Glatiramer acetate-specific T-helper 1- and 2-type
`cell lines produce BDNF: implications for multiple
`sclerosis therapy
`
`Brain (2002), 125, 2381–2391
`
`Tjalf Ziemssen,1 Tania Ku¨mpfel,1,2 Wolfgang E. F. Klinkert,1 Oliver Neuhaus3 and
`Reinhard Hohlfeld1,2
`
`Correspondence to: Dr R. Hohlfeld, Institute for Clinical
`Neuroimmunology, Klinikum Grosshadern, Ludwig
`Maximilians University, Marchioninistrasse 15,
`D-81366 Munich, Germany
`E-mail: hohlfeld@neuro.mpg.de
`
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`cells produce BDNF, we selected four GA-specific, long-
`term T-cell
`lines (TCLs), which were characterized
`according to their cytokine profile by intracellular
`double-fluorescence flow cytometry. Three TCLs (isol-
`ated from a normal subject) had the phenotypes TH1,
`TH1/TH0, and TH0; the fourth, derived from a GA-
`treated patient, had the phenotype TH2. To demon-
`strate BDNF production, we used a combination of RT-
`PCR (reverse transcription-polymerase chain reaction)
`and two specially designed techniques for BDNF protein
`detection: one was based on ELISA (enzyme-linked
`immunosorbent assay) of supernatants from co-cultures
`of GA-specific TCLs plus GA-pulsed antigen-presenting
`cells, and the other on the direct intracellular staining
`of BDNF in individual T cells and flow cytometric
`analysis. The different assays and different TCLs
`yielded similar, consistent results. All four GA-specific
`T-cell lines, representing the major different TH pheno-
`types, could be stimulated to produce BDNF.
`
`1Department of Neuroimmunology, Max Planck Institute of
`Neurobiology, Martinsried, 2Institute for Clinical
`Neuroimmunology and Department of Neurology, Klinikum
`Grosshadern, Ludwig Maximilians University, Munich,
`Germany and 3Department of Neurology, Karl-Franzens-
`University Graz, Austria
`
`Summary
`The clinical effects of glatiramer acetate (GA), an
`approved therapy for multiple sclerosis, are thought to
`be largely mediated by a T-helper 1 (TH1) to T-helper 2
`(TH2)
`shift of GA-reactive T-lymphocytes. Current
`theories propose that activated GA-reactive TH2 cells
`penetrate the CNS, release anti-inflammatory cytokines
`such as interleukin (IL)-4, IL-5 and IL-10, and thus
`inhibit neighbouring inflammatory cells by a mechanism
`termed ‘bystander suppression’. We demonstrate that
`both GA-specific TH2 and TH1 cells produce the neuro-
`trophin brain-derived neurotrophic factor (BDNF). As
`the signal-transducing receptor for BDNF,
`the full-
`length 145 tyrosine kinase receptor (trk) B, is expressed
`in multiple sclerosis lesions, it is likely that the BDNF
`secreted by GA-reactive TH2 and TH1 has neuro-
`trophic effects in the multiple sclerosis target tissue.
`This may be an additional mechanism of action of GA,
`and may be relevant for therapies with altered peptide
`ligands in general. To demonstrate that GA-reactive T
`
`Keywords: multiple sclerosis; altered peptide ligand (APL); immunotherapy; neuroprotection; glatiramer acetate
`
`Abbreviations: APL = altered peptide ligand; APC = antigen presenting cell; BDNF = brain-derived neurotrophic factor;
`ELISA = enzyme-linked immunosorbent assay; FACS = fluorescence-activated cell sorter; FITC = fluorescein
`isothiocyanate; GA = glatiramer acetate; IL = interleukin; MBP = myelin basic protein; PBMC = peripheral blood
`mononuclear cell; PMA = phorbol 12-myristate 13-acetate; RT-PCR = reverse transcription-polymerase cell reaction;
`TCL = T-cell line; TCR = T-cell receptor; TH1 = T-helper 1; TH2 = T-helper 2; trk = tyrosine-receptor kinase
`
`Introduction
`Glatiramer acetate (GA, copolymer 1, Copaxone(cid:226)) is a
`heterogeneous but standardized mixture of synthetic poly-
`peptides consisting of L-glutamic acid, L-lysine, L-alanine
`and L-tyrosine (average molecular mass, 6400 Da). GA has
`been known for a long time to have suppressive and
`ª Guarantors of Brain 2002
`
`protective effects in experimental autoimmune encephalo-
`myelitis, which can be induced in different species by various
