Selective Inhibition of T Cell Activation Via CD147 Through Novel Modulation of Lipid Rafts

Günther Staffler, Andreas Szekeres, Gerhard J. Schütz, Marcus D. Säemann, Elisabeth Prager, Maximilian Zeyda, Karel Drbal, Gerhard J. Zlabinger, Thomas M. Stulnig and Hannes Stockinger
J Immunol August 15, 2003, 171 (4) 1707-1714; DOI: https://doi.org/10.4049/jimmunol.171.4.1707

Abstract

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor α-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.

There is increasing evidence that the plasma membrane is organized in microdomains. This compartmentalization of the plasma membrane appears, on the one hand, to be important to separate reactive components (i.e., receptors and associated signal-transducing molecules) in unprimed resting cells, and, on the other hand, to cluster and concentrate them when determined for execution of signal transduction and function in activated cells (1, 2). These membrane compartments are relatively resistant to solubilization by many nonionic detergents. They contain GPI-anchored proteins, glycosphingolipids, sphingomyelins, cholesterol, Src family protein tyrosine-kinases and G proteins (3, 4) and are, therefore, also called GPI microdomains (1), glycosphingolipid-cholesterol rafts, detergent-insoluble glycolipid-enriched domains (5) or glycosphingolipid-enriched membrane domains (6).

The primary components responsible for the integrity of GPI microdomains have been shown to be cholesterol and saturated fatty acids mainly associated with sphingolipids, GPI proteins and Src kinases (7). Consequently, also these lipids play a major role to enable signaling. Reduction of the cholesterol content inhibited signaling via GPI-anchored proteins (8). Disruption of cholesterol-rich membrane domains in T cells by polyene antifungal agents decreased tyrosine phosphorylation of CD3-ζ, phospholipase Cγ1, and Ca2+ flux in response to stimulation using CD3 mAb OKT3 (2). In addition, it was shown that these antifungal agents also inhibited mAb-induced internalization of the GPI-anchored protein CD59 (9). Furthermore, it was recently shown that depletion of cholesterol using methyl-β-cyclodextrin induced uncontrolled T cell activation by transient tyrosine phosphorylation of multiple proteins, including ζ-associated protein 70 (ZAP-70),4 linker for activation of T cells (LAT), and phospholipase Cγ1 (10). However, not only a change of membrane cholesterol alters signaling and receptor function, but also modification of the saturation index of the fatty acids in the membrane. Shifting the index toward unsaturation by feeding cells with polyunsaturated fatty acids resulted in an inhibition of signal transduction, which is accompanied by a specific displacement of intracellular signaling molecules from microdomains (11).

With the exception of fractions of some transmembrane proteins including integrins (12), CD4 and CD8 (13, 14), most transmembrane proteins are excluded in the resting state of the cell from GPI microdomains (1). Upon receptor ligation/cell activation, however, several recent studies show association of the TCR, FcεR1, or FcαR with GPI microdomains and subsequent signal transduction (2, 15, 16, 17). The mechanisms ruling over association/dissociation of transmembrane receptors with microdomains are not clear yet. However, CD28 engagement induces redistribution and polarization of GPI microdomains into caps at the site of TCR engagement. Thus, CD28 appears to promote association of TCR with microdomains (18). The existence of regulators, which amplify association of molecules with GPI microdomains, implies that there might also exist receptors that trigger dissociation of molecules from microdomains, resulting in inhibition/deactivation of signal transduction. Searching for molecules that might execute negative regulating signals via modification of GPI microdomains, we found CD147 to be a potential candidate.

CD147, also known as M6 Ag (19), extracellular matrix metalloproteinase inducer (20), basigin or neurothelin (21, 22), is a 50- to 60-kDa type I transmembrane glycoprotein belonging to the Ig superfamily (19). CD147 is widely expressed on hemopoietic and nonhemopoietic cells. It is strongly up-regulated on T cells upon activation, indicating a function in T cell biology (19, 23). Indication of a potential negative regulatory function of CD147 in T cell regulation was shown by enhanced mixed lymphocyte responses of lymphocytes from CD147 knockout mice (24) and by a mAb that inhibited T cell proliferation (23). In this study, we show that triggering the CD147 molecule on T cells by this inhibitory mAb results in a characteristic modulation of microdomains, which is associated with impaired signaling for expression of the IL-2R α-chain CD25.

Materials and Methods

Cell preparation

Peripheral blood was taken from healthy donors and PBMCs were obtained by density gradient centrifugation using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). T cells were enriched by removing adherent cells on nylon wool (Robbins Scientific, Sunnyvale, CA) and purified by negative depletion of CD14-, CD16-, CD20-, and CD56-positive cells using the respective specific mAbs (CD14 mAb MEM-18, CD16 mAb MEM-154, CD20 mAb MEM97, CD56 mAb MEM-188, all of which were produced by Dr. I. Hilgert and Dr. V. Horejsi, Academy of Sciences of the Czech Republic, Prague, Czech Republic) and the MACS technique of Miltenyi Biotec (Bergisch Gladbach, Germany). Afterward, the purified T cells were washed and resuspended in complete RPMI 1640 medium supplemented with 10% heat-inactivated FCS (PAA, Linz, Austria), 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 18.75 μg/ml gentamicin sulfate.

