Multiple sclerosis: 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
`
`Oliver Neuhaus*, Cinthia Farina*, Alexander Yassouridis†, Heinz Wiendl*, Florian Then Bergh‡, Tatjana Dose‡,
`Hartmut Wekerle*, and Reinhard Hohlfeld*‡§
`
`*Department of Neuroimmunology, Max Planck Institute of Neurobiology, Am Klopferspitz 18A, 82152 Martinsried, Germany; †Department of Statistics,
`Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany; and ‡Institute for Clinical Neuroimmunology and Department of
`Neurology, Klinikum Grosshadern, Ludwig Maximilians University, Marchioninistrasse 15, 81366 Munich, Germany
`
`Communicated by Michael Sela, Weizmann Institute of Science, Rehovot, Israel, April 13, 2000 (received for review February 10, 2000)
`
`Copolymer 1 (COP), a standardized mixture of synthetic polypep-
`tides consisting of L-glutamic acid, L-lysine, L-alanine, and L-ty-
`rosine, has beneficial effects in multiple sclerosis and experimental
`autoimmune encephalomyelitis. We selected a panel of 721 COP-
`reactive T cell lines (TCL) from the blood of COP-treated and
`untreated multiple sclerosis patients and from healthy donors by
`using the split-well cloning technique. All TCL selected with COP
`proliferated in response to COP but not to myelin basic protein
`(MBP). Conversely, 31 control TCL selected with MBP proliferated
`in response to MBP but not to COP. We used intracellular double-
`immunofluorescence flow cytometry for quantitative analysis of
`cytokine production (IL-4, IFN-g) by the TCL. The majority of the
`COP-reactive TCL from untreated multiple sclerosis patients and
`normal donors predominantly produced IFN-g and, accordingly,
`were classified as T helper 1 cells (TH1). In contrast, the majority of
`the COP-reactive TCL from COP-treated patients predominantly
`(but not exclusively) produced IL-4 —i.e., were TH2 (P < 0.05 as
`assessed by using a suitable preference intensity index). Longitu-
`dinal analyses revealed that the cytokine profile of COP-reactive
`TCL tends to shift from TH1 to TH2 during treatment. Interestingly,
`although there was no proliferative cross-reaction, about 10% of
`the COP-reactive TCL responded to MBP by secretion of small
`amounts of IL-4 or IFN-g, depending on the cytokine profile of the
`TCL. These results are consistent with a protective effect of COP-
`reactive TH2 cells. It is hypothesized that these cells are activated
`by COP in the periphery, migrate into the central nervous system,
`and produce immunomodulatory cytokines after local recognition
`of MBP.
`
`Copolymer 1 (COP, glatiramer acetate, Copaxone) is a stan-
`
`dardized mixture of synthetic polypeptides consisting of
`L-glutamic acid, L-lysine, L-alanine, and L-tyrosine with a defined
`molar residue ratio of 0.14:0.34:0.43:0.1 and an average molec-
`ular mass of 4,700–11,000 Da. COP has beneficial effects on the
`clinical course and magnetic resonance imaging (MRI)-defined
`brain lesions of patients with multiple sclerosis (MS) (1–4).
`Furthermore, COP has suppressive and protective effects in
`experimental autoimmune encephalomyelitis (EAE) induced by
`various encephalitogens in different species (5–9), but not in
`other experimental autoimmune models (10). On the basis of
`extensive in vitro and in vivo studies in EAE, it has been proposed
`that COP acts by two basic mechanisms, (i) competition with
`myelin basic protein (MBP) at the MHC and T cell antigen
`receptor (TCR) level (11–14), and (ii) induction of T helper 2
`(TH2)-type regulatory T cells (13, 15, 16). Relatively little is
`presently known about the in vitro and in vivo effects of COP in
`the human immune system (14, 17–19).
`In the present study we isolated and analyzed a large panel
`of human COP-reactive T cell
`lines (TCL) by using the
`split-well cloning protocol (20). Consistent with previously
`
`7452–7457 u PNAS u
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`June 20, 2000 u vol. 97 u no. 13
`
`reported results in EAE animals (13), we found that treatment
`with COP induces a shift from a TH1-biased cytokine profile
`observed in COP-reactive TCL obtained from untreated MS
`patients and healthy donors, toward a TH2-biased profile
`observed in TCL obtained from COP-treated patients. Fur-
`thermore, 8–15% of the tested COP-reactive TCL responded
`to MBP by secretion of small amounts of either IL-4 (TH2 or
`TH0 lines) or IFN-g(TH1 or TH0 lines), although none of our
`COP-reactive TCL proliferated in the presence of MBP. The
`results indicate that the therapeutic effect of COP in MS may
`be related to a cytokine shift of COP-reactive T cells from TH1
`to TH2, and to a cross-reaction with MBP at the level of
`cytokine production.
