RESEARCH PAPER
`
`Multiple Sclerosis 2008; 14: 749–758
`
`Multiple sclerosis: glatiramer acetate induces
`anti-inflammatory T cells in the cerebrospinal fluid
`
`ALK Hestvik1, G Skorstad2, DA Price3, F Vartdal1 and T Holmoy1,2
`
`Glatiramer acetate (GA) is believed to induce GA-reactive T cells that secrete anti-inflammatory cyto-
`kines at the site of inflammation in multiple sclerosis (MS). However, GA-reactive T cells have not been
`established from the intrathecal compartment of MS patients, and intrathecal T cells may differ from T
`cells in blood. Here, we compared the phenotype of GA-reactive T cells from the cerebrospinal fluid
`(CSF) and blood of five MS patients treated with GA for 3-36 months, and in three of these patients
`also before treatment. From the CSF of these patients, all 22 T cell lines generated before and all 38 T
`cell lines generated during treatment were GA-reactive. GA treatment induced a more pronounced
`anti-inflammatory profile of GA-reactive T cell lines from CSF than from blood. While GA-reactive T cell
`clones from CSF were restricted by either human leukocyte antigen (HLA) -DR or HLA-DP, only HLA-
`DR restricted GA-reactive T cell clones were detected in blood. No cross reactivity with myelin proteins
`was detected in GA-reactive T cell lines or clones from CSF. These results suggest that a selected
`subset of GA-reactive T cells are present in the intrathecal compartment, and support an anti-inflam-
`matory mechanism of action for GA. Multiple Sclerosis 2008; 14: 749–758. http://msj.sagepub.com
`
`Key words: multiple sclerosis; immunology; glatiramer acetate; disease modifying therapies
`
`Introduction
`
`Glatiramer acetate (GA) has been shown to reduce
`both the relapse rate and the appearance of new
`central nervous system (CNS) lesions in multiple
`sclerosis (MS) [1,2]. GA is a polymer of the four
`amino acids most prevalent in myelin basic protein
`(MBP) and has been shown to bind several different
`human leukocyte antigen (HLA)-DR molecules [3].
`Antigen presenting cells (APCs) within or outside
`the CNS may be the primary target of GA-
`mediated immune modulation [4–6]. The mecha-
`nism of GA is thought to involve induction of
`anti-inflammatory GA-reactive T cells in secondary
`lymphoid organs [7]. Anergy induction of activated
`and potentially pathogenic T cells is thought to
`play a role as long-term GA treatment leads to a
`drop in the precursor frequency of GA-reactive
`T cells [8,9]. Within the CNS, GA-reactive T cells
`may cross-react with myelin proteins
`[10–12],
`dampen the inflammatory response by secretion of
`anti-inflammatory Th2 cytokines such as interleu-
`
`kin (IL)-4, IL-5, IL-10, and IL-13, [10,13,14], induce
`regulatory T cells [5], and stimulate reparative pro-
`cesses through secretion of neurotrophic factors
`[15–18].
`For these mechanisms to be relevant in MS, GA-
`reactive T cells must gain access to the CNS. Studies
`in mice have shown that GA-reactive T cells
`induced in the periphery penetrate the blood-brain
`barrier [13,14], and human GA-reactive T cells have
`been shown to migrate across an artificial blood-
`brain barrier in vitro [19]. However, because of diffi-
`culties in investigating migration of human T cells
`in vivo, current knowledge of human GA-reactive
`T cells stems from analyses conducted with lym-
`phocytes derived from blood.
`Studies in MS and other immune-mediated dis-
`eases have shown that T cells from the diseased
`organ may differ from T cells in blood [20–23]. To
`understand the effects of GA in MS, it is therefore
`important to investigate GA-reactive T cells from
`the intrathecal compartment. The aim of the pres-
`ent study was to compare GA-reactive T cells from
`
`1Faculty of Medicine, Institute of Immunology, Rikshospitalet-Radiumhospitalet, Oslo, Norway
`2Department of Neurology, Ullevål University Hospital, Oslo, Norway
`3Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK
`Correspondence to: Anne Lise K Hestvik, Institute of Immunology, University of Oslo, 0027 Oslo, Norway.