Mixed lymphocyte culture (MLC)

Responder PBMCs (4 × 104) were incubated with 4 × 104 irradiated (6000 rad) PBMCs from an unrelated donor in complete RPMI 1640 medium in the presence of various concentrations of the CD147 mAbs. Cultures were set up in round-bottom 96-well plates (Costar, Cambridge, MA) at 37°C in a 5% CO2 atmosphere for 4 days. Assays were performed in triplicates. Sixteen hours before harvesting, the cultures were pulsed with 1 μCi [3H]thymidine (NEN, Boston, MA). Incorporated radioactivity was measured by liquid scintillation counting.

T cell proliferation assay

Proliferation assays of highly purified T lymphocytes (1 × 105 cells/well) were performed in triplicates in 96-well flat-bottom tissue culture plates (Nunc, Roskilde, Denmark) in a final volume of 200 μl. Proliferation was induced by CD3 mAb OKT3 in combination with the CD28 mAb Leu-28 (BD Biosciences, San Jose, CA). CD3 mAb OKT3 was immobilized to the surface of the well. For this purpose, plates were incubated overnight at 4°C with 100 μl of an OKT3 (0.05 μg/ml in PBS) solution. After washing the precoated plates twice, we added T cells, which were suspended in complete RPMI 1640 medium containing Leu-28 (1 μg/ml) and different concentrations of CD147 mAbs. Alternatively, T cells were stimulated with PMA (10−7 M; Sigma-Aldrich, St. Louis, MO) plus ionomycin (1 mM; Sigma-Aldrich), or PMA in combination with immobilized OKT3 or Leu-28. For cytokine supplementation experiments, recombinant human IL-2 (kindly provided by the Novartis Research Institute, Vienna, Austria) was added as indicated. Proliferation was assessed as described above.

Capping

Purified T lymphocytes were stored overnight in complete RPMI 1640 medium without phenol red (1640 without PR). Afterward T cells were washed once, resuspended at 1 × 106 cells per 100 μl of 1640 without PR, and incubated on ice for 30 min with a Cy5-labeled Fab of mAb MEM-102 (10 μg/ml), specific for the GPI-anchored molecule CD48. After washing with ice-cold 1640 without PR, T cells were resuspended at 1 × 106 per 100 μl of 1640 without PR. To induce capping, T cells were incubated with 10 μg/ml OKT3 alone or in combination with 1 μg/ml Leu-28 for 30 min at 37°C in the presence or absence of 1 μg/ml of the indicated CD147 mAbs. Subsequently, cells were plated onto glass microscope cover slips and examined on a Carl Zeiss microscope (Zeiss, Oberkochen, Germany). The percentage of cells, which showed a cap, was assessed by analyzing the distribution of the fluorescence intensity on the plasma membranes of MEM-102-Cy5-stained cells using SPOT Advanced Software (Diagnostics Instruments, Sterling Heights, MI). Cells displaying a 3-fold increase of fluorescence intensity on one site of the plasma membrane were counted as cells with caps. Three hundred cells per sample were evaluated and the experiment was repeated once.

Analysis of protein tyrosine phosphorylation

Purified human T cells were rested overnight in complete RPMI 1640 medium. Before stimulation, T cells were incubated in RPMI 1640 medium supplemented with 1% FCS for 4 h at 37°C. Cells (3 × 106) were stimulated for 5 min at 37°C using CD3 mAb OKT3 (10 μg/ml) along with CD28 mAb Leu-28 (2 μg/ml) in the presence or absence of CD147 mAbs, or they were left unstimulated. The reaction was stopped by addition of ice-cold washing buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA). After centrifugation (2 min, 850 × g, 4°C), cells were immediately lysed for 30 min in ice-cold TBS (20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) containing 1% Nonidet P-40 (Pierce, Rockford, IL), 1 mM sodium orthovanadate, 20 mM NaF, 5 mM EDTA, and protease inhibitors (5 mM aprotinin, 5 mM leupeptin, 1 mM PMSF, and 1 μM pepstatin; all from Sigma-Aldrich). After centrifugation (5 min, 14,000 × g, 4°C), lysates were analyzed by nonreducing SDS-PAGE (12% gel) and immunoblotting using HRP-labeled anti-phosphotyrosine Ab 4G10 (1/4000; Upstate Biotechnology, Lake Placid, NY).

