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The Journal of Immunology, 2005, 175: 7650-7660.
Copyright © 2005 by The American Association of Immunologists

A Role for the Neuronal Protein Collapsin Response Mediator Protein 2 in T Lymphocyte Polarization and Migration1

Peggy Vincent*, Yves Collette{dagger}, Romain Marignier*, Carine Vuaillat*, Véronique Rogemond*, Nathalie Davoust*, Christophe Malcus{ddagger}, Sylvie Cavagna*, Antoine Gessain§, Irma Machuca-Gayet, Marie-Françoise Belin*, Tam Quach* and Pascale Giraudon2,*

* Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 433 and Institut Fédératif de Recherche 19, Faculté de Médecine R. Laënnec, Lyon, France; {dagger} INSERM Unité Mixte de Recherche (UMR) 599, Institut de Cancérologie de Marseille, Marseille, France; {ddagger} Laboratoire d’Immunologie-Hôpital Neurologique, Lyon, France; § Unité d’Epidémiologie et Physiopathologie des virus oncogènes, Institut Pasteur, Paris, France; and UMR 5161, Centre National de la Recherche Scientifique, Ecole Normale Supérieure de Lyon, Lyon, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The semaphorin-signaling transducer collapsin response mediator protein 2 (CRMP2) has been identified in the nervous system where it mediates Sema3A-induced growth cone navigation. In the present study, we provide first evidence that CRMP2 is present in the immune system and plays a critical role in T lymphocyte function. CRMP2 redistribution at the uropod in polarized T cells, a structural support of lymphocyte motility, suggests that it may regulate T cell migration. This was evidenced in primary T cells by small-interfering RNA-mediated CRMP2 gene silencing and blocking Ab, as well as CRMP2 overexpression in Jurkat T cells tested in a chemokine- and semaphorin-mediated transmigration assay. Expression analysis in PBMC from healthy donors showed that CRMP2 is enhanced in cell subsets bearing the activation markers CD69+ and HLA-DR+. Heightened expression in T lymphocytes of patients suffering from neuroinflammatory disease with enhanced T cell-transmigrating activity points to a role for CRMP2 in pathogenesis. The elucidation of the signals and mechanisms that control this pathway will lead to a better understanding of T cell trafficking in physiological and pathological situations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although differing in function and architecture, all cell types need to maintain a dynamic cell shape and adapt their morphology to stay responsive to a variety of stimuli. Most cell types polarize during the acquisition of a final phenotype, like neurons, or in a transient manner during the development of a specific function, like leukocytes. In fact, leukocytes develop their polarized morphology when they undergo migration, activation, and cell-to-cell interaction during immune response. This polarity acquisition requires drastic cell morphological changes and the permanent remodeling of intracellular architecture, leading in leukocytes to specialized protrusive membrane structures. These protruding cell surface structures may differ from cell to cell, e.g., filopodia and dendrites on dendritic cells, lamellipodia on monocytes, while uropods are typical of lymphocytes (1). In fact, the T lymphocyte polarization is characterized by the formation of two functionally and morphologically distinct poles: the leading edge, the advancing front of migrating cells, and the uropod, the trailing edge with motility and adhesive/deadhesive functions (2). Acquisition of a polarized morphology is the first requirement for a T cell to initiate migration at sites of immune defense and inflammation (2). Lymphocyte migration largely depends on adhesive interactions with recognition of chemoattractant gradients sensed by chemokine receptors concentrated at the leading edge. Although chemokines are master controllers of lymphocyte migration (3, 4), another class of chemoattractants has recently emerged, the semaphorin protein family (5, 6, 7).

First described in the nervous system (8, 9), semaphorins form a key protein family emerging in cellular communication involved both in the modulation of cellular morphology and in acquisition of functional protrusions leading to chemoattractant-mediated migration. They repel and collapse axonal growth cones to regulate the formation of neuronal connections during development and plasticity throughout adult life (reviewed in Refs.10, 11, 12). In addition to their role in directional guidance of cellular process, semaphorins inhibit or induce directional cell motility for a range of migrating cells, including oligodendrocytes, hemopoietic, myocardiac, and endothelial cells (5, 7, 13, 14, 15). Semaphorins signal through various transmembrane molecules, including neuropilin and plexin family members, CD72, integrin, and Tim2 (10, 16, 17), and trigger cytoskeleton rearrangement. The intracellular signaling machinery linking receptors to the cytoskeleton is still incompletely known, but several studies in neural cells have found that it converges on the phosphoprotein CRMP2 ((18), reviewed in Refs.10 and 19).

CRMP2 belongs to a family of five homologous members and was first identified as a mediator of semaphorin-induced growth-cone collapse (18). Downstream of semaphorin signal, they reorganize the cytoskeleton by controlling microtubule assembly (20, 21, 22), playing a crucial role in axonal outgrowth and neurite extension (10, 18, 23). Collapsin response mediator proteins (CRMP)3 are not solely limited to the signaling transduction of semaphorin guidance cues but are probably involved in multiple cellular events such as cell migration and differentiation (24, 25, 26). Thus, CRMP act as downstream effector of distinct extracellular signals to regulate cellular shape remodeling. Many of the signal transduction events underlying neuronal navigation are remarkably similar to those responsible for the chemotaxis of leukocytes. The presence of semaphorins and their receptors in immune cells (17, 27) led us to investigate the expression and potential function of CRMP in hemopoietic cells. In the present study, we have identified CRMP2 in T lymphocytes and demonstrated its involvement in both spontaneous and chemokine-induced T lymphocyte migration. An elevated CRMP2 level in activated T cells of patient suffering from neuroinflammation points to its possible role in pathogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

The human PBMC from healthy blood donors (HD) or infected patients (human T cell leukemia virus-I (HTLV-I)) were isolated by a Ficoll gradient and kept at –170°C. Alternatively, PBMC were treated with 1 µg/ml PHA in RPMI 1640 (Invitrogen Life Technologies) for 2 days and then cultured in RPMI 1640 supplemented with 2 g/L glucose, 50 µg/ml gentamicin, 10% human AB serum, and 20 U/ml IL-2, giving rise to CD4+ and CD8+ PHA blasts, termed primary T lymphocytes. Culture of primary T cells in IL-2, termed IL-2-polarized cells, resulted in ~30% of cells with a bipolar morphology. Experiments were performed on 4- to 8-day-old cultures. Cells were deprived of IL-2 (24 h) before any chemical treatment. The CD4+ T cell lines (CEM, Jurkat) and the B cell line Raji were cultured in the same medium without IL-2. The human neural cell line Dev (28) was cultured in DMEM supplemented with 10% heat-inactivated FCS.