`Email: a.l.k.hestvik@medisin.uio.no
`Received 9 August 2007; revised 2 December 2007; accepted 18 January 2008
`
`© SAGE Publications 2008
`Los Angeles, London, New Delhi and Singapore
`
`10.1177/1352458508089411
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`blood and cerebrospinal fluid (CSF) in terms of
`cytokine profile, HLA restriction,
`and cross-
`recognition properties before and after initiation of
`GA treatment.
`
`Materials and methods
`
`Patients
`
`Five patients (four women and one man) with
`relapsing-remitting MS (Expanded Disability Status
`Scale 1–2) gave informed consent to participate in
`this study. Study participants were treated by neu-
`rologists with no other involvement in the study
`and had received 20 mg GA daily for 0–36 months
`at inclusion. The study was approved by the local
`ethics committee.
`
`Antigens
`
`GA (copaxone©) was obtained from Teva Pharma-
`ceuticals Ltd (London, UK). In all, 531 15-mer pep-
`tides, overlapping by 10 amino acids spanning
`the complete sequences of MBP (18.5 kDa and
`21.5 kDa isoforms), protelipid protein (PLP), myelin-
`oligodendrocyte glycoprotein (MOG;
`including
`the β2 isoform), myelin-associated oligodendrocytic
`basic
`protein, myelin-associated
`glycoprotein,
`oligodendrocyte myelin glycoprotein, αβ-crystallin,
`S100β,
`and
`2′,
`3′-cyclic
`nucleotide
`3’-
`phosphodiesterase (partly a gift from N. Karandikar,
`UT Southwestern Medical Center, Dallas, Texas,
`USA) were synthesized using standard techniques
`(BioSynthesis, Texas, USA) at 44.8–100% purity
`(0.5% were below 50% pure and 12% were above
`90% pure) [24]. Peptides were dissolved in dimethyl
`sulfoxide (DMSC) at a concentration of 50 mg/mL
`and tested in pools of 8–10 peptides; each peptide
`was tested at a concentration of 2.5 μM. A chymo-
`trypsin digest of gluten extracted from wheat flour
`(gift from O. Molberg, Institute of Immunology, Rik-
`shospitalet
`Radiumhospitalet, Oslo, Norway),
`human MBP (Chemicon, California, USA), and a pep-
`tide from glutamic acid decarboxylase (GAD57–72)
`served as control antigens.
`
`Generation of T cell lines and T cell clones
`
`In all, 40,000–160,000 cells were obtained from
`23 mL CSF collected by lumbar puncture. No bleed-
`ing was observed during the procedures. To further
`avoid blood contamination, the first 2 mL of CSF
`were discharged. To minimize differences related
`to in vitro conditions, T cell lines from CSF and
`blood were cultured in parallel using identical pro-
`
`cedures. Briefly, 1 × 105 peripheral blood mononu-
`clear cells (PBMC) or 4000–6000 CSF cells were
`incubated with 105 irradiated (25 Gy) autologous
`PBMC, which had been pre-incubated overnight
`with 50 or 200 μg/mL GA. Cells were stimulated
`with 10 IU/mL IL-2 (RnDSystems, Minnesota, USA)
`on day 7 and restimulated on day 14 with 105 irra-
`diated autologous PBMC and 50 or 200 μg/mL GA.
`The T cell lines were expanded with irradiated het-
`erologous PBMC, 1 μg/mL phytohemagglutinin
`(Remel, Kansas, USA), and 10 IU/mL IL-2 on day
`20 and tested for specificity and cytokine produc-
`tion on day 28 with autologous PBMC preincubated
`overnight with 50 or 200 μg/mL GA. T cell cloning
`and determination of the HLA-isotype presenting
`GA were performed using procedures and monoclo-
`nal antibodies described previously [25].
`
`Proliferation assays
`
`T cells were tested by incubating 105 T cells sus-
`pended in 200 μL medium with 105 irradiated autol-
`ogous PBMC, which had been pre-incubated with
`antigen overnight in 100 μL medium. T cells were
`cultured for 72 h and proliferation tested on the
`basis of 3H-thymidine incorporation as described
`previously [25]. A proliferative response was scored
`as positive if counts per minute (cpm) of stimulated
`wells minus cpm of unstimulated wells (Δcpm)
`exceeded 1000 and the stimulatory index (SI)
`exceeded 3 [23].