`
`Materials and Methods
`Patients and Control Subjects. Blood was drawn with informed
`consent from 26 MS patients and 4 healthy donors. At the time
`of first sampling, 15 patients were treated with COP (20 mg s.c.
`per day; Teva Pharma, Kirchzarten, Germany). Six of the
`untreated patients were later started on COP; from these
`patients, only the data before treatment were included in the
`statistical analysis. All donors were HLA-typed (Table 1).
`
`Antigens. COP (batch 242992997, average molecular mass 7,000
`Da, and batch 242992899, average molecular mass 6,400 Da) was
`obtained from Teva Pharmaceutical Industries, Petah Tiqva,
`Israel. The two batches were cross-reactive with each other as
`assessed in a proliferation assay. MBP was purified from human
`brain by standard methods (21). Overlapping peptides covering
`the entire human MBP molecule were synthesized by using an
`automatic peptide synthesizer (431A; Applied Biosystems). The
`extracellular Ig-like domain of human myelin-oligodendrocyte
`glycoprotein (MOG), amino acids 1–125, was expressed in an
`Escherichia coli system as described before (22). As a recombi-
`nant control, rat S100b protein was expressed and prepared in
`the same way (23). Tetanus toxoid (TT) was kindly provided by
`Chiron Behring, Marburg, Germany. Tuberculin purified pro-
`
`Abbreviations: APC, antigen-presenting cells; COP, copolymer 1; EAE, experimental auto-
`immune encephalomyelitis; MBP, myelin basic protein; MOG, myelin-oligodendrocyte
`glycoprotein; MS, multiple sclerosis; PE, phycoerythrin; PPD, purified protein derivative; SI,
`stimulation index; TCL, T cell line(s); TCR, T cell antigen receptor; TH0, TH1, and TH2, T
`helper type 0, 1, and 2 cells, respectively; TT, tetanus toxoid.
`§To whom reprint requests should be addressed at: Institute for Clinical Neuroimmunology,
`Klinikum Grosshadern, Ludwig Maximilians University, Marchioninistrasse 15, 81366 Mu-
`nich, Germany. E-mail: hohlfeld@neuro.mpg.de.
`
`The publication costs of this article were defrayed in part by page charge payment. This
`article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
`§1734 solely to indicate this fact.
`
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`MEDICALSCIENCES
`
`Table 1. Basic characteristics of MS patients and healthy
`donors (HD)
`
`No.
`
`Initials
`
`Sex
`
`Age, yr
`
`Duration of
`disease, yr*
`
`EDSS*†
`
`HLA-DR
`type
`
`MS 1
`MS 2
`MS 3
`MS 4
`MS 5
`MS 6
`MS 7
`MS 8
`MS 9
`MS 10
`MS 11
`MS 12
`MS 13
`MS 14
`MS 15
`MS 16
`MS 17
`MS 18
`MS 19
`MS 20
`MS 21
`MS 22
`MS 23
`MS 24
`MS 25
`MS 26
`HD 1
`HD 2
`HD 3
`HD 4
`
`SSt
`HK
`AZ
`HM
`SW
`CS
`GJ
`WHa
`HC
`SZ
`LN
`BG
`UB
`US
`SH
`BS
`KR
`RD
`RM
`IB
`MB
`MC
`BK
`RR
`RO
`AF
`WH
`CH
`CB
`VV
`
`F
`M
`M
`F
`F
`F
`F
`F
`F
`F
`F
`F
`F
`F
`F
`M
`F
`M
`F
`F
`F
`F
`F
`M
`F
`F
`M
`M
`F
`F
`
`35
`34
`28
`25
`40
`42
`30
`43
`32
`34
`36
`33
`39
`35
`31
`21
`39
`40
`34
`48
`34
`30
`43
`28
`33
`18
`30
`31
`29
`34
`
`9
`8
`1
`2
`11
`4
`1
`1
`2
`4
`1
`4
`10
`2
`3
`1
`4
`17
`7
`20
`1
`3
`5
`2
`2
`2
`—
`—
`—
`—
`
`4.0
`1.0
`1.0
`1.0
`3.5
`1.0
`1.0
`2.5
`2.5
`2.5
`2.5
`1.5
`6.0
`1.0
`1.0
`1.5
`1.5
`2.5
`1.5
`4.0
`1.0
`5.0
`1.0
`4.0
`3.5
`1.0
`—
`—
`—
`—
`
`2, 5
`3, 12
`2, 7
`2
`2, 8
`4, 8
`4, 6
`2, 11
`11, 12
`2, 7
`1, 2
`4, 7
`4, 7
`7, 10
`3
`13, 14
`1, 7
`2
`7, 13
`7, 11
`2, 7
`3, 7
`2, 4
`2, 11
`7, 13
`10, 12
`2, 11
`11, 13
`4, 11
`4, 15
`
`*At the time of first sampling.