`encephalitogenic antigens (Teitelbaum et al., 1972; Webb
`et al., 1975; Teitelbaum et al., 1996; Sela, 1999). More
`recently, GA has also been shown to have beneficial effects
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`on the clinical course and MRI-defined brain lesions of
`patients with multiple sclerosis. As a result, GA is now
`approved for use in the immunomodulatory therapy of
`relapsing-remitting multiple sclerosis (Teitelbaum et al.,
`1997; Comi et al., 2001; Sela et al., 2001; Ziemssen et al.,
`2001)
`Among potential mechanisms, the initial T-helper 1 (TH1)
`type response of GA-treated patients was found to gradually
`shift to a T-helper 2 (TH2) type response (Miller et al., 1998;
`Duda et al., 2000; Gran et al., 2000; Neuhaus et al., 2000; Qin
`et al., 2000). TH1 cells characteristically produce a spectrum
`of ‘proinflammatory’ cytokines such as interferon (IFN)-g,
`interleukin (IL)-2 and IL-12. In contrast, TH2 cells produce
`TH2-type ‘anti-inflammatory’ cytokines, i.e. IL-4, IL-5, IL-6
`and IL-13 (Paul and Seder, 1994; Mosmann and Sad, 1996;
`Allen and Maizels, 1997). An intermediate type of T cell,
`called a TH0 cell, produces both TH1- and TH2-type
`cytokines. Current
`theories propose that
`the GA-specific
`TH2 cells, which are induced and constantly activated during
`treatment, migrate into the CNS and release their TH2-like
`cytokines locally (Aharoni et al., 2000). These cytokines are
`thought to have beneficial effects on the local inflammatory
`milieu and to inhibit the action of encephalitogenic T cells by
`‘bystander suppression’ (Neuhaus et al., 2001).
`Although plausible,
`this scenario may actually be an
`oversimplification as two unexpected and intriguing findings
`suggest. Human immune cells including T-lymphocytes,
`B-lymphocytes and monocytes can produce brain-derived
`neurotrophic factor
`(BDNF)
`(Besser and Wank, 1999;
`Kerschensteiner et al., 1999)—a potent neurotrophin that
`has profound effects on neuronal
`survival and repair
`(Thoenen, 1995; Barde, 1997). Moreover, the receptor for
`BDNF, gp145TrkB, is expressed in neurones and astrocytes
`in multiple sclerosis brain lesions (Stadelmann et al., 2002).
`These findings prompted us to ask the following questions:
`(i) can GA-reactive T lymphocytes produce BDNF, and if so,
`(ii) do TH1-type and TH2-type GA-specific T cells differ in
`their capacity to produce BDNF?
`To answer these questions, we first had to overcome two
`major technical obstacles: (i) adapting our culture system to
`prevent added GA from affecting the BDNF enzyme-linked
`immunosorbent assay (ELISA) and (ii) optimizing the
`intracellular detection of BDNF in individual T-lymphocytes.
`This enabled us to demonstrate formally that GA-specific
`TH1, TH2 and TH0 cells all have the capacity to produce
`BDNF. We therefore postulate that the beneficial effects of
`not only TH2-type, but also TH1-type GA-reactive T cells,
`might, at least partly, be due to their release of BDNF in
`multiple sclerosis lesions.
`
`Material and methods
`Subjects
`Blood samples were drawn from a GA-treated patient (B.K.)
`and a healthy donor (T.Z.) after their informed consent was
`
`given. The patient, a 47-year-old woman, had been diagnosed
`in 1993 to have relapsing-remitting multiple sclerosis. Her
`current Expanded Disability Status Scale (EDSS) (Kurtzke,
`1983) is 1. She has been essentially free of exacerbations
`since GA treatment was started in December 1998. Her
`human leukocyte antigen (HLA) class II phenotype is DR2/
`DR4. The HLA class II phenotype of the healthy volunteer
`(T.Z.), a 28-year-old postdoctoral fellow, is DR8/DR13. HLA
`typing was kindly performed by Drs E. Albert and S. Scholz,
`Department of
`Immunogenetics, University of Munich,
`Germany.
`
`Selection and culture of GA-specific T-cell lines
`(TCLs)
`GA-specific CD4+ TCLs were selected from peripheral blood
`mononuclear cells (PBMCs) using a split-well
`technique
`(Kitze et al., 1988; Pette et al., 1990; Neuhaus et al., 2000).