`
`Cytokine measurement
`
`Supernatants of T cells were frozen at −80°C for
`48 h after antigen stimulation. Concentrations of
`IL-4, IL-5, IL-10, IL-13, IL-17, tumor necrosis factor
`(TNF)-α, and interferon (IFN)-γ were measured with
`a custom-made multiplex antibody bead kit (Bio-
`source International
`Inc., Camarillo, California,
`USA) for Luminex™, according to the manufac-
`turer’s recommendations. The detection level for
`all cytokines was 3–5 pg/mL. Unstimulated T cells
`incubated with irradiated PBMC in the absence of
`antigen served as control. Irradiated PBMC incu-
`bated with antigen overnight produced no detect-
`able levels of the cytokines. Cytokine production
`was calculated as cytokine concentrations in super-
`natants from antigen-stimulated cells (T + APC + GA)
`minus unstimulated cells (T + APC).
`
`Statistics
`
`Cytokine ratios were compared with a two-way
`ANOVA analysis with patients as random effect in
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`Statistical Package for the Social Sciences (SPS) version
`15 (SPSS Inc., Chicago, Illinois, USA). To correct for
`deviation from the normal distribution, data were
`log transformed before analysis. The proportion of
`Th1-biased and Th2-biased GA-reactive T cell lines
`was analyzed using generalized estimating equation
`regression models in Statistical Analysis System (SAS)
`version 9.1 (SAS Inc., Cary, North Carolina, USA).
`
`Results
`
`GA-reactive T cells are present in the CSF and blood
`before and during GA treatment
`
`To establish whether GA-reactive T cells are present
`in the CSF of patients before and after initiation of
`treatment, 22 T cell lines from CSF and 36 T cell
`lines from blood were generated from patients 1–3
`before they started GA treatment, and 28 T cell
`lines from CSF and 31 T cell lines from blood of
`the same patients were generated after 3–6 months
`on treatment. All T cell lines generated both before
`and during treatment from both blood and CSF
`responded vigorously to GA both by proliferation
`
`GA-reactive T cells from the CSF of MS patient
`
`751
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`and cytokine production (Figure 1). Although the
`number of CSF cells is too low to allow calculation
`of the frequency of GA-specific T cells, the observa-
`tion that all T cell lines were GA-reactive indicates
`that the precursor frequency of GA-specific T cells
`in CSF both before and after initiation of GA treat-
`ment exceeds 1:5000.
`
`Th2-bias is more pronounced in GA-reactive T cell
`lines from CSF than from blood
`
`To establish cytokine profiles of GA-reactive T cells,
`concentrations of pro- and anti-inflammatory cyto-
`kines were measured in supernatants of proliferat-
`ing GA-reactive T cell lines. Using an IL-5/IFN-γ
`ratio of 1 as the cut-off between a predominant
`Th1 versus Th2 phenotype, 13 of 22 GA-reactive
`T cell lines from CSF and 11 of 36 GA-reactive
`T cell lines from blood were Th2-biased before treat-
`ment. After treatment, the proportion of predomi-
`nantly Th2-biased GA reactive T cell lines from the
`same patients was 25 of 28 (CSF) and 21 of 31
`(blood). Thus, short-term GA-treatment induced
`an increase in the ratio of Th2-biased cell lines in
`both CSF and blood. The increase was only
`
`Figure 1 Glatiramer acetate (GA)-reactive T cell lines (TCL) can be generated from the CSF of GA-treated and treatment
`naive individuals. Proliferative responses (Δcpm) against four-fold serial dilutions of GA from 800–0.2 μg/mL GA in three rep-
`resentative CSF T cell lines from patient 1 generated before GA-treatment (CSF TCL 1–3) and after 3 months on GA treatment
`(CSF TCL 4–6) are shown (A). None of the T cell lines displayed any proliferation in response to 20–0.003 μg/mL myelin basic
`protein or gluten in the same experiment (data not shown). Production of IL-5 and IFN-γ is shown for all cell lines (B).
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`significant in the CSF (P = 0.03), where a shift
`towards almost total dominance of Th2-biased GA-
`reactive T cells was observed.