`†EDSS, expanded disability status scale (31).
`
`tein derivative (PPD; batch RT49) was purchased from Statens
`Serum Institut, Copenhagen.
`
`Cell Culture and Isolation of TCL. All cell cultures were performed
`in RPMI medium 1640 (GIBCO) supplemented with 5% pooled
`and heat-inactivated human AB serum (German Red Cross,
`Baden-Baden) containing 2 mM glutamine, 100 unitsyml pen-
`icillin, 100 mgyml streptomycin (all from GIBCO), and 20 mgyml
`ciprofloxacin (Ciprobay; Bayer Vital, Leverkusen, Germany)
`and incubated at 37°C in an atmosphere of 5% CO2y95% air.
`Long-term TCL were selected from peripheral blood mononu-
`clear cells (PBMC) by using the split-well cloning technique as
`previously described (20).
`
`Proliferation Assay. Antigen-specific proliferation was deter-
`mined by [3H]thymidine incorporation at several restimulation
`(R) steps (minimum R2). Autologous PBMC x-irradiated with
`40 Gy (Stabiloplan 2; Siemens, Erlangen, Germany) were used
`as antigen-presenting cells (APC). APC (1 3 105 per well) were
`preincubated for 1 h in the presence or absence of various
`antigens (COP, 50 mgyml; MBP, 30 mgyml; MBP peptides, 10
`mgyml; MOG, 15 mgyml; S100, 15 mgyml; TT, 2 mgyml; PPD, 10
`mgyml) in 96-well round-bottom microtiter plates (Nunc) in
`duplicate. Phytohemagglutinin (PHA, 10 mgyml; Sigma) was
`used as a maximal stimulus. TCL cells were pooled, kept on ice
`for 3–4 h, and added to each well. After 48 h, [methyl-
`3H]thymidine (0.2–0.5 mCi per well; Amersham Buchler, Braun-
`schweig, Germany; 1 mCi 5 37 kBq) was added for another
`
`16–18 h. Cells were harvested and [3H]thymidine incorporation
`was measured with a direct b-counter (Matrix TM 96; Packard,
`Frankfurt). Note that this method yields only 20% of the counts
`obtained by standard liquid scintillation systems. Only TCL with
`a minimum of 300–500 absolute cpm and a minimum stimulation
`index (SI) of 3.0 (except TT-reactive TCL: SI $ 1.8) were taken
`into account. The median SI of the COP-reactive TCL was 44.7
`(range 3.0–1,826).
`The MHC restriction was determined by using blocking mAbs
`to HLA-DR (L243; American Type Culture Collection) or
`HLA-DQ (SPVL3; Immunotech, Marseille, France), which were
`preincubated with the APC for 45 min at a final concentration
`of 20 mgyml before adding the antigens.
`
`Cytokine Production. Antigen-induced production of IL-4 and
`IFN-gwas measured by ELISA (Endogen, Woburn, MA), using
`the supernatants of the proliferation assay. For this purpose,
`aliquots of 100 ml were removed from each well just before
`labeling with [methyl-3H]thymidine. Cytokine concentrations at
`least 2 SD above background were considered positive. Accord-
`ing to the manufacturer’s manuals, the lower limit of detection
`(sensitivity) was ,2 pgyml IL-4 or IFN-g.