`GA (batch 242992899, average molecular mass 6400 Da) was
`obtained from Teva Pharmaceutical Industries, Petah Tiqva,
`Israel.
`the
`Four GA-specific CD4+ TCL representatives of
`phenotypes TH1 (TZ-COP-1), TH1/0 (TZ-COP-3), TH0
`(TZ-COP-5)
`and TH2
`(BK-M6-COP-7) were
`used
`(Neuhaus et al., 2000). The protocols for fluorescence-
`activated cell sorter (FACS) phenotyping of the TCLs are
`described below. The TCLs TZ-COP-1, TZ-COP-3 and TZ-
`COP-5 were obtained from the healthy untreated subject T.Z.
`The TH2-type TCL, BK-M6-COP-7, was obtained from the
`GA-treated patient B.K. This TCL was originally described
`by Neuhaus et al. (2000).
`
`Stimulation of GA-specific TCL with GA-pulsed
`antigen presenting cells (APCs)
`In pilot experiments, the GA in the culture supernatants was
`occasionally observed to interfere with the BDNF ELISA,
`especially in the low range of BDNF concentrations
`(M. Kerschensteiner, W. Klinkert and T. Ziemssen, unpub-
`lished data). We therefore established a rigorous antigen-
`pulsing protocol to minimize these soluble GA concentra-
`tions. This protocol was used in all the experiments reported
`here. Thrombocyte-depleted APCs, X-irradiated with 40 Gy
`(Stabiloplan 2; Siemens, Erlangen, Germany), were incu-
`bated (‘pulsed’) with GA at a final concentration of 400 mg/ml
`for 4 h. The GA-pulsed APCs were washed twice before
`being used to stimulate the GA-specific TCLs. The same
`protocol was used for parallel proliferation assays.
`Proliferation was measured by [3H]thymidine uptake as
`described previously (Neuhaus et al., 2000).
`For proliferation and BDNF secretion (ELISA) assays, 105
`washed GA-specific TCL cells were stimulated in RPMI 1640
`medium supplemented with 5% foetal calf serum (FCS), 1%
`glutamine and 1% penicillin/streptomycin (all from Gibco
`BRL, Gaithersburg, MD, USA) with 9 3 104 GA-pulsed
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`APCs. Supernatants were removed after 72 h and analysed for
`BDNF concentrations by ELISA (see below). For prolifer-
`ation assays, parallel cultures were labelled after 48 h with
`[3H]thymidine (0.2–0.5 mCi per well; Amersham Buchler,
`Braunschweig, Germany; 1 mCi = 37 kBq) and harvested 16–
`18 h later. [3H]thymidine incorporation was measured with a
`direct b-counter
`(Matrix TM 96; Packard, Frankfurt,
`Germany). For reverse transcription-polymerase chain reac-
`tion (RT-PCR) analysis of BDNF transcription, RNA was
`extracted (see below) from cell pellets after 24 h of incuba-
`tion with GA (50 mg/ml).
`
`Phenotypic characterization of the GA-specific
`TCL by flow cytometry
`TCLs were stained with monoclonal antibodies directed
`against CD3 (mouse IgG1, biotinylated;
`Immunotech,
`Marseille, France) plus
`streptavidin-phycoerythrin (PE)
`(PharMingen, San Diego, CA, USA), CD4 (mouse IgG1,
`PE-labelled; PharMingen) and CD8 (mouse IgG1, fluorescein
`isothiocyanate (FITC)-labelled; Becton Dickinson, San Jose,
`CA, USA) or the corresponding non-immune isotype controls
`[mouse IgG1, biotin- or FITC-labelled (PharMingen); PE-
`labelled (Becton Dickinson)]. The T-cell receptor (TCR) Vb
`(variable region) repertoire was analysed using monoclonal
`antibodies that recognize the following subfamilies: Vb2,
`Vb3, Vb3.1, Vb5.3, Vb7, Vb7.1, Vb8, Vb9, Vb11, Vb12,
`Vb13.1, Vb13.2, Vb13.6, Vb14, Vb17, Vb18, Vb20,
`Vb21.3, Vb23 (Immunotech) and Vb5a, Vb5b, Vb6.7
`(T-cell Diagnostics, Woburn, MA, USA). Monoclonal anti-
`bodies and isotype controls [mouse IgG1 (Becton Dickinson);
`mouse IgG2a and IgG2b (Cymbus, Chandlers Ford,
`Hampshire, UK)] were visualized with a FITC-labelled goat
`anti-mouse IgG antibody (Jackson ImmunoResearch, West
`Grove, PA, USA). The stained cells were analysed using a
`FACScan (Becton Dickinson).