`No consensus exists regarding the threshold ratios
`that define human T cells as Th1 or Th2. In addition
`to comparisons based on a potentially arbitrary clas-
`sification into Th1-biased and Th2-biased T cell lines
`it is therefore also important to compare absolute
`ratios of pro- and anti-inflammatory cytokines pro-
`duced by GA-reactive T cell lines from blood and
`CSF before and during GA treatment. Figure 2
`depicts the secretion of IL-5, IL-10, and IL-13 com-
`pared with IFN-γ produced by GA-reactive T cell
`
`lines before and during treatment for patients 1–3.
`Results for the secretion of IL-5, IL-10, and IL-13
`compared with IFN-γ and TNF-α in patients 1–3 com-
`bined are given in Figure 3. These results show that i)
`in T cells from CSF, GA treatment induced a highly
`significant increase in the secretion of IL-5, IL-10,
`and IL-13 compared with IFN-γ and TNF-α; ii) in T
`cells from blood, GA-treatment induced a highly sig-
`nificant increase in the secretion of IL-5, IL-10, and
`IL-13 compared with IFN-γ but not compared with
`TNF-α; iii) after 3–6 months on GA treatment, there
`is a prominent and significant Th2-polarization of
`GA-reactive T cells from both CSF and blood,
`
`Figure 2 Short-term glatiramer acetate (GA) treatment induces a Th2-bias of GA-reactive T cells, which is most prominent in
`CSF. Each data point represents the indicated cytokine ratio for individual GA-reactive T cell lines before and after 3 months
`(patient 1) or 6 months (patient 2 and 3) of treatment.
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`Figure 3 Glatiramer acetate (GA) treatment induces a pronounced and significant increase in the ratio between anti- and
`pro-inflammatory cytokines produced by GA-reactive T cells from CSF and blood. GA-reactive T cell lines were established
`from CSF and blood of patients 1–3 before and 3–6 months after initiation of GA treatment. Each data point represents the
`indicated cytokine ratio for one GA-reactive T cell
`line 48 h after GA stimulation. Lines represent mean ratios. *P < 0.05;
`**P < 0.01; ***P < 0.005. The absolute cytokine concentrations (pg/mL, mean ± SD) in all cell lines (CSF and blood, before
`and after combined) were 2339, 3045 (interferon [IFN-γ]), 1981, 2703 (tumor necrosis factor [TNF-α]), 3024, 4209 (IL-5),
`1188, 1385 (IL-10), and 3680, 3916 (IL-13). The mean absolute value for unstimulated cells, all cytokines combined was
`15 pg/mL, SD = 31 pg/mL.
`
`which is more pronounced in T cells from CSF. The
`Th2-polarization of GA-reactive T cells was evident
`in each of the three patients.
`IL-17 has recently been identified as an impor-
`tant pro-inflammatory cytokine [26]. In CSF, IL-17
`production was detected in 5 of 21 GA-reactive
`T cell lines during GA treatment compared with 10
`of 28 before treatment. In blood, IL-17 production
`was detected in 11 of 31 GA-reactive T cell lines dur-
`ing GA treatment compared with 18 of 37 before
`treatment. These differences were not significant
`(results not shown).
`Patients 1–3 had only received GA for 3–6 months.
`To assess the impact of prolonged treatment, we estab-
`lished 10 GA-reactive T cell lines from CSF and 23 GA-
`reactive T cell lines from blood obtained from two
`patients who had received GA for 24 and 36 months
`(patients 4 and 5). Interestingly, GA-reactive T cell
`lines from both CSF and blood of these patients dis-
`played a significantly stronger Th2-bias than GA-
`reactive T cells from the short-term treated patients
`
`(Figure 4a). Although based on few patients, these
`results
`suggest
`that
`long-term treatment
`further
`enhances the Th2-polarization of GA-reactive T cells,
`which is most pronounced in CSF.
`Combining results for all GA-reactive T cell lines
`generated after initiation of GA treatment from
`patients 1–5 further confirms that GA-reactive
`T cells from CSF display a more pronounced anti-
`inflammatory profile than GA-reactive T cells from
`the blood (Figure 4b). In sum, the mean IL-5/IFN-γ
`ratio of GA-reactive T cells from CSF was 35.8 (95%
`CI: 20.4–51.6), compared with 9.6 (95% CI:
`6.3–12.8)
`for GA-reactive T cells
`from blood
`(P < 0.005).
