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Specific Marker of Sézary CellsDetection of Extracellular Vimentin as a SC5 mAb Represents a Unique Tool for the

Bensussan and Anne Marie-CardineCapdevielle, Laurence Conraux, Pascual Ferrara, Armand Delphine Huet, Martine Bagot, Denis Loyaux, Joël

http://www.jimmunol.org/content/176/1/652doi: 10.4049/jimmunol.176.1.652

2006; 176:652-659; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/176/1/652.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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SC5 mAb Represents a Unique Tool for the Detection ofExtracellular Vimentin as a Specific Marker of Sezary Cells1

Delphine Huet,* Martine Bagot,* Denis Loyaux,† Joel Capdevielle,† Laurence Conraux,†

Pascual Ferrara,† Armand Bensussan,2,3* and Anne Marie-Cardine2*

Circulating malignant Sezary lymphocytes result from a clonal proliferation of memory/activated CD4�CD45RO� T lymphocytesprimarily involving the skin. Recently, the CD158k/KIR3DL2 cell surface receptor has been identified to phenotypically charac-terize these cells. We previously described a mAb termed SC5 that identifies an unknown early activation cell membrane molecule.It is expressed selectively by T lymphocytes isolated from healthy individuals upon activation, and by circulating Sezary syndromelymphocytes. In addition, we found that SC5 mAb was reactive with all resting T lymphocytes once permeabilized, indicating thatSC5 mAb-reactive molecule might present distinct cellular localization according to the T cell activation status. In this study, weshow for the first time that SC5 mAb recognizes the intermediate filament protein vimentin when exported to the extracellular sideof the plasma membrane of viable Sezary malignant cells. We demonstrate that SC5 mAb is unique as it reacts with both viablemalignant lymphocytes and apoptotic T cells. As vimentin is also detected rapidly at the cell membrane surface after normal Tlymphocyte activation, it suggests that its extracellular detection on Sezary cells could be a consequence of their constitutiveactivation status. Finally, as a probable outcome of vimentin cell surface expression, autoantibodies against vimentin were foundin the sera of Sezary syndrome patients. The Journal of Immunology, 2006, 176: 652–559.

C utaneous T cell lymphomas (CTCL)4 are a heteroge-neous group of lymphomas primarily involving the skin(1). Mycosis fungoides (MF) and Sezary syndrome (SS)

are the two major clinical variants of CTCL. MF, the mostcommon form, is characterized by erythematous patches,plaques, or tumors of the skin constituted of clonally derivedmalignant T lymphocytes that phenotypically resemble mature/memory T cells. SS, a more aggressive leukemic and erythro-dermic variant of CTCL, is characterized by the presence of aclonal CD4�CD45RO� T lymphocyte population with atypicalcerebriform nuclei in the skin, lymph nodes, and peripheralblood (2). To understand the pathogenesis and to facilitate themolecular diagnosis of Sezary cells, a gene expression analysisapproach was undertaken and revealed the selective expressionof the tyrosine kinase receptor EphA4 and of the transcriptionfactor Twist (3), an overexpression of JunB (4), and a completeloss of Bcl2 (5). Furthermore, using a modified representationaldifference analysis, Su et al. (6) reported the aberrant mRNAand protein expression of T-plastin in Sezary cells. A differentapproach to better understand the pathogenesis of Sezary cells,

and to distinguish them from normal reactive CD4� lympho-cytes, was to identify characteristic cell membrane receptors.Thus, the immunophenotypic analysis of peripheral blood Tcells from patients with SS has revealed a frequent loss of Tlymphocyte receptor expression, such as CD7 or CD26, whencompared with normal T lymphocytes (7–12). Recently, we re-ported the presence of the CD158k/KIR3DL2 inhibitory cellmembrane receptor for HLA-A alleles on different CTCL linesestablished from the skin or the blood of patients with SS (13).Furthermore, we found in a large group of patients with SS astrong positive correlation between the percentage of CD158k�

blood lymphocytes, detected by flow cytometry, and the per-centage of Sezary cells evaluated by cytomorphology (14). In-terestingly, in situ analysis revealed that CD158k is expressedby cutaneous malignant lymphocytes from SS patients, but notfrom patients with early MF (15). This identified CD158k as thefirst specific cell surface marker available for the evaluation ofthe circulating tumoral burden, and for the follow-up of patientswith SS. In addition to CD158k, we further evidenced the pres-ence, on the cell surface of circulating Sezary cells, of a struc-ture that was recognized by a mAb that we called SC5 (16, 17).However, in contrast to CD158k, which expression is highlyrestricted to a minor circulating NK and CD8� T cell subset, theSC5-reactive molecule was found to be expressed in the cyto-plasm of all circulating lymphocytes, and at the cell surface ofnormal T lymphocytes following activation. Thus, we postu-lated that this nonidentified protein might correspond to an in-tracellular structure in normal resting T lymphocytes, and thatits surface membrane expression increased rapidly after activa-tion, as reported for CD152 (18, 19), and for a heterogeneousfamily of lysosome-associated membrane proteins (20).