`
`Intracellular flow cytometry analysis of cytokine
`profile and BDNF production
`Intracellular flow cytometry of the TCLs was performed 8–
`10 days after restimulation in the absence of viable APCs.
`GA-specific TCL were stimulated with phorbol 12-myristate
`13-acetate (PMA, 2.0 mg/ml) and ionomycin (250 pg/ml)
`(both from Sigma, St Louis, MO, USA) for 3 h (cytokine
`profile) or 12 h (BDNF production); the last 2 h (cytokine
`profile) or 6 h (BDNF production) in the presence of
`monensin (2 mmol/l; Sigma). The T cells were then washed
`with phosphate-buffered saline (PBS) fixed with 4%
`paraformaldehyde (Merck, Darmstadt, Germany) and perme-
`abilized with 0.1% saponin/PBS (Sigma). For the character-
`ization of the cytokine profile, the T cells were then stained
`using appropriate concentrations of monoclonal antibody
`directed
`against
`IL-4
`(mouse
`IgG1,
`PE-labelled;
`PharMingen) and IFN-g
`(mouse IgG1, FITC-labelled;
`
`BDNF production by GA-specific TCL
`
`2383
`
`PharMingen) or the corresponding isotype controls [mouse
`IgG1, PE-labelled (Becton Dickinson); mouse IgG1, FITC-
`labelled (Immunotech)]. For the detection of intracellular
`BDNF production, activated and non-activated T cells were
`stained with a chicken IgY antibody against human BDNF or,
`as an isotype control, with a chicken control immunoglobulin
`IgY (both Promega, Madison, WI, USA). IgY, the 180 kDa
`chicken IgG homologue, can be produced in chickens against
`certain biological antigens that fail
`to elicit a humoral
`immune response in other mammals due to species related-
`ness. The antibody is highly specific for BDNF. A rabbit anti-
`chicken Ig antibody (FITC-labelled; Promega) was used as
`secondary antibody.
`The untransfected murine ecotropic packaging line
`GP+E86 was used as a negative control for intracellular
`BDNF FACS staining. The packaging line transfected with
`the retroviral vector pLXSN into which BDNF cDNA was
`cloned (kindly provided by R. Kramer, Max-Planck-Institute
`of Neurobiology, Martinsried, Germany) served as a positive
`control (Flugel et al., 2001).
`The stained cells were analysed using a FACScan (Becton
`Dickinson). On a dot plot showing forward and side scatter,
`lymphoid cells were gated for further analysis. Dead cells
`were excluded by gating.
`
`Quantification of BDNF protein secretion in
`culture supernatants by ELISA
`BDNF protein concentrations were determined in duplicate
`using
`a
`sensitive
`sandwich ELISA as
`described
`previously (Kerschensteiner et al., 1999). In brief, 96-well
`flat-bottomed plates were coated with the chicken anti-human
`BDNF IgY antibody (Promega) in 0.025M NaHCO3 and
`0.025M Na2CO3 (pH 8.2). Recombinant human BDNF
`(used as standard; Research Diagnostics, Flanders, PA,
`USA) was used in serial dilutions and cell supernatants in
`1 : 2 dilutions. Bound BDNF was detected by incubating
`plates with a mouse anti-human BDNF antibody (Research
`Diagnostics) followed by peroxidase-conjugated goat anti-
`mouse IgG (Dianova, Hamburg, Germany). The plates were
`developed using a 3,3¢,5,5¢-tetramethyl-benzidine liquid
`substrate system (Sigma); the optical density was determined
`at 450 nm.
`
`RT-PCR analysis of BDNF transcription
`Total cellular RNA was extracted using the RNA extraction
`system of Qiagen (Hilden, Germany) with DNase digestion.
`The RNA (1 mg) was transcribed with oligo(dt) primers,
`Superscript(cid:226) Reverse Transcriptase (both Gibco BRL) and
`dNTP (MBI Fermentas, St Leon-Rot, Germany). All PCR
`in a total volume of 50 ml
`reactions were carried out
`containing 2 U Taq polymerase (Qiagen), 200 mM of each
`dNTP, and 15 pmol of each primer for 35 PCR cycles with an
`annealing temperature of 60(cid:176)C. The correct size of the bands
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`challenged with myelin basic protein (MBP) as was pre-
`viously reported for murine and human GA-specific T cells
`(data not shown) (Aharoni et al., 1998; Neuhaus et al., 2000).
