Structure and function of lipid rafts in human activated T cells
Shizue Tani-ichi1,
Koji Maruyama1,
Nami Kondo1,
Masakazu Nagafuku1,
Kazuya Kabayama2,
Jin-ichi Inokuchi2,
Yukiko Shimada3,
Yoshiko Ohno-Iwashita3,
Hideo Yagita4,
Sunao Kawano1 and
Atsushi Kosugi1,5
1 Department of Immunobiology, Medical Technology and Science, Osaka University Graduate School of Medicine, 1-7, Yamadaoka, Suita, Osaka 565-0871, Japan
2 Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan
3 Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
4 Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
5 Core Research for Evolutional Science and Technology program, Japan Science and Technology Corporation, Saitama, Japan
Correspondence to: A. Kosugi; E-mail: kosugi{at}nature.email.ne.jp
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Abstract
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Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, have been shown to function as signaling platforms in T cells. Surface raft expression is known to be increased in human T cells upon activation, and this increased raft expression may account for efficient signaling capability and decreased dependency for co-stimulation in effector and/or activated T cells. However, raft-mediated signaling ability in activated T cells remains to be clarified. In this study, we analyzed the structure and function of lipid rafts in human activated T cells. We demonstrated that raft protein constituents are dramatically changed after activation along with an increase in lipid contents. T cells stimulated with anti-CD3 plus anti-CD28 antibodies showed an increase not only in surface monosialoganglioside GM1 expression but also in total amounts of raft-associated lipids such as sphingomyelin, cholesterol and glycosphingolipids. Raft proteins increased after activation include Csk, Csk-binding protein and Fyn, the molecules known to be involved in negative regulation of T cell activation. Consistent with the increase in expression of these proteins, TCR-mediated Ca2+ response, a response dependent on raft integrity, was clearly inhibited in activated T cells. Thus, the structure and function of lipid rafts in human activated T cells seem to be quite distinct from those in naive T cells. Further, human activated T cells are relatively resistant to signaling, at least transiently, by TCR re-stimulation even though their raft expression is increased.
Keywords: Cbp, cholesterol, GM1, negative regulation, TCR signaling
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Introduction
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Lipid rafts, highly organized microdomains in the plasma membrane, play important roles in various cellular functions (13). In T lymphocytes, some of the key proteins involved in TCR signaling such as Src-family kinases, Lck and Fyn and an adaptor molecule, linker for activation of T cells (LAT), are constitutively localized in rafts, which enable these microdomains to act as signaling platforms that facilitate intermolecular association and the propagation of signal transduction cascades (4). Indeed, it was reported that TCR signaling in LAT-negative T cell lines is severely impaired and cannot be reconstituted by transfection with LAT mutated at residues required for raft localization (5, 6). These results clearly demonstrated that raft localization of signaling proteins is indispensable for T cell activation. Rafts are highly enriched in cholesterol and sphingolipids, and these lipid components are critical for maintaining raft integrity (1). Cholesterol depletion from T cell membranes using drugs such as methyl-ß-cyclodextrin (MßCD) is known to inhibit TCR-mediated T cell activation presumably due to disruption of raft structure (7, 8).
Upon activation, surface raft expression is known to be increased in human T cells. Several investigators demonstrated that in response to TCRCD28 ligation, surface expression of ganglioside GM1 (GM1), a component of T cell membrane rafts, is remarkably up-regulated in human peripheral blood T cells (9, 10). This difference in surface raft expression between naive and activated T cells may underlie the differences in responsiveness and in dependence of co-stimulation in these T cells. However, although GM1 is generally considered to be a reliable raft marker, it is one out of hundreds of complex glycosphingolipids (GSLs) found in rafts and does not necessarily represent rafts per se on T cell membranes. In fact, GM3 is the major ganglioside on the plasma membrane of human peripheral blood lymphocytes and appears to play a functional role in raft-mediated T cell activation (11, 12). Moreover, although human naive T cells express only low levels of GM1 on the cell surface, mouse T cells show higher GM1 levels irrespective of activation state (13), suggesting that GSL expression on lymphocytes is heterogeneous between different species. These observations suggest that the present usage of GM1 as a uniform raft marker in immunological research should be reconsidered.
In the present study, we try to define the structure and function of lipid rafts in human activated T cells using several raft markers. We investigated the changes of raft protein and lipid components in activated T cells as compared with those in naive T cells. We also examined whether the changes in raft structure after activation influence the signaling activity through the TCR and co-receptor CD28. We demonstrated that raft protein constituents are dramatically changed after activation along with the increase in lipid contents. Raft proteins increased after activation include Csk, Cbp (Csk-binding protein) and Fyn, the molecules known to be involved in negative regulation of T cell activation (1417). As a result, it was observed that human activated T cells are relatively resistant to signaling by re-stimulation even though their raft expression is increased.
