Brain, Vol. 124, No. 4, 705-719,
April 2001
© 2001 Oxford University Press
Treatment of multiple sclerosis with Copaxone (COP)
Elispot assay detects COP-induced interleukin-4 and interferon-
response in blood cells
1 Department of Neuroimmunology, Max Planck Institute of Neurobiology, Martinsried, 2 Institute for Clinical Neuroimmunology and Department of Neurology, Klinikum Grosshadern, Ludwig Maximilians University, 3 Department of Statistics, Max Planck Institute of Psychiatry, Munich, 4 Marianne-Strauss-Klinik, Berg, Germany
Correspondence to:
Dr R. Hohlfeld, Institute for Clinical Neuroimmunology, Klinikum Grosshadern, Ludwig Maximilians University, Marchioninistrasse 15, D-81366 Munich, Germany E-mail: hohlfeld{at}neuro.mpg.de
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Abstract |
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Copolymer-1 (Copaxone or COP) inhibits experimental allergic encephalomyelitis and has beneficial effects in multiple sclerosis. There is presently no practical in vitro assay for monitoring the immunological effects of COP. We used an automated, computer-assisted enzyme-linked immunoadsorbent spot assay for detecting COP-induced interferon-
(IFN-)- and interleukin-4 (IL-4)-producing cells and a standard proliferation assay to assess the immunological response to COP in peripheral blood mononuclear cells from 20 healthy donors, 20 untreated multiple sclerosis patients and 20 COP-treated multiple sclerosis patients. Compared with untreated and healthy controls, COP-treated patients showed (i) a significant reduction of COP-induced proliferation; (ii) a positive IL-4 Elispot response mediated predominantly by CD4 cells after stimulation with a wide range of COP concentrations; and (iii) an elevated IFN- response partially mediated by CD8 cells after stimulation with high COP concentrations. All three effects were COP-specific as they were not observed with the control antigens, tuberculin-purified protein or tetanus toxoid. The COP-induced changes were consistent over time and allowed correct identification of COP-treated and untreated donors in most cases. We propose that these criteria may be helpful to monitor the immunological response to COP in future clinical trials. multiple sclerosis; immunotherapy; copaxone; autoimmune T cells; cytokine response
Ab = antibody; CD = cluster of differentiation; COP = copolymer-1; Elispot = enzyme-linked immunoadsorbent spot (assay); FACS = fluorescence-activated cell sorter; IFN = interferon; IL = interleukin; PBMC = peripheral blood mononuclear cells; PPD = tuberculin purified protein; SEB = staphylococcal enterotoxin B; SI = stimulation index; TH = T helper; TT = tetanus toxoid
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Introduction |
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Copolymer-1 (Cop-1, Copaxone, glatiramer acetate; COP) is a mixture of synthetic polypeptides composed of the four amino acids, L-alanine, L-glutamic acid, L-lysine and L-tyrosine in a defined molar ratio of 0.43 : 0.14 : 0.33 : 0.1 and with an average molecular weight of 4700 to 11 000. COP is one of the currently approved agents for immunomodulatory therapy of multiple sclerosis (Wolinsky, 1995
; Arnon, 1996; Noseworthy et al., 1999). In contrast to interferon (IFN)-ß, COP is believed to selectively down-modulate the immune response to myelin autoantigens, especially to myelin basic protein (Sela et al., 1990; Arnon, 1996). In previous clinical trials, the efficacy of COP was demonstrated by monitoring clinical changes (Bornstein et al., 1991; Johnson et al., 1995, 1998) and more recently, disease activity by means of brain MRI (Mancardi et al., 1998; Ge et al., 2000; Comi et al., 2001). Thus far, however, there are no practical laboratory techniques available to assess the immunological effects of COP treatment.
We have demonstrated recently that COP-reactive T-cell lines from COP-treated patients are preferentially T helper (TH) type-2, whereas COP-specific T-cell lines from untreated patients and healthy controls are predominantly TH1 (Neuhaus et al., 2000). However, the isolation of COP-specific T-cell lines is technically demanding and time-consuming, and hence inappropriate for routine monitoring of treatment effects during clinical trials. In the present study we used a simple, automated enzyme-linked immunoadsorbent spot (Elispot) assay to detect interleukin-4 (IL-4) and IFN- in short-term (18 h) cultures of freshly isolated peripheral blood mononuclear cells (PBMC).