In this study, we report that the SC5 mAb-reactive molecule,located on the extracellular side of the plasma membrane of acti-vated normal T lymphocytes and of viable malignant SS cells, cor-responds to the class III intermediate filament protein vimentin. Moreimportantly, we identified SC5 mAb as a unique tool for the detection

*Institut National de la Sante et de la Recherche Medicale Unite 659, University ofParis XII, Medical School of Creteil, Henri Mondor Hospital, Department of Der-matology, Creteil, France; and †Sanofi-Aventis, Labege, France

Received for publication June 30, 2005. Accepted for publication October 20, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Institut National de la Sante et de la Recherche Medi-cale and Grants 4604 (to A.B.) and 4220 (to M.B.) from the Association pour laRecherche contre le Cancer.2 A.B. and A.M.-C. are senior authors on this paper.3 Address correspondence and reprint requests to Dr. Armand Bensussan, InstitutNational de la Sante et de la Recherche Medicale Unite 659, Faculte de Medecine deCreteil, 8 rue du General Sarrail, F-94010 Creteil Cedex. E-mail address:Armand.Bensussan@im3.inserm.fr4 Abbreviations used in this paper: CTCL, cutaneous T cell lymphoma; LSP1, lym-phocyte-specific protein 1; MF, mycosis fungoides; PI, propidium iodide; SS, Sezarysyndrome.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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of vimentin at the cell membrane surface of viable lymphocytes, andtherefore discuss whether this intermediate filament protein could betargeted as a tumor or an inflammatory Ag for mAb therapy.

Materials and MethodsPatients

After informed consent and approval by an ethics committee (Comite Con-sultatif de Protection des Personnes dans la Recherche Biomedicale, HenriMondor), sera and PBMC were isolated from blood samples of 10 patientswith CTCL. The patients had not been previously treated withchemotherapy.

Cells and cell lines

PBMCs were isolated by the technique of Ficoll-Isopaque (Pharmacia)density gradient centrifugation. To obtain T cell lines, cells were main-tained in RPMI 1640 supplemented with 2 mM L-glutamine, 100 �g/mlpenicillin/streptomycin (Invitrogen Life Technologies), 10% heat-inacti-vated human serum (Jacques Boy Institute), and 100 IU/ml human rIL-2(produced by Sanofi-Aventis). The mycoplasma-free HUT78 cell line wasgrown in RPMI 1640 containing 10% FCS (Perbio Science Europe) andantibiotics.

Antibodies

The mouse SC5 mAb (IgM) was raised in our laboratory (Institut Nationalde la Sante et de la Recherche Medicale Unite 659) against the leukemicNK cell line YTindi, as described elsewhere (17). SC5 hybridoma culturesupernatant was used for Western blot analysis. The purified anti-vimentingoat polyclonal Abs (C-20) were generated against a peptide located in theC terminus part of human vimentin (sc-7557; Santa Cruz Biotechnology),while the anti-vimentin mAb (clone V9, IgG1; Oncogene Research Prod-ucts) was obtained by immunization of mice with purified vimentin. BothAbs were used at 1 �g/ml for Western blot analysis. The anti-TCR�� mAb(IP26, IgG1) was locally produced (Institut National de la Sante et de laRecherche Medicale Unite 659) and can be purchased from eBioscience.The PE-conjugated anti-TCRV�23 mAb was purchased from BeckmanCoulter.

Flow cytometry

Double cell staining was performed according to standard procedure.Briefly, cells (4 � 105) were incubated with SC5 mAb (purified fromhybridoma culture supernatant) for 30 min at 4°C, followed by goat anti-mouse IgM FITC Abs (1 �l/test; Beckman Coulter). After washes, a sec-ond labeling with the anti-TCR�� mAb (hybridoma culture supernatant)and goat anti-mouse IgG1 PE (1 �l/test; Beckman Coulter) was realized.Alternatively, PE-conjugated anti-TCRV�23 mAb was used (10 �l/test).

For single labeling, cells were left untreated or fixed for 30 min at 4°Cin PBS/4% paraformaldehyde. When indicated, an additional step of per-meabilization was performed in PBS/0.1% Triton X-100. Cells were thenlabeled with SC5 mAb or anti-vimentin Ab, followed by the appropriateFITC-conjugated secondary Ab.

Detection of apoptotic cells was performed by incubating the cells witheither annexin V directly coupled to FITC (5 �l/test; BD Biosciences) orpropidium iodide (PI; 1 �g/test; Sigma-Aldrich), according to the manu-facturer’s recommendations.