Multiprobe RNase protection assay

Purified human T cells were rested overnight in RPMI 1640 medium supplemented with 10% FCS. Cells (4 × 107) were stimulated by plate-bound OKT3 (immobilized at 0.1 μg/ml) along with soluble Leu-28 (0.5 μg/ml) alone or in the presence of soluble CD147 mAbs (1 μg/ml). After a 12-h incubation in a humidified atmosphere at 37°C, T cells were harvested. Subsequently, total RNA was isolated using TRIreagent (Sigma-Aldrich) according to the manufacturer’s instructions and used in the standard RNase protection assay (BD PharMingen, San Diego, CA). In brief, the template sets hCK-1 and hCR-1 were used to synthesize [α-32P]UTP (Amersham Biosciences, Buckinghamshire, U.K.) containing riboprobes (in vitro transcription kit; BD PharMingen) that were hybridized with 4.0 μg total RNA and treated with a mixture containing RNase A+T1. Samples were denatured and loaded onto 0.4-mm, 6% polyacrylamide/urea gels. After electrophoresis, gels were transferred to filter papers, dried under vacuum, and analyzed by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Each band was normalized by comparison of its intensity value to the value of the housekeeping gene L32. The normalized values were used to quantify expression of the individual mRNAs from CD147 mAb-treated cells as percent change in comparison to CD3/CD28-stimulated cells without CD147 mAbs.

Immunofluorescence analysis of CD25

Highly purified T cells were activated as described before (see T cell proliferation assay) in the presence or absence of soluble CD147 mAbs. Analysis of cell surface expression of CD25 was performed 24 h after culture initiation. For this purpose, T cells were incubated with a directly labeled CD25 mAb (10 μg/ml, FITC-labeled CD25; BD Biosciences) for 30 min on ice in PBN (PBS, containing 1% BSA and 0.02% NaN3) and afterward washed twice with PBN. To prevent nonspecific binding of the mAb to Fc receptors, the cells were preincubated for 30 min with 4 μg/ml human Ig on ice. Fluorescence was analyzed on a FACScan flow cytometer (BD Biosciences). DNA staining using ethidium bromide excluded dead cells from the analysis.

Cytokine assay

Highly purified T cells were stimulated using mAb OKT3 along with mAb Leu-28 (as described in the T cell proliferation assays) in the presence or absence of soluble CD147 mAbs. Alternatively, as a control for inhibition of IL-2 secretion, CD3/CD28-stimulated T cells were incubated with cyclosporin A (1 μg/ml; Sigma-Aldrich). Cell culture supernatants were harvested 24 h following activation. IL-2 secretion was assayed by a sandwich ELISA using a commercial ELISA kit (R&D Systems, Minneapolis, MN).

Analysis of glycosphingolipid-enriched membrane domain

Freshly isolated highly purified T lymphocytes (1 × 107 cells/ml) were stimulated for 20 min at 37°C in complete RPMI 1640 medium (10 ml) using CD3 mAb OKT3 (5 μg/ml) alone or in combination with 5 μg/ml of different CD147 mAbs, or they were left unstimulated. Alternatively, unstimulated T cells were treated for 20 min at 37°C with 5 μg/ml of the indicated CD147 mAbs alone. The reaction was stopped by adding a 3-fold volume of ice-cold washing buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM EDTA). After a short centrifugation (2 min, 850 × g, 4°C), the cells were immediately lysed for 30 min on ice in 0.25 ml of lysis buffer consisting of TBS (20 mM Tris-HCl (pH 7.5) and 150 mM NaCl), 1% Brij-58 (Pierce), 2 mM EDTA, and protease inhibitors (5 mM aprotinin, 5 mM leupeptin, 1 mM PMSF, and 1 mM pepstatin; all from Sigma-Aldrich). After centrifugation (30 s, 14,000 × g, 4°C), the cell lysates were adjusted to 40% (w/v) sucrose by adding an equal volume of a 80% sucrose solution (TBS containing 80% w/v sucrose, 2 mM EDTA, and protease inhibitors). These preparations were placed on top of a 60% sucrose layer in a centrifuge tube (Sorvall Instruments-DuPont, Wilmington, DE). On the top of this, layers of 20, 10, and 5% sucrose were placed. After ultracentrifugation (180,000 × g, 16 h, 4°C), 375-μl fractions were collected from the top. Aliquots of each fraction were diluted in a 4× gel loading SDS buffer. Proteins were separated by nonreducing SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). After a 1-h incubation in blocking buffer (20 mM Tris-HCl (pH 7.5), 0.15 mM NaCl, and 3% BSA), membranes were probed with the respective mAbs (mouse anti-human mAbs: lck-01 to p56lck, TS2/18 to CD2, MEM-102 to CD48, MEM-43/5 to CD59, MEM-M6/1 to CD147 (all produced by Dr. I. Hilgert and Dr. V. Horejsi), rabbit mAbs SC-1239 to CD3ζ (kindly provided by Dr. L. E. Samelson, National Institute of Child Health and Human Development, Bethesda, MD), anti-ZAP-70 mAb (Transduction Laboratories, Lexington, KY), and anti-LAT mAb (Upstate Biotechnology) washed in washing buffer (20 mM Tris-HCl (pH 7.5), 0.15 mM NaCl, and 0.1% Tween 20) and incubated for 1 h with HRP-conjugated secondary Abs: Either goat anti-mouse IgG (Sigma-Aldrich) or goat anti-rabbit IgG (Bio-Rad, Richmond, CA). Proteins were visualized using the ECL detection system (Roche Applied Science, Mannheim, Germany) and a Lumi-Imager (Roche Applied Science).

