Glatiramer acetate (Copaxone®) induces degenerate, Th2-polarized immune responses in patients with multiple sclerosis (original) (raw)
The in vitro proliferative response of PBMCs to GA decreases upon in vivo administration of GA. PBMCs were isolated from 7 patients with RR MS before and at various times after subcutaneous administration of GA. At each time point tested, primary and secondary in vitro proliferation and cytokine assays in the presence and absence of GA were performed. We found that before treatment, there was a significant proliferative response as measured by [3H]thymidine incorporation to GA in all 7 patients, with an average stimulation index (SI) in vitro of 24.8 ± 1.1; the average Δ cpm was 37,241 ± 1,766 cpm. Additionally, all of a total of 170 independently derived T-cell lines stimulated in primary in vitro culture with GA proliferated in response to the antigen (data not shown). After treatment with GA 20 mg subcutaneously daily for 3, 6 and 12 months, the proliferative response as measured by SI and Δ cpm significantly decreased (Figure 1a) (P < 0.001), although, as expected, individual patients varied in their response to GA (Figure 1b).
The proliferative response to GA is decreased on average after daily injections of GA. The antigen-specific proliferative response of 20 or 30 primary T-cell lines induced with 40 μg/mL GA as described in Methods was measured by split-well assay for each patient at each time point. Before and at 6 months of treatment, all 7 patients could be tested; at 3 and 12 months, data for 5 patients were obtained. (a) Each panel represents data from an individual patient. Squares represent mean ± SEM proliferation in Δ cpm of the GA-specific response compared with the no-antigen control. Background levels of the [3H]thymidine incorporation for all patients of the no-antigen condition were 1,747 ± 111 before treatment, and 3,286 ± 175, 3,785 ± 262, and 3,509 ± 239 at 3, 6, and 12 months of treatment, respectively. The numbers in the figure indicate the SI over the no-antigen control for each time point. (b) The mean stimulation index SI ± SEM for all T-cell lines from all patients tested at each time point is shown.
In vitro–generated GA-reactive T-cell lines deviate toward a Th2-cytokine profile upon treatment with GA. Having demonstrated that the proliferative response to GA changed after in vivo subcutaneous administration of GA, we next examined whether the cytokine profile also changed. The cytokine response was measured for the prototypic Th1 and Th2 cytokines IFN-γ and IL-5 by ELISPOT and sandwich ELISA in a total of 590 T-cell lines generated before and at various times after GA injection in all 7 patients. As shown in Figure 2, a and b, compared with the values detected before treatment, the average IFN-γ secretion to GA measured by either ELISA or ELISPOT was significantly decreased (P < 0.001 by Tukey’s honest statistical difference test) after treatment for 3, 6, and 12 months, except for the measurement by ELISPOT at 3 months, which did not reach significance, and the ELISA measurement at 6 months, which only reached a significance level of _P_ < 0.01. The GA-dependent IFN-γ secretion as determined by ELISPOT as Δ spots between the cells tested with antigen and the no-antigen control was 104 ± 10 before treatment, 132 ± 18 at 3 months, 28 ± 4 at 6 months, and 18 ± 3 at 12 months. IFN-γ secretion measured by sandwich ELISA in Δ pg/mL was 1,405 ± 150 before treatment, 222 ± 38 at 3 months, 797 ± 185 at 6 months, and 5.9 ± 46 at 12 months. When patients were analyzed individually, 5 of the 7 patients had statistically significant decreases in IFN-γ secretion (_P_ < 0.001) by ELISPOT or sandwich ELISA (data not shown). The levels of IFN-γ secretion were correlated with the decreased proliferative capacity in these patients (_r_2 of > 0.8 for ELISA values in all patients tested, measurement by ELISPOT correlated less well with _r_2> 0.5 in 4 patients). This is in accordance with previous observations that the proliferative and IFN-γ responses are correlated (25).
