JNDR_0-0-00-Ziemssen.qxp 9/9/2008 12:32 PM Page 1
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`ORIGINAL ARTICLE
`
`Presence of glatiramer acetate-specific TH2 cells in the
`cerebrospinal fluid of patients with multiple sclerosis 12 months
`after the start of therapy with glatiramer acetate
`
`Tjalf Ziemssen, MD
`Heinz Reichmann, MD
`Hauke Schneider, MD
`
`ABSTRACT
`
`The proposed mechanism of action of glatiramer acetate
`(GA) from animal models is that GA-specific T cells enter
`the CNS to act there. To evaluate GA-specific T cells in
`patients with multiple sclerosis (MS), we tried to culture GA-
`specific T cell lines from the CSF of the identical patients
`with MS before and during GA therapy. Before treatment
`T cell lines could not be obtained, whereas after at least
`1 year of GA treatment 12 GA-specific T cell lines secreting
`TH2 cytokines and BDNF could be generated from the CSF.
`Key words: multiple sclerosis, glatiramer acetate,
`mechanism of action
`
`INTRODUCTION
`
`The current mechanism of action of glatiramer acetate
`(GA), approved for use in the immunomodulatory ther-
`apy of relapsing-remitting multiple sclerosis (MS), pro-
`poses that activated GA-specific TH2 cells penetrate the
`CNS and release anti-inflammatory cytokines and neu-
`rotrophic factors in situ.1 Until now it is only known from
`animal experiments that activated GA-specific T cells are
`capable of entering the brain.2 Studies involving areas
`beyond the blood-brain barrier are not available.
`To demonstrate that GA-specific T cells are able to enter
`the human CNS compartment we cultured GA-specific
`T cells from the CSF of three identical patients with MS
`before treatment and at least 1 year after start of GA treat-
`ment, which were characterized in more detail.
`
`METHODS
`
`Three patients with relapsing-remitting MS underwent
`CSF puncture as part of their diagnostic procedure. All
`three received GA as their first immunomodulatory
`therapy. Twelve months after start of GA therapy, they
`received an additional CSF puncture with informed
`
`consent of the patients and approval of the ethical board.
`These patients on GA who enrolled in this longitudinal
`pilot study did not demonstrate any relapse or progres-
`sion after starting their GA therapy.
`After centrifugation of the CSF, 103 CSF cells were co-
`cultured with 2 ⫻ 105 irradiated autologous antigen pre-
`senting cells (APC) in the presence of GA (50 ␮g/mL) and
`IL-2 (2 U/mL) according to Neuhaus et al.3 For our proto-
`col, at least 100 wells were plated for T cell culture. T
`cells were cultured for 14 to 21 days until the next restim-
`ulation with GA (50 ␮g/mL) and 2 ⫻ 105 irradiated autol-
`ogous APC. Until analysis, GA-specific T cell cultures
`underwent 5 to 7 rounds of restimulation.
`For analysis, 105 washed GA-specific TCL cells
`were stimulated with 104 GA- or myelin-basic protein
`(MBP)-pulsed irradiated autologous PBLs. Supernatants
`were analyzed after 72 h for BDNF (Promega) and
`cytokine concentrations (interferon (IFN)-␥ and inter-
`leukin (IL)-4) (Becton Dickinson) by ELISA. For prolifera-
`tion assays, parallel cultures were labeled after 48 h with
`[methyl3H]thymidine (0.2 to 0.5 ␮Ci per well; Amersham
`Buchler) and harvested 16 to 18 h later. [3H]Thymidine
`incorporation was measured with a direct ␤-counter. The
`intracellular flow cytometry analysis of BDNF production
`was performed 8 to 10 days after restimulation.4
`
`RESULTS
`
`We were not able to generate GA-specific T cell lines
`from the CSF of the three patients with MS before GA
`treatment. Although we cultured at least 100 wells for GA
`T cell culture of CSF cells, no GA-specific T cell lines
`could be obtained. Using the same protocol 12 months
`later, we were able to generate 12 GA-specific CSF T cell
`lines in the same patients. All 12 T cell lines were GA-
`specific as demonstrated by antigen specific proliferation.
`Eleven of 12 GA-specific cell lines demonstrated a TH2 or
`TH0 cytokine phenotype, and only one T cell line GA2-3
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`had a TH1 phenotype (Table 1). After stimulation with
`GA, all 12 GA-specific T cell lines secreted BDNF in vari-
`ous concentrations. For the T cell line GA2-4, we could
`demonstrate cross-reactivity with MBP at the level of
`cytokine and BDNF production, but not at the level of
`proliferation (Figure 1a).