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Methods |
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Patients and control subjects
Blood was drawn after informed consent from 20 healthy donors, 20 untreated multiple sclerosis patients, and 20 COP-treated multiple sclerosis patients (Table 1
). The untreated multiple sclerosis patients had received no immunosuppressive or immunomodulatory treatment for at least 3 months preceding the study. The group of untreated patients included 10 relapsing–remitting, seven secondary progressive, and three primary progressive patients (Table 1). The group of COP-treated multiple sclerosis patients (20 mg/day COP subcutaneously) consisted of 20 relapsing–remitting patients (Table 1). All patients were clinically stable at the time of sampling. All donors were HLA (human leukocyte antigen)-typed (Dr E. Albert, Department of Immunogenetics, University of Munich, Germany). The proportion of DR2-positive patients was similar in the untreated (12 out of 20) and COP-treated (13 out of 20) multiple sclerosis patients.
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Antigen stimulation and proliferation assay
PBMC were isolated on a discontinuous density gradient (Lymphoprep, Nycomed, Oslo, Norway). Viable cells were counted with Trypan Blue (Sigma-Aldrich, Deisenhofen, Germany) and resuspended in culture medium (RPMI 1640 supplemented with 5% foetal calf serum, 1% glutamine and 1% penicillin/streptomycin; Gibco, Karlsruhe, Germany). One batch of foetal calf serum was used throughout the study.
PBMC (1 x 105 cells/well) were cultured in 96-well microtitre plates for 5 days in the presence of one of the following antigens: COP (6.25, 12.5, 25, 50 and 100 µg/ml; batch 242992899; Teva Pharmaceutical Industries, Petah Tiqva, Israel), tetanus toxoid (TT, 4 µg/ml; Chiron-Behring, Marburg, Germany), tuberculin purified protein (PPD, 20 µg/ml; Statens Serum Institut, Copenhagen, Denmark) and staphylococcal enterotoxin B (SEB, 1 µg/ml; Toxin Technology, Sarasota, Fla., USA) as a positive control. [3H]Thymidine (0.2 µCi/well) was added during the last 18 h of culture. Cells were harvested and [3H]thymidine incorporation was measured using a direct ß-counter (Matrix 9600, Packard, Frankfurt, Germany). All experiments were performed in triplicate. Standard deviations were below 20% of the mean. The results were expressed as absolute counts per minute and as stimulation index (SI = ratio of the counts obtained in the presence of antigen and the counts in the absence of antigen). In all experiments, the counts in the presence of SEB were >5000.
Elispot assays
The Elispot assays were performed in parallel with the proliferation tests and analysed with an automated imaging system and appropriate computer software (KS ELISPOT automated image analysis system; Zeiss, Jena, Germany). Briefly, 96-well polyvinylidene difluoride plates (Millipore, Eschborn, Germany) were coated at 4°C overnight with 10 µg/ml capture antibody [anti-IFN- antibody (Ab) clone 1-D1K; Mabtech, Nacka, Sweden; or with anti-IL-4 Ab clone MP4–25D2, Pharmingen, Hamburg, Germany]. The plates were then washed and blocked with culture medium for 1 h at 37°C. PBMC (2 x 105 cells/well for the IFN- and 4 x 105 for the IL-4 Elispot assay) were cultured for 18 h at 37°C and 5% CO2. For each subject, quadruplicate wells were exposed to the same antigens used in the proliferation assay. After culture, the plates were washed and incubated first with 1 µg/ml biotinylated detector Ab (anti-IFN- Ab clone 7-B6–1 or anti-IL-4 Ab clone 12–1; Mabtech), then with 1 : 1000 streptavidin-alkaline phosphatase (Mabtech), and finally with BCIP/NBT (bromo-chloro-indolyl phosphate/nitroblue tetrazolium; Sigma-Aldrich). The frequency of cytokine-producing, antigen-reactive cells was expressed as the difference between the mean number of spots after antigen stimulation and the mean background for each experiment. Overall, the mean absolute spot background was ~10 spots (9.4 ± 2.3) for the IFN- assay and ~5 spots (5.3 ± 2) for the IL-4 assay. A value equal to zero was assigned to spot frequencies smaller than the mean background of the individual assay plus 2 SD. All standard deviations were below 20% of the mean. In all experiments, stimulation with SEB induced more than one out of 200 IFN-- or IL-4-producing cells.