`Irradiated APCs alone did not proliferate, regardless of
`whether they were pulsed with GA or not (Fig. 2). When T
`cells were added to unpulsed APCs,
`there was a small
`background proliferation that was at least six times lower than
`the proliferation in the presence of GA-pulsed APCs (Fig. 2,
`right columns).
`The results of the BDNF ELISA indicate that the irradiated
`APCs (that is, PBMCs containing monocytes, T cells and
`B cells) produced small but clearly detectable amounts of
`BDNF (Fig. 2, left columns in left panels), although they did
`not proliferate (Fig. 2, left columns in right panels). There
`was a tendency for higher BDNF production by GA-pulsed
`APCs alone. All GA-specific TCLs showed an increased
`BDNF production after incubation with GA-pulsed APCs
`(Fig. 2, right columns in left panels).
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`RT-PCR analysis of BDNF transcription in
`GA-specific TCL
`The transcription of BDNF mRNA in GA-specific T-cells
`was analysed by RT-PCR. In contrast to the BDNF protein
`secretion assay, the cells were harvested after a culture period
`of 24 h for RNA extraction and reverse transcription. The
`expression of BDNF was examined using RT-PCR in
`comparison with the housekeeping gene b-actin. There was
`no detectable contamination by genomic DNA, i.e. there were
`no bands in the negative BDNF and b-actin controls using
`RNA samples processed in the absence of reverse transcrip-
`tase (Fig. 3). Consistent with the BDNF protein data shown in
`Fig. 2, RT-PCR revealed weak bands in the absence of GA
`and stronger bands in the presence of GA (Fig. 3).
`
`Detection of BDNF protein in GA-specific
`T-cells by intracellular flow cytometry
`To confirm that the GA-specific T-cells are the source of the
`GA-induced BDNF release, we developed a new intracellular
`staining technique suitable for flow cytometry and FACS
`analysis of intracellular BDNF production. This method
`allows the analysis of BDNF production by individual
`unstimulated und stimulated T cells. Since the analysis was
`performed at least 8–10 days after the last restimulation with
`antigen and irradiated APCs, the cultures contained only T
`cells in the absence of viable APCs. Before intracellular
`staining and FACS analysis, the T cells were stimulated with
`ionomycin and PMA; this mode of stimulation does not
`require the presence of APCs (Dayton et al., 1994). A BDNF-
`transfected and non-transfected murine retroviral packaging
`line was used as the positive and negative controls (Fig. 4, top
`panels).
`This new method was able to detect BDNF produced by the
`GA-specific TCLs, even without stimulation (left panels in
`
`2384
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`T. Ziemssen et al.
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`was determined by comparison with a DNA mass standard
`(SM0403, MBI Fermentas). RNA samples incubated in the
`absence of reverse transcriptase were used as negative
`controls to exclude genomic contamination. The primer
`sequences were as follows: BDNF forward 5¢-AGCGTG-
`AATGGGCCCAAGGCA-3¢
`(position 208–228); BDNF
`5¢-TGTGACCGTCCCGCCCGACA-3¢
`reverse
`(position
`570–551); b-actin
`5¢-CCTCGCCTTTGCCGA-
`forward
`TCC-3¢ (position –8 to 9); and b-actin reverse 5¢-GGATCT-
`TCATGAGGTAGTCAGTC-3¢ (position 623–604).
`
`Results
`Phenotypic characterization of GA-specific
`TCLs
`To analyse the production of BDNF by GA-specific T cells
`after antigen challenge in vitro, four GA-specific long-term
`TCLs were used—one TCL obtained from the GA-treated
`multiple sclerosis patient (B.K.) and three TCLs from the
`healthy donor (T.Z.). The TCLs were selected with the split-
`well cloning technique (Kitze et al., 1988; Pette et al., 1990).
`FACS analysis showed that all GA-specific TCLs had a
`CD3+CD4+CD8– phenotype (data not shown). The TCL from
`the multiple sclerosis patient (BK-M6-COP-7) was pre-
`viously characterized as part of another study (Neuhaus et al.,
`2000). The other three TCLs were analysed as to their TCR
`Vb usage. A panel of 23 different anti-TCR-Vb antibodies
`was used to demonstrate the oligoclonal nature of the TCLs.