Two-dimensional gel electrophoresis and mass spectrometryprotein sequencing

PBMCs, cultured for 3 days in the presence of PHA (3 �g/ml; Sigma-Aldrich), were washed in PBS and resuspended in Triton X-100 lysis buffer(20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM Navanadate, 10 mM NaF, 1 mM PMSF, 1 �g/ml aprotinin, and 1 �g/mlleupeptin). Following incubation for 1 h at 4°C and centrifugation, post-nuclear lysates were subjected to a preclearing step on protein A-Sepharosebeads. Immunoprecipitation was performed using SC5 mAb, or an isotype-matched mAb as negative control, and protein G-Sepharose beads. Afterwashes, precipitated proteins were released in 1% Triton X-100 lysis buffersupplemented with 8 M urea at 37°C for 5 min. Samples were resolved bytwo-dimensional gel electrophoresis (isoelectrofocusing in the first dimen-sion, followed by a SDS-8% PAGE in the second dimension), and proteinswere detected by silver nitrate staining of the gel. Protein spots of interestwere in-gel digested with trypsin, according to the method of Shevchenkoet al. (21) on a MassPrep robot (Waters). Extracted peptide mixtures wereanalyzed by a Voyager-DE STR MALDI-TOF instrument (Applied Bio-systems) and NanoHPLC (Switchos/Ultimate Pump; Dionex) on line with

an ESI-QTOF instrument (Waters). Mascot (Matrix Sciences) and Protein-Lynx Global server (Matrix Sciences) softwares were used together forSwiss- and National Registar-National Center for Biotechnology Informa-tion-protein data bank searches.

Generation of Flag-tagged vimentin constructs

The cDNA coding for a C-terminal Flag-tagged vimentin was generated byPCR amplification of the full-length coding region of vimentin. The re-sulting PCR product was purified and ligated into the pcDNA3.1 vector,according to the manufacturer’s recommendations (Invitrogen Life Tech-nologies). Similarly, the vimentin-deleted constructs were raised by PCRamplification of the coding regions corresponding to aa 101–466 (�1–100), 139–466 (�1–138), and 228–466 (�1–227), fused to a C-terminalFlag tag. All cDNA constructs were further inserted into pcDNA3.1 ex-pression vector.

Generation of GST-vimentin fusion protein

Total RNAs were extracted from the human CTCL cell line HUT78, andvimentin cDNA was generated by RT-PCR. The PCR product was clonedinto the pGEX4T1 vector (Amersham Biosciences) at BamHI and EcoRIsites. The cDNA sequence was confirmed by double-stranded sequencinganalysis.

Expression and purification of GST-vimentin protein were essentiallyperformed according to the manufacturer’s procedure and as previouslydescribed (22). Where indicated, the fusion protein was subjected to di-gestion with thrombin before analysis by SDS-PAGE and Western blotting.

Immunoprecipitation and immunoblotting

Cells (2 � 107 per sample) were lysed in 1% Nonidet P-40 lysis buffer andprocessed as described above. After preclearing on protein A-Sepharosebeads, lysates were incubated with SC5, anti-vimentin (clone V9), or iso-type-matched irrelevant mAb. Immunoprecipitates were collected on pro-tein G-Sepharose beads and separated by SDS-8% PAGE under nonreduc-ing conditions. Proteins were electrotransferred on nitrocellulosemembrane and subjected to immunoblotting using SC5 or anti-vimentinAbs and HRP-conjugated secondary Abs. Detection was realized with anECL detection system, according to the manufacturer’s recommendations(Amersham Biosciences).

For the mapping of SC5 mAb recognition site, COS 7 cells were trans-fected with the indicated plasmid using a DEAE-dextran transfectionmethod (23). After 48 h of culture, cells were harvested and lysed. Re-combinant proteins were immunoprecipitated using an anti-Flag mAb andprotein G-Sepharose beads, and resolved by SDS-12% PAGE under non-reducing conditions. Immunoblotting was performed as described above.

Confocal immunofluorescence microscopy

HUT 78 cells were placed in V-shaped 96-well plates (3 � 105cells/well).After a washing step in PBS, cells were fixed in PBS/4% paraformalde-hyde/0.025% glutaraldehyde. For intracellular staining, cells were perme-abilized with PBS/0.1% Triton X-100. After quenching with 50 mMNH4Cl, and saturation of unspecific sites with PBS/1% BSA, cells wereincubated with SC5 and anti-vimentin (C-20) Abs. After washes, cells weresubsequently incubated with goat anti-mouse PE (Beckman Coulter) anddonkey anti-goat FITC (Jackson ImmunoResearch Laboratories) secondaryAbs. Cells were transferred onto coverslips, and slides were mounted usingMowiol (Calbiochem). Analysis of cell labeling was performed on a Zeissconfocal microscope (LSM510; Zeiss).

ResultsSC5 mAb stains circulating SS lymphocytes and the Sezary cellline HUT78

The SC5 mAb raised against the functional cytotoxic NK tumorcell line YTindi was initially selected for its reactivity with boththe immunizing tumor cell line and a minor population of normalPBLs (Fig. 1A, left panel). We previously reported that the PBMCsreactive with SC5 mAb, corresponding to 5–10% of gated lym-phocytes, included CD4�, CD8�, as well as CD56� cells (17).Most of these SC5� circulating lymphocytes belonged to the ac-tivation/memory cell subset coexpressing CD45RO. We also dem-onstrated that the expression of the SC5 mAb-reactive moleculewas highly increased in peripheral blood T cells from patients withSS (16, 17), as shown in Fig. 1A (right panel), with PBMC froma representative patient presenting �90% of malignant CD4�

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cells. Furthermore, we found that the Sezary cell line HUT78,which was weakly recognized by an anti-TCR�� mAb, was alsodimly labeled by SC5 mAb (Fig. 1B). Thus, circulating SS T lym-phocytes and the HUT78 cell line both presented an increasedreactivity toward SC5 mAb when compared with normal resting Tcells.