Preparation of CD4- and CD8-enriched cell populations and fluorescence-activated cell sorter (FACS) analysis
For some Elispot and proliferation experiments, CD4 (CD = cluster of differentiation) and CD8-enriched cell populations were prepared from PBMC by positive or negative selection with immunomagnetic beads coated with anti-CD4 or anti-CD8 antibodies (Dynal, Hamburg, Germany) according to the manufacturer's instructions. The selected cell populations were analysed by FACS (fluorescence-activated cell sorter). Briefly, 105 cells were labelled with anti-CD4 mAb [monoclonal antibody clone SK3, peridinin chlorophyll protein (PerCP)-labelled], anti-CD8 mAb (clone SK1, fluorescein isothiocyanate (FITC)-labelled), or with the corresponding isotype controls [mouse IgG1 labelled with PerCP or FITC and analysed on a FACScan using Cell-Quest software (all from Becton Dickinson, Heidelberg, Germany)]. The composition of the different cell preparations was as follows: CD4-enriched cells = 78% CD4 cells, 4% CD8 cells; CD-8 depleted cells = 40% CD4 cells, 10% CD8 cells; CD8-enriched cells = 4% CD4 cells, 75% CD8 cells; CD4-depleted cells = 2% CD4 cells, 55% CD8 cells.
The positively selected cells were stimulated with COP and with irradiated autologous PBMC (105 cells/well) as antigen presenting cells. In this case, the number of spots generated by the antigen presenting cells alone after antigen stimulation was subtracted as background. The negatively selected (depleted) cell preparations could be stimulated without additional antigen presenting cells as they still contained intrinsic antigen presenting cells.
Statistical analysis
To test the significance of the proliferative response and of the Elispot response to COP, TT and PPD in the three groups of subjects, MANOVA (multivariate analysis of variance) was applied. Univariate F-tests and tests with contrasts were performed to identify variables, groups or concentrations with meaningful contributions to the observed effects.
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Results |
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PBMC from COP-treated patients show a significant reduction of COP-induced proliferation
We analysed the proliferative response to COP of freshly isolated PBMC from 20 COP-treated multiple sclerosis patients and compared it with the response of 20 healthy donors and 20 untreated multiple sclerosis patients. In dose–response experiments, T cells from treated patients showed a significant reduction of proliferation both in terms of absolute counts and stimulation index to all but the highest concentration of COP (tests with contrasts, P < 0.01) (Fig. 1A
). To assess whether COP treatment also affected T cells with other specificity, we measured the proliferative response to the recall antigens TT and PPD in the same subjects. As shown in Fig. 1B, there was no significant difference in the T-cell response to TT and PPD between the three groups.
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COP-treated patients show a differential increase of COP-reactive IL-4-producing and IFN-
-producing cellsIn parallel with the proliferation tests, we performed Elispot assays to detect COP-induced production of a typical TH1 cytokine (IFN-
) and a typical TH2 cytokine (IL-4) (Fig. 2A). The Elispot plates were read with an automated imaging system and the images were analysed with appropriate computer software (Zeiss, Jena, Germany).
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The IFN-
Elispot detected a response to COP in treated patients as well as control donors, and it was not significantly different at the low concentrations (6.25, 12.5 and 25 µg/ml COP; ~30 responder cells per 200 000 PBMC). In contrast, at concentrations >25 µg/ml (Fig. 2A
), there was a strongly significant reaction in the treated population (tests with contrasts, P < 0.0001). The frequency of COP-reactive, IFN--producing cells could reach up to 400 responder cells per 200 000 seeded cells at the highest concentration of COP (100 µg/ml).
The IFN- Elispot response was thus specific for COP, since there was no difference between the groups with the control antigen TT (Fig. 2B, middle panel; P = 0.09). The response to another control antigen, PPD, differed between the healthy subjects and the multiple sclerosis population (tests with contrasts, P < 0.05, when compared with untreated and COP-treated multiple sclerosis patients, respectively), but not between untreated and COP-treated multiple sclerosis patients (P = 0.9) (Fig. 2B, lower panels).