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Methods
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Cells
Human peripheral blood T lymphocytes were isolated from fresh heparinized blood of healthy donors or buffy coats using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ, USA) density gradient separation followed by magnetic depletion of non-T cells (MACS Pan T cell isolation kit; Miltenyi Biotec, Auburn, CA, USA). Buffy coats from healthy donors were provided by the Osaka Red Cross Blood Center (Osaka, Japan). Human peripheral blood T cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 50 µM 2-mercaptoethanol, penicillin (100 units ml1) and streptomycin (100 µg ml1) at 37°C in a humidified atmosphere of 5% CO2.
Antibodies and reagents
The following antibodies were used: OKT3 (American Type Culture Collection, Manassas, VA, USA), anti-CD3
mAb; TN228 (18), anti-CD28 mAb; HRP-conjugated cholera toxin B (CTx-B) (Sigma-Aldrich, St Louis, MO, USA); anti-flotillin-1 (BD Biosciences, San Diego, CA, USA); anti-flotillin-2 (BD Biosciences); MOL171 (19), anti-Lck mAb; anti-Fyn mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-LAT polyclonal antibody (pAb) (Upstate Biotechnology, Lake Placid, NY, USA); anti-Cbp pAb (provided by Okada, Osaka University) (15); anti-Csk pAb (Santa Cruz Biotechnology); PY20 (BD Biosciences), anti-phosphotyrosine mAb and anti-phospho-Lck (Tyr 505) pAb (Cell Signaling Technology, Beverly, MA, USA). Avidin was purchased from SigmaAldrich. FITC-conjugated avidin and FITC-conjugated CTx-B were purchased from BD Biosciences and SigmaAldrich, respectively. Biotinylated
-toxin (BC
), biotin-conjugated cholesterol probe, was described elsewhere (20). OKT3 and TN228 were conjugated with biotin N-hydroxysuccinimide ester (Sigma). Protein was measured by BCA Protein Assay Kit (Pierce, RockFord, IL, USA).
Reverse transcriptionPCR
Peripheral blood T cells were cultured with or without anti-CD3/CD28 for 0, 24, 48, 72 and 96 h. Total RNA was first extracted from the cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA), then 2 µg of total RNA were reverse transcribed using SuperScript II RNase H Reverse Transcriptase (Invitrogen). A total of 0.3 µg of cDNA products were used for PCR reaction to analyze the mRNA expression of sphingolipid synthases as previously described with slight modifications (21).
Isolation of raft fraction and immunoblotting analysis
Raft fractions were prepared as described (22). A total of 1 x 108 cells were washed in PBS containing 5 mM sodium orthovanadate and 5 mM EDTA and then were lysed with 1 mM 4-(N-morpholine) ethanesulfonic acid (MES)-buffered saline (25 mM MES, pH 6.5 and 150 mM NaCl) containing 1% Triton X-100, 10 µg ml1 aprotinin, 10 µg ml1 leupeptin, 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium orthovanadate and 5 mM EDTA. The lysate was homogenized with 20 strokes of a Dounce homogenizer, gently mixed with an equal volume of 80% sucrose (w/v) in MES-buffered saline and placed in the bottom of a 14 x 95-mm clear centrifuge tube (catalog number 344060, Beckman, Fullerton, CA, USA). The sample was then overlaid with 6.5 ml of 30% sucrose and 3.5 ml of 5% sucrose in MES-buffered saline and centrifuged at 200 000 x g using a Beckman SW 40Ti rotor at 4°C for 18 h. Following centrifugation, 12 1-ml fractions (excluding the pellet) were collected from the top of the gradient. Aliquots corresponding to fractions 4 and 5 were combined as raft fractions. Immunoblotting analysis was performed as previously described (22).
Lipid analysis
Lipid analysis was performed as described (23). The chloroform-rich phase from Folch partition was separated into neutral and acidic lipid fractions by using a DEAE-Sephadex A-25 column (acetate form, 2.4-ml bed volume; Amersham Pharmacia Biotech). The fractions were dried and subjected to methanolic 0.1 M NaOH for ester cleavage. After neutralization, solutions were desalted with Sep-Pak C18 reverse-phase cartridges (Waters Associates, Walling Ford, Oxon, UK) and lipids were eluted. Acidic lipids were separated by silica gel high-performance thin-layer chromatography (HPTLC) plates with chloroform : methanol : 0.5% CaCl2 (55 : 45 : 10) and detected with orcinol-sulfuric acid reagent. Neutral lipids were separated by HPTLC half developed with chloroform : methanol : water (65 : 25 : 4), then dried, re-developed with hexane : diethyl ether : acetic acid (50 : 50 : 1) and detected with 3% cupric acetate8% phosphoric acid reagent.