`
`GA-specific T cells from CSF are restricted by either
`HLA-DR or HLA-DP
`
`To investigate the characteristics of individual GA-
`specific T cells, we cloned GA-reactive T cell lines
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`Figure 4 Glatiramer acetate (GA)-reactive T cell lines from patients treated long-term (patients 4 and 5) display more pro-
`nounced Th2-bias than GA-reactive T cell
`lines from patients treated short-term (patients 1–3) with GA (A). Following
`3–36 months of GA treatment, GA-reactive T cells from the CSF of patients 1–5 display a more pronounced Th2-bias than
`T cells from blood (B). Concentrations of cytokines were measured in supernatants of proliferating GA-reactive T cell lines
`48 h after stimulation with GA. Each data point represents the IL-5/IFN-γ ratio for one cell
`line. *P < 0.05; **P < 0.01;
`***P < 0.005. The absolute IFN-γ and IL-5 concentrations (pg/mL, mean/SD) for all CSF and blood T cell lines from patient 4
`and 5 were 800, 1253 and 5361, 1874, respectively (For patients 1–3, see legend Figure 3).
`
`from patients 4 and 5 by limiting dilution. The
`cloning frequency was less than 5% making it
`more than 95% probable that each cell culture was
`monoclonal [27]. Forty-eight of 90 T cell clones
`from patient 4 and 154 of 379 T cell clones
`from patient 5 specifically recognized GA (mean
`Δcpm = 130239 and mean SI = 341 for 20 tested
`clones). Proliferative responses of representative
`T cell clones with and without HLA-blocking anti-
`bodies and HLA restriction are shown in Figure 5.
`Interestingly, all 10 tested T cell clones from blood
`were restricted by HLA-DR, whereas HLA-DP
`restricted T cells were cloned from CSF of both
`patients. One of five CSF T cell clones from patient
`4 and three of five CSF T cell clones from patient 5
`were restricted by HLA-DP.
`
`Cross-reactivity with myelin proteins
`
`GA-reactive T cells from mice and human blood
`have been reported to cross-recognize GA and mye-
`lin proteins, especially MBP [10,12,28,29]. To our
`knowledge, cross-reactivity in human T cells has
`not been confirmed at a clonal level. To investigate
`whether cross-reactivity can be detected at a clonal
`level, 20 GA-specific T cell clones (10 from CSF and
`10 from blood) from patient 4 and 5 were tested for
`proliferation against 531 peptides spanning most
`myelin proteins suggested to be associated with
`MS [30] and for cytokine production against 146
`peptides spanning MBP, MOG, and PLP. Because all
`T cell lines previously had produced either IFN-γ or
`IL-5 in response to GA, these two cytokines were
`used to assess cross-reactivity at the cytokine level.
`
`None of the 20 T cell clones showed any signs of
`proliferation or cytokine production in response to
`the peptide library. The T cells responded vigor-
`ously to GA in the same experiment (mean Δcpm:
`64,098; mean IFN-γ concentration 3400 pg/mL and
`mean IL-5 concentration 2330 pg/mL). The T cell
`clones were also tested against complete MBP.
`MBP did not elicit any proliferation, but we were
`able to detect a small amount of IFN-γ production
`in one T cell clone from blood (10 pg/mL elicited by
`MBP versus 350 pg/mL elicited by GA). MBP did not
`elicit any detectable cytokine production in T cell
`clones from CSF.
`GA-reactive T cell clones may represent a biased
`subpopulation of the total GA-reactive T cell reper-
`toire. To test if cross-reactive T cells were repre-
`sented in the polyclonal GA-reactive T cell lines,
`all 117 GA-reactive T cell lines from patients 1–3
`were tested for cross-reactivity against complete
`MBP. Neither MPB nor the control protein (gluten)
`elicited any detectable proliferation or cytokine
`production. The T cells responded vigorously to
`GA in the same experiment (mean IL-5 concen-
`tration 3900 pg/mL; mean IFN-γ concentration
`1880 pg/mL; mean Δcpm > 100,000).