The IL-4 Elispot also detected a striking difference between the treated and untreated subjects (Fig. 2A, right panels). No IL-4 response was found in 15 out of 20 healthy donors and in 14 out of 20 untreated patients. In contrast, 17 out of 20 COP-treated patients showed a positive IL-4 response (as defined in Methods). The increase of the frequency of COP-reactive IL-4-producing T cells in the COP-treated patients was statistically significant (tests with contrasts, P < 0.0001).
The dose–response profile of the IL-4 Elispot differed markedly from the dose-response profile of the IFN- Elispot in that it was apparent at low COP concentrations; it did not increase at higher concentrations (Fig. 2A). Again, the treatment-induced IL-4 response was COP-specific, since it was not observed with the recall antigens TT and PPD (P = 0.8 for both antigens, Fig. 2B, middle and lower panels).
Figure 3 shows the relationship between the IFN- and IL-4 Elispot responses to different concentrations of COP for each subject (black dots). At low COP concentrations (Fig. 3, top panels), a consistent difference between the COP-treated and control subjects was observed only with the IL-4 Elispot, but not with the IFN- Elispot (shift of the black dots along the y axis). Increasing COP concentrations had no effect on the frequencies of cytokine-producing cells in the controls, but the IL-4 response persisted in the COP-treated multiple sclerosis patients and was accompanied by an increasing frequency of IFN- producing cells (shift of the black dots to the right at higher concentrations).
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The COP-induced IFN-
Elispot response is best observed in patients who are treated for >6 monthsTo assess whether the IL-4 and IFN-
Elispot responses were related to the duration of COP treatment, we examined six additional COP-treated patients and then divided the entire group into short-term (<6 months, six subjects) and long-term (>6 months, 20 subjects) treated patients. As shown in Fig. 4, the strong IFN- response at 100 µg/ml COP was most obvious in the long term treated patients (upper panel), whereas COP-induced IL-4 production was already seen after a few weeks of treatment (lower panel). Nineteen of the 20 long-term treated patients were clinical responders (defined here as patients who showed a reduction in the frequency of exacerbations during treatment compared to the frequency before treatment; see Table 1). The single non-responder (S.H., open triangle in Fig. 4) showed hardly any IL-4 and IFN- Elispot responses and thus behaved like the untreated controls. We were unable to recruit additional clinical non-responders, since treatment is usually discontinued in such patients.
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Both CD4 and CD8 cells contribute to the COP-induced cytokine response
To compare the contribution of CD4 and CD8 T cells to the COP-induced cytokine response, we repeated the Elispot assays with cell preparations enriched in CD4 or CD8 cells, prepared by positive or negative selection (see Methods) from PBMC of a COP-treated patient (R.R.) who showed strong IFN-
and IL-4 responses with unselected PBMC. We stimulated the positively selected (CD4- or CD8-enriched) populations (105 cells/well, in triplicate) with COP in the presence of irradiated autologous PBMC (105 cells/well) as antigen presenting cells.
As shown in Fig. 5, both CD4-enriched cells (open columns in upper panels) and CD8-enriched cells (open columns in lower panels) produced IFN- at the low (6.25 µg/ml) COP concentration. At 100 µg/ml, the contribution of CD8-enriched cells was greater than that of CD4-enriched cells (~300 IFN- spots per 105 CD8-enriched cells versus 100 spots per 105 CD4-enriched cells). Depletion of the CD8 cells reduced the IFN- response. In contrast, IL-4 production was mainly seen in the CD4-enriched and CD8-depleted cell preparations (Fig. 5, upper right). At the high concentration, a significant IL-4 response was also seen in the CD8-enriched population (open column, lower right). Similar results were obtained in a second COP-treated patient (patient B.G.; data not shown).
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The immunological response to COP is conserved over time
To assess the longitudinal stability of the immunological response to COP, we repeated the proliferation and Elispot assays at different time points in individual donors. A representative set of experiments is shown in Fig. 6
. This patient (C.S.) was examined 7, 11, 13, and 13.23 months after beginning COP treatment. At all time points, the proliferative reactivity to COP remained low, the increase in the IFN- Elispot response was confirmed and the IL-4 Elispot response was positive as in most other COP-treated patients. The relative stability of the response to COP was confirmed in six other COP-treated patients, one healthy donor and one untreated multiple sclerosis patient (Table 2).