Identification of vimentin as the Ag recognized by the SC5 mAb

We previously reported that stimulation of PBMCs with PHA re-sulted in an increased cell staining with SC5 mAb when comparedwith resting cells, a maximum detection level being observed after72–96 h of activation (17). To identify SC5 mAb-reactive protein,large-scale SC5 immunoprecipitates were obtained from lysates ofPHA-treated cells. Immunoprecipitations with an isotype-matchedmAb were done in parallel as control (data not shown). The im-munoprecipitates were further subjected to two-dimensional gelelectrophoresis, and proteins were detected by silver staining of thegel. Comparison between SC5 and control mAb protein patternsallowed the specific detection of three protein spots in SC5 mAbimmunoprecipitate (Fig. 2). Each protein spot was cut out from thegel, digested with trypsin, and purified, and the resulting peptideswere subjected to sequencing. Data-based assisted analysis of theobtained amino acid sequences led to the identification of all threepolypeptides. Thus, the 96-kDa protein was found to correspond tothe spectrin superfamily cytoskeleton protein �-actinin 4 (24). Fur-thermore, the proteins with an apparent molecular mass of 60 kDa,and close isoelectric point of 4.7 and 5.1, were identified as thelymphocyte-specific protein 1 (LSP1) (25) and vimentin, respec-tively. Note that the 70-kDa protein was identified as nonspecifi-cally precipitated murine serum albumin.

The molecular characterization of the proteins present in SC5immunoprecipitate demonstrated that the Ab recognizes a protein

complex in PHA-activated cells that at least encompasses �-acti-nin 4, LSP1, and vimentin. To further assess which of thesepolypeptides was specifically recognized by SC5 mAb, immuno-precipitates were prepared from HUT78 cell lysates using com-mercially available Abs directed against each of the identified pro-teins. An anti-vimentin Ab (C-20), raised toward the C-terminalmoiety of the protein, was first tested (Fig. 3A). Immunoprecipi-tations with a control irrelevant Ab, and with SC5 mAb, were donein parallel. Immunoblot analysis with SC5 mAb allowed the de-tection of two protein bands at 53 and 60 kDa in the anti-vimentinprecipitate (C-20). After stripping and reprobing of the blot withthe anti-vimentin Ab (C-20), an identical protein pattern was ob-tained. Conversely, Western blot analysis of SC5 mAb immunecomplex with SC5 or anti-vimentin Ab resulted in the visualizationof a broad signal in the 50- to 60-kDa range (Fig. 3A). In contrast,no signal was obtained when probing an anti-LSP1 or �-actinin 4immunoprecipitate with SC5 mAb (Fig. 3B).

To definitely assess the specificity of SC5 mAb for vimentin, acDNA construct coding for a GST-vimentin fusion protein wasgenerated. Following expression and purification, a thrombin di-gest was performed and the resulting cleaved polypeptide was sep-arated by means of SDS-PAGE. Immunoblot analyses were thenrealized using SC5 mAb or two anti-vimentin Abs of distinct or-igin (Fig. 3C). An identical recognition profile, with the detectionof a 50- to 60-kDa protein, was obtained independently of the Abused for revelation. Altogether, these results demonstrated thatSC5 mAb was directed against vimentin.

SC5 mAb specifically recognized vimentin when located on theouter side of the plasma membrane

The identification of SC5 mAb as an anti-vimentin Ab, combinedwith our previous observation that SC5 immunostaining was pos-itive on malignant Sezary cells (16, 17), suggested that vimentinmight present an atypical location at the plasma membrane in thesecells. To further investigate this possibility, immunofluorescenceconfocal microscopy was performed on the HUT78 cell line (Fig.4A). In a first set of experiments, cells were fixed and doublestained with SC5 (red) and anti-vimentin (green) Abs (Fig. 4A, leftpanel). An overlay of the two labelings (yellow) showed that bothAbs exerted a similar reactivity toward vimentin. Indeed, an im-munostaining polarized at one edge of the cell, and which appearedto be tightly associated with the plasma membrane, was constantly

FIGURE 1. Sezary cells present an increased reactivity toward SC5mAb. A, PBMCs were isolated by density gradient centrifugation from theblood of a healthy donor (left panel) or of a representative SS patient (rightpanel). Double labeling was performed by incubation of the cells with SC5and anti-TCR�� mAb, followed by goat anti-mouse IgM FITC and IgG1PE secondary Abs, or anti-TCRV�23 PE when indicated. Isotype-matchedcontrols were used for delimiting the positive regions in the two colorhistograms. B, Double staining of the Sezary cell line HUT78 was per-formed as in A.

FIGURE 2. Purification of proteins coprecipitated by SC5 mAb. Ly-sates from PHA-activated PBMCs were subjected to SC5 precipitation.Proteins were separated by two-dimensional gel electrophoresis and de-tected by silver nitrate staining of the gel. The protein spots of interest werecut out from the gel and further processed for peptide sequencing. Theidentity of each polypeptide, as well as the position of the IgM H and Lchains, and of the nonspecifically precipitated murine serum albumin areindicated.