`
`Characterization of the Cytokine Profile by Intracellular Double-
`Fluorescence Flow Cytometry. The cytokine profile of the TCL was
`analyzed 8–10 days after restimulation in the absence of viable
`APC. COP-reactive TCL cells were stimulated with phorbol
`12-myristate 13-acetate (PMA, 2.5 mgyml) and ionomycin (250
`ngyml; both from Sigma) for 3 h, the last 2 h in the presence of
`the glycoprotein secretion blocker monensin (2 nmolyml; Sig-
`ma). The T cells were then washed with PBS, fixed with 4%
`paraformaldehyde (Merck), and permeabilized with 0.1% sapo-
`ninyPBS (Sigma). The T cells were stained by using appropriate
`concentrations of mAbs directed against IL-4 [8D4–8, phyco-
`erythrin (PE)-labeled; PharMingen] and IFN-g [B27, fluores-
`cein isothiocyanate (FITC)-labeled; PharMingen] or the corre-
`sponding isotype controls (mouse IgG1 PE-labeled, Becton
`Dickinson; mouse IgG1 FITC-labeled, Immunotech).
`The cytokine profile was analyzed with a FACScan (Becton
`Dickinson). Data from 5,000 cells were accumulated and the
`results were analyzed as dot plots representing the relative
`fluorescence intensity (Fig. 1). On a dot plot showing forward
`and sideward scatter, lymphoid cells were gated for further
`analysis (Fig. 1). Note that for unknown reasons, dead cells
`stained positive with the anti-IL-4 mAb and had to be excluded
`by gating. To define the predominant cytokine profile of each
`TCL, the following algorithm was applied (cf. Fig. 1): Only cells
`positive for IFN-g, IL-4, or both were considered ‘‘activated.’’ If
`a single-positive fraction (i) exceeded 50% of all ‘‘activated’’
`cells, and (ii) was at least 20% higher than the other single-
`positive fraction, the line was defined ‘‘TH1’’ (IFN-g) or ‘‘TH2’’
`(IL-4). All other TCL were designated as ‘‘TH0.’’ TCL with less
`than 100 ‘‘activated’’ events were not taken into account. Five or
`more TCL per donor were considered representative.
`
`Phenotypical Characterization of TCL by Flow Cytometry. TCL were
`stained with labeled mAbs directed against CD3 (UCHT1,
`FITC-labeled; DAKO), CD4 (RPA-T4, PE-labeled, PharMin-
`gen), and CD8 (DK25, FITC-labeled; DAKO) and the corre-
`sponding isotype controls described above. The TCR Vb (vari-
`able region) repertoire was analyzed by using mAbs recognizing
`the following subfamilies: Vb2, Vb3, Vb5.3, Vb7, Vb8, Vb9,
`Vb11, Vb12, Vb13.6, Vb14, Vb16, Vb17, Vb18, Vb20, Vb21.3,
`Vb22, Vb23 (all Immunotech), Vb3.1, Vb5a, Vb6.7 (T-Cell
`Diagnostics, Woburn, MA), Vb5b (T-Cell Sciences, Cambridge,
`MA), Vb7.1 (Labgen, Frankfurt). mAbs and isotype controls
`(mouse IgG1, Becton Dickinson; mouse IgG2a and IgG2b,
`Cymbus, Chandlers Ford, U.K.) were visualized with an FITC-
`
`Neuhaus et al.
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`PNAS u
`
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`Cytokine profile of COP-reactive TCL analyzed by intracellular dou-
`Fig. 1.
`ble-fluorescence flow cytometry. (Upper Left) Dot plot of scatter parameters.
`(Upper Right) Isotype controls of one representative TCL. (Lower) Cytokine
`profiles of three representative TCL. Dot-plot events in the single-positive and
`double-positive quadrants were added. They represent ‘‘activated’’ cells. The
`numbers represent the percentage of events in each quadrant relative to the
`total number of activated cells. TH1, TH0, and TH2 assignments were made
`according to the algorithm described in the text.
`
`labeled goat anti-mouse IgG antibody (Jackson ImmunoRe-
`search, West Grove, PA).