Reagents

The following monoclonal mouse Abs were used: anti-vimentin (DakoCytomation), anti-ezrin (Sigma-Aldrich), anti-CD2 and anti-c-myc (Santa Cruz Biotechnology), and anti-GAPDH (Chemicon International); PE-conjugated anti-CD4, anti-CD8, anti-CD69, anti-CD45RO, anti-HLA-DR, and anti-VLA4 (Immunotech); and anti-plectin (Santa Cruz Biotechnology). The following polyclonal sera were used: goat anti-GST (Amersham Biosciences) and rabbit anti-CRMP2 Abs, and were directed against a C-terminal epitope (C-ter: 558–572 residues) or peptide 4 epitope (pep4: 454–465 residues) and purified with these peptides. They specifically recognize CRMP2 either as recombinant protein (25) or as native protein identified in brain tissue by sequencing. Adsorption of anti-pep4 Ab on peptide 4 (2 µg/µl Ab, 37°C, 1 h) abolished the positive signal in immunochemistry and Western blot assay (see Fig. 1B) performed on neural and T cells. The following secondary Abs were used: peroxidase-conjugated F(ab')2 goat anti-rabbit IgG (H+L) or goat anti-mouse or rabbit anti-goat (Biosys); and Alexa488- and Alexa546-conjugated F(ab')2 anti-rabbit or anti-mouse Abs (Molecular Probes). The following chemokines (CXCL12 (10 ng/ml), CCL2 (20 ng/ml), CXCL10 (20 ng/ml), and CCL5 (100 ng/ml) (R&D Systems)), cytokines (TNF-{alpha}, IFN-{gamma}, and IL-1{beta} (10 ng/ml; R&D Systems)), and semaphorins (Sema3A (R&D Systems) (100 nM), Sema3F (100 nM), and Sema7A (100 nM)) were used.



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  		FIGURE 1. CRMP2 is detected in T lymphocytes at the mRNA and protein levels. A, CRMP2 mRNAs detection by RT-PCR and Southern blotting with CRMP2-specific probe (GAPDH was used as control) and quantification by real-time RT-PCR (LightCycler, normalization to {beta}-actin expression) in primary T cells from several blood donors, in the CD4+ T cell lines Jurkat and CEM, and in the neural cell line Dev. B, Western blot analysis of CRMP2 using Abs directed against two different epitopes (pep4, C-ter) in lane 1 (primary T cells) and lanes 2 and 3 (control T cell lines Jurkat and CEM (GAPDH used as internal control)). CRMP2 is also detected with anti-c-myc Abs in Jurkat T cells transfected with a c-myc-tagged CRMP2 plasmid. Adsorption of CRMP2 Ab with peptide 4 (track 5) extinguished the specific signal detected in T cells (track 4).

 
Sema3F and 7A supernatant production

293-T subconfluent cells were transfected in optiMEM medium with a soluble, Myc-tagged, Sema3F- and 7A-encoding vector (a gift from Exelis, Lyon, France) or with an empty pSecTagA-encoding vector for the Myc tag alone (Invitrogen Life Technologies) as described previously (29). Forty-eight hours posttransfection, the secreted protein containing supernatants were harvested and concentrated on Vivaspin 50,000 Kd column (Vivascience), semaphorin (Sema) concentration was estimated by gel electrophoresis, followed by Coomassie blue staining. Inactivated Sema was obtained by heating at 100°C for 10 min.

Plasmids and constructs

c-myc-tagged CRMP2 plasmid (a gift from Dr. K. Kaibuchi, Nagoya, Japan) has been described previously (30). To obtain the plasmid EGFP-CRMP2, the cDNA fragments of CRMP2 were amplified by PCR and subcloned into pEGFP-C1 expression vector. The fusion proteins GST and GST-CRMP2 were synthesized as followed: the human CRMP2-coding sequence was cloned in the EcoRI and XhoI cloning sites of pGEX 6P1 vector (Amersham Biosciences) in frame with the GST protein. GST protein production is as follows: one night preculture of Escherichia coli (BLR21 plysS) transformed either with the pGex plasmid or with pGex/CRMP2 was diluted at 1/10 and grown for 2 h at 37°C. Fusion protein expression was induced by adding 0.1 mM isopropyl {beta}-D-thiogalactoside and incubation was continued for 2.5 h at 25°C. Bacterial pellet was resuspended in cold TENGN (50 mM Tris (pH 7.4), 1 mM EDTA, 100 mM NaCl, 10% glycerol, and 0.5% Nonidet P-40) with antiproteases and lysozyme (0.5 mg/ml) and incubated 15 min on ice. The soluble extract was obtained by sonication followed by centrifugation at 6000 x g for 10 min. GST proteins were purified on glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Small-interfering RNA (siRNA)-mediated gene silencing

Two of five 21-oligonucleotide siRNA duplex (Qiagen), siRNA-1 and siRNA-4, chosen to target the human CRMP2 and one siRNA negative control duplex (Eurogentec OR-0030-NEG05) were used in primary T cells. Preliminary experiments have shown the ability of siRNA-4 to reduce CRMP2 expression in neurons and Jurkat T cells (immunofluorescence, Western blotting), while siRNA-1 was less effective and siRNA-scramble had no effect. siRNA (3 µg) was mixed with 100 µl of primary T cells and nucleoporation immediately performed (Nucleofector; Amaxa Biosystems). The transfected cells were added to 2 ml of RPMI 1640 (10% human sera, 100 µg/ml streptomycin and penicillin) and CRMP2-suppressed T cells visualized by immunostaining after 24 h (paraformaldehyde fixation 4%, 30 min, +4°C) and Western blotting on total cell lysates: siRNA-1, 5'-AAGCCGUGAAUCGUGCCAUCA-3'; and siRNA-4, 5'-AAGAUGGGUUGAUCAAGCAAA-3'.

