermediate order cells preferentiallyinduced INF-g and IL-6 production compared with high ordercells that produced more IL-4 and IL-10 (Fig. 3G). CD4+ T cellscharacterized by low lipid order did not proliferate or produce IL-2 in response to TCR stimulation; furthermore, they were moreprone to cell death following activation (Fig. 3H) but were rescuedby 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 sur-face markers (CD45RA, CD27, CD25, and CD69) and multipara-meter flow cytometry is shown in Supplemental Fig. 2. The results

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 superantigen-

loaded 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.

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revealed that high order cells were predominantly naive/nonactivated cells whereas intermediate order cells had a moreactivated/effector memory phenotype (Supplemental Fig. 2C–E).Analysis of the low order population revealed that a high per-centage were CD45RA+, but they also revealed a more activatedphenotype expressing high levels of CD69 and CD25 (Supple-mental Fig. 2E), further supporting their “anergic-like” profile(20). In support of the phenotyping profiles, in vitro TCR trig-gering using soluble anti-CD3 at a range of concentrations togetherwith costimulation with anti-CD28 (1 mg/ml) reduced globalmembrane order and led to an increase in the intermediate and lowpopulations (Supplemental Fig. 3A, 3B). This effect was inhibited

by the addition of protein tyrosine kinase inhibitor PP2 (Supple-mental Fig. 3C), indicating that signaling via TCR-associatedkinases mediates an effect on membrane order.The results described above suggest that membrane lipid order

could represent a phenotypic marker that reflects the functionalcapability of T cells with a gradient ranging from T cells with highmembrane order with a naive phenotype, which proliferate robustlyand produce IL-2 and IL-4, to intermediate order cells that havea memory phenotype, proliferate weakly, but produce IFN-g, andfinally to low membrane order T cells that have an activatedphenotype, are unresponsive to TCR stimulation, and are moreprone to apoptosis (Table I).

Table I. 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 +++/+++ ++/+++ 2/2Proliferation +++ ++ 2IL-2 production +++ ++ NDIL-4/IFN-g production +++/+ +/+++ NDApoptosis 2 2 +Associated membrane lipidsCholesterol +++ +++ ++GM1 + ++ +++GM3 ++ ++ ++

+++, 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 vol-

unteers (n = 10) and patients with active SLE

(n = 10), RA (n = 10) and SS (n = 5). Repre-

sentative 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 pro-

liferation by thymidine incorporation (C) and

IFN-g production by intracellular staining (D).

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Differential membrane order in patients with autoimmunerheumatic disease

To relate CD4+ T cell membrane order to in vivo function inhumans, we investigated autoimmune T cells, characterized bychronic activation, isolated from patients with RA, SLE, and SS,compared with healthy volunteers. The representative dot plotsand cumulative data (Fig. 4A, 4B) show that patients with activelupus and RA have a significant expansion of the intermediateorder population compared with healthy volunteers. Interestingly,those patients with expanded intermediate order populations hadlower proliferation (Fig. 4C) and increased production of INF-g inresponse to in vitro TCR stimulation (Fig. 4D) compared withhealthy controls. These results imply that membrane order couldplay an important role in determining the response of T cells toTCR stimulation in vivo.

Th cell phenotypes are associated with specific plasmamembrane lipid order

Previous reports examining polarized Th cell populations revealeddifferences in IS formation between Th1 and Th2 phenotypes (21,22). We reasoned that the distinct patterns of IS formation andalterations in proliferation and cytokine production seen in ex vivointermediate and high order CD4+ populations could be connectedwith different function in human Th cell subsets.

To examine this possibility we cultured FACS-sorted naiveT cells under Th1- or Th2-polarizing conditions (Fig. 5A) and thenlabeled the differentiated cells with ANE to evaluate their plasmamembrane 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 culturedunder Th1-polarizing conditions assume an intermediate orderphenotype (lower GP) (Figs. 5B, 5C, 6A). Successful Th1 differ-entiation was confirmed by the production of high levels of IFN-gand expression of the transcription factor T-bet (Fig. 5D). Incontrast, CD4+ T cells cultured under Th2-polarizing conditionsretained a relative high membrane order (higher GP) (Figs. 5B,5C, 6A), produced high levels of IL-4, and exhibited increasedlevels 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 assessedas described previously. Th1 cells with intermediate lipid orderformed less stable IS compared with Th2 cells with higher lipidorder (Fig. 6A), thus confirming the aforementioned findings inFig. 2A. Furthermore, Th1 cells formed T cell/APC conjugatesmore quickly (Fig. 6C, 6D) but had reduced proliferation (Fig. 6E)compared with the Th2 cells, again corroborating the results de-scribed previously (Fig. 3A–D).