GA-specific secretion of cytokines is polarized toward a Th2 response after daily injections of GA. The GA-specific secretion of the cytokines IL-5 and IFN-γ was measured in T-cell lines by 2 methods: ELISPOT (a) and ELISA (b) assays. Each symbol represents the difference of spots counted or Δ pg/mL measured in split-well assays between the GA (20 μg/mL) condition and the no-antigen control. The limits of detection were 1 spot and 10 pg/mL, respectively. Numbers represent the percentage of T-cell lines in each quadrant with a minimum difference in spots of twice the SD of the negative controls for IL-5 and IFN-γ, respectively.
GA-specific IL-5 secretion was, on average, not statistically significantly altered during treatment with GA. By ELISPOT assay, the average IL-5 secretion in Δ spots was 10 ± 3 before, 10 ± 2 at 3 months, 11 ± 2 at 6 months, and 6 ± 1 at 12 months after the initiation of treatment. When measured by sandwich ELISA as Δ pg/mL, IL-5 secretion was 1,484 ± 266 before treatment; 1,940 ± 554 at 3 months; 1,738 ± 343 at 6 months; and 1,146 ± 303 at 12 months after the initiation of treatment. One patient had a sustained statistically significant decrease in IL-5 (P < 0.001), and 1 had a sustained statistically significant increase (P < 0.001) in IL-5 secretion as measured by ELISPOT during treatment.
To examine further the cytokine pattern of PBMCs from patients before and after treatment with GA, T-cell lines from all subjects were grouped into Th0, Th1, and Th2 subsets, based on their cytokine profile. T-cell lines were considered positive for a cytokine when the difference of the GA condition was increased at least 2-fold over the SD of the no-antigen controls. Thus, values over background considered positive were 19 spots for IFN-γ and 10 spots for the IL-5 ELISPOT assay, and 138 pg/mL for IFN-γ and 485 pg/mL for the IL-5 ELISA assays. When measured by ELISPOT before treatment, 46% of all T cells evaluated were characterized as Th1 (Figure 2a). During the course of treatment, there was an increased proportion at 3 months of Th0-type T-cell lines and at 6 and 12 months of Th2-type T-cell lines. In parallel with the decreased proliferative response, an increasing proportion of T-cell lines that did not secrete either IFN-γ or IL-5 in response to GA was seen with treatment. Measurement by ELISA appears to be slightly more sensitive in this assay in which 39% of T-cell lines secreted Th0 cytokines and 41% secreted Th1 cytokines before treatment (Figure 2b). A similar shift toward a Th2 response as with the ELISPOT assay with a decrease of Th0 and a simultaneous increase of Th2-type T-cell lines at 3, 6, and 12 months of treatment was seen. When classifications of T-cell lines into Th0, Th1, and Th2 were performed under less-stringent conditions (minimum difference of 20 and 10 ELISPOTS for IFN-γ and IL-5, respectively, or 200 pg/mL in ELISA assays), a cytokine shift toward Th0/Th2 could also be confirmed (data not shown).
As the magnitude of the IL-5 response was uniformly low, especially as measured by ELISPOT, it was important to reconfirm the apparent Th2 deviation with GA treatment by a means other than measuring IL-5. Toward this end, 3 new patients with RR MS were recruited to the study, and serial measurements of IFN-γ and IL-13 secretion were made before and after the initiation of daily subcutaneous GA injections. IL-13 was chosen as the candidate Th2 cytokine because there are no IL-13 receptors on T cells to consume the secreted cytokine. Primary in vitro T-cell lines generated in the presence of no antigen and of 1.0, 10, and 100 μg/mL GA were examined at 30,000 PBMCs per well each with the 4 antigen concentrations. Thus, equal numbers of cells were tested in the secondary stimulation, whereas in the previous assay with equal aliquots of primary cell lines, the number of cells tested decreased, on average, with treatment owing to lower expansion in the primary cultures. A marked increase in IL-13 secretion in 2 of 3 patients after 3 months of therapy was observed. IFN-γ secretion and the proliferative response were stable or decreased when compared with the pretreatment values (Figure 3).
IL-13 secretion is increased after daily injections of GA. Primary T-cell lines were set up in 10 identical wells each in the presence of no antigen or with 1.0, 10, and 100 μg/mL GA and cultured as described in Methods. Identical wells were pooled on day 11, and 30,000 T cells each were restimulated with no antigen or 1.0, 10, and 100 μg/mL GA pulsed on 100,000 autologous APCs. Cytokines were measured in supernatants by ELISA after 48 hours as described, and proliferation was measured by [3H]thymidine incorporation. Asterisks point at values that were above the upper limit of detection of the IL-13 assay of 10,000 pg/mL.