Results

Down-regulation of T cell alloresponsiveness by CD147 mAb MEM-M6/6

Among a panel of 13 CD147 mAbs, we found one, termed MEM-M6/6, which inhibited CD3 mAb OKT3 activation of human T cells enriched from peripheral blood. Epitope mapping analysis revealed that MEM-M6/6 is a mAb directed to a unique epitope located in the membrane proximal Ig-like domain of CD147 (23).

To examine the inhibitory capacity of MEM-M6/6 on T cells in more detail, we first analyzed the response of T lymphocytes in the presence of MEM-M6/6 in a MLC. As shown in Fig. 1⇓A, MEM-M6/6 inhibited the T cell proliferative response in MLCs up to 90%, whereas all other CD147 mAbs did not effect T cell proliferation. MEM-M6/1 representing these nonfunctional CD147 mAbs served as isotype-matched control in this study.

FIGURE 1.
FIGURE 1.

Influence of CD147 mAb MEM-M6/6 on T cell proliferation. A, PBMCs were mixed with irradiated PBMCs from an unrelated donor in the presence or absence of different concentrations of the CD147 mAbs MEM-M6/6 or MEM-M6/1. Proliferation was determined on day 4 following stimulation as described in Materials and Methods. This figure shows the [methyl-3H]TdR uptake in cpm (mean ± SD) of four independent experiments using cells from different donors. B, Highly purified T cells were stimulated with a CD3 mAb and a CD28 mAb (OKT3 and Leu-28, respectively) in the presence of various concentrations of soluble CD147 mAbs MEM-M6/6 and MEM-M6/1 or with medium alone. Proliferation was determined 72 h after starting the culture as described in A. This figure shows the [methyl-3H]TdR uptake in cpm (mean ± SD) of five independent experiments using cells from different donors. C, T lymphocytes were stimulated with PMA alone, PMA plus ionomycin (Iono), PMA plus immobilized CD3 mAb OKT3, or with PMA plus CD28 mAb Leu-28. In all experiments, cells were cultured with two different concentrations (1 or 0.3 μg/ml) of soluble CD147 mAbs MEM-M6/1 and MEM-M6/6 or with medium alone. Proliferation was determined as described in A. This figure shows the [methyl-3H]TdR uptake in cpm (mean ± SD) of three independent experiments using cells from different donors.

Down-regulation of TCR/CD3 plus CD28-induced proliferation of highly purified T lymphocytes by CD147 mAb MEM-M6/6

To analyze whether the inhibitory potential of mAb MEM-M6/6 relies upon interaction with the CD147 molecule on T cells or on accessory cells, we highly purified peripheral blood T lymphocytes and challenged them with CD3 mAb OKT3 plus CD28 mAb Leu-28 in the presence of MEM-M6/6. The high purity of the T lymphocytes was underlined by their unresponsiveness when both stimulatory mAbs were provided in soluble form (25). Proliferation of these highly purified T lymphocytes was obtained only when both mAbs were present and at least OKT3 was immobilized. Fig. 1⇑B shows that soluble MEM-M6/6 also significantly decreased proliferation (up to 70–80%) of these T cell preparations in contrast to control mAb MEM-M6/1.These results indicate that MEM-M6/6 exerts its function upon interaction with the CD147 molecule expressed on T cells. Maximal down-regulation was seen with concentrations between 1 μg/ml and 0.1 μg/ml of the mAb. Increasing or decreasing the concentration of MEM-M6/6 gradually abrogated the inhibitory affect (the same was found in the MLC). This is in line with earlier data (23) and indicates that an appropriate Ag:Ab ratio is necessary to mediate the inhibitory effect.

CD147 mAb MEM-M6/6 does not inhibit proliferation of T lymphocytes upon bypassing early signaling cascades

Next, we analyzed the inhibitory effect of MEM-M6/6 when PMA and the Ca2+ ionophore ionomycin were used for stimulation. As shown in Fig. 1⇑C, MEM-M6/6 could not inhibit proliferation of these T cell cultures. Since T cells could also be stimulated by cross-linking CD3 or CD28 in the presence of PMA, we analyzed whether MEM-M6/6 affects T cell activation induced by these stimulants. None of these stimulations were blocked by MEM-M6/6 (Fig. 1⇑C). Inasmuch as PMA alone induces only a weak proliferation in this T cell preparation, these findings show that certain signals can be transmitted via CD3 as well as CD28 in the presence of CD147 mAb MEM-M6/6. Furthermore, the experiment indicates that CD147 targeting disturbs early T cell signaling events, which can be bypassed by PMA.

CD147 mAb MEM-M6/6 inhibits redistribution and clustering of membrane microdomains upon TCR/CD3 triggering

A prerequisite for efficient induction of membrane proximal signaling events via TCR is the redistribution and clustering of membrane microdomains to the site of TCR engagement (18, 26, 27). Therefore, we studied whether CD147-induced negative regulation of T cell proliferation is associated with an alteration of membrane microdomain reorganization. For this purpose, highly purified T lymphocytes were stained using Cy5-labeled mAb MEM-102, which specifically recognizes the GPI-anchored coreceptor CD48. GPI-anchored molecules are highly associated with microdomains and, thus, are regarded as markers for lipid rafts.