`
`Statistical Analysis. Each line was assigned one of three numbers,
`namely 21 for the TH1 lines, 0 for the TH0 lines, and 11 for the
`TH2 lines. For each individual donor, a ‘‘preference intensity
`index’’ (I) was calculated for the TCL isolated from the donor
`according to the formula:
`~11! 3 nTH2 1 ~0! 3 nTH0 1 ~21! 3 nTH1
`nTH2 1 nTH0 1 nTH1
`
`I 5
`
`,
`
`where nTH2, nTH0, and nTH1 denote the total number of TH2-,
`TH0-, and TH1-type TCL isolated from a given individual at a
`particular time. In this way, I is a metric variable expressing the
`proportion of TH1 TCL with respect to TH2 TCL independent
`of the absolute number of TCL obtained per donor. I varies
`between two extreme values, 21 (when all TCL are TH1) and
`11 (when all TCL are TH2). For comparing the three groups
`(treated vs. untreated MS patients vs. healthy donors), a one-
`factorial analysis of covariance (ANCOVA) was applied with the
`age of the donors as covariate. If a significant group effect was
`observed, post hoc tests (tests with contrasts) were applied to
`identify pairs of groups with significant preference intensity
`differences. a 5 0.05 was accepted as nominal level of signifi-
`cance and corrected for the post hoc tests according to the
`Bonferroni procedure to keep the type I error # 0.05.
`
`Results
`Isolation and Characterization of COP-Reactive TCL. We plated a total
`of 3,031 wells and isolated a panel of 721 COP-reactive TCL
`(23.8%). One hundred and sixty TCL were isolated from 1,054
`plated wells (15.2%, untreated MS patients), 300 TCL from
`1,155 plated wells (26.0%, COP-treated patients), and 90 lines
`from 330 plated wells (27.3%, healthy donors). In addition, 171
`TCL could be established after onset of COP treatment from 492
`plated wells (34.8%, previously untreated patients). All COP-
`
`Proliferative response of a representative COP-reactive and a MBP-
`Fig. 2.
`reactive TCL. TCL were stimulated with COP, MBP, various control antigens
`(MOG, S-100b, and TT), or the T-cell mitogen phytohemagglutinin (PHA).
`There was no detectable cross-reaction between COP and MBP at the level of
`proliferation. Ag, antigen.
`
`reactive TCL showed a proliferative response to COP (mean SI
`78.2, median SI 44.7, range 3.0–1,826), but none of the tested
`TCL proliferated significantly in response to MBP, MBP pep-
`tides, or any other antigens tested (MOG, S100b, and TT) (Fig.
`2). Vice versa, 31 MBP-reactive TCL established from 2 un-
`treated patients (25 lines) and 2 healthy donors (6 lines) prolif-
`erated in response to MBP (mean SI 223, median SI 109, range
`5.3–1,044) but not to COP (Fig. 2). Interestingly, thus far, we
`have been unable to select MBP-specific TCL from 474 plated
`wells cultured from COP-treated patients, whereas TT-reactive
`TCL (16 lines) and PPD-reactive TCL (17 lines) could be easily
`isolated from both treated and untreated donors.
`The complete phenotype, HLA restriction, and TCR usage
`were analyzed only in a subset of TCL. With the exception of two
`TCL which were composed of approximately equal numbers of
`CD41 and CD81 cells, the analyzed TCL (n 5 40) were
`predominantly CD41 (mean 87.4%, median 93.4%). The tested
`TCL (n 5 12) were restricted by HLA-DR as assessed by 42–99%
`inhibition with an anti-HLA-DR mAb. TCR-Vb expression
`(analyzed in 10 TCL) was heterogeneous.
`
`Cross-Sectional Analysis: Effect of COP Treatment on the Cytokine
`Profile of COP-Reactive TCL. We analyzed the cytokine profile of
`693 of our COP-reactive TCL (150 TCL from untreated patients,
`284 TCL from COP-treated patients, 90 TCL from healthy
`donors, 169 TCL from previously untreated patients after onset
`of COP treatment) by double-fluorescence flow cytometry at
`various restimulation steps (mostly R2 and R3). To minimize the
`influence of culture conditions, we strictly kept all TCL under
`identical conditions. Longitudinal comparisons of the cytokine
`profile of individual TCL at different restimulation steps showed
`that despite small fluctuations, the predominant cytokine profile
`remained stable: Of nine TCL that were followed up to five times
`(R2–R16), seven TCL strictly kept their predominant cytokine
`profile, whereas two TCL shifted from TH1 (R3) to TH0
`(R8–R14).
`To further validate this method, we assessed the COP-induced
`secretion of IFN-g or IL-4 in a subset of 164 TCL by ELISA.