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 per-

centage of cells with intermediate and

high order membranes, *p # 0.05

Th1 versus Th2. To confirm pheno-

type differentiation, cells and super-

natants 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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Manipulation of membrane order modulates T cell function

The results described so far suggest that the ability of T cells torespond to TCR stimulation might be associated with mechanismsthat control membrane order. Therefore, to further understand therelationship between lipid order and T cell function we used theoxysterol 7KC, which has been shown to reduce lipid order at theT 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 membraneorder as shown by reduced GP values (Supplemental Fig. 4A) andan increase in the number of intermediate and low order cells(Supplemental Fig. 4B). TCR-induced proliferation (SupplementalFig. 4C) and IL-2 production (data not shown) were also inhibitedas described previously (14). Having established that 7KC could

reduce global membrane order in primary T cells, we tested itsability to influence T cell function in FACS-sorted ex vivo CD4+

intermediate and high order populations. Cells were treated with7KC or left untreated for 30 min and IS formation was assessed byIRM as before. 7KC treatment of intermediate order T cells didnot influence their membrane order or their ability to formattachments to Ab-coated coverslips (Fig. 7A, 7B). However, whenhigh order T cells were treated with 7KC, their membrane orderwas reduced (shown in the GP images in Fig. 7A), accompaniedby a significant reduction in the area of cell/coverslip contact (Fig.7A, 7B). Despite this, 7KC treatment did not significantly in-fluence the number of cells within each population interactingwith 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 theintermediate 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 byintermediate order T cells. Taken together, these results provideevidence that changes in membrane order could influence T cellplasticity in terms of cytokine production and proliferation.

DiscussionOne of the determining properties of lipid raft microdomains is thatthey form areas of lipid order in biological membranes that help tocompartmentalize signaling molecules (18, 25). Lipid order affectsmembrane fluidity; relative disordered membranes support free/random movement of protein molecules within the membrane,whereas movement of molecules within more ordered membranemay be restricted (26). However, controversy has surrounded therole, and very existence, of these regions mainly because theyhave been difficult to visualize by microscopy (9, 10). The use ofmembrane probes such as ANE and LAURDAN (12, 27) has gonesome way to resolving some of these difficulties, and the impor-tance of membrane order in the formation of stable IS in bothT cell lines and in primary T cells has been established (13, 14,16). In this study, by labeling human CD4+ T cells with ANE, wego further and reveal that ex vivo T cells are heterogeneous interms of global plasma membrane lipid order, and three distinctpopulations can be discerned. Importantly, we found that not onlycan the overall level of T cell plasma membrane lipid order predictthe stability and pattern of IS formation, but it can also predictT cell function in terms of proliferative ability and profile of cy-tokine production. Moreover, we were able to manipulate T cellfunction by changing membrane lipid order. These results offera possible mechanism by which T cells could control functionalplasticity and raise the possibility that therapeutic targeting ofmembrane lipid order could direct immune cell activation, helpingto correct abnormal immune responses in conditions such as au-toimmunity.High lipid order at the IS has been shown by several groups,

mainly in Jurkat T cell lines. Combining imaging of the IS withLAURDAN labeling has shown that, at the T cell/APC interface,lipid order is high compared with low order in the surroundingmembrane (14, 15). These results corroborate the wealth of dataassociating lipid microdomains with T cell activation (1, 2, 4, 18,28, 29). Additionally, the results indicate that global membranelipid order, not just order at the IS, plays a role in shaping theinteraction between a T cell and APC. This could provide anadditional mechanism influencing how T cells relate with othercells in their immediate environment and may ultimately de-termine T cell differentiation and function. High order T cellsformed IS with a large surface area that resembled the classical