Intracellular calcium analysis
Resting or activated T cells were incubated with 3 µM Indo-1 AM (Molecular Probes, Eugene, OR, USA) for 30 min at 37°C in HBSS (Sigma) containing 1% FCS, 1 mM CaCl2 and 1 mM MgCl2. In some experiments, Indo-1 AM-labeled cells were then incubated with MßCD. Intracellular calcium analysis was performed on a BD LSR (BD Biosciences). Indo-1 was excited by a HeCd laser (325 nm, 8 milliwatts). Indo-1 emission was detected using a 380 nm (violet) low-pass filter and a 510/20 nm (blue) bandpass filter. To determine the relative intracellular calcium concentration, Indo-1-loaded cells were warmed to 37°C, analyzed for 1530 s to establish baseline calcium levels and stimulated by the addition of biotinylated OKT3 (0.3 µg ml1), biotinylated TN228 (10 µg ml1) or biotinylated OKT3 (0.3 µg ml1) plus biotinylated TN228 (5 µg ml1). After the addition of these antibodies, surface CD3 or CD28 was cross-linked by the addition of avidin (10 µg ml1). Acquisition was continued in real time up to 204 s.
MßCD treatment and cholesterol reconstitution
For cholesterol depletion, cells were incubated with 2 mM MßCD (SigmaAldrich) in HBSS containing 0.2% BSA for 20 min at 37°C. MßCD-treated cells were washed twice with HBSS containing 60 µg ml1 water-soluble cholesterol (SigmaAldrich) and 0.2% BSA to neutralize any residual MßCD. To reconstitute cholesterol of MßCD-treated cells, 150 µg ml1 water-soluble cholesterol in HBSS containing 0.2% BSA was added to the cells and incubated at 37°C for 30 min. Cells were washed with HBSS containing 0.2% BSA twice and then used for all experiments.
Cholesterol staining
To analyze cholesterol expression on the cell surface, BC
derived from perfringolysin O (
-toxin) was used (24). T cells were incubated with BC
(20 µg ml1) in HBSS containing 0.2% BSA at room temperature for 20 min. After washing twice with HBSS containing 0.2% BSA, the cells were stained with FITC-conjugated avidin and analyzed by flow cytometry.
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Results
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Changes of surface GM1 and cholesterol expression in human peripheral blood T cells upon activation
Previous studies demonstrated that human peripheral blood T cells are induced to express high levels of surface GM1 when cross-linked with anti-CD3 plus anti-CD28 (9, 10). To investigate whether the up-regulation of surface GM1 is associated with changes in the expression of other raft-lipid components, we analyzed surface cholesterol expression in activated T cells using BC
, a digested form of
-toxin. The
-toxin (perfringolysin O) is a thiol-activated cytolysin produced by Clostridium perfringens and is known to bind cholesterol-rich membrane microdomains on the cell surface (20). Consistent with previous reports, anti-CD3 plus anti-CD28 stimulation clearly increased surface GM1 expression in T cells, whereas stimulation with anti-CD3 or anti-CD28 alone did not change GM1 levels (Fig. 1A). Although cholesterol was highly expressed on the cell surface in resting T cells, the expression levels were clearly augmented by cross-linking T cells with anti-CD3 plus anti-CD28 (Fig. 1A). Surface GM1 and cholesterol expression was increased from 48 h after stimulation and continued to be at high levels at 120 h (Fig. 1B and data not shown). However, surface cholesterol returned to the level of resting T cells shortly after termination of CD3CD28 ligation, whereas surface GM1 remained at high levels (Fig. 1C), suggesting that raft-lipid constituents are dynamically altered in activated T cells. Together, our data confirm the previous observation that surface raft expression is increased by TCRCD28 co-stimulation in human T cells.