`Virtually all (98%) of the tested TCL showed a COP-induced
`cytokine response.
`Fig. 3 shows the overall cytokine profiles of the complete panel
`of our COP-reactive TCL. Comparing the three groups (treated
`
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`Fig. 3. Overview of the cytokine profiles of COP-reactive TCL of healthy donors (A), untreated MS patients (B), and COP-treated MS patients (C). Each column
`under patients’ initials represents a panel of COP-reactive TCL isolated at one time point. Red indicates TH1, gray TH0, and green TH2. In C, the duration of
`treatment is indicated at the top. Large arrows indicate intraindividual longitudinal comparisons before and during COP treatment. Small arrows indicate
`intraindividual longitudinal comparisons after various times of COP treatment. (D) Comparison of the preference intensity indices I (calculated as described in
`the text) in untreated MS patients (left, r) and healthy controls (left, (cid:140)), and COP-treated MS patients (right, r). I , 0 indicates a TH1 bias and I . 0 a TH2 bias,
`independent of the absolute number of TCL obtained per donor. Open squares represent mean preference intensity indices (6SD) of untreated donors (left) and
`COP-treated patients (right). Lines indicate intraindividual comparisons of six patients (only the data before treatment were included in the statistical analysis).
`
`vs. untreated MS patients vs. healthy donors), the analysis of
`covariance revealed a significant effect [F(2, 29) 5 10.26,
`significance of F 5 0.001]. Although the donors were not
`age-matched, ‘‘age’’ as a covariate did not seem to play a role.
`The mean preference intensity index in COP-treated MS pa-
`tients (I 5 10.23) was significantly higher (indeed positive and
`thus skewed to TH2) than in untreated MS patients (I 5 20.42)
`and untreated control subjects (I 5 20.65) (tests with contrasts,
`P , 0.05). By pooling untreated patients and healthy donors and
`subsequently comparing the mean preference intensity of the
`combined group (I 5 20.48) with the treated patients’ group
`(I 5 10.23) by ANCOVA, significant differences were observed
`[F (1, 29) 5 20.40, significance of F , 0.0001]. The results suggest
`that treatment with COP induces a shift from TH1 (COP lines
`from untreated patients and healthy donors) toward TH2 (COP-
`treated MS patients). Clearly, the data do not allow us to decide
`whether this change occurs at the level of the cell population,
`individual cells, or both.
`The TH2-inducing effect was specific for COP, as it was not
`seen with PPD- and TT-reactive TCL from two treated patients
`
`(one example is shown in Fig. 4). After 6 months of COP
`treatment, the COP-reactive TCL from the donor CS were either
`TH2 or TH0 (cf. Fig. 3), whereas 3 of 3 PPD- and 2 of 2
`TT-reactive TCL were TH1 (Fig. 4). Moreover, while 14 of 15
`COP-reactive TCL from the donor SH (after 15 months of
`treatment) were TH2 and 1 was TH0, all of this donor’s 4 PPD-
`and 3 TT-reactive TCL were TH1.
`
`Longitudinal Analysis: Change of the Cytokine Profile of COP-Reactive
`TCL During Treatment of Individual Patients. To further corroborate
`the results obtained by cross-sectional analysis, we investigated
`some patients longitudinally, that is, before and after various
`time periods of COP treatment (Fig. 3).
`Cytokine profile before and after treatment. TCL from one
`patient (GJ) had an unbiased cytokine profile before treatment
`(preference intensity I 5 20.08), which almost completely
`shifted toward TH2 after 1 month of treatment (I 5 10.92).
`After 3 months of treatment, the cytokine profile shifted back to
`TH0 (I 5 20.10) (Fig. 3). Another patient, CS, kept a TH1-
`biased cytokine profile from before treatment (I 5 20.38)
`
`Neuhaus et al.
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`PNAS u
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`Cytokine profile of representative COP-, PPD- and TT-reactive TCL
`Fig. 4.
`obtained from one COP-treated patient (month 6) and analyzed by intracel-
`lular double-fluorescence flow cytometry. The numbers represent the per-
`centage of events in each quadrant relative to the total number of activated
`cells. TH1 and TH2 assignments were made according to the algorithm de-
`scribed in the text (cf. Fig. 1).