Cross-reactivity of GA-reactive T-cell lines is increased upon treatment with GA. Cross-reactivity of GA-induced T-cell lines to combinatorial peptide libraries derived from the immunodominant MBP 84-102 peptide and a completely randomized 13mer library were performed to determine degeneracy of GA-specific T cells (Figure 4). Before treatment with GA, there was minimal cross-reactivity in either the proliferative or the cytokine responses. There was only 1 instance when antigen cross-reactivity was observed, in the proliferative response to the 93R90X combinatorial peptide library in patient 7. In striking contrast, 6 of the 7 patients demonstrated an increased number of cross-reactive T-cell lines after therapy to the combinatorial peptide libraries. However, no dominantly cross-reactive APL emerged from this analysis, consistent with the degenerate immune responses we observed.
Cross-reactivity of GA-reactive T-cell lines is increased after daily injections of GA. Percentages of the GA-induced T-cell lines cross-reacting to each APL tested at each time point are shown for the 7 patients encoded by gray scale. Proliferative IFN-γ and IL-5 responses were examined for all T-cell lines and are represented separately in the top, middle, and lower third. A minimum SI of 2 and a difference of 2 SD over the background was required for classification as a cross-reactive T-cell line.
In vitro T-cell reactivity to the immunodominant epitope MBP 84-102 is not significantly altered during GA therapy. In contrast to the results from primary in vitro cultures with GA, which is well approximated by a normal distribution of responses owing to the high precursor frequency of responsive T cells, the reactivity of primary T-cell lines to MBP 84-102 was low and as such not normally distributed. Therefore, nonparametric statistics were used for analysis. No significant change over time of treatment was seen for the MBP 84-102–specific proliferative responses (Figure 5a) by Mann-Whitney U tests. Further analysis included percentage of MBP 84-102–specific T-cell lines, which were not significantly changed over the course of treatment.
(a). The proliferative response to MBP 84-102 is not altered after daily injections of GA. A total of 900 T-cell lines were generated in response to MBP 84-102. The proliferative response in Δ cpm is given for each of 30 or 40 lines tested at each time point (circles, Δ cpm on left axes). On the right axes, the percentage of positive lines determined by a minimum 2.5-fold increase over background and a minimum difference of 1,500 cpm is shown (line). The mean background was 4,662 ± 138. (b). No changes occur in the cytokine response to MBP 84-102 after daily injections of GA. Lines having a minimum difference to the negative control of 2 SD, i.e., 70 spots for IFN-γ and 19 spots for IL-5, were considered positive for the respective cytokine.
Analysis of cytokine secretion in response to MBP 84-102 by Mann-Whitney U test also did not reveal any significant differences during the course of treatment with one exception: IL-5 measured by ELISPOT at 12 months tested significantly decreased (P < 0.01) compared with pretreatment values. When the cytokines were analyzed for each patient individually, no significant change in the frequency of Th0-, Th1-, and Th2-cytokine–secreting lines was seen (Figure 5b), regardless of the stringency of the criteria used for classification. No cross-reactivity was seen between MBP 84-102–induced T cell lines and GA in vitro when judged by these criteria.
GA treatment does not change tetanus toxoid–specific in vitro T-cell responses. To examine the effects of GA treatment on the primary in vitro T-cell response to an unrelated recall antigen, PBMCs from 6 patients with RR MS who were undergoing GA therapy were analyzed with tetanus toxoid. A dose-dependent proliferative response to 0.3 μg/mL, 3 μg/mL, and 30 μg/mL of tetanus toxoid as well as to 0.1 μg/mL and 1 μg/mL of the PHA control was observed at all time points. Compared with the pretreatment values, no significant differences in the tetanus toxoid–specific proliferative responses were seen during treatment with GA as determined by the Student’s t test (data not shown). Thus, as with MBP 84-102, subcutaneous treatment with GA did not alter immune responses to a common recall antigen.