`As characteristically seen with TCLs selected with the split-
`well protocol (Kitze et al., 1988; Pette et al., 1990), each line
`reacted predominantly with one monoclonal antibody from
`the panel of anti-TCR Vb antibodies (Fig. 1). The three
`investigated TCLs used different Vb elements. The fact that
`the TCL populations predominantly stained positive for only
`the 23 Vb elements indicated that
`one of
`they were
`oligoclonal (Fig. 1).
`Fig. 1 shows the cytokine profile of the TCLs for IL-4 and
`IFN-g. Whereas the TCLs TZ-COP-1 and TZ-COP-3 dis-
`played a TH1 or TH1/TH0 phenotype, TZ-COP-5 cells
`produced both IL-4 and IFN-g (TH0 phenotype). As
`described previously (Neuhaus et al., 2000), the TCL from
`the GA-treated multiple sclerosis patient (BK-M6-COP-7)
`had a stable TH2 cytokine profile (Fig. 1).
`
`GA-induced proliferation and BDNF protein
`secretion by GA-specific TCLs
`To demonstrate the ability of GA-specific T cells to secrete
`BDNF upon stimulation, we quantified the amount of BDNF
`in supernatants of GA-stimulated T cells and, in parallel,
`assessed the proliferative response to GA (Fig. 2). All GA-
`specific TCLs showed a specific proliferative response to GA
`with stimulation indices ranging between 6.5 and 21.0 over
`several re-stimulations. None of the tested GA-specific TCLs
`showed a proliferative response or cytokine production when
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`Fig. 1 Phenotypical characterization of the GA-specific TCLs (TZ-COP-1, TZ COP-3, TZ-COP-5, BK-M6-COP-7) as to their Vb TCR
`usage (left panels: fine lines represent isotype controls and bold lines represent the indicated TCR Vb antibodies) and their cytokine
`secretion profile analysed by intracellular double-fluorescence cytometry (middle panels with isotype controls; right panels with the
`cytokine profile). ND* = not done; the Vb TCR usage of the published GA-specific T cell line BK-M6-COP-7 was not determined
`(Neuhaus et al., 2000).
`
`Fig. 4; fine lines represent isotype controls). After stimulation
`with ionomycin and PMA,
`there was an increase of
`intracellular BDNF production (Fig. 4, right panels). The
`results of this intracellular assay of BDNF expression are
`consistent with the results of the ELISA and RT-PCR
`analysis.
`
`Discussion
`Technical aspects of the study
`We clearly show that GA-specific, activated TH0, TH1 and
`TH2 cells produce the neurotrophic factor BDNF—not only
`at the transcriptional (mRNA) level by RT-PCR, but also at
`the protein level—using the newly developed assays for
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`Fig. 2 BDNF secretion (left panels) and proliferation (right panels) of the examined GA-specific TCLs (TZ-COP-1, TZ-COP-3, TZ-COP-
`5, BK-M6-COP-7) after restimulation with GA. Irradiated autologous unpulsed and GA-pulsed APCs were analysed alone and in co-
`culture with the examined TCL as described in Material and methods. Filled columns indicate mean of duplicates and vertical lines
`indicate individual measurements. Different ordinate scales are used. The statistical significance of the difference between BDNF
`production of (unpulsed APC+TCL) versus (GA-pulsed APC+TCL) was P < 0.0003 (t-test; all TCL included).
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`To show conclusively that the BDNF is produced by the
`GA-specific T cells rather than the APCs, we optimized its
`detection in individual T cells in the absence of APCs. Similar
`assays are widely used for the intracellular staining of various
`cytokines, including IL-4 and IFN-g (see Fig. 1); the T cells
`are typically stimulated with ionomycin and PMA [an
`antigen-independent maximal stimulus for T cells (Dayton
`et al., 1994)] before being fixed and permeabilized. In
`preliminary experiments with a series of monoclonal and
`polyclonal antibodies to BDNF, we obtained the best results
`with a highly specific chicken anti-human BDNF antiserum
`(Fig. 4) that also detects BDNF in ELISA and specifically
`labels a murine retroviral packaging cell line transfected with
`BDNF-cDNA. Together with the results of RT-PCR and
`ELISA, these results formally establish that human GA-
`specific T cells of different TH types can indeed produce
`BDNF.