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detected. An underneath diffuse intracellular labeling, linked to theone detected at the plasma membrane, was also observed.

A second set of experiments was done, in which a step of per-meabilization of the cells was added after fixation and before im-munostaining (Fig. 4A, right panel). A complete and homogeneousstaining of the plasma membrane was observed with both Absunder these conditions. This membrane-bound staining was asso-ciated to a deeper cytoplasmic labeling, when compared with theone observed in fixed and nonpermeabilized cells. These observa-tions demonstrated that, according to the cell treatment (fixationalone vs fixation and permeabilization), one could distinguish amembrane-bound vimentin, presenting a polarized location, fromthe total pool of cellular vimentin.

One limitation of the preparation of cells for their observationby confocal microscopy relies on the use of a fixative agent beforeimmunoanalysis. This therefore did not allow us to establishwhether the immunofluorescent signal was associated with theouter or the inner face of the plasma membrane. To overcome thisdifficulty, flow cytometry analyses were conducted using SC5mAb as well as two distinct anti-vimentin Abs (Fig. 4B). In agree-ment with the confocal microscopy data, all three Abs were found

FIGURE 3. SC5 mAb is directed against vimentin. A, Immunoprecipi-tations were performed on HUT78 cell lysates using an irrelevant Ab (con-trol), SC5 mAb, or purified goat anti-vimentin Ab C-20. After separationby SDS-PAGE, proteins were first detected by Western blotting using SC5mAb. The nitrocellulose membrane was then stripped and subjected to asecond step of revelation with the anti-vimentin Abs C-20. B, Immuno-precipitations were performed on lysates of PHA-activated normal PBMCsusing purified SC5, anti-LSP1, or anti-�-actinin 4 mAb. Immune com-plexes were separated by SDS-PAGE, and proteins were detected by West-ern blotting using SC5 mAb. C, Vimentin was expressed as a GST fusionprotein and digested with thrombin. The cut protein was then immunoblot-ted using SC5 mAb or the anti-vimentin Abs V9 or C-20. Protein detectionwas made with the appropriate HRP-conjugated secondary Ab and an ECLdetection system.

FIGURE 4. SC5 mAb recognizes a pool of vimentin located on theouter face of the plasma membrane. A, The Sezary cell line HUT78 waseither fixed (left panel) or fixed and permeabilized (right panel) beforeimmunostaining. Double labeling was performed using SC5 mAb (red) andthe anti-vimentin Abs C-20 (green). An overlay of both staining is shown(yellow) as well as a light microscopy view of the cell field. B, Flowcytometry analyses were performed on HUT78 cells that were subjected toa step of fixation alone (upper panel), fixation and permeabilization (mid-dle panel), or left untreated (lower panel) before labeling. Cell staining wasthen performed with SC5 mAb or the commercial anti-vimentin Abs V9 orC-20, followed by the appropriate FITC-conjugated secondary Abs, as indi-cated. Mouse (for SC5 and V9) or goat (for C-20) control Igs were used asnegative controls. C, Cell surface immunolabeling was performed on resting orPHA-activated lymphocytes from a healthy donor, or alternatively on Sezarycells isolated from a representative SS patient. Cell staining was performedwithout any pretreatment of the cells, with SC5 mAb or the commercial anti-vimentin Abs V9 or C-20, as described in B. Mouse (for SC5 and V9) or goat(for C-20) control Igs were used as negative controls.

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to similarly label HUT78 cells when previously fixed, or fixed andpermeabilized (Fig. 4B, upper and middle panels). However, astriking difference was observed when the immunostainings wereperformed on nontreated cells. Thus, while the cell labeling re-mained negative with both anti-vimentin Abs, a positive signal wasstill obtained with SC5 mAb (Fig. 4B, lower panel). A similar setof experiments performed on nontreated resting or PHA-activatednormal PBMC, and on lymphocytes isolated from a representativeSS patient (Fig. 4C), definitely confirmed the unique specificity ofSC5 mAb. Indeed, as expected, the V9 (middle panel) and C-20(right panel) anti-vimentin Abs remained unreactive with the celltypes tested when not subjected to a fixation step. In contrast, SC5mAb exhibited almost no stain with freshly isolated PBMCs froma healthy donor, but it showed an increased reactivity toward theirPHA-activated counterparts, and more importantly with the Sezarymalignant cells obtained from a SS patient (Fig. 4C, left panel).Altogether, these results demonstrated that, unlike commerciallyavailable Abs, SC5 mAb specifically reacts with a peculiar fractionof vimentin. The observation that this pool of vimentin was con-centrated at one edge of the cell, and its association with the ex-tracellular leaflet of the plasma membrane, strongly suggested thatvimentin was exported from the intracellular to the extracellularenvironment in both normal activated T cells and malignant SSlymphocytes.

