Interferon-beta therapy reduces CD4+ and CD8+ T-cell reactivity in multiple sclerosis

Abstract

Therapy with interferon-beta (IFN-beta) has well-established clinical effects in multiple sclerosis (MS), albeit the immunomodulatory mechanisms are not fully understood. We assessed the prevalence and functional capacity of CD4+ and CD8+ T cells in healthy donors, and in untreated and IFN-beta-treated MS patients, in response to myelin oligodendrocyte glycoprotein (MOG). The proportion of CD45RO+ memory T cells was higher in MS patients than in healthy donors, but returned to normal values upon therapy with IFN-beta. While CD45RO+ CD4+ T cells from all three groups responded to MOG in vitro, untreated patients showed augmented proliferative responses compared to healthy individuals and IFN-beta treatment reduced this elevated reactivity back to the values observed in healthy donors. Similarly, the response of CD45RO+ CD8+ T cells to MOG was strongest in untreated patients and decreased to normal values upon immunotherapy. Overall, the frequency of peripheral CD45RO+ memory T cells ex vivo correlated with the strength of the cellular in vitro response to MOG in untreated patients but not in healthy donors or IFN-beta-treated patients. Compared with healthy individuals, responding CD4+ and CD8+ cells were skewed towards a type 1 cytokine phenotype in untreated patients, but towards a type 2 phenotype under IFN-beta therapy. Our data suggest that the beneficial effect of IFN-beta in MS might be the result of the suppression or depletion of autoreactive, pro-inflammatory memory T cells in the periphery. Assessment of T-cell subsets and their reactivity to MOG may represent an important diagnostic tool for monitoring successful immunotherapy in MS.

Figure 1

Frequencies of CD45RO⁺ T cells in healthy donors (n =14) and in untreated (n = 15) and IFN-β-treated (n = 16) MS patients. Data shown are mean values and SEM for expression of CD45RO in CD3⁺ lymphocytes, and CD4⁺ and CD8⁺ T cells in each group. *P < 0·05; **P < 0·01.

Figure 2

Pattern of typical T-cell responses in a non-responding (a) and a responding (b) MS patient. PBMC were labelled with CFSE and incubated with MOG for 6 days, after which half of the supernatant was replaced by fresh medium supplemented with IL-2. After a total of 10 days, cells were harvested, and the CFSE fluorescence intensity was analysed. Upper panels are dot plots showing the signals for CFSE and CD4 or CD8 within the CD3⁺ gate. Lower panels are projections of the CFSE signals onto histograms gated on CD3⁺ CD4⁺ and CD3⁺ CD8⁺ lymphocytes, respectively, showing the percentage of residual undivided, i.e. non-proliferating CFSE^hi cells.

Figure 3

Time–course experiment showing a typical proliferative response of autoreactive CD45RO⁺ CD4⁺ and CD8⁺ T cells. CFSE-labelled PBMC from an untreated MS patient were incubated in the presence of MOG for 6 days, after which half of the supernatant was replaced by fresh medium supplemented with IL-2. At the time-points indicated, proliferation was assessed as dilution of the CFSE signal. Numbers indicate the percentage of CFSE^lo CD45RO⁺ cells among CD4⁺ and CD8⁺ T cells, respectively.

Figure 4

MOG-driven T-cell responses in healthy donors (n = 14) and in untreated (n = 15) and IFN-β-treated (n = 14) MS patients. For conventional thymidine uptake assays, PBMC were incubated with medium alone (med.), or in the presence of MOG (MOG). After 6 days, [³H]thymidine was added to each well, and cells were collected 6 hr later. Data shown represent the mean radioactivity in c.p.m. from cultures set up in triplicate. For flow cytometric assays, CFSE-labelled PBMC were cultured with or without MOG for 6 days, after which half of the supernatant was replaced by fresh medium supplemented with IL-2. After a total of 10 days, cells were harvested, and the CFSE fluorescence intensity was analysed. Data show individual proliferative responses as mean percentage of undivided, i.e. non-proliferating CFSE^hi cells within the indicated CD3⁺ lymphocytes gates from cultures set up in triplicate. Error bars indicate the mean values ± SEM for each group. *P < 0·05; **P < 0·01; ***P < 0·001.

Figure 5

Correspondence of the initial frequency of CD45RO⁺ cells in untreated MS patients ex vivo (i.e. on day 0) with the MOG-specific response of CD4⁺ and CD8⁺ T cells, shown as percentage of divided, i.e. proliferating, CFSE^lo cells on day 10 (n = 15). Correlations were analysed by applying Spearman's rank correlation coefficients.

Figure 6

Mean intracellular cytokine production by CD4⁺ and CD8⁺ T cells from healthy donors (n = 13) and from untreated (n = 13) and IFN-β-treated (n = 8) MS patients. PBMC were cultured in the presence of MOG or TT for 6 days, after which half of the supernatant was replaced by fresh medium supplemented with IL-2. On day 10, the cells were re-stimulated with irradiated antigen-pulsed PBMC in IL-2-containing medium. T cells were stained after another 3 days, and intracellular cytokines were analysed. Bars indicate the mean values + SEM for IFN-γ and IL-4 production by CD4⁺ or CD8⁺ T cells in each group. *P < 0·05; **P < 0·01.

Figure 7

Ratios of IFN-γ : IL-4 expression in MOG-stimulated CD4⁺ and CD8⁺ T cells. Data show ratios in individual healthy donors (n = 13) and in untreated (n = 13) and IFN-β-treated (n = 8) MS patients. Error bars indicate the geometric means + SEM for each group. *P < 0·05; **P < 0·01.