`
`Implications for the presumed mechanism of
`action of GA
`Our findings have obvious implications for the presumed
`mechanism of action of GA. According to current theory, the
`beneficial effects of GA are mainly mediated by a population
`of GA-reactive TH2 cells. GA treatment induces a gradual
`shift of GA-reactive T cells from TH1 to TH2 (Miller et al.,
`1998; Neuhaus et al., 2000; Duda et al., 2000a; Farina et al.,
`2001). Their constant activation by daily immunization
`enables them to enter the CNS (Wekerle et al., 1986;
`Hickey et al., 1999). Indeed, transferred GA-reactive T cells
`have been directly demonstrated in the CNS of recipient mice
`(Aharoni et al., 2000). It is further assumed, that after local
`recognition of cross-reactive myelin degradation products,
`the GA-specific TH2 cells are stimulated to secrete anti-
`inflammatory cytokines, which in turn induce bystander
`suppression in neighbouring encephalitogenic T cells
`(Aharoni et al., 1997; Aharoni et al., 1998; Neuhaus et al.,
`2001). Our present findings imply that the locally activated
`GA-reactive TH2 cells produce not only protective TH2
`cytokines, but also BDNF. They further indicate that GA-
`specific TH1 cells, which are reduced but are still present in
`GA-treated patients (Neuhaus et al., 2000; Farina et al.,
`2001), could also act as a local source of BDNF.
`BDNF is one of the most potent factors supporting
`neuronal survival and regulating neurotransmitter release
`and dendritic growth (Thoenen, 1995; Lewin and Barde,
`1996; Barde, 1997). Several studies have shown that BDNF
`can rescue injured or degenerating neurones and induce
`axonal outgrowth, remyelination and regeneration (Yan et al.,
`1992; Gravel et al., 1997). Moreover, it can also protect axons
`from elimination during development, or from degeneration
`after axotomy, or in experimental neurodegenerative disease
`(Mitsumoto et al., 1994). Most known BDNF functions are
`signalled via the full-length gp145 tyrosine kinase receptor
`(trk) B (Bothwell, 1995); intriguingly, it is found in neurones
`
`Fig. 3 The BDNF transcription by unstimulated and GA-
`stimulated GA-specific TCLs (TZ-COP-1, TZ-COP-3, TZ-COP-5,
`BK-M6-COP-7) was examined by RT-PCR analysis of BDNF
`transcripts. The transcription of the housekeeping gene b-actin was
`used for comparison. RNA samples transcribed in the absence of
`reverse transcriptase were used as negative controls to exclude
`genomic contamination.
`
`BDNF secretion and synthesis of BDNF in individual T cells.
`The results from all assays consistently showed a low level of
`basal secretion of BDNF by GA-specific T cells and an
`increase of BDNF production after stimulation. For our
`analysis, we selected four well-characterized prototypic GA-
`specific TCLs. Similar
`results were obtained with >20
`additional GA-specific TCLs (data not shown).
`In pilot experiments, the presence of soluble GA in culture
`supernatants sometimes caused elevated ELISA readings for
`BDNF (M. Kerschensteiner, W. Klinkert and T. Ziemssen,
`unpublished data). To prevent that, we pre-pulsed the APCs
`with GA and washed them, before co-culturing with our
`highly purified long-term GA-specific oligoclonal TCL. In
`this culture system, the irradiated PBMC produced small
`amounts of BDNF even in the absence of GA-specific TCLs
`(Fig. 2). This was expected because the irradiated PBMC
`preparations contained T cells, and GA strongly stimulates T
`cells from naı¨ve human subjects (Duda et al., 2000a; Duda
`et al., 2000b; Farina et al., 2001). Although proliferation of
`the irradiated PBMC was abolished completely, some GA-
`stimulated BDNF production evidently persists (Fig. 2).
`When the GA-specific TCLs were added, BDNF production
`increased and was highest in the presence of GA-pulsed
`APCs. Qualitatively similar results were obtained in the
`parallel proliferation assays. All four tested TCLs yielded
`consistent results in all assays (Fig. 2).
`
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`Fig. 4 Intracellular BDNF production of unstimulated (left panel) and PMA- and ionomycin-stimulated (right panels) GA-specific TCLs
`(TZ-COP-1, TZ-COP-3, TZ-COP-5, BK-M6-COP-7). Fine lines represent isotype controls and bold lines represent the anti-BDNF-
`antibody. The untransfected and BDNF-transfected murine ecotropic packaging line GP+E86 was used as the negative and positive control
`for the intracellular BDNF FACS staining.