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Fig. 1. Changes of surface GM1 and cholesterol expression in human peripheral blood T cells upon activation. (A) Human peripheral blood T cells were treated with the following antibodies coated to the plate for 72 h: 10 µg ml1 anti-CD3 (top panel), 5 µg ml1 anti-CD28 (middle panel) or 10 µg ml1 anti-CD3 plus 5 µg ml1 anti-CD28 (bottom panel). The cells were stained with FITC-conjugated CTx-B (left) or with BC followed by FITC-conjugated avidin (right), and analyzed by flow cytometry. The shaded histogram represents cells stained with FITC-conjugated avidin as a negative control. The bold and dotted lines represent cells with and without stimulation, respectively. (B) Peripheral blood T cells were treated with anti-CD3 plus anti-CD28 for 0, 24, 48 or 72 h and analyzed by flow cytometry as described in (A). (C) Peripheral blood T cells were treated with anti-CD3 plus anti-CD28 for 72 h. Cells were then washed with PBS and cultured in media without antibodies for 24, 48 or 72 h as indicated. Surface GM1 and cholesterol were analyzed as described in (A).
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Changes of lipid constituents in human T cells upon activation
We next examined whether the changes of surface raft expression is associated with a net increase of raft-lipid constituents in a cell. We isolated total cellular lipids from resting T cells or T cells activated with anti-CD3/CD28 stimulation and analyzed them by HPTLC. HPTLC analysis using acidic lipid fractions revealed that the major GSL expressed in human peripheral blood T cells was GM3, and GM3 was clearly increased after activation (Fig. 2A). Two other orcinol-positive bands were detected in the acidic fractions, the one between GM2 and GM1 and the other between GD3 and GD1a. These two GSLs are likely to be sialosyl lactohexaosyl ceramide and sialosyl paragloboside, respectively, as previously reported (12). The amounts of these GSLs seemed to be unchanged by activation. When we analyzed the neutral lipid fractions, we were able to detect the expression of sphingomyelin (SM), lactosylceramide (LacCer), glucosylceramide (GlcCer), ceramide (Cer) and cholesterol both in resting and activated T cells. However, the levels of these lipids were apparently up-regulated in activated T cells (Fig. 2A). Because lipids and proteins in membrane rafts are known to be fractioned into a Triton X-100-insoluble floating fraction, known as the raft or detergent-resistant membrane fraction by sucrose density gradient centrifugation. Next, we used this method to prepare raft fractions from resting and activated T cells and analyzed raft-associated lipids by HPTLC. Again, the amounts of cholesterol, Cer and GlcCer were greatly augmented in activated T cells as compared with those in resting T cells. Thus, these results demonstrated that anti-CD3/CD28 stimulation induced an increase not only in surface expression but also in total amounts of raft-associated lipids.

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Fig. 2. Changes of lipid constituents in human T cells upon activation. (A) Human peripheral blood T cells were stimulated with anti-CD3 plus anti-CD28 or cultured without stimulation for 72 h. Neutral and acidic lipids obtained from these cells were separated by HPTLC. Neutral lipids were visualized with 3% cupric acetate8% phosphoric acid (left). Acidic lipids were visualized with orciniol-sulfuric acid (right). The positions of lipid standards are indicated on the left of each panel. The arrow heads on the right panel indicate sialosyl lactohexaosyl ceramide (black) and sialosyl paragloboside (white), respectively. (B) T cells with or without stimulation were lysed and subjected to sucrose density gradient centrifugation. Aliquots corresponding to fractions 4 and 5 (the raft fractions) were combined. Neutral lipids from the raft fractions were separated and visualized as described in (A).
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Because the increased expression of GM1 was linked to the total contents of raft-associated lipids including GSLs, we next investigated changes in the mRNA expression of enzymes involved in the biosynthetic pathway of GM1 upon T cell activation. Ganglioside GM1 is synthesized by consecutive addition of monosaccharides to Cer by multiple sphingolipid synthases (Fig. 3A). We examined the mRNA expression of these synthases in response to anti-CD3/CD28 stimulation over a 4-day period. As shown in Fig. 3(B and C), there exist three patterns of mRNA expression changes: increase (LacCer and GA2/GM2/GD2 synthases), decrease (asialo GM1/GM1/GD1b synthase) and unchanged (GlcCer, GM3 and GD3 synthases) after activation. Of these, the expression of GA2/GM2/GD2 synthase was remarkably induced after activation. The expression of this enzyme may facilitate a transition from GM3 to GM2 in the biosynthetic pathway of gangliosides, resulting in the increased expression of GM1 in activated T cells.

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Fig. 3. mRNA expression of sphingolipid synthases in resting and activated T cells. (A) The biosynthetic pathway of sphingolipids. (B) Human peripheral blood T cells were stimulated with or without anti-CD3 plus anti-CD28 for 0, 24, 48, 72 or 96 h. Total RNA was isolated from the cells and mRNA expression of sphingolipid synthases as indicated was analyzed by reverse transcriptionPCR. (C) Relative changes of mRNA expression in activated T cells (solid triangle) or in T cells cultured without stimulation (open circle) compared with that in resting T cells (0 h) were analyzed by densitometric scanning of the bands shown in (B).