CD69 immunomagnetic bead separation

Full protocol details are obtained from datasheets from MACS (Miltenyi Biotec). To select a highly pure population of CD69+ cells, PBMC (5 x 106) were labeled with anti-CD69 mAb (20 min, 4°C), washed in medium, and incubated (20 min, 4°C) with anti-CD69 magnetic microbeads. Cells were washed and suspended in 500 µl of medium and put through magnetic columns, giving ~2 x 105 purified cells ready to use in transmigration assay.

Transmigration assay

T cell transmigration was performed in triplicate with Jurkat T cell line and primary T cells in microtranswells (Boyden chamber, Costar, 3- or 5-µm diameter pore size membrane), as described previously (31). T cell preparations (3 x 105 cells/well) were added in the upper chambers and incubated at 37°C (2–18h for Jurkat T cells; 1.5–2h for primary T cells). Chemokines were added in the lower compartment: CXCL12 (10 ng/ml) for Jurkat cells, and CCL2, CXCL10 (20 ng/ml), CCL5 (100 ng/ml), and CXCL12 (10 ng/ml) for primary T cells. Semaphorins Sema3A, 3F, and 7A (100 nM) were added in the lower compartment to test their chemoattractive or chemorepulsive capacity. Assay was performed on CRMP2-transfected Jurkat cells and primary T cells, either untreated or treated with CRMP2-siRNA- and CRMP2-blocking Ab. Anti-CRMP2-blocking Ab (25) (pep4 Ab: 7, 3, and 1.5 µg/ml), anti-GAPDH (irrelevant Ab directed against an internal protein, same range), anti-rabbit and anti-mouse Ig (Ig isotype control, same range) were added to primary T cell culture 30 min at 37°C before transmigration. Preliminary experiments (Ref.25 and data not shown) showed that Abs spontaneously enter the cell in a dose-dependent manner and could be detected as intracytoplasmic protein in fixed cells (cold acetone, 5 min) by immunostaining with Alexa-anti-Ig species, as soon as 30 min and are still present at 18 h following treatment (34.3 ± 1.6, 25.2 ± 4.8, and 17.5 ± 0.7% positive cells in culture treated with 7, 3, and 1.5 µg/ml Ab, respectively). The migratory T cells in the lower chambers and total T cells in well without insert membrane were counted under microscopy.

Statistical analysis

The number of migratory and polarized T cells were counted with photonic microscopy (15–20 microscope fields; two or three independent experiments). The data are expressed either as mean number of migratory or polarized T cells per field or as a percentage of the total number of cells ± SE to the mean (± SEM or SD). Student’s t test was used as appropriate.

Immunocytochemistry

CRMP2 was detected by immunofluorescence either on T cells fixed (PFA 2%; 1 min) and cytospun (600 rpm-5 min) or on IL-2-polarized primary T cells adhered on collagen I-coated slides (20 µg/ml) to preserve cellular structure and polarized shape. Vimentin, ezrin, CD2, and plectin were detected by immunofluorescence on IL-2-polarized T cells adhered on collagen I-coated slides. After fixation (acetone –20°C; 5 min), intracellular proteins were detected by incubating slides with specific Ab diluted in phosphate buffer (1 h, 37°C) and after a 10-min wash incubated with Alexa 488- or 546-conjugated anti-mouse or anti-rabbit IgG Ab (1 h, room temperature). In all experiments, nuclear staining was performed using a fluorescent DNA intercalant, 4',6'-diamidino-2-phenylindole (Boehringer Mannheim).

Flow cytometry assay

Ab raised against pep4 epitope has been optimized for flow cytometry on immune cells. PBMC or primary T cells were stained with PE-conjugated anti-CD4, -CD8, -CD69, -CD45RO, -HLA-DR, and -VLA4 Ab (20 min, 4°C; Immunotech). After washing and fixation (1% paraformaldehyde, 30 min, 4°C), cells were stained with anti-CRMP2 peptide4 Ab (30 min, 4°C) in a permeabilization buffer (1% saponin; Sigma-Aldrich). After washing (0.05% saponin buffer), Ag/Ab complexes were revealed after incubation (30 min, 4°C) with fluorescein-conjugated anti-mouse F(ab')2 Ab fragments (Roche). Cell immunostaining was analyzed on a Coulter XL flow cytometer using XL software (Beckman Coulter). Fluorescence was measured with an excitation wavelength of 488 nm and an emission wavelength of 515 and 615 nm. Nonspecific staining was excluded by isotypic control gating. In addition, control experiment has been performed. Knocking-down CRMP2 expression in Jurkat T cells with specific siRNA decreased the CRMP2 signal, while it was enhanced in cells transfected with c-myc-CRMP2 plasmid.

Western blotting

Total proteins were measured by Lowry assay (Bio-Rad). Protein samples (10–20 µg) were separated under reducing conditions by SDS-PAGE and transferred to nitrocellulose membrane (BA85; Schleicher & Schuell Microscience). Membranes were incubated in blocking solution (PBS, 0.1% Tween 20, 5% nonfat dried milk, 1 h), then incubated (overnight, 4°C) with specific Ab. After washing, samples were incubated (1 h, room temperature) with anti-IgG Ab conjugated to HRP, and proteins were revealed using a chemiluminescence detection kit (Covalab).

Plasmid transfection

Jurkat T cells (10 x 106) cultured in RPMI 1640 were transfected with c-myc-tagged CRMP2, GFP-CRMP2, or pEGFP-C1 plasmids by electroporation using 250 V, 950 µF (Bio-Rad) and 25 µg of DNA/sample. The percentage of transfection was evaluated by immunostaining using anti-c-myc Ab or GFP fluorescence (transfection efficacy: 10–15% of total cells).