`
`Discussion
`
`In the current study, we examined GA-reactive T
`cells from the CSF of humans and provided longitu-
`dinal information about the intrathecal immune
`response against GA at the clonal level. The princi-
`pal findings are that (i) GA-reactive T cells are pres-
`ent in the intrathecal compartment of MS patients
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`Figure 5 Glatiramer acetate (GA)-specific T cells cloned from CSF and blood of patients treated long-term with GA (patients
`4 and 5) display divergent HLA-restriction. Proliferative responses (in Δcpm) against GA and a control peptide GAD57–72 of
`two representative CSF T cell clones are given in Figure 5A. Figure 5B shows proliferative responses to GA (in cpm) of the
`same T cell clones with or without monoclonal antibodies against HLA- DR, -DQ, -DP and HLA class I. Medium control repre-
`sents T cells and antigen-presenting cells in the absence of GA.
`
`before and after initiation of GA-treatment; (ii) GA
`treatment induces a Th2-bias of GA-reactive T cells
`from CSF and blood; and iii) following treatment,
`the Th2-bias is more pronounced in GA-reactive T
`cells from CSF than from blood. These results sup-
`port an anti-inflammatory T cell-mediated mecha-
`nism of action for GA.
`Resting T cells are not able to penetrate the intact
`blood-brain barrier, whereas activated T cells seem
`to gain access to the CNS regardless of their specific-
`ity [31]. The finding that GA-reactive T cells were
`present in the CSF before treatment, therefore,
`suggests that some GA-reactive T cells exist in an
`activated state in the absence of treatment. Accord-
`ingly, previous studies have shown high frequen-
`cies of GA-reactive memory T cells in the blood of
`treatment-naive individuals [32,11].
`GA is quickly degraded upon subcutaneous
`administration, making it unlikely that GA reaches
`the CNS in sufficient concentrations to induce Th2-
`polarization within the CNS [33]. Although GA-
`
`reactive T cells were present in the CSF before treat-
`ment, the observation that GA treatment induced
`an increasing Th2-polarization in both CSF and
`blood is compatible with the notion that a Th2-
`shift is induced in the periphery, and that a subpop-
`ulation of GA-reactive T cells subsequently gains
`access to the CNS. An alternative or additional
`explanation is the migration of type II monocytes
`to the CNS and induction of Th2 T cells at the site
`of inflammation [5,6]. The Th2-shift in blood of the
`short-term treated patients may be less pronounced
`than previously observed in long-term treated
`patients [12], and it is therefore possible that the
`more pronounced Th2 induction observed in CSF
`is a transient phenomenon. However, the marked
`Th2-bias in CSF compared with blood in the two
`long-term treated patients suggests that there is a
`persistent difference in favor of a more pronounced
`Th2-bias in CSF compared with blood.
`intrathecal
`Several observations
`suggest
`that
`GA-reactive T cells are not a random selection of
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`GA-reactive T cells from blood. First, GA-reactive
`T cells from CSF were more Th2-biased than
`GA-reactive T cells from blood, possibly reflecting
`that GA induces Th2-skewing earlier in CSF than
`in blood or a preferential migration of strongly
`Th2-biased T cells to the CSF. Second, GA treatment
`imposed a more pronounced reduction in TNF-α
`secretion in GA-reactive T cells from CSF than
`from blood. Third, GA-specific T cells from the
`CSF were restricted by either HLA-DP or HLA-DR,
`whereas only HLA-DR restricted T cells were
`detected in blood. HLA restriction of GA-specific
`T cells from blood has previously been extensively
`studied, and HLA-DP restriction has to our knowl-
`edge not been reported [3,34]. The finding of
`HLA-DP restricted GA-reactive T cells in CSF was,
`therefore, highly unexpected and supports that a
`subset of GA-reactive T cells selectively accesses
`the intrathecal compartment.
`In this study, we only detected GA-reactive CD4+
`T cells. GA may also have an effect on CD8+ T cells
`as shown by clonal expansion of GA-reactive CD8+
`T cells from the blood of patients receiving GA [35].
`This difference may be related to different method-
`ological approaches. There are few reports on
`cloned GA-specific T cells [34] and to our knowl-
`edge no GA-specific CD8+ T cell clones have been
`established. The method applied in this study has
`previously allowed generation of T cell lines and
`clones with several specificities from the limited
`number of CD4+ T cells in CSF of MS patients
`[22,36], but there is currently no adequate method
`to establish antigen specific CD8+ T cell lines and
`clones from CSF and we can, therefore, not exclude
`that CD8+ GA-reactive T cells are present in the
`intrathecal compartment.