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Taken together, these results demonstrated that the increase of surface GM1 expression in activated T cells was not due to simple movement of GM1 from the cytoplasm to the plasma membrane as suggested previously (9, 10), but resulted from the activation of raft-lipid biosynthesis. Our results also indicated that the lipid contents of membrane rafts in human activated T cells were quite distinct from those in naive T cells.
Changes of raft protein constituents in human T cells upon activation
We next analyzed changes of protein expression in rafts upon T cell activation. We focused on three groups of well-known raft-resident proteins in T cells: the transmembrane adaptor proteins, LAT and Cbp (Csk-binding protein); Src-family tyrosine kinases, Lck and Fyn and flotillins, flotillin-1 and flotillin-2. The importance of LAT, Cbp and Src-family kinases in T cell activation has already been well established (2527). Flotillins are considered to be a structural protein that assists in raft assembly in T cells (28). First, we examined changes of protein expression using whole-cell lysates. As shown in Fig. 4(A), Fyn, but not Lck, was greatly increased in T cells stimulated with anti-CD3 plus anti-CD28 for 72 h, and this increase was maintained during the following 24-h culture without stimulation. In contrast, the expression of Csk was not changed significantly with activation. Next, we analyzed raft-associated proteins using raft fractions from resting and activated T cells. As shown in Fig. 4(B), the expression of flotillins, especially flotillin-2, in rafts was greatly induced upon activation, suggesting that not only raft lipids but also basic raft components are increased in general in activated T cells. The increased expression of flotillin-2 in rafts was not caused merely by an elevated level of total protein contents in activated T cells because total protein contents were only 1.2-fold higher in activated than in resting T cells (Fig. 4E). Using confocal microscopy, a previous study demonstrated that Lck in human CD4+ T cells was translocated from the intracellular compartment to membrane rafts in the plasma membrane in response to anti-CD3/CD28 stimulation (10). Consistent with this finding, Lck was translocated from the non-raft into the raft fractions upon activation although total amounts appeared not to be changed (Fig. 4B). In contrast, the expression of Fyn was clearly induced in activated T cells. Although Fyn was barely detectable in resting T cells, its expression was increased by 14-fold on an average in two independent experiments after stimulation (Fig. 4B and data not shown). Thus, two Src-family kinases showed quite different behavior in terms of their expression and localization upon activation.

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Fig. 4. Expression of protein constituents in rafts from anti-CD3/CD28-treated or untreated T cells. Human peripheral blood T cells were stimulated with (+) or without () anti-CD3 plus anti-CD28 for 72 h, then washed with PBS and cultured without antibodies for 24 h. These cells and freshly isolated resting T cells (0 h) were lysed with 1% TritonX-100 lysis buffer and total cell lysates were electrophoresed and immunoblotted with specific antibodies for Lck, Fyn and Csk (A). T cells were stimulated with (+) or without () anti-CD3 plus anti-CD28 for 72 h. The cells were lysed with MES-buffered saline containing 1% Triton X-100, and the lysates were subjected to equilibrium gradient centrifugation. An aliquot of each fraction was electrophoresed and immunoblotted with specific antibodies as indicated (B), anti-phosphotyrosine antibody (PY20; C) or anti-phospho-Lck (Y505; D). Protein contents of each fraction were measured by BCA Protein Assay Kit (E).
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LAT, a key adaptor molecule in TCR signaling, was shown to be increased in rafts by anti-CD3/CD28 stimulation (9). However, the LAT expression was more dramatically increased in the non-raft soluble fraction in our experiments (Fig. 4B). In contrast, Cbp, another transmembrane adaptor protein, was markedly increased in rafts. We have previously demonstrated that Fyn is essential for tyrosine phosphorylation of Cbp (17), and the increased expression of Fyn and Cbp might augment Cbp phosphorylation in lipid rafts in activated T cells. Indeed, when we analyzed tyrosine phosphorylation of cellular proteins in the raft fraction, we detected phosphorylated protein likely to be Cbp (8085 kDa) in activated T cells (Fig. 4C). With regard to other tyrosine-phosphorylated proteins, proteins likely to be Lck and/or Fyn (5560 kDa) and extracellular signal-regulated protein kinase (ERK) (4244 kDa) were observed in activated T cells. Given the enhanced tyrosine phosphorylation of Cbp, we assessed whether Csk was recruited to rafts upon T cell activation. We demonstrated that anti-CD3/CD28 stimulation clearly increased the amount of Csk protein in the raft fraction. To further confirm the function of Csk, we analyzed the phosphrylation level of a C-terminal residue in Lck (Lck Tyr 505) using a phosphospecific antibody (Fig. 4D). We observed that Tyr 505 phosphorylation in the raft fractions was apparently increased in activated T cells. Together, these results indicated that raft protein composition was dramatically changed in response to T cell stimulation. Of note, the amounts of Fyn, Cbp and Csk, the proteins involved in negative regulation for T cell activation, were remarkably increased in activated T cells.