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The in vitro response to COP allows identification of COP-treated patients
We next asked whether the strong COP-induced IFN-
response could be used as a potential marker for the detection of treatment effects in a double-blind prospective analysis. To this end, we analysed six samples from COP-treated patients, five from untreated patients, and two from healthy subjects. Of these 13 samples, 10 could be correctly assigned to either the COP-treated or control group. Of the remaining three samples, one came from an untreated patient and two from long-term treated patients who failed to show a strong IFN- response.
To further improve the criteria for a positive immunological response to COP, we considered two additional COP-induced in vitro effects: the reduction of proliferation and the stimulation of IL-4 production by T cells. Specifically, we assessed the following three criteria to define the in vitro response to COP: (i) strong IFN- response at high concentrations of COP (
50 spots per 2 x 105 PBMC at 100 µg/ml COP); (ii) positive IL-4 response at two or more concentrations of COP; and (iii) reduced proliferation to COP (SI <2.5 at three or more COP concentrations). In a retrospective analysis of our initial experiments (Figs 1 and 2), eight COP-treated patients met all three criteria, four additional COP-treated patients were positive for (i) and (ii) and five other treated patients met the criteria for the proliferation assay and for one cytokine response (Table 3). Thus, 17 out of 20 COP-treated patients could be identified on the basis of these proposed immunological criteria, whereas only three of 20 untreated patients and none of the 20 healthy donors met the criteria (false positives). We expect that the immunological response to COP measured by Elispot and proliferation assays will help in future studies to identify `immunological responders' to COP and to assess whether and how the immune response to COP relates to the clinical response.
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Discussion |
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We analysed the proliferation and cytokine response to COP directly ex vivo in PBMC isolated from COP-treated multiple sclerosis patients and untreated controls. Besides the previously reported reduction in proliferation, we have demonstrated that PBMC from COP-treated patients show a treatment-related, COP-induced differential TH1 and TH2 cytokine response that is detected by an automated Elispot assay. While the COP-induced IFN-
response is observed only at high concentrations of COP, the IL-4 response is seen over a wide range, including low concentrations of COP. Furthermore, while the IL-4 response at low concentrations is mainly mediated by CD4 cells, the IFN- response is partially mediated by CD8 cells. These observations have implications for the monitoring of COP treatment in future clinical trials and for a better understanding of the mechanisms of action of COP.
Assay for monitoring COP treatment
Practical laboratory techniques to assess the immunological response to COP have been lacking until now. It was noted in previous clinical trials that the proliferative response of PBMC to COP is reduced in COP-treated patients (Teitelbaum et al., 1994). This has been confirmed recently in a small study involving seven COP-treated multiple sclerosis patients (Duda et al., 2000). However, no control antigen or control group was included in that study. Our present results indicate that the reduced proliferation is specific to COP, since it is not seen with the recall antigens PPD and TT, and is related to COP treatment, since it is not seen in the control groups.
More striking than the changes in proliferation are the COP-induced changes observed with our Elispot assay. Elispot assays have been used for many years to demonstrate T-cell responses in freshly isolated (ex vivo) cell preparations. Until recently, however, the Elispot system was limited by the subjective evaluation, which relied on the visual counting of individual spots. In contrast, modern imaging techniques now allow an automated, objective analysis and precise quantification. Thus, a high degree of reliability and reproducibility can be achieved, and the results can be analysed with appropriate computer software.