Protein interaction assay (pull-down GST-CRMP2)

One hundred microliters of Jurkat cell lysate prepared as above was added either to 80 µl of GST-CRMP2 protein fusion or to 80 µl of GST protein coupled with glutathione-Sepharose 4B (Pharmacia Biotech) (1 h at 4°C). GST-CRMP2 and GST beads were washed four times (50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, and 0.5% Nonidet P40), and proteins bound to CRMP-2 or to GST beads alone were eluted. GST (in GST beads), CRMP2 (in GST-CRMP2 beads), vimentin, and ezrin (in GST and GST-CRMP2 beads) were revealed by Western blotting.

mRNA analysis

Total RNA was extracted using RNA PLUS (Qbiogen). Following DNase digestion (DNase I; Ambion), reverse transcription was performed using 500 ng of total treated RNA incubated with 100 ng of oligo(dT)12–18 primers (10 min, 70°C; Pharmacia Biotech) then for 42°C at 90 min for first strand synthesis. cDNA were further amplified by PCR with different sets of primers (see Table I) and revealed by Southern blot analysis (30, 23, and 25 cycles of 95°C for 1 min, 62°C for 1 min and 15 s, and 72°C for 1 min and 30 s, with a final elongation step of 72°C for 9 min; ([32P]ATP) specific internal probes for CRMP2, Robocycler Stratagene, PhosphorImager; Molecular Dynamics). Controls consisted of samples without reverse transcription or without RNA and were negative. Alternatively, cDNA were amplified by real-time PCR. LightCycler PCR mixtures contained 10 µl of QuantiTect SYBR Green PCR Master Mix (QuantiTect SYBR Green PCR kit; Qiagen), 2.5–5 mM MgCl2, 0.4 µM of each primer oligonucleotide, and 5 µl of template DNA in a final volume of 20 µl. For amplification of the three target sequences, shorter primer pairs than those for RT-PCR were designed from GenBank sequences (see Table I), and their specificity was verified using FASTA3. The following thermocycler protocol was applied: initial denaturation at 95°C for 15 min to activate the HotStarTaq DNA polymerase, and 48 PCR cycles consisting of heating at 20°C/s to 94°C with a 10-s hold, annealing at 20°C/s to 60°C with a 20-s hold, and elongation at 20°C/s to 72°C with a 10-s hold. Fluorescence values from capillary were measured during every cycle at 521 nm. All expression data were normalized to {beta}-actin expression level from the same sample. Relative quantification using Real-Quant allows us to compare the relative expression level of CRMP2 with regard to a reference cell type (arbitrary fluorescence value = 1).


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  		Table I. Oligonucleotide primers used in the RT-PCR and LightCycler PCR assays

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CRMP2 is present in immune cells, in particular in T lymphocytes

CRMP2 mRNA was detected by RT-PCR followed by Southern blotting using a specific probe (Table I), both in blood donors’ primary T cells and in the CD4+ T cell line CEM, as well as in Dev, a human neural cell line (28) (Fig. 1A). However, further quantification of mRNA by real-time PCR showed that CRMP2 was expressed at much lower levels in T cells than in neural cells (Fig. 1A). CRMP2 expression in T cells was further confirmed at the protein level with two specific anti-CRMP2 Abs directed against two distinct epitopes (pep4, position 454–465; C-ter, position 558–572). Western blotting performed on primary T cells and CD4+ T cell lines (Fig. 1B) detected two major products with apparent molecular mass of 58 and 62 kDa, also present in a neural cell line, Dev, confirming the expression of CRMP2 in T lymphocytes. The 62-kDa product, detected with the Ab directed against the C-terminal part of CRMP2 (C-ter Ab), corresponds to the native protein (theoretical molecular mass of 62.5 kDa). The 58-kDa band, detected by an anti-peptide4 Ab (pep4 Ab), likely results from proteolytic cleavage (J. Honnorat, unpublished data). Adsorption of anti-CRMP2 pep4 Ab with peptide 4 abolished the positive signal in T cells (tracks 4 and 5). These two major CRMP2 products were also revealed by the tag immunodetection (anti-c-myc Ab) in Jurkat T cells transfected with c-myc-CRMP2 plasmid (Fig. 1B). Proteolytic CRMP2 products of lower mass (50 and 40 kDa) could also be detected (Figs. 4 and 6). Protein analysis in two-dimensional electrophoresis and sequencing of immunoblotted positive products by mass spectrometry further confirmed both the presence of eight CRMP2 forms in T lymphocytes and the specificity of anti-CRMP2 Ab (data not shown). Several CRMP2 forms with different molecular mass have been reported previously in neural cells (21, 22). Similar protein levels associated with different levels of mRNAs in T and neural cell lines suggest a more efficient translation or stability of CRMP2 in T cells compared with neural cells.



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  		FIGURE 4. CRMP2 binds to the cytoskeleton, particularly to vimentin, an intermediate filament protein. A, Double-immunostaining of CRMP2 (green) and vimentin (red) on IL-2-polarized T cells revealed their coredistribution at the uropod. B, The interaction of CRMP2 with vimentin is detected by GST pull-down assay in vitro. GST-CRMP2 fusion proteins or GST alone, immobilized on glutathione-Sepharose 4B beads, were incubated with Jurkat T cell lysate. Vimentin (56 kDa) and ezrin (75 kDa) were detected in the lysate by Western blotting. As expected, CRMP2 and GST (26 kDa, data not shown) were detected in GST-CRMP2 or GST beads, respectively. Vimentin and ezrin were immunoblotted in total lysate (input), in GST-CRMP2, and in GST eluates (output). Whereas ezrin binds neither to GST-CRMP2 nor to GST alone, vimentin binds specifically to CRMP2 but not to GST alone.

 


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  		FIGURE 6. Decrease in CRMP2 expression correlates with decreased migratory rate of primary T cells. A, Primary T cells were transfected with two specific siRNA, oligonucleotides siRNA-1 and siRNA-4, and one CRMP2-nonspecific siRNA-control and analyzed for transmigration toward a mix of chemokines (CCL2, CXCL10, CCL5, and CXCL12). Immunofluorescence (anti-pep4 CRMP2 Ab and 4',6'-diamidino-2-phenylindole nuclear staining) showed that siRNA-1 and siRNA-4 decrease CRMP2 expression but at a much lesser degree for siRNA-1. Western blot and RT-PCR analyses on total cell lysates confirmed the decreased CRMP2 expression. B, siRNA-mediated CRMP2 silencing (siRNA-1, siRNA-4) in primary T cells correlates with the decrease in the number of migratory T cells toward chemokines. C, Polarization of primary T cells was evaluated following CRMP2 silencing and blockade. CRMP2 gene silencing using siRNA-4 and siRNA-1 reduced the number of polarized T cells. Uropod formation was analyzed in T cells treated with CRMP2-specific siRNA compared with untreated cells. D, Primary T cells were pretreated 30 min with anti-CRMP2-blocking Ab and assayed for transmigration. Abs were detected in the T cell cytoplasm (Ig detection by immunofluorescence). Treatment with anti-CRMP2 Ab (pep4 Ab: 1.5, 3, and 7 µg/ml) clearly reduced the number of migratory T cells while the irrelevant Ab (anti-GAPDH, same range) had no effect. Data presented here are representative of four experiments in immunofluorescence, three in Western blotting, five in T cell migration, two in polarization study, four in CRMP2-blocking Ab and transmigration, and three in siRNA and transmigration.