Raft-dependent TCR signaling in human activated T cells
It has been well established that the process of TCR-mediated signaling is dependent upon raft structure and function (4). We next examined whether the changes of raft expression and structure in activated T cells modulate TCR-mediated signal transduction. Human peripheral blood T cells were stimulated with anti-CD3 plus anti-CD28 for 72 h, cultured without stimulation for 24 h to recover TCR expression on the cell surface and then re-stimulated either with anti-CD3, anti-CD28 or anti-CD3 plus anti-CD28 to examine Ca2+ mobilization. As shown in Fig. 5(A), Ca2+ response was clearly detected in resting T cells stimulated either with anti-CD3 or anti-CD3 plus anti-CD28. In contrast, Ca2+ response was greatly reduced in activated T cells as compared with that in resting T cells. The percentages of cells in which Ca2+ mobilization was observed by anti-CD3 or anti-CD3/CD28 stimulation were 75.4 ± 0.6 (mean ± SD, n = 3) and 83.5 ± 2.3, respectively, at the resting stage, whereas the percentages of cells were 31.6 ± 9.0 and 57.3 ± 3.2 after activation. Interestingly, although anti-CD28 stimulation did not induce Ca2+ mobilization in resting T cells, it appreciably induced Ca2+ response in activated T cells (Fig. 5A). The increase of the response by CD28 engagement could be, at least in part, due to an increase of CD28 expression in activated T cells (Fig. 5A). Together, although surface raft expression was increased, TCR signaling seemed to be inhibited in human activated T cells.

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Fig. 5. Signaling activity in anti-CD3/CD28-treated or untreated human T cells. Human peripheral blood T cells were treated with (+) or without () anti-CD3 plus anti-CD28 for 72 h, washed with PBS and then cultured without antibodies for 24 h (A) or 72 h (B). The cells were then loaded with Indo-1-AM and tested for their ability to mobilize calcium after cross-linking of cell surface CD3 and/or CD28 using biotinylated anti-CD3 (0.3 µg ml1), biotinylated anti-CD28 (10 µg ml1) or biotinylated anti-CD3 (0.3 µg ml1) plus biotinylated CD28 (5 µg ml1), followed by avidin (10 µg ml1). The dot plot of cells shows the ratio of Indo-1 violetblue fluorescence. The percentage of responding cells is indicated in the graph. Surface expression of CD3, CD28, GM1 and cholesterol in untreated (dotted lines) or activated (bold lines) T cells were analyzed by flow cytometry as described in Fig. 1.
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To examine whether the inhibition of TCR signaling in activated T cells continues after termination of TCR stimulation, T cells were stimulated with anti-CD3 plus anti-CD28 for 72 h, cultured without stimulation for 72 h, and then re-stimulated to check Ca2+ response. In this culture protocol, we observed that activated T cells substantially recovered their reactivity in response to anti-CD3 or anti-CD3/CD28 stimulation (Fig. 5B). Yet, activated T cells continued to express a high level of CD28 and show increased Ca2+ response by anti-CD28 stimulation (Fig. 5B). Together, these results demonstrated that the reduced ability for TCR signaling in activated T cells was transient and gradually recovered after T cells were released from TCR engagement. Although we observed that Ca2+ response was reduced in activated T cells, this reduction seemed not to result in the decrease of T cell response in general since T cells which were stimulated for 72 h, cultured without stimulation for 24 h and then re-stimulated with anti-CD3 plus anti-CD28 can proliferate and produce IL-2 as efficiently as resting T cells with anti-CD3/CD28 stimulation (data not shown).
Since TCR-mediated Ca2+ response has been shown to be initiated in raft microdomains and require their integrity (7), we believe that anti-CD3-induced Ca2+ response is an indicator of raft function in T cells. However, because a recent report suggested that TCR-dependent Ca2+ signaling was not affected by raft disorganization (29). We decided to re-address this point. Cholesterol is an essential component of membrane rafts, and extraction of cholesterol from cell membrane using MßCD has been widely used as a method that disrupts raft function (8). We were able to treat human T cells with 2 mM MßCD without affecting cell viability, and this treatment resulted in severe reduction in
-toxin binding by flow cytometry (Fig. 6, the lowest row). We found that MßCD clearly inhibited Ca2+ mobilization induced by anti-CD3/CD28 stimulation both in resting and activated T cells (Fig. 6). The inhibition of Ca2+ response by MßCD appeared not to be due to non-specific effects on T cells such as depletion of intracellular Ca2+ stores as reported previously (29) because addition of cholesterol to MßCD-treated T cells almost completely reversed the inhibitory effects of MßCD. Thus, we confirm that TCR-mediated Ca2+ response is dependent upon raft integrity and represents raft function in human T cells.