Several earlier studies indicated that a cytokine shift from TH1 to TH2 occurs in COP-treated multiple sclerosis patients (Miller et al., 1998; Neuhaus et al., 2000; Duda et al., 2000). However, none of these studies suggested a practical laboratory assay suitable for the routine monitoring of the immunological response to COP. We too have demonstrated elsewhere that COP-specific T-cell lines from COP-treated patients are predominantly TH2, whereas T-cell lines from untreated patients and normal controls are predominantly TH1 (Neuhaus et al., 2000). However, long-term T-cell lines cannot be used for the monitoring of COP treatment, because their isolation and propagation are far too technically demanding and time consuming. Similar limitations apply to COP-specific short-term T-cell lines, which have been used by other investigators to demonstrate a cytokine shift (Duda et al., 2000). In contrast, the automated, computer-assisted Elispot assay used in our present study seems to provide a practical and reproducible system for monitoring the immunological response to COP. Compared with the previous studies with COP-specific T-cell lines (Duda et al., 2000; Neuhaus et al., 2000; Qin et al., 2000), the Elispot assay described here has the advantage that it makes use of freshly isolated PBMC, which are cultured directly ex vivo for a short period of time (18 h). This allows direct and quick sampling of cell populations that would be inevitably changed or lost during prolonged culture in vitro. Furthermore, the treatment-related changes that can be observed with the Elispot assay seem to be more pronounced than the changes observed with the proliferation assay.
Differential IFN- and IL-4 cytokine responses
We found that PBMC from the majority of COP-treated patients produce IFN- and IL-4 in response to in vitro stimulation with different concentrations of COP. While the COP-induced IFN- response was apparent only at high concentrations of COP, the IL-4 response was already seen at low concentrations of 6.25 µg/ml. The COP-specific TH2 cytokine response is consistent with previous observations in multiple sclerosis patients (Neuhaus et al., 2000; Duda et al., 2000). In animal models, immunization with COP induces splenic TH2 cells, which are responsible for experimental allergic encephalitis suppression and resistance to disease induction (Aharoni et al., 1993; Aharoni et al., 1997). However, the pronounced IFN- response seen at high concentrations of COP is a new observation not previously reported. Interestingly, this response is partially mediated by CD8 cells, as indicated by our experiments with CD8-enriched and CD4-depleted cell populations obtained by magnetic bead sorting. In contrast to the IFN- response, the IL-4 response is mainly mediated by CD4 cells at low concentrations of COP.
One of the reasons why the COP-induced CD8 response has not been detected previously may be that the earlier studies employed long-term (Neuhaus et al., 2000) or short-term (Duda et al., 2000; Qin et al., 2000) COP-specific T-cell lines. However, the standard techniques for culturing T-cell lines select for CD4 T cells. Furthermore, our comparison of the IFN- Elispot and the proliferation responses (Figs 1 and 2) suggests that most of the IFN--producing cells do not proliferate but presumably die (C. Farina, unpublished observations) and are therefore difficult to propagate and characterize as T-cell lines.
Although COP-reactive CD8 cells have not been reported previously in multiple sclerosis or experimental allergic encephalitis, a recently published abstract indirectly points in a similar direction. The COP-induced proliferation of PBMC can be inhibited by both anti-MHC (major histocompatability complex) class I and anti-MHC class II antibodies (Ragheb and Lisak, 2000). This observation would be consistent with a mixed, MHC class I-restricted (CD8 cell-mediated) and MHC class II-restricted (CD4 cell-mediated) response to COP. Further experiments are needed to characterize the COP-reactive CD8 cells. For functional characterization, it will probably be necessary to establish and study COP-reactive CD8 T-cell lines in animal models, or to use appropriate knock-out mice.
Implications for the mechanism of action of COP
Although the IFN- response seen at high concentrations of COP may seem unexpected, it does not contradict the present concepts of COP action (reviewed by Neuhaus et al., 2001). According to these concepts, it is assumed that COP induces a population of TH2-like T cells which cross-react with myelin basic protein (Teitelbaum et al., 1988; Sela et al., 1990; Arnon, 1996) and perhaps other myelin antigens. These cells are activated by the daily subcutaneous injection of COP. Both the soluble antigenic nature of COP and its local presentation by dermal dendritic cells may favour such a TH2 response after prolonged in vivo application. The activated COP-reactive T cells enter the central nervous system, where they are restimulated by locally processed myelin autoantigens, secrete TH2-like cytokines such as IL-4 and IL-13 and thereby suppress autoaggressive, pathogenic T cells via a bystander mechanism (Hohlfeld, 1997; Aharoni et al., 1998; Weiner, 1999).