 
Flow cytometry analysis performed on PBMC of healthy donors (HD) showed that CRMP2 was expressed in 97–98% lymphocytes (Fig. 2A, one example, signal gated on lymphocyte population with structural criteria; mean fluorescence intensity (MFI) = 24.7 ± 3.2). A double-labeling experiment detected CRMP2 in both CD4+ and CD8+ T cell populations. Strategy using specific siRNA previously shown to reduce CRMP2 detection by Western blotting and immunochemistry (Fig. 6A) clearly reduced the CRMP2 signal intensity (MFI) detected by flow cytometry in Jurkat T cells and primary lymphocytes (Fig. 2B). Further analysis has shown CRMP2 expression in several immune cells, including monocyte/macrophages (flow cytometry on CD14+ cells) and B cell line (Western blotting on Raji cells) (data not shown), indicating that CRMP2 expression is not restricted to the T lymphocyte populations and may function in a variety of hemopoietic cells.



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  		FIGURE 2. CRMP2 detection in T lymphocytes using flow cytometry. A, Cell populations expressing CRMP2 characterized by flow cytometry in PBMC from one HD with signal gated on total lymphocyte population and after double-labeling with anti-pep4-CRMP2 Ab and the T cell markers CD4 and CD8. B, Treatment with CRMP2 siRNA-4 was followed by reduction of the global fluorescence intensity in Jurkat T cells (decrease in the CRMP2+ cells frequency) and primary T lymphocytes (decrease in CRMP2 MFI values) compared with treatment with control siRNA.

 
CRMP2 redistributes at the uropod in polarized T cells

In neural cells, CRMP2 is localized in different cell compartments, including cytoskeleton, endocytic vesicles, mitochondria, and nucleus (20, 21, 32, 33). Immunofluorescence experiments performed on cytospun (cell shape not conserved) Jurkat T cells and primary T lymphocytes (PHA blasts) (Fig. 3A) showed that CRMP2 preferentially localized in the cytoplasm as punctuate dots. Further observations with confocal microscopy confirmed its major cytoplasmic localization and also revealed the presence of nuclear labeling, as previously described in neural cells (20). CRMP2 silencing by treatment of T cells with CRMP2-siRNA resulted in a decreased fluorescence signal (Fig. 6A) substantiating the specificity of CRMP2 immunodetection.



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  		FIGURE 3. CRMP2 localization in unpolarized and polarized T lymphocytes. A, Immunocytochemistry (pep4 CRMP2 Ab) and fluorescence microscopy on fixed (PFA 2%) then cytospun primary T cells or Jurkat T lymphocytes. Confocal microscopy confirmed CRMP2 cytoplasmic and nuclear localization in unpolarized T cells. B and C, Cell polarization in IL-2 cultured primary T cells allowed to adhere on collagen-1 coated slides to preserve cellular structure and polarized shape. B, Immunostaining (pep4 CRMP2 Ab): CRMP2 redistribution at one cell pole in polarized T cell. C, Double-immunostaining of Ezrin/CRMP2: coredistribution at the uropod (arrow) (arrow with dotted line: unpolarized cells).

 
It has been shown that T cell-polarizing agents, including IL-2, are responsible for the establishment of two different regions in the cell, the leading edge, at the front and the uropod, at the rear of polarized cells (2). When IL-2-polarized T cells were allowed to adhere on a collagen-I-coated coverslip to preserve cellular structure and shape, an immunofluorescence study detected CRMP2 redistribution at one pole in polarized T cells (Fig. 3B). This bipolar remodeling is known to redistribute the chemosensory receptors, actin and myosin II, at the leading edge while ezrin, CD2, and ICAMs segregate at the uropod. An immunofluorescence study confirmed the polar pattern of CD2, ezrin, and actin expression in our polarized cell system (data not shown) and further identified the pole of CRMP2 distribution as the uropod since double-labeling of CRMP2 and ezrin showed their colocalization (Fig. 3C). These data suggested the implication of CRMP2 in T lymphocyte motility, as T cell polarization is required for its directional migration (review in Ref.34).

During migration, a drastic reconfiguration of the cytoskeleton occurs, including collapse of vimentin intermediate filaments, and increases deformability of T cells, a process essential for their migration through constricted spaces (35). Because CRMP2 regulates cytoskeleton dynamics in neural cells (20, 22, 36), its role in cytoskeletal reorganization during T cell migration was suspected. Immunochemistry experiments performed on polarized T cells detected vimentin at the uropod, as already reported (37), and its coredistribution with CRMP2 (Fig. 4A). CRMP2 also coredistributed with the cytolinker plectin (data not shown), a molecule that bridges vimentin with cellular structures and acts in lymphocyte polarization (37). As CRMP2 interaction with vimentin never had been reported, we further investigated this association by using GST-CRMP2 fusion protein and vimentin pull-down assay. Clearly, CRMP2 bound to cell-derived vimentin (Fig. 4B) but not to ezrin, a protein colocalized with CRMP2 at the uropod (Fig. 3C) and known as actin/filament plasma membrane linker. Taken together, these findings suggest that CRMP2 could function as an adaptor protein in cytoskeleton rearrangement during T lymphocyte polarization and preferentially segregate with vimentin at the uropod.