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Fig. 6. Effect of cholesterol depletion and reconstitution on the signaling activity in anti-CD3/CD28-treated or untreated human T cells. Human peripheral blood T cells were treated with or without anti-CD3 plus anti-CD28 for 72 h, washed with PBS and cultured without antibodies for 24 h. The cells were then loaded with Indo-1-AM and then incubated with or without 2 mM MßCD at 37°C for 20 min. After MßCD treatment, the cells were washed and incubated with water-soluble cholesterol (150 µg ml1) at 37°C for 30 min for cholesterol reconstitution. Calcium mobilization was measured as described in Fig. 5. Surface cholesterol was stained with FITC-conjugated CTx-B or with BC followed by FITC-conjugated avidin and analyzed by flow cytometry (bottom panel). Bold black, dotted and grayish lines represent untreated, MßCD-treated and cholesterol-reconstituted cells, respectively.
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Discussion
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Lipid rafts, specialized membrane microdomains enriched in sphingolipids and cholesterol, function as signaling platforms in lymphocyte activation (8). Rafts in lymphocytes are known to be involved in the signals mediated through TCR, BCR, Fc receptor and some cytokine receptors (4). The common method of analyzing membrane rafts depends on reagents that can interact with raft-localized lipids. Because cholesterol is an indispensable component of raft structure, reagents that react with membrane cholesterol could be powerful probes for raft research. Filipin, a fluorescent polyene antibiotic from Saccharomyces filipinensis, forms a multimeric globular complex with cholesterol in the cell membrane and has been used to stain cholesterol of T cells (13). We used BC
in this study. We have previously demonstrated that BC
could be superior to filipin for monitoring rafts in living cells owing to its selectivity for cholesterol-rich membrane domains and minimization of non-specific effects on membrane integrity (20). Another reagent that is much more common than the cholesterol probes in raft research is CTx-B subunit which binds ganglioside GM1. CTx-B binds to GM1 with a strong affinity and has been widely used to detect rafts in biochemical, flow cytometry and microscopic analysis. However, in contrast to cholesterol expression, GM1 expression on the cell surface is heterogeneous between different species. For example, although human naive T cells express only low levels of GM1 on the cell surface, mouse naive T cells are very rich in surface GM1 (10, 13). Moreover, GM1 is one out of hundreds of complex GSLs and its expression is relatively low as compared with other gangliosides in lymphocytes (12). Thus, it seems that the present primary usage of GM1 as a raft marker could not be due to its importance for raft structure and expression, but simply due to the availability of the reagent.
Previous studies have demonstrated that human naive T cells increase surface raft expression in response to TCRCD28 ligation (9, 10). However, these studies only utilize GM1 as a raft marker and, therefore, do not completely prove the increase of raft expression in activated T cells. Using BC
as a cholesterol probe, we confirmed that surface raft expression is indeed increased by TCRCD28 co-stimulation in human T cells (Fig. 1). Moreover, we found that activated T cells show an increase not only in surface expression but also in total amounts of raft-associated lipids as compared with naive T cells (Fig. 2). Although every lipid constituent of membrane rafts, cholesterol, SM and GSLs, seemed to be augmented upon activation (Fig. 2), it is not clear whether individual raft microdomains have the same lipid composition between naive and activated T cells. Further, the contribution of each lipid to raft-mediated signaling in lymphocytes, if any, remains to be clarified. Garofalo et al. reported that GM3, the main ganglioside in human T cells, is significantly associated with 70-kD zeta-associated protein (ZAP-70) in rafts upon activation and may be involved in the process of T cell activation (12). However, we have demonstrated that GSL depletion from lipid rafts using a specific GSL synthesis inhibitor does not impair TCR signal transduction (24). Further studies are necessary to define a specific role of each raft-associated lipid on lymphocyte activation.