Mapping of SC5 mAb recognition site

The observation that the anti-vimentin Abs used in this study ap-parently displayed different binding abilities toward the intra- andextracellular fractions of vimentin prompted us to map their cor-responding recognition site. As the commercially available AbC-20 was generated against a peptide located within the C-terminalportion of the protein, we focused our attention on the specificityof recognition of V9 and SC5 mAb. cDNA constructs coding forfull-length vimentin or deletion mutants, fused to a C-terminalFlag tag, were generated (Fig. 5A). Anti-Flag immunoprecipitateswere prepared from lysates of transiently transfected COS cellsand further analyzed by immunoblotting using V9 or SC5 mAb(Fig. 5B). We observed that the vimentin mutants presenting adeletion of the N-terminal head (mutant �1–100) alone or togetherwith the 1A domain (mutant �1–138) were still detected by V9 orSC5 mAb. Moreover, the truncation of the full N-terminal half ofthe protein (mutant �1–227) resulted in a molecule recognized byV9 mAb, but which showed no more reactivity with SC5 mAb. Wetherefore concluded that both C-20 and V9 Abs are directedagainst the C-terminal moiety of vimentin, while SC5 mAb rec-ognition site is part of the N-terminal half of the protein, and moreprecisely corresponds to a region located in the 1B domain.

SC5 mAb is reactive with apoptotic T lymphocyte cell line

One of the earliest cellular events upon apoptosis is the appearanceof phosphatidylserine residues at the cell membrane, which can berevealed by the binding of annexin V. To determine whether thepresence of vimentin at the cell surface of SS lymphocytes is aconsequence of an apoptotic cell status, we simultaneously testedannexin V and SC5 binding on the HUT78 cell line. The PI uptake,reflecting the necrotic cell status, was also estimated. The resultsshown in Fig. 6A indicated that the HUT78 cells were negative forannexin V binding, and for PI uptake, whereas they presented asignificant amount of extracellular vimentin as identified by SC5mAb. These data demonstrated that vimentin was detected at thecell surface of viable Sezary cells, and that this location did notderive from a process of cell death.

We next investigated whether the relocation of vimentin fromthe intracellular compartment to the extracellular matrix could be

an inducible event specific to apoptotic cells. A T lymphocyte cellline, developed by long-term growth of normal peripheral blood Tlymphocyte in the presence of IL-2, was tested for annexin V andSC5 labeling. We observed that this cell line showed no reactivitywith SC5 mAb under basal conditions of cell growth (Fig. 6B). Incontrast, a high number of cells was found to be positively labeledwith SC5 mAb when cells were deprived of IL-2 for 4 days (Fig.6C). Importantly, 45% of the SC5-positive cells were also stainedby annexin V. These results indicate that besides its reactivity withviable Sezary malignant cells and activated T lymphocytes, theanti-vimentin SC5 mAb might serve as an apoptotic marker asefficiently as annexin V.

Autoantibodies against vimentin are present in the serum of SSpatients

The unusual location of vimentin on the extracellular side of theplasma membrane of SS lymphocytes led to the possibility of anautoantibodies production by Sezary patients. This hypothesis wastested by performing Western blot analysis on vimentin using theserum corresponding to 10 Sezary patients. The serum of two pa-tients was found to recognize vimentin, at a dilution of 1/100 (Fig.7). In contrast, no signal was obtained when using the serum ofhealthy donors (Fig. 7, control lane), inferring the specificity of thedetection. In addition, Abs against vimentin were detected for athird patient when the serum was used at a 1/50 dilution for im-munoblotting (data not shown).

DiscussionSezary cells correspond, in most patients, to lymphocytes exhib-iting a CD4�CD45RO� phenotype. It is generally admitted that

FIGURE 5. A, Schematic representation of the vimentin constructs usedfor transfection. C-terminal Flag-tagged full-length (FL) or deleted vimen-tin constructs were ligated in frame in pcDNA3.1 expression vector. Thevarious domains of vimentin are as depicted. B, COS cells were transfectedwith the empty expression vector pcDNA3.1 (Ctrl) or with the indicatedvimentin construct. After lysis, immunoprecipitation was performed usingan anti-Flag mAb, and proteins were resolved by SDS-12% PAGE. Theimmunoprecipitated proteins were first detected by immunoblot analysisusing SC5 mAb (top panel). The blot was then stripped and reprobed withV9 mAb (middle panel). A second dehybridization step was performedbefore analysis with the anti-Flag mAb (bottom panel). Arrows indicate theposition of full-length or truncated vimentin.