CRMP2 is involved in chemokine-directed T lymphocyte transmigration

The potential involvement of CRMP2 in T lymphocyte polarization and migration was investigated using CRMP2 overexpression (Fig. 5) and blockade (Fig. 6). Spontaneous and chemoattractant-directed migratory rate of primary human T lymphocytes was evaluated in a transmigration assay (Transwell system (31)). In preliminary experiments, comparative analysis of CRMP2 expression was performed by immunofluorescence on T cells isolated from a HD, tested for transmigration assay, and taken out from the lower chamber (migrating T cells) and the upper one (total cells before transmigration) (Fig. 5A). CRMP2 was heterogeneously expressed in the total cell population while all migrating cells highly expressed CRMP2, suggesting an enhanced migration capacity of these later ones. In addition, 85.7% migrating cells displayed a polarized morphology vs 5.4% in the total cell population. To substantiate the involvement of CRMP2 in T cell migration, its expression was elevated by transfection of Jurkat T cells with EGFP- or c-myc-tagged CRMP2 constructs. Elevated CRMP2 expression was detected in T cells that expressed the CRMP2 transgene by the tag immunodetection (c-myc; Fig. 5B). Elevated expression of CRMP2 in Jurkat T cells either as a c-myc- or an EGFP-tagged fusion protein clearly affected their spontaneous and chemoattractant-directed transmigration (table in Fig. 5B). Enhanced rate of spontaneous transmigration was detected in CRMP-2-overexpressing Jurkat cells as compared with control cells (EGFP alone). Migratory rate, also evaluated in a transmigration assay toward CXCL12 (stromal cell-derived factor 1), a chemoattractant for Jurkat T cells (38), was enhanced in a same range in CRMP2-tranfected cells.



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  		FIGURE 5. CRMP2 overexpression in T lymphocytes enhances their migratory rate. A, Comparative immunostaining of primary T cells tested in transmigration assay and taken out from the lower chamber (migrating cells) or the upper one (total cells): heterogeneous CRMP2 level expression in the total cell population before transmigration, while all migrating cells highly expressed CRMP2. B, Jurkat T cells were transfected with EGFP- or c-myc-tagged CRMP2 plasmids to overexpress CRMP2 or with empty EGFP-plasmid as control. Overexpression of CRMP2 (tagged c-myc) is visualized in transfected cells double-immunolabeled with anti-CRMP2 (green) and anti-c-myc (red) Ab. Migratory rate of transfected Jurkat T cells evaluated in a transmigration assay (0.5-µm pore membrane, with or without CXCL12): the percentage of migratory T cells vs total cell number (seeded cells) is reported from five distinct experiments. Overexpression of CRMP2 (EGFP or c-myc-tagged) enhances migratory rate compared with untransfected cells (representative experiments of seven).

 
In additional experiments, a CRMP2 blockade was performed using siRNA-mediated gene silencing in primary T lymphocytes, and cells were subsequently examined for their migratory rate toward a gradient of human chemokines known to be expressed in inflammatory situation (CCL2, CXCL10, CCL5, and CXCL12 (39)). Among five tested CRMP2-siRNA oligonucleotides, siRNA-4 and siRNA-1 significantly reduced CRMP2 expression in Jurkat T cell line and in cultured human neurons as well. siRNA-4 is the most efficient sequence for CRMP2 silencing (data not shown). Tested in primary T cells, siRNA-4 dramatically reduced CRMP2 expression evaluated by immunofluorescence, Western blotting, and RT-PCR, while siRNA-1 was a less efficient sequence (Fig. 6A). A decrease in CRMP2 expression clearly correlated with a decrease in T cell migratory rate toward the chemokine gradient (Fig. 6B, a representative experiment out of three independent ones is shown). A CRMP2-nonspecific-siRNA (control siRNA) had no significant effect on T cell migration.

Additional blocking experiments were performed to further support this hypothesis. We tested a specific blocking anti-CRMP2 Ab (pep4 Ab), previously shown to enter neural cells and reduce CRMP2-mediated Sema-3A signaling (25). Primary T cells were treated with the pep4-Ab Ab, with anti-GAPDH Ab used as irrelevant Ab directed against an internal protein or with isotypic Ab controls. Immunofluorescence analysis detected Abs in the cytoplasm (see Materials and Methods and Fig. 6D). Treatment with anti-CRMP2 Ab clearly reduced in a dose dependent manner the rate of primary T cell transmigration: the 40% decrease is consistent with the percent of cells transduced with the Ab (Fig. 6D, a representative experiment out of three independent ones is shown). The irrelevant Ab (Fig. 6D) and isotypic controls (data not shown) had no effect. These data confirm the involvement of CRMP2 in T cell migration. As T cell polarization is a prerequisite for migration, we also tested the effect of CRMP2 silencing (siRNA) on the acquisition of the bipolar morphology induced by the polarizing agent IL-2. The number of T cells with a uropod was clearly reduced following these treatments (Fig. 6C), indicating the involvement of CRMP2 in T cell polarization. Overall, these observations strongly suggest a functional role for CRMP2 in polarization and in spontaneous or chemokine-directed T cell migration.

CRMP2 is not involved in the reduced T cell chemokinesis induced by semaphorins

The previous observation that semaphorins can modulate migration of monocytes (5), the fact that immune semaphorins may bind T lymphocytes (5, 40) and the presence of semaphorin receptors on T cells (Refs.17 and 27 and our unpublished observation) led us to investigate the effect of Sema3A, Sema3F and Sema7A on primary T cell transmigration. These semaphorins were added in the lower compartment of the transmigration system in place of chemokines. A T cell chemotaxis was triggered by chemokines, as shown by elevated number of migrating T cell compared with spontaneous migrating ones. In contrast, a negative effect on T cell chemokinesis, reminding the "repulsive" signals evoked by semaphorins in the CNS, was evidenced for Sema7A, Sema3A, and Sema3F (100 nM) (reduction by four times) (Fig. 7). Although siRNA-1 and siRNA-4 treatment reduced the chemokine-mediated transmigration, CRMP2 silencing in T cells did not revert the negative effect of the semaphorins. Thus, CRMP2 acted in T lymphocytes downstream the chemokine signal, but was not required for semaphorin-induced negative effect.



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  		FIGURE 7. Role of Sema3A, 3F, and 7A and involvement of CRMP2 in T cell transmigration. IL-2-deprived (24 h) primary T cells tested for transmigration toward semaphorin or chemokine gradients. A, When added in the lower compartment of the transmigration system, chemokines induced a T cell chemotaxis (attractive effect) compared with medium alone (0, spontaneous transmigration). By contrast, a negative effect of Sema7A, Sema3A, and Sema3F (100 nM) on T cell transmigration was evidenced (heat-inactivated semaphorins and control supernatant did not modify the T cell migration, not shown). CRMP2 silencing by siRNA-1 and -4 reduced the T cell chemokine-induced chemotaxis but did not revert the negative effect of semaphorins.