`The dissimilarities between GA-reactive T cells
`from CSF and blood can hardly be explained by dif-
`ferences in APCs or other in vitro factors because
`identical procedures were used to generate T cells
`from both compartments. The mechanisms recruit-
`ing a particular subset of GA-reactive T cells to the
`CSF are not clear, but several possibilities exist.
`Restimulation of GA-reactive T cells by cross-
`recognition of myelin antigens within the CNS
`could explain expansion of a particular subset of
`GA-reactive T cells within the CNS [13]. However,
`GA-reactive T cells from blood and CSF in this
`study displayed almost no cross-reactivity. Previous
`studies have shown that a proportion of human
`GA-reactive T cell lines from blood cross-recognize
`MBP [11,32]. Moreover, GA treatment has been
`found to induce degenerated T cell responses with
`broad
`cross-reactivity,
`suggesting
`that
`cross-
`recognition might also occur outside the CNS [12].
`Cross-recognition of myelin proteins can, therefore,
`hardly explain the differences between GA-reactive
`T cells from CSF and blood. Another possibility is
`
`that a selected subpopulation of GA-reactive T cells
`penetrates the blood-brain barrier. T cells from CSF
`display a more activated phenotype than T cells
`from blood [20,23], and differences in cytokine pro-
`files could reflect differences in activation status.
`However, a putative difference in activation status
`between GA-reactive T cells in blood and CSF
`would be expected to decline during prolonged
`treatment and could hardly explain the selective
`finding of HLA-DP restricted T cells in CSF. Further
`studies of expression of chemokine receptors and
`adhesion molecules on T cells from blood and CSF
`may contribute to our understanding of
`these
`intriguing findings. The expression patterns of
`such molecules are, however, sensitive to in vitro
`changes during cell culture and are difficult to
`establish directly ex vivo with the limited numbers
`of T cells available from CSF.
`
`Several studies on T cells from mice and human
`blood have shown cross-reactivity between GA and
`MBP. In one study, some GA-reactive T cell lines
`proliferated vigorously against MBP [32]. In other
`studies, evidence of cross-reactivity has been absent
`or limited to cytokine secretion [10,12,28,29,37]. In
`this study, we primarily studied cross-reactivity
`between GA and myelin proteins at a clonal level,
`whereas previous studies have studied polyclonal
`T cell lines [11,12]. The strict approach adopted
`here may have contributed to the finding of very
`limited cross-reactivity in this study. By definition,
`cross-reactivity of T cells means that one T cell
`receptor
`recognizes two different antigens and
`must, therefore, be established at a clonal
`level
`[38,39]. Because of limited cell numbers,
`it was
`only possible to test the extensive panel of myelin
`protein peptides at one concentration, and cross-
`reactivity between GA and myelin protein peptides
`with low affinity for the relevant HLA molecule or
`GA-reactive T cell receptor can, therefore, not be
`excluded. It must, however, be kept in mind that
`only 10–17% of polyclonal GA-reactive T cell lines
`from MS patients cross-recognized MBP in previous
`studies
`[11,32]. The limited finding of cross-
`reactivity at the clonal
`level
`in this study was,
`therefore, not unexpected and agrees with recent
`findings showing that cross-reactivity of adoptively
`transferred T cells
`from GA-treated mice was
`not necessary for the therapeutic benefit in recipi-
`ent mice [5]. These findings support previous
`suggestions that GA may also be effective in inflam-
`matory diseases outside the CNS [40,41].
`
`an anti-
`support
`In summary, our data
`inflammatory mechanism of action for GA at the
`site of the pathogenic process in MS and highlight
`the need to study intrathecal T cells in MS patients.
`
`Multiple Sclerosis 2008; 14: 749–758
`
`http://msj.sagepub.com
`
`Page 8 of 10
`
`YEDA EXHIBIT NO. 2059
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`Acknowledgements
`
`The project has received financial support from
`Sanofi Aventis, The Odd Fellow Organization of
`Norway, The Multiple Sclerosis Society of Great
`Britain and Northern Ireland (grant 589–00), The
`National MS Society, and The National Institute of
`Health (grant AI53439 and AI65463). Professor
`Magne Thoresen at Department of Biostatistics at
`the University of Oslo is acknowledged for statisti-
`cal support. Nitin J. Karandikar at UT Southwestern
`Medical Center, Dallas, USA is acknowledged for
`providing synthetic peptides and purity data.