First, we analyzed the distribution of the microdomain-localized CD48 molecules in unstimulated as well as TCR/CD3-stimulated T lymphocytes. Unstimulated cells showed a diffuse or ring pattern of plasma membrane staining (Fig. 2⇓; unstim), whereas stimulation of T lymphocytes resulted in clustering of CD48 molecules to polarized surface caps in ∼60% of cells (Fig. 2⇓; stim).

FIGURE 2.
FIGURE 2.

Inhibition of TCR/CD3-mediated redistribution of the CD48 molecule by mAb MEM-M6/6. Purified peripheral blood T lymphocytes were stained with Cy5-labeled mAbs to CD48 (MEM-102) and washed. Afterward CD3 mAb OKT3 was added for 30 min at 37°C in the presence (stim + MEM-M6/6 or stim + MEM-M6/1) or absence (stim) of CD147 mAb MEM-M6/6 or MEM-M6/1. Cells were also incubated at 37°C without addition of CD3 or CD147 mAbs (unstim).

Then, we analyzed a possible influence of MEM-M6/6 on TCR/CD3 induced cap formation of CD48. As can be seen in Fig. 2⇑, the number of cells showing a cap after stimulation dropped from ∼60 to ∼20% when T cells were stimulated in the presence of MEM-M6/6 (stim + MEM-M6/6), but not when the cells were incubated with the nonfunctional CD147 control mAb MEM-M6/1 (Fig. 2⇑; stim + MEM-M6/1). This result shows that CD147 mAb MEM-M6/6 disturbs the reorganization of GPI-anchored CD48 molecules upon T cell activation.

Effect of CD147 mAb MEM-M6/6 on transcription of cytokines and cytokine receptors

To get a hint of which signaling pathways are affected by CD147-mediated membrane alteration, we performed RNase protection assays on purified human T cells. We analyzed mRNA expression of different cytokines as well as cytokine receptor subunits. As shown in Fig. 3⇓, the expression of IL-2 mRNA as well as the IL-2Rα mRNA was strongly induced upon T cell stimulation. In parallel, also the expression of mRNAs coding for IFN-γ, IL-9, IL-15, IL-10, IL-4, IL-5, IL-2Rβ, common γ-chain, IL-4Rα, and IL-15R were induced or enhanced. In contrast, IL-7Rα mRNA, which seems to be expressed strongly on unprimed T cells, is down-regulated upon stimulation (28, 29).

FIGURE 3.
FIGURE 3.

Effect of CD147 mAb MEM-M6/6 on the expression of cytokines and cytokine receptors. Total RNA was isolated from unstimulated as well as from stimulated human T lymphocytes treated with or without CD147 mAbs. Samples were analyzed by RNase protection assays using template set hCK-1 (A) and hCR-1b (B) (see Materials and Methods). Expression of housekeeping genes L32 and GAPDH are shown as a measure of equal RNA loading. This experiment is representative of three independent experiments. Lane 1, Unstimulated T cells; lane 2, OKT3/Leu-28-stimulated T cells; lane 3, stimulated T cells with MEM-M6/1; and lane 4, stimulated T cells with MEM-M6/6.

T cells stimulated in the presence of MEM-M6/6 showed a unique cytokine and cytokine receptor pattern. Expression of the IL-2Rα chain was reduced by 66% compared with the control (Table I⇓). Furthermore, the mAb moderately inhibited expression of IL-4R α-chain (27% reduction) and inhibited down-regulation of IL-7R α-chain, but did not influence the expression of other cytokine receptors (Fig. 3⇑B and Table I⇓). Concerning cytokines, IFN-γ and IL-15 mRNAs were blocked by 40%, those of IL-9, IL-10, IL-4, and IL-5 by >70%. MEM-M6/6 had no effect, however, on expression of IL-2 (Fig. 3⇑A and Table I⇓). In summary, MEM-M6 treatment blocks expressions of cytokines with the exception of IL-2 but has little influence on cytokine receptor expression except for IL-2Rα.

View this table:
Table I.

Quantification of bands in Fig. 3⇑ by PhosphorImagera

Targeting the CD147 molecule by mAb MEM-M6/6 does not inhibit secretion of IL-2

Next, we analyzed whether CD147 mAb MEM-M6/6-induced modulation of cytokine and cytokine receptor expression is not only seen at the mRNA but also at the protein level. We concentrated on IL-2, which is the most important growth factor for T cells and the IL-2 receptor α-chain CD25, which is required for action of IL-2. As can be seen in Fig. 4⇓A, surface expression of CD25 was inhibited up to 80% by MEM-M6/6. In contrast, secretion of IL-2 was not significantly diminished by MEM-M6/6 (Fig. 4⇓B), although the proliferation of the T cells in these cultures was reduced up to 60–70% compared with the controls (cf Fig. 1⇑B). IL-2 secretion was also not influenced by the nonfunctional control mAb MEM-M6/1, but it was inhibited when we treated cells of the same preparation with cyclosporin A. Consistent with the impaired expression of CD25, addition of exogenous IL-2 did not significantly restore T cell proliferation in MEM-M6/6-treated T cells (Fig. 4⇓C). These results demonstrate that triggering the CD147 protein by mAb MEM-M6/6 does not lead to a general block of T cell signaling but rather may interfere with particular T cell signaling pathway(s).