 
Heightened CRMP2 expression in T lymphocytes isolated from patients with neuroinflammatory disease

High migratory rate and aberrant trafficking of T cells across the CNS barriers is a hallmark of neuroinflammatory diseases, (41, 42, 43, 44). To substantiate the physiological role of CRMP2 in motility of T lymphocytes, we took advantage of the chronic activation and high transmigration activity displayed by T cells in patients infected with retrovirus HTLV-I (45, 46, 47). HTLV-I infection is associated with inflammation and immunological studies have revealed infiltration of immune cells, including T lymphocytes, in various organs and tissues (48, 49). HTLV-I-associated myelopathy (HAM/TSP), a neuroinflammatory disease, may develop in a small percent of infected patients (50).

CRMP2 expression was examined with flow cytometry in PBMC isolated from HD (n = 8) and compared with PBMC of HTLV-I-infected patients (n = 14). Six of these patients suffered from HAM/TSP, the others were asymptomatic virus carriers (AC). To avoid experimental bias, PBMC of one individual in each group (HD, HAM/TSP, AC) was examined in the same experiment. In some cases, flow cytometry analysis was performed twice for a same individual and gave similar results. As expected, flow cytometry analysis detected CRMP2 in 97.4–98.1% of lymphocytes (gating on lymphocyte population) (Fig. 8A); however, difference in MFI revealed that CRMP2 expression was enhanced in neurological patients (MFI = 24.7 ± 4 in HD, 25.8 ± 2 in AC, and 37.1 ± 4 in HAM/TSP patients) (Fig. 8B and Table II). Using double labeling and flow cytometry, CRMP2 was examined in T cell subsets bearing the early and late activation markers, CD69+ and HLADR+, respectively, the activated memory T lymphocyte marker CD45RO+ and the migration marker VLA4+, and compared with CRMP2 level in CD4+ and CD8+ cell populations. Data from Table II revealed that, in all individuals examined, CRMP2 level was always higher in activated T cells, in particular in the CD69+ cell subset. Interestingly, activated T cells of neurological patients displayed higher CRMP2 expression compared with activated T cells of healthy donors or of HTLV-I-infected asymptomatic carriers. Thus, compared with the CRMP2 level found in CD69+, HLA-DR+, CD45RO+, and VLA-4+ cell subsets of healthy donors, there was a 1.6-fold increase in CD69+ and HLA-DR+ cell subsets and a 1.3-fold increase in CD45RO+ and VLA-4+ cell subsets of patient suffering from neurological disease. These variations were all statistically significant (p = 0.02–0.007). The highest CRMP2 level found in activated T cells probably supports the elevated CRMP2 level detected in total lymphocyte population in neurological patients. To substantiate the functional pertinence of our observations, activated T cells bearing the CD69 marker were selected from PBMC of one healthy donor and two HTLV-I-infected patients with elevated CRMP2 level and further analyzed in transmigration assay (Fig. 8C). This preliminary study detected an heightened migratory rate of selected cells from infected patients vs healthy donor. Reduced migration of T cells in primary culture from HTLV-I-infected patient (Cib) by CRMP2-blocking Ab (Fig. 8D) confirmed the involvement of CRMP2 in transmigration of HTLV-I-infected cells.



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  		FIGURE 8. Heightened CRMP2 expression in T lymphocytes of patients with neuroinflammatory disease. Flow cytometry analysis of CRMP2 in PBMC from HD (n = 8) and compared with PBMC of HTLV-I-infected patients (n = 14). Six of these patients suffered from HAM/TSP, the others being AC. A, Representative examples of CRMP2 expression for each group and one isotypic control superposed in the same flow cytometry panel. The MFI is enhanced in the neurological patient HAM-TSP-1. B, Average of CRMP2 (MFI) in each group: increased CRMP2 level in neurological patients. C, Activated cells (CD69+) selected from HD (HD-1) and HTLV-I-infected patients (HT-1, HT-2) tested in transmigration assay toward chemokines: elevated migratory rate for HT cells. D, Treatment of T cells cultured from infected patients (Cib) with CRMP2-blocking Ab (reduced their migratory rate in a dose-dependant manner (Ab 1/20, 1/100, and 1/500).

 

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  		Table II. CRMP2 expression detected by flow cytometry in various cell subsets in PBMC from healthy donors and inflammatory patients with viral infection

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A function for CRMP2 in regulation of cytoskeletal reorganization has become well established in the CNS. This phosphoprotein may act as downstream component of semaphorin-plexin signals transduction pathway, regulating migration of either the entire neuron or its nerve growth cone (19). In addition, CRMP2 cellular localization has been shown to be crucial in establishing and maintaining neuronal polarity (30, 36). The fact that many of the signal transduction events underlying neuronal navigation are remarkably similar to those responsible for the chemotaxis of leukocytes led us to examine the presence and function of CRMP2 in T cell polarization and migration. Its presence in immune cells, in particular T lymphocyte, extends its function beyond that of a neural semaphorin transducer. In the present study, we show that CRMP2 is expressed efficiently in T lymphocytes freshly isolated from blood donor and that its expression depends of the activated status of the cell. Various experiments, including modulation of expression and knocking-down CRMP2 message, have demonstrated the role of CRMP2 in T cell migration.