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the lack of CD7 and CD26 expression characterizes the malignantlymphocytes, although controversial reports were made concern-ing the former receptor (14). Until now, and apart from the iden-tification of specific TCRV� rearrangements, only the KIR3DL2/CD158k cell membrane structure was described to phenotypicallyidentify skin and blood malignant Sezary cells, its expression cur-rently allowing their efficient isolation from the patients’ blood(14). To further identify cell surface receptors expressed by themalignant CD4� CTCL cells, we developed a mAb called SC5.We previously established that it recognized a plasma membraneAg, which detection was increased on PBMCs from patients withSS, when compared with PBMCs isolated from healthy individuals(16, 17). In addition, we observed that the SC5 mAb-reactive mol-ecule was mainly located in the cytoplasm of normal resting Tlymphocytes, and that it rapidly became detectable at the cell sur-face upon T cell activation (16, 17). Our first attempts to charac-terize the SC5 mAb-corresponding molecule, by performing im-munoprecipitation on lysates of cell surface biotinylated activatedT lymphocytes, led to the detection of a 96-kDa protein (17). Inthis study, we report further biochemical studies indicating thatSC5 mAb precipitates a protein complex containing a 96-kDa andtwo 60-kDa proteins from PHA-activated lymphocytes (Fig. 2).These polypeptides were identified by peptide sequencing as �-ac-tinin 4, LSP-1, and vimentin, respectively. In contrast to what weinitially thought, we established that the SC5 mAb is in fact di-rected against the 60-kDa molecule, exerting an isoelectric point of5.1 and corresponding to vimentin. In light of these new data, thepreviously detected 96-kDa protein most likely corresponded tothe coprecipitated �-actinin 4. The generation of a GST-vimentinfusion protein provides definitive evidence for SC5 mAb specific-ity toward vimentin. Indeed, the use of SC5 mAb in Western blotanalysis allows the specific detection of vimentin as efficiently asknown anti-vimentin Abs (Fig. 3).

Unlike the commercial anti-vimentin Abs tested, SC5 mAb ex-erted a unique ability of Ag recognition. Thus, identical patterns ofimmunolabeling were obtained on the Sezary cell line HUT78when fixed and/or permeabilized, regardless of the anti-vimentinAb used for detection (Fig. 4). However, a striking difference wasobserved when cells were not subjected to any treatment beforeimmunostaining. Under such conditions, SC5 mAb was the onlyAb able to interact with a pool of vimentin otherwise not detectedby the commercial Abs (Fig. 4, B and C). We therefore establishedthat, while recognizing intracellular vimentin, SC5 mAb could alsospecifically target a fraction of the protein located on the outer faceof the plasma membrane. By generating vimentin deletion mu-tants, we established that, while V9 and C-20 Abs were directedagainst regions present within the C-terminal moiety of the pro-tein, SC5 mAb recognition site was located in the N-terminalhalf of the molecule (Fig. 5). This therefore suggests that theprotein sequence recognized by SC5 mAb was exposed in boththe intra- and extracellular form of the protein, while the onetargeted by the commercial Abs became unaccessible in thesoluble form, most likely as a consequence of conformationalmodifications. Because SC5 mAb was found to specifically re-act with viable Sezary cells or activated T lymphocytes (16, 17),the detection of extracellular vimentin on living cells using thisAb might represent a powerful tool for the identification ofmalignant transformed lymphocytes.

It is well established that vimentin is an intermediate filamentprotein that participates to the formation of the intracellular cy-toskeletal structure. The comparison of the confocal microscopydata obtained on fixed cells vs fixed and permeabilized cells evi-denced the existence of a vimentin pool that is polarized at oneedge of the Sezary cells (Fig. 4A). Because this immunofluorescentsignal was observed with both SC5 mAb and the purified poly-clonal goat anti-vimentin Abs C-20, it cannot only reflect the pres-ence of vimentin on the external side of the cell membrane. Rather,it might also correspond to a pool of protein undergoing a trans-location from the intracellular to the extracellular compartment.The detection of vimentin on the outer leaflet of the plasma mem-brane of Sezary cells might thus result from a dynamic and in-duced process of exocytosis. Interestingly, it has been recentlyreported that vimentin, which is highly abundant in human acti-vated macrophages, is secreted from ex vivo long-term culturedmonocyte-derived macrophages (26). The pool of secreted vimen-tin was then involved in the settings of two major activated mac-rophage functions, namely bacterial killing and the generation ofoxidative metabolites. Furthermore, the release of vimentin in the

FIGURE 6. SC5 mAb labels apoptotic T lymphocytes. A, HUT78 cellswere double labeled with SC5 mAb and either PI (left panel) or annexin VFITC (right panel). The percentages of single- and double-labeled cells areindicated for each cell group. B and C, An IL-2-dependent T cell line wasobtained by long-term culture of PBMCs in the presence of IL-2. Immu-nolabeling was then performed with SC5 mAb and annexin V FITC oncells grown with IL-2 (B) or after induction of apoptosis by deprivation ofIL-2 for 4 days (C).

FIGURE 7. Presence of anti-vimentin autoantibodies in the serum of SSpatients. Purified GST-vimentin was subjected to digestion with thrombinand separated by means of SDS-PAGE. After transfer onto a nitrocellulosemembrane, samples were subjected to immunoblotting using SC5 mAb orsera prepared from the blood of SS patients (serum 1 and 2). The controlcorresponds to the use of the serum obtained from a healthy individual, andis representative of 10 tested. Protein revelation was performed with per-oxidase-conjugated secondary Abs and an ECL detection system.