FIGURE 4.
FIGURE 4.

CD147 mAb MEM-M6/6 inhibits surface expression of CD25, but does not affect IL-2 secretion. A, T cells were stimulated as described in Fig. 1⇑B with (stim + MEM-M6/6) or without (stim) CD147 mAb MEM-M6/6 or with control mAb MEM-M6/1 (stim + MEM-M6/1; each 0.5 μg/ml). Twenty-four hours after activation, cells were harvested and surface expression of CD25 was analyzed by immunostaining using a directly labeled CD25 mAb and flow cytometry. Thin lines represent histograms received with isotype control mAbs. This experiment is representative of three independent experiments. B, Highly purified T lymphocytes were stimulated as described in Fig. 1⇑B. Supernatants were taken 24 h following stimulation and the cytokine content was determined by ELISA. The cytokine concentrations in the supernatants are shown in percent to the medium control (100% = 1770 pg/ml). Comparable results were obtained in five independent experiments. C, Highly purified T cells were stimulated as described in Fig. 1⇑B in the presence of exogenous rIL-2. Proliferation was determined 72 h after starting the culture as described in Fig. 1⇑. This experiment is representative of five independent experiments.

CD147 mAb MEM-M6/6 does not affect major protein tyrosine phosphorylation upon TCR/CD3 T cell stimulation

One of the earliest signaling events that follow TCR engagement is tyrosine phosphorylation of several proteins. As shown in Fig. 5⇓, CD3 and CD28 treatment resulted in a strong tyrosine phosphorylation of proteins in a zone of 50 and 60 kDa corresponding to the Src kinases lck and fyn, as well as a 36- to 38-kDa band corresponding to LAT. Importantly, we did not see a difference in protein tyrosine phosphorylation when T lymphocytes were stimulated in the presence or absence of MEM-M6/6. Moreover, MEM-M6/6-treated and untreated T cells exhibited comparable calcium fluxes after anti-CD3 cross-linking (data not shown). Thus, the major signaling molecules required for expression of IL-2 (30, 31) are not influenced by MEM-M6/6. This is in agreement with the undisturbed expression of IL-2 shown before.

FIGURE 5.
FIGURE 5.

CD147 mAb MEM-M6/6 does not impair protein tyrosine phosphorylation upon TCR triggering. Highly purified T cells were incubated for 10 min on ice with medium alone or with medium supplemented with CD147 mAb MEM-M6/6 or MEM-M6/1. Afterward, cells were left unstimulated (unstim) or were stimulated (stim) by mAb OKT3 for 5 min on 37°C. Subsequently, cells were lysed and tyrosine phosphorylated proteins were analyzed using Western blotting. Similar results were obtained with four other experiments.

CD147 triggering by mAb MEM-M6/6 modulates T cell microdomains

As shown before by fluorescence microscopy, CD147 mAb MEM-M6/6 modulated mAb OKT3 induced cap formation of the GPI-anchored coreceptor CD48. Because CD48 is known as marker for microdomains (1, 4, 5, 11, 13, 32), one might suggest that association of microdomains and TCR is impaired by MEM-M6/6 treatment. However, CD147 triggering did not alter expression of IL-2 that requires phosphorylation of microdomain-associated lck and LAT (30, 31). Thus, these results suggested that CD147 mAb MEM-M6/6 did not block association of microdomains and TCR in general but rather caused a unique alteration of TCR-associated microdomains. To study this, we isolated microdomains by sucrose gradient centrifugation (7, 13). According to the enrichment of CD48 in fractions 2–5 (11, 13, 15) and the localization of CD2 mainly in the high-density fractions 7–8 as described before (33), we concluded that the buoyant fractions 2–5 of the sucrose gradient shown in Fig. 6⇓A contain microdomains.

FIGURE 6.
FIGURE 6.

CD147 mAb MEM-M6/6 mediates displacement of the GPI-linked molecules CD48 and CD59 from lipid rafts of T lymphocytes. A, Resting T lymphocytes were lysed in a buffer containing the nonionic detergent Brij-58. Lysates were subjected to sucrose density gradient centrifugation. Aliquots of the individual fractions were mixed with 4× nonreducing SDS sample buffer and a quarter of each fraction was loaded on 12% SDS-polyacrylamide gels. Proteins were detected by Western blotting using specific mAbs. This experiment is representative of three independent ones. B, T lymphocytes were stimulated in the presence of either mAb MEM-M6/1 (stim + M6/1) or MEM-M6/6 (stim + M6/6), or without addition of CD147 mAbs (stim). Subsequently, sucrose density gradient centrifugation and Western blotting analysis were performed as described in A. Comparable results were obtained with five independent experiments. C, T lymphocytes were treated either with mAb MEM-M6/1 (M6/1) or MEM-M6/6 (M6/6) alone. Subsequently, sucrose density gradient centrifugation and Western blotting analysis were performed as described in A. Comparable results were obtained with three independent experiments. GEM, Glycosphingolipid-enriched membrane protein.