`To confirm that the CSF-derived GA-specific T cells are
`the source of the GA-induced BDNF release, we used
`intracellular staining technique suitable for flow cytometry
`and FACS analysis of intracellular BDNF production. This
`method allows the analysis of BDNF production by indi-
`vidual unstimulated und stimulated T cells. After stimula-
`tion with ionomycin and PMA, there was an increase of
`intracellular BDNF production. For all 12 GA-specific CSF-
`derived T cell lines, we were able to demonstrate intracel-
`lular BDNF production (given in Figure 1b for GA2-4). Our
`results here were in line with those obtained in studies on
`peripheral blood demonstrating a significant TH1-TH2-
`shift regarding GA-specific T cells (data not shown).
`
`DISCUSSION
`
`Results from animal and human in vivo studies indi-
`cate that GA induces regulatory T cells of the T-helper
`
`2- (TH2)-type in the peripheral immune system outside
`the CNS.1 When spleen cells from GA-treated mice were
`adoptively transferred into syngenic animals, these cells
`protected the animals from EAE induced by different CNS
`antigens.5 Further support for the proposed protective
`role of GA-specific regulatory T cells comes from the
`demonstration that GA-specific TH2 cells are present in
`the CNS of GA-treated mice.2 Some of these GA-specific
`TH2 cells are reactivated in the CNS because they cross
`react with locally presented myelin autoantigens.3,5 The
`GA-specific T cells secrete anti-inflammatory cytokines
`such as IL-4, which, in turn, dampens the activity of any
`nearby autoaggressive T cells.6 In addition to this anti-
`inflammatory bystander suppression, Aharoni et al.1
`demonstrated that GA treatment led to sustained aug-
`mentation in the expression of neurotrophic factors like
`BDNF in various brain regions as demonstrated by histo-
`logical analysis of immunostained brain sections.
`Until now, it was not known whether GA-specific T
`cells can migrate into the CNS in patients with MS who
`have been treated with this agent. Kim et al.8 demonstrated
`that human TH1 and TH2 GA-reactive T cells could
`migrate across an artificial blood-brain barrier in vitro. In
`this pilot longitudinal study, we were able to demonstrate
`
`Table 1. Proliferation (cpm), IFN-␥␥, IL-4, BDNF secretion (pg/mL) of the examined GA-specific T cell lines
`derived from CSF culture of three GA-treated patients with MS (GA1-3)
`
`Patient
`
`T cell
`line
`
`Proliferation
`
`IFN-␥␥
`
`IL-4
`
`BDNF
`
`TH profile
`
`No antigen
`
`GA
`
`No Antigen
`
`GA
`
`No Ag
`
`GA
`
`No antigen
`
`GA
`
`GA1
`
`GA1-1
`
`100 ⫾ 12
`
`3234 ⫾ 678
`
`GA1-2
`
`155 ⫾ 22
`
`5223 ⫾ 987
`
`10 ⫾ 5
`
`28 ⫾ 7
`
`17 ⫾ 4
`
`5 ⫾ 2
`
`45 ⫾ 5
`
`34 ⫾ 9
`
`7 ⫾ 2 65 ⫾ 21
`
`GA1-3
`
`300 ⫾ 50
`
`6213 ⫾ 1245
`
`8 ⫾ 12
`
`123 ⫾ 34 11 ⫾ 3 77 ⫾ 11
`
`GA2
`
`GA2-1
`
`232 ⫾ 27
`
`3563 ⫾ 543
`
`35 ⫾ 17
`
`44 ⫾ 12 15 ⫾ 5 55 ⫾ 21
`
`GA2-2
`
`117 ⫾ 23
`
`2356 ⫾ 765
`
`23 ⫾ 5
`
`33 ⫾ 5
`
`8 ⫾ 3
`
`49 ⫾ 7
`
`GA2-3
`
`521 ⫾ 276 2365 ⫾ 490
`
`1345 ⫾ 445 2145 ⫾ 251
`
`834 ⫾ 327 2542 ⫾ 398
`
`1500 ⫾ 211 2532 ⫾ 654
`
`467 ⫾ 127 1589 ⫾ 265
`
`TH2
`
`TH2
`
`TH0
`
`TH2
`
`TH2
`
`TH2
`
`99 ⫾ 34
`
`1200 ⫾ 234
`
`28 ⫾ 12
`
`33 ⫾ 8
`
`11 ⫾ 4 77 ⫾ 12
`
`1278 ⫾ 398 2000 ⫾ 112
`
`GA2-4
`
`115 ⫾ 50
`
`2377 ⫾ 432
`
`44 ⫾ 3
`
`55 ⫾ 9
`
`15 ⫾ 6
`
`55 ⫾ 3
`
`GA3
`
`GA3-1
`
`234 ⫾ 37
`
`4519 ⫾ 399
`
`8 ⫾ 4
`
`7 ⫾ 5
`
`9 ⫾ 5
`
`45 ⫾ 2
`
`GA3-2
`
`334 ⫾ 44
`
`5200 ⫾ 522
`
`11 ⫾ 9
`
`234 ⫾ 7
`
`12 ⫾ 3
`
`15 ⫾ 6
`
`GA3-3
`
`212 ⫾ 12
`
`3789 ⫾ 687