`Danny Douek is acknowledged for provision of
`reagents for the peptide synthesis.
`
`Reference
`
`1. Johnson, KP, Brooks, BR, Cohen, JA, et al. Copolymer 1
`reduces relapse rate and improves disability in relapsing-
`remitting multiple sclerosis: results of a phase III multi-
`center, double-blind placebo-controlled trial. The Copol-
`ymer 1 Multiple Sclerosis Study Group. Neurology 1995;
`45: 1268–1276.
`2. Johnson, KP, Brooks, BR, Cohen, JA, et al. Extended use
`of glatiramer acetate (Copaxone) is well tolerated and
`maintains its clinical effect on multiple sclerosis relapse
`rate and degree of disability. Copolymer 1 Multiple Scle-
`rosis Study Group. Neurology 1998; 50: 701–708.
`3. Fridkis-Hareli, M, Strominger, JL. Promiscuous binding of
`synthetic copolymer 1 to purified HLA-DR molecules. J
`Immunol 1998; 160: 4386–4397.
`4. Sanna, A, Fois, ML, Arru, G, et al. Glatiramer acetate
`reduces lymphocyte proliferation and enhances IL-5 and
`IL-13 production through modulation of monocyte-
`derived dendritic cells in multiple sclerosis. Clin Exp
`Immunol 2006; 143: 357–362.
`5. Weber, MS, Prod’homme, T, Youssef, S, et al. Type II
`monocytes modulate T cell-mediated central nervous
`system autoimmune disease. Nat Med 2007; 13: 935–943.
`6. Kim, HJ, Ifergan, I, Antel, JP, et al. Type 2 monocyte and
`microglia differentiation mediated by glatiramer acetate
`therapy in patients with multiple sclerosis. J Immunol
`2004; 172: 7144–7153.
`7. Ziemssen, T, Schrempf, W. Glatiramer acetate: mechan-
`isms of action in multiple sclerosis. Int Rev Neurobiol
`2007; 79: 537–570.
`8. Ragheb, S, Abramczyk, S, Lisak, D, Lisak, R. Long-term
`therapy with glatiramer acetate in multiple sclerosis:
`effect on T cells. Mult Scler 2001; 7: 43–47.
`9. Chen, M, Conway, K, Johnson, KP, Martin, R, Dhib-
`Jalbut, S. Sustained immunological effects of Glatiramer
`acetate in patients with multiple sclerosis treated for over
`6 years. J Neurol Sci 2002; 201: 71–77.
`10. Aharoni, R, Teitelbaum, D, Sela, M, Arnon, R. Copolymer
`1 induces T cells of the T helper type 2 that crossreact
`with myelin basic protein and suppress experimental
`autoimmune encephalomyelitis. Proc Natl Acad Sci U S A
`1997; 94: 10821–10826.
`11. Neuhaus, O, Farina, C, Yassouridis, A, et al. Multiple scle-
`rosis: comparison of copolymer-1-reactive T cell lines
`from treated and untreated subjects reveals cytokine
`shift from T helper 1 to T helper 2 cells. Proc Natl Acad
`Sci U S A 2000; 97: 7452–7457.
`
`GA-reactive T cells from the CSF of MS patient
`
`757
`
`12. Duda, PW, Schmied, MC, Cook, SL, Krieger, JI, Hafler,
`DA. Glatiramer acetate (Copaxone) induces degenerate,
`Th2-polarized immune responses in patients with multi-
`ple sclerosis. J Clin Invest 2000; 105: 967–976.
`13. Aharoni, R, Teitelbaum, D, Leitner, O, Meshorer, A, Sela,
`M, Arnon, R. Specific Th2 cells accumulate in the central
`nervous system of mice protected against experimental
`autoimmune encephalomyelitis by copolymer 1. Proc
`Natl Acad Sci U S A 2000; 97: 11472–11477.
`14. Aharoni, R, Meshorer, A, Sela, M, Arnon, R. Oral treat-
`ment of mice with copolymer 1 (glatiramer acetate)
`results in the accumulation of specif