`
`in the immediate vicinity of multiple sclerosis plaques as well
`as
`in reactive astrocytes
`in multiple sclerosis lesions
`(Stadelmann et al., 2002). Therefore, T-cell-derived BDNF
`could directly act on target cells expressing the appropriate
`trkB receptor.
`
`BDNF has been immunolocalized in inflammatory cells in
`active multiple sclerosis lesions (Stadelmann et al., 2002).
`This indicates that, even in untreated multiple sclerosis
`patients, inflammatory cells might have a beneficial (neuro-
`protective) effect
`in addition to their
`(probably major)
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`detrimental role (Hohlfeld et al., 2000). Indeed, the concept
`of ‘neuroprotective autoimmunity’ has attracted considerable
`attention and is supported by experimental evidence from
`various animal models (Kipnis et al., 2000; Schori et al.,
`2001; Schwartz, 2001). It is conceivable that
`there is a
`complex interplay between detrimental and beneficial factors,
`and mediators in the inflammatory milieu of multiple
`sclerosis lesions. BDNF imported by GA-reactive T cells
`might help tip the balance in favour of the beneficial,
`neuroprotective influences. Indeed, in a rat neurodegenerative
`model, Kipnis and colleagues showed that rat immune cells
`stimulated by immunization with GA secreted neurotrophic
`factors upon restimulation with GA (Kipnis et al., 2000).
`The following scenario would accommodate both the
`findings in experimental autoimmune encephalomyelitis and
`the observations in human multiple sclerosis. GA-specific
`activated T cells can pass through the blood-brain barrier.
`Inside the CNS, some GA-specific T cells cross-react with
`products of local myelin turnover presented by local APCs. In
`our previous studies, 8–15% of GA-reactive TCLs cross-
`reacted with MBP as indicated by cytokine secretion, but not
`proliferation (Neuhaus et al., 2000). Recognition of cross-
`reactive antigens at the lesion site seems to be necessary for
`reactivation of the protective T cells and for their neuropro-
`tective effect. In rat models, only MBP- and GA-specific, but
`not ovalbumin-specific, T cells confer neuroprotection
`(Moalem et al., 1999). After reactivation in situ, GA-specific
`TH2-like regulatory T cells would not only provide anti-
`inflammatory cytokines like IL-4, IL-5, IL-13, and trans-
`(TGF)-b,
`forming
`growth
`factor
`but
`also BDNF.
`Furthermore, TH1-like GA-reactive T cells, which are
`reduced but still present in GA-treated patients (Neuhaus
`et al., 2000; Farina et al., 2001), would also have the capacity
`to release BDNF in multiple sclerosis lesions.
`
`Implications for therapy with other altered
`peptide ligands
`The above concept may be extended to other types of
`immunomodulatory therapy, especially with ‘altered peptide
`ligands’
`(APLs). By definition,
`they are derived from
`immunogenic peptide antigens by the selective alteration of
`one or more T-cell receptor-contacting residues. A good
`example is an APL designed from the myelin basic protein
`peptide 83–99 (Bielekova et al., 2000; Genain and Zamvil,
`2000; Kappos et al., 2000). This APL has been tested in
`clinical trials in multiple sclerosis patients. Despite several
`adverse effects, there was some evidence for partial efficacy
`and for a TH1-to-TH2 shift during treatment with low doses
`of the APL (Bielekova et al., 2000; Crowe et al., 2000;
`Kappos et al., 2000). The rationales for APL and GA
`treatment are very similar: APLs are able to expand
`populations of TH0 and TH2 cells that have specificity for
`the APL itself, but they can also cross-react with the native
`peptide. Thus, T cells specific for an APL analogue of myelin
`
`BDNF production by GA-specific TCL
`
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`
`antigen will be able to penetrate the CNS, where they can
`down-regulate encephalitogenic inflammatory cells via ‘by-
`stander
`suppression’
`(Steinman, 1996; Hohlfeld, 1997;
`Nicholson et al., 1997; Genain and Zamvil, 2000). It should
`be noted, however, that TH2 responses can in principle
`contribute to myelin damage (Genain et al., 1996).
`Extrapolating from our present results to APL therapies in
`general, we would expect that APL-specific TH2 and TH1
`cells are also capable of producing BDNF (as do GA-specific
`TH2 and TH1 cells). However, for APL therapy to work, it is
`obviously cru