`
`33 ⫾ 5
`
`29 ⫾ 6
`
`19 ⫾ 2
`
`77 ⫾ 5
`
`GA3-4
`
`120 ⫾ 23
`
`2245 ⫾ 234
`
`9 ⫾ 4
`
`15 ⫾ 6
`
`5 ⫾ 3
`
`57 ⫾ 3
`
`GA3-5
`
`176 ⫾ 10
`
`5467 ⫾ 469
`
`13 ⫾ 4
`
`23 ⫾ 4
`
`7 ⫾ 2
`
`63 ⫾ 4
`
`TH2
`
`TH2
`
`TH1
`
`TH2
`
`TH2
`
`TH2
`
`223 ⫾ 98
`
`812 ⫾ 233
`
`499 ⫾ 129 1736 ⫾ 321
`
`677 ⫾ 381 2987 ⫾ 455
`
`324 ⫾ 136 1566 ⫾ 361
`
`551 ⫾ 269 2001 ⫾ 471
`
`431 ⫾ 233 1991 ⫾ 287
`
`Values are mean of duplicates with standard deviation. The statistical significance between BDNF production without and with
`GA was p ⬍ 0.0004 (t-test).
`
`2
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`Journal of Neurodegeneration and Regeneration 1:1 (cid:132) Fall 2008
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`Figure 1. (a) IL-4 and BDNF production (pg/mL) by GA-specific T cell line GA2-4 measured by ELISA in supernatants of
`the same assay using no antigen, GA (50 ␮␮g/mL) and MBP (30 ␮␮g/mL). (b) Intracellular BDNF production of unstimulated
`(left panel) and PMA- and ionomycin-stimulated (right panels) GA-specific T cell line GA2-4. Fine lines represent isotype
`controls and bold lines represent the anti-BDNF-antibody.
`
`for the first time that GA-specific T cells of TH2/TH0 phe-
`notype are present in the CSF compartment of GA-treated
`patients after 12 months of GA treatment, whereas no GA-
`specific T cell lines could be obtained from the CSF com-
`partment. Although GA is a polyclonal mitogen for T cell
`responses, no T cell responses could be demonstrated in
`the CSF compartment of untreated patients with MS.
`This longitudinal study is only a pilot study with three
`enrolled patients. Beyond this limitation, these data are
`important and presented for the first time, which should
`be the basis for detailed longitudinal analysis of GA-
`specific T cell responses in the CSF. It has to be clarified
`whether peripheral immunological changes that are spe-
`cific for GA like TH1-TH2-shift or GA-specific antibodies
`correlate with immunological changes in the CSF com-
`partment, which is the only accessible compartment
`
`close to the CNS. The results obtained from the CSF
`should be representative of the CNS as we have already
`demonstrated that GA-specific T cells can enter the CNS
`and CSF in a comparable quantity using the murine
`model of experimental autoimmune encephalomyelitis.
`
`Tjalf Ziemssen, MD, Neuroimmunological Laboratory,
`Neurological Clinic, Technical University of Dresden,
`Dresden, Germany, ziemssen@web.de.
`
`Heinz Reichmann, MD, Neurological Clinic, Technical
`University of Dresden, Dresden, Germany.
`
`Hauke Schneider, MD, Neuroimmunological Laboratory,
`Neurological Clinic, Technical University of Dresden,
`Dresden, Germany.
`
`Journal of Neurodegeneration and Regeneration 1:1 (cid:132) Fall 2008
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`ACKNOWLEDGMENTS
`This study was supported by an unrestricted grant of Teva
`Pharmaceutical Industries, Ltd, Israel.
`
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`4. Ziemssen T, Kumpfel T, Klinkert WE, et al.: Glatiramer
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`
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`6. Aharoni R, Kayhan B, Eilam R, et al.: Glatiramer acetate-
`specific T cells in the brain express T helper 2/3 cytokines and
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`
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`MYLAN PHARM. v YEDA
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