The increased raft expression in effector T cells has been interpreted as a phenomenon which accounts for the fact that effector and memory T cells have more efficient TCR signaling machinery than naive T cells (10). However, we demonstrated that activated T cells have poor TCR signaling ability, at least transiently, than naive T cells even though their raft expression is clearly up-regulated (Fig. 5). Using MßCD, we monitored TCR-induced Ca2+ influx as raft-dependent signaling ability in this study. A previous study raises a serious concern about this assay as an evaluation method for raft function. Pizzo et al. reported that inhibition of CD3-induced Ca2+ influx by MßCD could be due to non-specific effects involving depletion of Ca2+ stores and plasma membrane de-polarization in Jurkat and normal human T cells (29). They indicated that the inhibition is mainly due to non-specific depletion of intracellular Ca2+ stores and plasma membrane de-polarization. However, the inhibitory effect on TCR-induced Ca2+ influx with MßCD treatment was recovered by cholesterol reconstitution in our study (Fig. 6). Although it is possible that cholesterol addition to MßCD-treated T cells influences Ca2+ signaling independent of the recovery of raft structure, cholesterol addition to T cells without MßCD treatment failed to change TCR-induced Ca2+ influx (data not shown). Therefore, cholesterol itself does not seem to have the ability to enhance Ca2+ mobilization under our experimental conditions. With regard to the mechanism of the effect of MßCD on Ca2+ mobilization, Kabouridis et al. showed that the inability of cells pre-treated with MßCD to mobilize intracellular Ca2+ is due to inhibition of inositol-1,4,5, trisphosphate generation (8). Phosphatidylinositol 4,5-bisphosphate (PIP2) is known to be enriched in lipid rafts (30, 31). Because phospholipase C
(PLC
) is shown to be catalytically active even after MßCD treatment in T cells, it was suggested that PLC
1 might fail to hydrolyze PIP2 efficiently due to its dilution through the bulk plasma membrane (8). Since a number of non-raft roles have been indicated for cholesterol including alteration of the physical properties of cellular membranes (32), the results obtained by cholesterol-depletion experiments need to be carefully evaluated.
In this study, we demonstrated that raft protein constituents were dramatically changed after activation (Fig. 4). Although a previous study showed that human CD4+ T cells exhibit higher levels of raft-associated Lck in an activated state than in a resting state (10), we found a remarkable increase in the expression of another Src-family kinase, Fyn, in the raft fractions. We have previously demonstrated that Fyn, but not Lck, has its kinase activity in lipid rafts and plays a critical role in phosphorylation of Cbp, an adaptor protein involved in the regulation of Src-family kinase activity (17). Upon phosphorylation, Cbp can associate with Csk. By its interaction with Cbp, Csk localizes into rafts and phosphorylates the C-terminal tyrosine of Src-family kinases to down-regulate their catalytic activity (33). Our data in this study clearly demonstrated that the amounts of Fyn, Cbp, Csk and phosphrylated-Cbp were clearly up-regulated in lipid rafts upon activation (Fig. 4B and C). As a result, the phosphorylation of Lck Tyr 505, a C-terminal Tyr residue in Lck, was predominantly detected in the raft fraction in activated T cells (Fig. 4D). It is very likely that the increased amounts and activity of signaling proteins involved in negative regulation for activation underlie the decreased Ca2+ signaling in activated T cells. A previous report demonstrated that Csk localized to lipid rafts via its interaction with Cbp is constitutively inhibiting activation in resting T cells, and transient release from the Csk-mediated inhibition leads to T cell activation (33). This study indicates that the CbpCsk system is also important for controlling an activation threshold in activated T cells.
Finally, although the model of lipid rafts in the plasma membrane has attracted much recent attention, a number of studies are beginning to highlight problems with this model. It is undoubtedly important to develop an experimental system which elucidates a role for lipid rafts under physiological conditions. One potential approach for this could be the modification of raft organization in vivo using gene-targeting technology. We are currently generating and investigating mice lacking a basic component of rafts (i.e. raft-associated lipids) using this technology.
 |
Abbreviations
|
---|
BC | biotinylated -toxin |
Cer | ceramide |
CTx-B | cholera toxin B |
ERK | extracellular signal-regulated protein kinase |
GlcCer | glucosylceramide |
GSL | glycosphingolipids |
HPTLC | high-performance thin-layer chromatography |
LacCer | lactosylceramide |
LAT | linker for activation of T cells |
MßCD | methyl-ß-cyclodextrin |
MES | 4-(N-morpholino) ethanesulfonic acid |
PIP2 | phosphatidylinositol 4,5-bisphosphate |
PLC | phospholipase C |
pAb | polyclonal antibody |
SM | sphingomyelin |
ZAP-70 | 70-kD zeta-associated protein |
 |
Notes
|
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Transmitting editor: A. Singer
Received 22 July 2004,
accepted 10 March 2005.
 |
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