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extracellular environment was found to be associated to its loca-tion on the extracellular side of the membrane of monocyte-de-rived macrophages. More recently, Xu et al. identified the endo-thelial cell-specific Ab PAL-E as an anti-vimentin Ab (27). PAL-EmAb shares some common features with SC5 mAb. Indeed,PAL-E was found to stain live endothelial cells (28), and this la-beling was polarized along the luminal endothelial surface (29,30), thus resembling what we observed upon SC5 labeling of Sez-ary cells. Further investigation established that blood endothelialcells exerted an unexpected vimentin metabolism, because the pro-tein was expressed and secreted as a dimer. Strikingly, PAL-E wasfound to be reactive with dimeric vimentin, but hardly detected theprotein in its monomeric form. In addition, further investigationdemonstrated that the commercial V9 mAb was also able to detectthe vimentin dimers expressed by endothelial cells, or followingsecretion. At this point, it is important to mention that we neverdetected vimentin as a dimeric protein following immunoprecipi-tation with SC5, V9, or C-20 Abs on lysates from Sezary cells orfrom activated normal T lymphocytes. In addition, only solublemonomeric vimentin was detected from the culture medium ofHUT78 Sezary cell line after immunoprecipitation (data notshown). Thus, while PAL-E specifically allows the detection of adimeric pool of vimentin processed by blood endothelial cells, SC5mAb remains the only mAb allowing the detection of extracellularvimentin at the surface of viable Sezary cells or of activated nor-mal T lymphocytes. Further studies will now be needed to deter-mine the role of secreted vimentin in the pathophysiology of SS.

Several hypotheses were made to explain how vimentin couldbe secreted, although it lacks a signal sequence. It was proposedthat its highly positively charged N-terminal sequence could reactwith the endoplasmic reticulum hydophobic core of the lipid bi-layer and direct the protein into membranes (26). In addition, theC terminus moiety of vimentin contains a di-acidic motif precededby a Y-X-X-� motif (in which � is a hydrophobic residue), knownto be required for the selective export of some proteins from theendoplasmic reticulum into the Golgi apparatus and found in manymembrane-associated proteins (26). Although allowing the detec-tion of the entire intracellular vimentin pool by confocal micros-copy or biochemical approaches, the conventional anti-vimentinAbs did not highlight the presence of extracellular vimentin onSezary cells. Similarly, the quantification of vimentin transcriptswithin the cells would not have answered the question of vimentinrelocation from the cytoplasm to the outer side of the plasma mem-brane. Because of its original specificity, SC5 mAb currently rep-resents a unique tool for the detection of vimentin translocation inSezary cells. It should be mentioned that another cytoplasmic pro-tein, T-plastin, involved in the regulation of actin assembly andcellular motility, has also been identified as a potential Sezarycell-specific marker (6), pointing to a strong involvement of cy-toskeleton-related proteins in Sezary cells.

Finally, we found that, apart from its ability to react with ex-tracellular vimentin on viable Sezary cells, SC5 mAb is also ca-pable of detecting cell surface vimentin on apoptotic T lympho-cytes. Apoptosis is characterized by cellular and nuclear shrinkage,cytoplasmic blebbing, condensation of nuclear chromatin, andfragmentation of nuclear DNA (31). Thus, besides the analysis ofapoptosis-associated proteins (32), the detection of vimentin at thecell surface could represent an additional tool to evaluate an earlystage of cell death. It is important to emphasize that the reactivityof SC5 mAb with Sezary cells is not a consequence of their apo-ptotic status. Indeed, we demonstrated that these cells were neg-ative for annexin V binding and PI uptake (Fig. 6A), and that theyfailed to react with Abs directed against cytoplasmic molecules,such as �-actinin 4 (data not shown).

In the past few years, the serological identification of recombi-nantly expressed genes has increasingly been used to screen newtumor Ags. This serological identification of recombinantly ex-pressed genes method has been demonstrated to be a powerful toolto identify Ags such as tumor-suppressor genes, oncogenes, can-cer-testis genes, and differentiation Ags (33, 34). Interestingly, vi-mentin has been isolated as a potential CTCL-associated Ag (35).In this study, our findings clearly confirm that vimentin representsa tumor Ag in SS patients. Interestingly, they also suggest that thepresence of anti-vimentin autoantibodies in the serum of these pa-tient might result from the presence of the protein at the Sezary cellsurface rather than from its overexpression, as reported for prostatecarcinoma (36) and endometrial neoplasm (37). However, furtherstudies are needed to determine whether the presence of autoanti-bodies is concomitantly associated with the detection of solublevimentin in the serum of Sezary patients.

In conclusion, we report for the first time that vimentin is lo-cated at the cell membrane surface of malignant Sezary cells andapoptotic T lymphocytes. This observation was rendered possibleby using the unique specificity of the anti-vimentin SC5 mAb. Wefurther suggest that anti-vimentin autoantibodies are present in theserum of SS patients, which might represent a useful marker fordiagnosis or prognostic purposes.

AcknowledgmentsWe thank Abdelali Jalil and Fatia Mami-Chouaib (Institut National de laSante et de la Recherche Medicale Unite 487, Gustave Roussy Institute,Villejuif, France) for their help with confocal microscopy cell examination,and Jacques Bertoglio and Catherine Froin (Institut National de la Sante etde la Recherche Medicale Unite 461, Chatenay-Malabry, France) for theirassistance with the generation of the GST-vimentin construct.

DisclosuresThe authors have no financial conflict of interest.

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