According to this scenario, a cytokine shift of COP-reactive CD4 T cells from TH1 to TH2 represents a major mechanism of action of COP. Indeed, the COP-induced, CD4 cell-mediated IL-4 response seen at low concentrations of COP supports this concept. However, it is presently open to question whether the IFN- Elispot response seen after stimulation with high COP concentrations is relevant in vivo. In fact, concentrations exceeding 50 µg/ml applied in vitro are probably far above the dose within the human body after subcutaneous application of the drug. Therefore, it is possible that the observation of an increased IFN- response in vitro may have no relevance in vivo.
We note that the steep increase in IFN- production seen at high concentrations of COP in vitro is not accompanied by an increase in proliferation. Therefore, even if we assume that COP can induce a similar IFN- response in vivo, it seems very unlikely that the responding cells proliferate and expand in number during the course of treatment. However, we cannot completely rule out that the IFN--producing cells observed in vitro have some role in vivo. Theoretically, they might even contribute to the beneficial effects of COP treatment. A purely speculative, but interesting, possibility would be that the IFN--producing cells secrete some additional cytokine or factor that is beneficial. For example, we have recently found that human immune cells, including CD8 T cells, may be stimulated to secrete functionally active brain-derived neurotrophic factor (Kerschensteiner et al., 1999; Hohlfeld et al., 2000). Furthermore, COP-specific rat T-cell lines (albeit presumably of the CD4 type) have been shown to produce brain-derived neurotrophic factor and to confer neuroprotection after adoptive transfer into rats with experimental crush injury of the optic nerve (Kipnis et al., 2000).
As regards the specificity of the Elispot response, our results strongly suggest that both the COP-induced IFN- and IL-4 responses are indeed COP-specific, as both were absent when the control antigens PPD and TT were used. This confirms and extends our previous findings that the COP-induced TH2 shift observed with T-cell lines is COP-specific (Neuhaus et al., 2000).
Criteria for identifying COP-treated patients on the basis of in vitro assays
The IFN- Elispot response had a striking correlation with COP treatment (Fig. 2). Using this test alone, we could correctly assign 10 out of 13 blinded samples to COP-treated patients or control donors. Together with the additional criteria of a positive IL-4 Elispot response and decreased proliferation, 17 of the 20 COP-treated patients were identified, whereas only three of the 20 untreated patients and none of 20 healthy controls gave a (false) positive result.
Importantly, longitudinal analyses of the responses of several patients and controls show that the COP-induced Elispot response is quite robust and stable over time. Therefore, the proposed criteria (elevated IFN- response, positive IL-4 response, and decreased proliferation) should be useful for identifying `immunological responders' to COP (see below).
Relationship between the clinical and immunological responses to COP
Our data suggest that the daily injection of COP leads to detectable changes in the cytokine response and proliferation of PBMC. Thus, the criteria described above may help to define an immunological response profile related to COP treatment. On the basis of these criteria, the majority of our COP-treated patients would qualify as `immunological responders', but a few appear to be `immunological non-responders' (see Table 3). We are well aware, however, that a prospective analysis with a larger number of patients is required to clearly define the sensitivity and specificity of the assay. The present data do not allow us to establish a correlation between the immunological response to COP and the clinical course of the disease.
Clearly, the question of whether and how the immunological response to COP relates to the clinical response falls outside the scope of the present study. Evaluation of the clinical response requires a rigorous prospective study design, including regular clinical and MRI assessments. This can only be done in the context of a formal clinical trial. The recently started study of oral Copaxone (CORAL) may provide an opportunity to evaluate different criteria for the immunological response to COP, although it is possible that the mechanism of action of oral COP differs from the mechanism of subcutaneously administered COP.
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We wish to thank H. Wekerle, M. Sela, R. Arnon, D. Teitelbaum, N. Tarcic and J. Benson for helpful comments on the manuscript, N. König (Marianne-Strauss Hospital, Berg) for providing clinical samples, E. Albert and S. Scholz (Department of Immunogenetics, University of Munich) for HLA-typing and M. Sölch for excellent technical assistance. This work was supported by TEVA Pharma/Hoechst Marion Roussel. O.N. is a postdoctoral fellow supported by the Deutsche Forschungsgemeinschaft. The Institute for Clinical Neuroimmunology is supported by the Hermann and Lilly Schilling Foundation.
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References |
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Received August 21, 2000. Revised November 14, 2000. Accepted November 24, 2000.
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