`
`during at least 2 months of treatment (I 5 20.42). After 6
`months of treatment, a slight shift toward TH2 was observed (I 5
`10.19). Four other patients (WHa, HC, SZ, and LN) showed no
`significant shift after 1–3 months of treatment.
`To exclude that the longitudinal changes of the cytokine
`profile were related to the source of APC (autologous APC from
`untreated patients vs. autologous APC from treated patients),
`we compared five COP-reactive TCL from an untreated patient
`(GJ) restimulated with COP presented by (i) frozen APC that
`had been collected before treatment, and (ii) fresh autologous
`APC obtained after 4 weeks of treatment. The cytokine profile
`of these TCL were virtually identical (four lines were TH2, one
`line was TH1), independent of the source of APC (data not
`shown).
`Cytokine profile during prolonged treatment. Additional TCL
`could be obtained from individual patients after various inter-
`vals of prolonged treatment (Fig. 3). In patient RO, there was a
`trend for a shift from TH1 to TH2 (the preference intensity
`index I was 20.14 after 1 month and 10.42 after 6 months of
`treatment) which virtually shifted back to TH1 after 9 months of
`treatment (I 5 20.10) (Fig. 3). Another patient (RR) showed the
`opposite trend during therapy. At month 1 and 2, the cytokine
`profile was biased toward TH2 (I 5 10.67), at month 9, it was
`TH0-dominated (I 5 20.13), whereas at month 12, the cytokine
`profile became TH1 (I 5 20.48). The cytokine profiles of MC,
`BK, and AF remained TH2 up to 9 months of treatment.
`
`Cytokine Secretion of COP-Reactive TCL After Cross-Stimulation with
`MBP. As mentioned before, none of the tested human COP-
`reactive TCL showed a proliferative response when challenged
`with MBP, and vice versa. Previous observations in EAE dem-
`onstrated that COP-reactive TCL from mice treated with COP
`also did not proliferate in the presence of MBP, but some
`COP-reactive TCL produced IL-4 when stimulated with MBP
`(13). On the basis of these results, we tested whether human
`COP-reactive TCL can also be induced to cytokine secretion by
`stimulation with MBP. We found that, indeed, several COP-
`reactive TCL responded to MBP by secretion of low amounts of
`IL-4 or IFN-g, depending on the predominant cytokine profile
`of the TCL. We tested 111 TCL for IL-4 secretion, and 53 TCL
`for IFN-g secretion. Of these, 9y111 (8.1%) and 8y53 (15.1%)
`responded to MBP by significant (.2 SD above background)
`production of IL-4 or IFN-g, irrespective of the source of the
`TCL (treated and untreated MS patients, healthy donors) (Fig.
`5). Conversely, 1 of 7 tested MBP-specific TCL responded to
`COP by production of IL-4 (5.5 pgyml vs. 0 pgymg in the negative
`control). Furthermore, 2 of 12 tested COP-reactive TCL re-
`
`Proliferative response and cytokine production by two COP-reactive
`Fig. 5.
`TCL. The left vertical axis denotes proliferation (gray bars), and the right
`vertical axis denotes cytokine secretion (black bars) measured by ELISA in
`supernatants of the same assay. Ag, antigen. (Upper) IL-4 secretion by a TH2
`COP-reactive TCL. (Lower) IFN-gsecretion by a TH0 COP-reactive TCL. Asterisks
`denote cytokine levels greater than 2 SD above background. The lower limit
`of detection (sensitivity) of the cytokine ELISAs is ,2 pgyml IL-4 or IFN-g.
`
`sponded to MOG by production of IFN-g (27.0 vs. 3.7 and 18.1
`vs. 3.3 pgyml, respectively).
`Discussion
`Our analysis of a large panel of COP-reactive human TCL
`revealed that the cytokine profile of the COP-reactive TCL tends
`to shift from TH1 before treatment to TH2 during treatment.
`Furthermore, about 10% of our COP-reactive TCL responded to
`MBP by cytokine secretion but not proliferation. Both obser-
`vations are remarkably consistent with previously reported
`results in EAE (13, 15).