FIGURE 6. IS formation and proliferation in Th1- and Th2-differenti-

ated 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-dif-

ferentiated 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 la-

beled 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 cocul-

tured with superantigen-loaded APC for 48 h before either staining for Ki-

67 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

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bulls-eye pattern of IS formation, with peripheral supramolecularactivation complex (SMAC) and distal SMAC regions of attach-ment, whereas intermediate order cells did not form distinct distalSMAC regions, had a smaller area of attachment, and interactionswere more rapid. Low order cells did not form a distinct IS patternof attachment visible by IRM, although they did form conjugateswith APC (3). It is possible that differences in global membraneorder could discriminate between cells that form long-lived stableIS that last several hours and the more dynamic and transientcontacts lasting a few minutes, also known as kinapses (30). Thesedifferences could also account for the striking variation in pro-liferative ability between T cells with low, intermediate, and highlipid order since stable IS formation is required for full T cellactivation (31), although further work is needed to confirm thisproposal.The different patterns of IS formation in the three lipid order

populations were characterized by a distinctive arrangement ofCD3 and accumulation of tyrosine phosphorylated proteins at theT cell/APC interface and subsequently by altered T cell function.These differences most likely reflect altered strength and organi-zation of TCR-associated signaling events at the IS (31). Stabilityof IS formation in naive, effector, and regulatory T cell subsets ispartly determined by differential accumulation of signaling pro-teins, including protein kinase C (PKC)-u and Wiskott–Aldrichsyndrome protein, that influence actin polymerization in the pe-ripheral and distal SMAC regions of the synapse (3, 32). Differ-

ential membrane lipid order in T cells could regulate these (andother) key signaling events by controlling the clustering ofmembrane proteins at the IS, thereby shaping subsequent T cellfunction. It is known, for example, that accumulation of PKC-u atthe IS is essential for Th2 cell development and production ofIL-4 (33), and that altered localization of key signaling moleculesat the IS, including PKC-u and CTLA-4, controls regulatoryT cell function (34, 35). It remains to be determined whethermolecules such as PKC-u preferentially accumulate at the IS inthe high order cells, thereby contributing to more robust pro-liferation. The unresponsive nature of low order cells, togetherwith their inability to partition CD3 and the accompanying lowlevels of protein tyrosine phosphorylation at the IS, is suggestiveof a tolerized or terminally differentiated phenotype (19, 36).In vivo-tolerized cells are able to form conjugates with APC butare unable to mobilize TCR, PKC-u, or lipid raft clustering at theimmune synapse and do not induce tyrosine phosphorylation ofsignaling proteins (37). It seems likely therefore that low plasmamembrane order could be a hallmark of tolerized T cells. Thesecells were also more prone to apoptosis. It is plausible that dif-ferential membrane order could influence multiple T cell intra-cellular signaling pathways, including cell survival and apoptosis(38), as indicated by recent work showing that T cell apoptosis isassociated with differential localization of PKC family proteinswith membrane lipid microdomains (39). It remains to be seenwhether lipid order plays a role in such mechanisms, and detailed

FIGURE 7. Manipulation of membrane order

modifies T cell function. FACS-sorted CD4+ in-

termediate 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. Repre-

sentative IRM and corresponding GP images. GP

panels: red, high order; green/blue, low order. Scale

bars, 5 mm. B, Cumulative results from three experi-

ments showing the contact area (pixels) of each cell

from the IRM images. *p = 0.02 high order no treat-

ment versus 7KC. C, Proliferation in untreated and

7KC-treated intermediate and high order T cells

assessed by thymidine incorporation. Cytokine pro-

duction assessed by intracellular staining for IFN-g in

intermediate and high order T cells treated with and

without 7KC. Representative dot plots (D) and cumu-

lative data from five experiments are shown (E). **p =

0.001 high versus high7KC, p = 0.008 high versus in-

termediate.

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biochemical analysis of downstream signaling pathways followingimmune activation will reveal important information to answerthese questions. Taken together, the results presented in this studygo some way to support the original lipid microdomain hypothesisin that they provide a “platform” to support or influence differ-ential T cell signaling (4). The concept that T cells have differentlevels of membrane lipid order provides an extra level of subtletyto the role that they play during T cell activation. However, notethat further work is needed to fully establish the relationship be-tween membrane lipid microdomains and plasma membrane or-der.Differential patterns of IS formation have been described pre-