According to the general model of directed migration of leukocytes toward a chemoattractant gradient, T cells acquire a polarized morphology and initiate crawling as a result of activation and chemotactic stimuli (2). Such a polarization involves cytoskeleton reorganization, pushing the membrane out at the propulsive pole of the leading edge and pulling it in at the rear at the uropod, a flexible structure where adhesion molecules are clustered with microtubules and intermediate filaments (34, 51). Although F-actin concentrates preferentially at the leading edge, the motor protein myosin II, the microtubule organizing center, and the intermediate filament vimentin are packed in the uropod. Continuous movement of the cell is possible because this network is uninterruptedly regenerated by assembly at the leading edge and disassembly at the rear (52). A complex and still not completely defined array of signaling cascades are induced, including calcium, cAMP, PI3K, tyrosine kinases and Rho family of GTPases, regulating cytoskeletal rearrangement, integrin-dependent adhesions, and cell displacement (2, 53). The specific relocalization of CRMP2 during polarization and its ability to bind and colocalize with vimentin support the idea of CRMP2 as a potential partner of these cascades. First, CRMP2 was enriched at the uropod, indicating that this is its primary site of action during T cell crawling. This data is consistent with CRMP2 enrichment in the distal part of the growing axon and its role in growth cone navigation (21). Second, specific inhibition of CRMP2 indicates that T cells require CRMP2 to establish their polarized morphology. This data are consistent with the crucial role of CRMP2 subcellular localization for determination of the fate of neurites to axon vs dendrites in the CNS (review in Ref.36). Third, CRMP2 is a good substrate for the Rho-kinase in brain (54), and several studies have shown the major role for this kinase and associated Rho GTPases in the control of leukocyte polarity (review in Refs.1 and 53). At last, CRMP2 probably does not act downstream from semaphorins during T cell migration as knocking down its expression did not modify the semaphorin-directed repulsive effect. In fact, the involvement of CRMP2 in T cell polarization and subsequent spontaneous and chemokine-directed migration suggests a variety of potential transmembrane and intracellular molecular partners remaining to be identified. The great variety of CRMP2 subcellular localizations and molecular interactors identified in neural cells during axonal differentiation and growth support this idea (32, 33, 55).

Our data have shown that CRMP2 may participate in the cell re-shaping required for T cell locomotion. Coordinated rearrangement of actin, microtubules, and vimentin occurs in motile T cell, but it has been proposed that the actin cytoskeleton is the most important element in driving the morphological changes. In addition, the compaction of intermediate filament network without disassembly during polarization appears to be an efficient and rapid means of increasing cell deformability. In fact, vimentin collapse is necessary to achieve the rapid conversion of lymphocyte cytoarchitecture from a semirigid to a highly deformable state, facilitating T cell migration across tissue (35). The association of CRMP2 with vimentin and their codistribution at the uropod suggest that CRMP2 might be a partner for this intermediate filament in uropod structure during migration.

It is now clear that the CNS is under constant immune surveillance and that interactions between the CNS and immune system occur both in the healthy organism and in pathological situations. The intact CNS parenchyma regularly contains small numbers of T cells. However, activated T cells are known to display a high migratory rate in patients suffering from neuroinflammatory and demyelinating diseases such as multiple sclerosis and HAM/TSP (42, 46) and to be recruited within sites of inflammation (48, 56, 57). Leukocyte recruitment is orchestrated by a series of coordinated interactions with endothelial and neural cells, involving several families of chemotactic and adhesion factors. To date, chemokines are the main known chemoattractants in leukocytes migration, with the ability to act at distance to direct leukocyte trajectories (58). The list of lymphocyte migration controllers and their receptors has expanded recently (3, 4) and include members of the semaphorin family. A regulatory effect of Sema4D/CD100 on immune cell migration has been demonstrated previously (5). This semaphorin inhibits the spontaneous and chemokine-induced migration of monocytic and B cell lineages. This observation and the fact that CRMP2 is known as a transducer of semaphorin signal led us to investigate the effect of three distinct semaphorins in T cell migration and the involvement of CRMP2. Sema7A functions both in the immune system as a potent monocyte activator and in the nervous system by promoting axon outgrowth (59, 60). Sema3A is involved in axonal and oligodendroglial process outgrowth in the CNS (25, 61). Sema3F acts as a chemorepulsive cue for neuron projections (62) and inhibits metastasis in lung cancer and melanoma (14, 63). We show that, tested as potential chemorepulsive or attractive molecules, these semaphorins reduced the T cell rate of spontaneous migration, giving a sort of "stop" signal to migrating cells. This observation supports the hypothesis that semaphorins, which are highly expressed by activated T cells (reviewed in Ref.27), might participate in the close signaling between immune cells to modulate lymphocyte migration either in the bloodstream, lymphoids organs, or within the inflamed tissues. However, our siRNA strategy aimed to reduce CRMP2 expression does not modify the negative effect of semaphorins on T cell migration. Studies on PBMC show that CRMP2 expression is elevated in activated T cells, CD69+, and HLA-DR+ of healthy donors, but a prominent expression is detected in patients suffering from a neuroinflammatory disorder, HAM/TSP. Similar observation was made on patients suffering from multiple sclerosis another neuroinflammatory disease. In addition, enhancement of CD69+ and HLA-DR+ cells in primary T cell culture following TCR engagement (anti-CD3 Ab) clearly paralleled heightened CRMP2 expression (R. Marignier, manuscript in preparation). Interestingly, HAM/TSP patients currently display a chronic activation status and a high migratory rate of T cells associated with an enhanced secretion of chemokines and semaphorins (45, 46, 64, 65, 66). Our data suggest that, following T cell activation at the periphery, CRMP2 could promote the chemokine-directed T cells trafficking, favoring their recruitment to inflamed CNS then participating in the T cell-mediated impairment of neural cell survival and function, as described previously (66, 67). Preliminary data on the migratory rate of activated cells selected from HTLV-I-infected patients support this hypothesis.

In conclusion, the present data on CRMP2 in T lymphocytes shed new light on its likely role in cell fate, motility, and on its cellular function outside the CNS and defines a promising field of research in immune system.


   Acknowledgments
 
We thank Dr. K. Kaibuchi, (Nagoya, Japan) for providing c-myc-tagged CRMP2 plasmid.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from French Agencies of Research on Multiple Sclerosis (Association pour la Recherche sur la Sclerose en Plaques, Ligue Française contre la Sclerose en Plaques, Association Française contre les Myopathies) and INSERM foundations. Back

2 Address correspondence and reprint requests to Dr. Pascale Giraudon, U433 INSERM, Faculty of Medicine R. Laennec, rue G. Paradin, 69372 Lyon Cedex 08, France. E-mail address: giraudon{at}lyon.inserm.fr Back

3 Abbreviations used in this paper: CRMP2, collapsin response mediator protein 2; AC, asymptomatic virus carrier; HAM/TSP, human T cell leukemia virus-I-associated myelopathy/tropical spastic paraparesis; HD, healthy donor; HTLV-I, human T cell lymphotropic virus type I; MFI, mean fluorescence intensity; siRNA, small-interfering RNA. Back

Received for publication April 13, 2005. Accepted for publication September 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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