Upon stimulation via the TCR/CD3 complex, 40–50% of the CD3ζ chain was detected in the low-density fractions and incubation with MEM-M6/6 did not alter its distribution (Fig. 6⇑B). (18, 34). Next, we studied the distribution of signaling molecules known to be associated with membrane-proximal TCR signaling events, such as LAT, p56lck, and ZAP-70 in the gradient fractions. As shown in Fig. 6⇑B, LAT was found in all fractions but peaked in the low-density fractions 2–5. The same pattern was seen in fractions derived from TCR-stimulated T cells triggered with MEM-M6/6. MEM-M6/6 presence also did not change the distribution pattern of p56lck, a substantial proportion of which was found in the fractions corresponding to microdomains. Although it was reported that ZAP-70 colocalizes with phosphorylated CD3ζ chains in microdomains upon T cell triggering (2, 15), we did not detect ZAP-70 in the low-density fractions of these cells and CD147 engagement did not alter its distribution (Fig. 6⇑B). We tested also the distribution of the CD147 molecule. In TCR/CD3-stimulated cells, a small part was found in fraction 5 but the majority was contained in the high-density fractions 7 and 8 and the intermediate fraction 6. Incubation of both CD147 mAbs, MEM-M6/6 and MEM-M6/1, resulted in a concentration of CD147 in the high-density fractions 7 and 8.

When analyzing density gradient distribution of the GPI-anchored protein CD48 in OKT3-stimulated T cells, we found a strong shift from the low-density fractions toward the higher density ones upon MEM-M6/6 treatment (Fig. 6⇑B): CD48 could not be found any longer in fraction 2 but became visible in the intermediate fraction 6 and, in particular, in the high-density fractions 7 and 8. In comparison, the distribution of CD48 in sucrose fractions of stimulated T cells incubated with the nonfunctional CD147 mAb or without any mAb was not altered. This experiment therefore shows that CD147-induced inhibition of T lymphocyte proliferation is accompanied by a displacement of the GPI-anchored protein CD48 from microdomains. In addition to CD48, a proportion of CD59, another GPI-anchored protein, was reproducibly shifted to high-density fractions in MEM-M6/6-treated cells (Fig. 6⇑B).

Next, we analyzed whether the changes in the molecular composition of lipid rafts are induced by MEM-M6/6 treatment alone or dependent on TCR/CD3 stimulation. As shown in Fig. 6⇑C, a comparable shift from low- to high-density fractions of the GPI-anchored molecules could also be observed when T cells were triggered by CD147 mAb MEM-M6/6 without stimulation using OKT3. Also in this approach, MEM-M6/6 did not alter the distribution of LAT. The control mAb MEM-M6/1 did not show any effect.

Discussion

During the last decade, it became more and more evident that the compartmentalization of the plasma membrane into microdomains is one of the main principles to control signaling of cell surface receptors. Today, two types of microdomains are relatively well defined: one enriched in tetraspan proteins (35), the second, the better characterized one, concentrate GPI-proteins, glycosphingolipids, sphingomyelins, and cholesterol and thus is called glycosphingolipid-enriched microdomain, GPI microdomain or lipid raft. These lipid rafts are enriched on the one hand in GPI-anchored molecules, known to act as costimulatory molecules, and, on the other hand, in molecules involved in signal transduction such as LAT, protein tyrosine kinases, and G proteins. In resting T cells, this conglomerate of extracellular accessory and intracellular signaling molecules is physically separated from transmembrane receptors including the TCR/CD3 complex, which is located in the more phospholipid-rich bulk of the cell membrane. Recently, several publications showed that TCR engagement led to association of the TCR with lipid rafts and to their redistribution to form dens caps. As a result of this physical contact, raft-resident signaling molecules couple triggered TCRs with more downstream signaling pathways, resulting finally in clonal expansion of T cells. A prerequisite for transmitting signals via lipid rafts is their integrity. As soon as the molecular organization is disordered (e.g., by fish oils or depletion of cholesterol), signal transduction is disturbed, resulting in blockade of T cell proliferation.

In this report, we provide evidence that specific triggering of the T cell activation-associated CD147 molecule by mAb MEM-M6/6 inhibits TCR/CD3-induced reorganization of parts or subtypes of lipid rafts into dense caps. While leaving expression and secretion of IL-2 untouched, this blockade of membrane reorganization is accompanied by impaired expression of CD25, resulting in reduced sensitivity to IL-2. MEM-M6/6, which recognizes a unique epitope on the CD147 molecule (23), blocks TCR-induced proliferation of T cells. Remarkably, MEM-M6/6 was not able to interfere with the clonal expansion of T cells when early signaling events associated with rafts were bypassed by PMA. Earlier studies showed that CD147 mAbs inhibited U937-dependent T cell proliferation (36) and T cell proliferation in a MLC (37). These studies claimed that the mAbs exerted their effects on accessory cells, because inhibition was seen when U937 cells were prepulsed with the mAbs. Indeed, we also have indication that MEM-M6/6 can influence accessory cell function (data not shown). However, the results shown here with highly purified T cells clearly demonstrate that MEM-M6/6 can directly affect T cell function. Inasmuch as proliferation in MLCs of lymphocytes from CD147 knockout mice was significantly greater than that of lymphocytes from wild-type littermates (24), a negative regulatory role of CD147 is indicated in vivo and suggests that MEM-M6/6 acts rather as an agonist mimicking a negative signal than as an antagonist inhibiting a positive one.