`An important technical aspect of our study is that we analyzed
`the cytokine profiles of the COP-reactive TCL by intracellular
`double-fluorescence flow cytometry. This allowed us to precisely
`quantify the cytokine profile of each individual TCL and assign
`a preference intensity index to each patient, ranging on a
`continuous scale from 21 (100% TH1 lines) to 11 (100% TH2
`lines). Parallel determinations with ELISA in a subset of TCL
`were consistent with the flow cytometry data. Although the
`observed cytokine shift was most obvious on cross-sectional
`analysis, our (limited) longitudinal data support the idea that a
`cytokine shift occurs during treatment. It is important to note,
`however, that the observed shift is a statistical phenomenon:
`individual patients showed a TH1- rather than TH2-biased
`cytokine profile despite prolonged treatment with COP. Fur-
`thermore, some COP-treated patients seem to shift back from a
`TH2 profile to a TH0 or even a TH1 profile. Yet, the cytokine
`data extend previous observations in COP-treated MS patients
`who showed increased levels of IL-10, transforming growth
`factor b(TGF-b), and IL-4 in peripheral blood cells (19). In our
`study, we selected one prototypical TH1 cytokine (IFN-g) and
`one TH2 cytokine (IL-4). Assessment of additional cytokines,
`
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`Neuhaus et al.
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`YEDA EXHIBIT NO. 2068
`MYLAN PHARM. v YEDA
`IPR2015-00644
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`

`
`ing IL-4, IL-5, IL-6, and IL-10 but not IL-2 or IFN-gin response
`to COP (13, 15). As in our study, some of the mouse COP-
`reactive T cells cross-reacted to MBP by cytokine secretion but
`not proliferation (13). The observed cross-reaction between
`COP and MBP at the cytokine level is apparently not unique to
`MBP but could also be observed with another myelin autoan-
`tigen, MOG, in a few TCL.
`Regarding the possible mechanism of COP in vivo, it is known
`that COP binds efficiently to MHC class II molecules (11–13),
`and it competes with MBP at both the MHC class II and TCR
`levels (14, 17, 18). However, because it seems unlikely that
`significant amounts of COP can reach the central nervous
`system, these in vitro effects probably do not explain the clinical
`effects observed in vivo.
`The following hypothetical scenario would accommodate both
`the previously reported EAE results and our observations in
`COP-treated patients: Chronic s.c. application of COP induces
`COP-reactive TH2 cells, which are able to cross the blood–brain
`barrier because they are activated (28). Inside the central
`nervous system, the COP-reactive T cells are confronted with
`products of myelin turnover presented by local APC (29). Some
`of the COP-reactive T cells react to MBP by secretion of
`protective cytokines such as IL-4. This might exert suppressive
`bystander effects on other inflammatory cells (13, 16, 30).
`
`MEDICALSCIENCES
`
`We are grateful to Drs. J. Haas and U. Augustin (Jewish Hospital,
`Berlin), Dr. N. Ko¨nig (Marianne-Strauß-Hospital, Berg, Germany), and
`Dr. C. Zimmermann (Institute for Clinical Neuroimmunology, Univer-
`sity of Munich, Germany) for providing clinical samples. We thank Drs.
`E. Albert and S. Scholz (Department of Immunogenetics, University of
`Munich) for the HLA-typing and Dr. L. Jiang and Ms. M. So¨lch for
`excellent technical assistance. This work was supported by Teva Pharmay
`Hoechst Marion Roussel. O.N. and H. Wiendl are postdoctoral fellows
`supported by the Deutsche Forschungsgemeinschaft. The Institute for
`Clinical Neuroimmunology is supported by the Hermann and Lilly
`Schilling Foundation.
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`
`such as IL-5, -6, -10, -12, and -13, lymphotoxin, or TGF-bwould
`probably not provide much additional information, and was
`simply not feasible, owing mainly to restrictions in cell numbers.
`It will be interesting to establish in future studies whether the
`observed COP-induced TH2 shift is related to the clinical
`response. However, correlation with clinical outcome measures
`requires prospective studies with proper quantitative assessment
`of clinical scores and quantitative MRI data. An important
`question in this regard is whether the TH2 shift can help to
`differentiate between clinical
`‘‘responders’’ and ‘‘nonre-
`sponders’’ to treatment. Furthermore, recent observations indi-
`cate that the beneficial effect of COP as detected by MRI
`requires several months to develop (3). Our data are consistent
`with such a delayed effect. It will also be interesting to see
`whether and to what extent the observed MRI effects are
`paralleled by the COP-induced TH2 shift.
`The influence of COP treatment on the cytokine profile seems
`to be COP-specific, as it was not observed in PPD- or TT-
`reactive T