viously in helper T cell subsets (40). Specifically, Th1-polarizedcells preferentially cocluster TCR and IFN-g receptor with lipidrafts (41) and form a more compact IS compared with Th2 cellsthat favor multifocal IS formation (21, 22). However, although wefound a correlation between Th1/Th2 IS formation and membraneorder, it was difficult to relate these differences to the earlierreports (21, 22). We show that the human Th2-like, high order cellsform IS that more closely resemble a classical bulls-eye pattern asopposed to Th2 cells generated in TCR-transgenic mice thatformed a multifocal IS. These disparities may be explained bydifferences in methodology used and differences in lipid contentbetween mouse and human lymphocytes (42); furthermore, thepattern of IS formation in the transgenic Th2 model was dependenton the concentration of antigenic stimulation, which was not con-sidered in this study since it is difficult to assess in ex vivo hu-man T cells (22). We also report differences in proliferation be-tween the Th1-polarized/intermediate order T cells that proliferateweakly compared with Th2-polarized/high order T cells that ex-hibit strong proliferation. Interestingly, increased proliferation inTh2-polarized cells has been identified previously (43).Although we could establish a clear relationship between mem-

brane order and T cell cytokine production in polarized T cellpopulations, in ex vivo, nonmanipulated CD4+ cells, the patternwas similar although less clear-cut. Intermediate order cells pro-duced more IFN-g whereas high order cells produced more IL-4and IL-10. One possibility is that changes in membrane lipid ordercould represent a mechanism by which T cells fine-tune their so-called “functional plasticity” (44, 45). Indeed, when we artificiallymanipulated membrane order we were able to influence IS for-mation and proliferation, and we skewed cytokine production to-ward IFN-g production in high order cells. Interestingly, althoughwe were able to influence the function of high order cells by re-ducing membrane order with 7KC, intermediate order cells wererelatively unaffected. We were also unable to influence the func-tion of intermediate order cells by increasing cholesterol content(data not shown). This could be because membrane order does notchange by simply adding cholesterol exogenously, or it may in-dicate that factors other than cholesterol content are important inthese cells. Interestingly, cholesterol and molecules that controlcellular cholesterol homeostasis have been recognized recently toaffect T cell function (46); therefore, more detailed analysis ex-amining the balance between cellular cholesterol and oxidizedforms of cholesterol in membranes (including 7KC) may proveimportant in understanding T cell functional plasticity. Alterna-tively, glycosphingolipid species have distinct roles in T cellfunction, are integral components of lipid microdomains, and playa role in T cell signaling events (38, 47, 48). We reveal differentialglycosphingolipid expression in low, intermediate, and high orderT cells that may contribute to their functional heterogeneity (49),and thus further dissection of the lipid content of these populationscould help identify mechanisms linking plasma membrane order toT cell function. The results presented in this study show that higher

membrane order is associated with higher levels of membranecholesterol as might be expected since cholesterol gives structureto the membrane by allowing lipids to become tightly packed (50,51). However, CTB binding to GM1 was associated with the lessordered populations, suggesting that membrane lipid order doesnot reflect lipid microdomains as they are described in the litera-ture. More detailed analysis of membrane lipid content using moreaccurate methods to determine lipid species will help improve ourunderstanding of the role played by membrane lipids in T cellactivation and function.The relevance of T cell membrane order was assessed in a range

of patients with autoimmune rheumatic disease. Patients with lupusand RA are characterized by chronic immune cell activation. Wereveal that these patients have increased numbers of T cells withintermediate membrane order, which was associated with reducedproliferation and increased production of proinflammatory cyto-kine IFN-g. Lupus T cells are characterized by altered expressionof raft-associated lipids, defective raft-associated signaling, andaltered patterns of IS formation when compared with T cells fromhealthy volunteers (52, 53). The enrichment of the intermediateorder population may reflect the altered lipid composition of T cellmembranes in patients with lupus. Whether this is a result ofchronic activation by endogenous Ags is unclear. A more detailedexamination of lipids in T cell membranes from patients withautoimmune disease is underway.To conclude, we show in this study that membrane lipid order

is heterogeneous in primary T cells and associated with distinctTh1-like, Th2-like, and tolerogenic characteristics. However, howplasma membrane lipid order relates to the previously describedlipid raft microdomains remains unclear. Artificial manipulation oflipid order influenced IS formation, which in turn may influence theability of membrane proteins to cluster at the IS and shape T cellfunction. This raises the possibility that therapeutic targeting ofmolecules controlling membrane lipid order could regulate im-mune activation and help control abnormal immune responses inconditions such as autoimmunity (29).

AcknowledgmentsWe 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.

DisclosuresThe authors have no financial conflicts of interest.

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