We observed blockade of the reorganization of the membrane through CD147 upon TCR/CD3 triggering by using fluorescence microscopy. CD48 proteins clustered into dense caps upon TCR/CD3 triggering and this effect was even more outspoken by costimulation with CD28 (data not shown). Remarkably, MEM-M6/6 inhibited this reorganization of CD48 (Fig. 2⇑), suggesting an inhibition of lipid rafts with TCR/CD3 association by CD147 triggering. However, biochemical analysis of lipid rafts revealed that this assumption was only partially true.

When lipid rafts were isolated by detergent lysis followed by density gradient centrifugation, in accord with previous studies (8, 32, 38), the GPI-anchored proteins CD48 and CD59 were found exclusively in the lipid raft fractions. In contrast, transmembrane molecules such as CD2 and CD147 were mainly excluded from these fractions. As shown before (2, 15, 33, 39), upon TCR triggering the TCRζ chain could be found up to 50% in the low-density fractions, indicating association of TCR/CD3 with lipid rafts. The inhibitory mAb MEM-M6/6 had no influence on this redistribution as well as on the distribution of the Src kinase lck and the linker proteins LAT. However, MEM-M6/6 induced translocation of CD48 and CD59 from the low-density fractions of TCR/CD3-stimulated as well as of unstimulated T lymphocytes to fractions of higher density. Whether CD48 and CD59 are associating with novel microdomains of higher density or as single molecules with the bulk of the membrane is obscure at the moment. Alternatively, they could be part of a subunit of lipid rafts, which appeared for the first time under this experimental setting. Evidence for the existence of various subtypes of lipid rafts and microdomains was shown in Th1 vs Th2 cells (40) and uropod vs leading edge associated rafts of migrating T cells (41). Furthermore, GM1, which is besides GPI-anchored proteins used as another marker for lipid rafts, is not expressed on the surface of resting T cells (18, 42). In contrast, CD48 and CD59 are abundantly observed on the surface of the latter T cells, indicating differences between lipid rafts containing GPI proteins or GM1.

The importance of GPI-anchored proteins in T cell activation is documented by several publications. Clustering of GPI-anchored proteins by mAbs to CD48, CD55, CD59, and CD73, Thy-1, Ly-6, and sgp-60 readily affects T cell activity usually by the induction of T cell proliferation or IL-2 production (9, 43, 44, 45, 46). In contrast, under some experimental conditions mAb binding to GPI-anchored proteins led to inhibition (46, 47, 48) or to a modulation of T cell activation (49). Moreover, T cells lacking GPI-anchored proteins, including GPI-deficient T cells derived from patients with paroxysmal nocturnal hemoglobinuria (50, 51) and T cells derived from CD48 −/− mice (52), were severely impaired in their activation via the TCR. Consistent with these observations, similar defects in T cell responsiveness were seen after enzymatic removal of GPI-linked proteins from normal T lymphocytes (53). Thus, it is tempting to speculate that the CD147-mediated depletion of the GPI proteins CD48 and CD59 from lipid rafts as found by our experiments is underlying the blockade of expression of CD25. This assumption is supported by the finding that triggering of CD59 in CD3-deficient T cells leads to expression of CD25 but not of IL-2 (54), which points to a link between CD59 signaling and CD25 induction.

In conclusion, our results provide evidence that triggering of a potential negative regulating receptor on T cells causes reorganization of lipid rafts that is accompanied by modulation of signaling and cell activation. It is the task of future studies to unveil the different types and subtypes of microdomains and their role in receptor signaling at and across the plasma membrane.

Acknowledgments

We thank Vaclav Horejsi and Ivan Hilgert for providing a number of mAbs, which were invaluable for this study. We thank Thomas Baumruker and Eva Prieschl for their expert assistance in performing the multiprobe RNase protection assay.

Footnotes

  • ↵1 This work was supported by the Competence Center for Biomolecular Therapeutics, by the GEN-AV program of the Austrian Federal Ministry of Education, Science and Culture, and by grants SFB00503, P15025-B08, and P13507-B01 from the Austrian Science Fund.

  • ↵2 Current address: Intercell, Campus Vienna Biocenter 6, A-1030 Vienna, Austria.

  • ↵3 Address correspondence and reprint requests to Dr. Hannes Stockinger, Institute of Immunology, University of Vienna, Brunner Strasse 59, A-1235 Vienna, Austria. E-mail address: hannes.stockinger{at}univie.ac.at

  • ↵4 Abbreviations used in this paper: ZAP-70, ζ-associated protein 70; LAT, linker for activation of T cells; PR, phenol red.

  • Received November 6, 2002.
  • Accepted June 17, 2003.

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