Expression of GM-CSF in T Cells Is Increased in Multiple Sclerosis and Suppressed by IFN-β Therapy

Abstract

Multiple sclerosis (MS) is an autoimmune disease of the CNS. Studies in animal models of MS have shown that GM-CSF produced by T cells is necessary for the development of autoimmune CNS inflammation. This suggests that GM-CSF may have a pathogenic role in MS as well, and a clinical trial testing its blockade is ongoing. However, there have been few reports on GM-CSF production by T cells in MS. The objective of this study was to characterize GM-CSF production by T cells of MS patients and to determine the effect of IFN-β therapy on its production. GM-CSF production by peripheral blood (PB) T cells and the effects of IFN-β were characterized in samples of untreated and IFN-β-treated MS patients versus healthy subjects. GM-CSF production by T cells in MS brain lesions was analyzed by immunofluorescence. Untreated MS patients had significantly greater numbers of GM-CSF(+)CD4(+) and CD8(+) T cells in PB compared with healthy controls and IFN-β-treated MS patients. IFN-β significantly suppressed GM-CSF production by T cells in vitro. A number of CD4(+) and CD8(+) T cells in MS brain lesions expressed GM-CSF. Elevated GM-CSF production by PB T cells in MS is indicative of aberrant hyperactivation of the immune system. Given its essential role in animal models, abundant GM-CSF production at the sites of CNS inflammation suggests that GM-CSF contributes to MS pathogenesis. Our findings also reveal a potential mechanism of IFN-β therapy, namely suppression of GM-CSF production.

Figure 1. Human PB T cells express GM-CSF

PBMCs from healthy donors (n=15) were activated with PMA + ionomycin + Golgiplug (PMA/iono/GP), stained and analyzed by flow cytometry. A) Representative flow cytometry dot-plots for gated CD4⁺ and CD8⁺ T cells from one donor. B) Summary (%) for GM-CSF⁺ CD4⁺ and CD8⁺ T cells among total CD4⁺ and CD8⁺ T cells. C) Representative histogram for T-bet expression in GM-CSF⁺IFN-γ⁺ and GM-CSF⁺IFN-γ⁻ CD4⁺ and CD8⁺ T cells from one donor (n=5). D) Representative histogram for CCR10 expression in GM-CSF⁺IFN-γ⁻, GM-CSF⁺IFN-γ⁺, GM-CSF⁻IFN-γ⁺, and GM-CSF⁻IFN-γ⁻ CD45RA⁻CD4⁺ cells. Graph on the right shows the percentage of CCR10⁺ cells in CD45RA⁻CD4⁺ T cells (mean ± SEM, n=6).

Figure 2. Untreated MS patients have more GM-CSF-producing T cells than healthy individuals and IFN-β-treated MS patients

PBMCs from healthy donors (n=15), untreated MS patients (n= 15), and IFN-β-treated MS patients (n=10) were activated with PMA/iono/GP, stained and analyzed by flow cytometry. A) Collective data (%) of GM-CSF⁺ CD4⁺ and CD8⁺ T cells among total CD4⁺ and CD8⁺ T cells. B) IFN-γ expression by CD4⁺ and CD8⁺ T cells. C) and D) flow cytometry analysis of GM-CSF expression in IFN-γ⁺ and IFN-γ⁻ T cells. * p < 0.05, ** p < 0.01, *** p < 0.001, NS: not significant, (One-way ANOVA).

Figure 3. IFN-β suppresses GM-CSF production by T cells from healthy donors

PBMCs and purified CD4⁺CD45RA⁻ T cells from healthy donors were stimulated with anti-CD3 and anti-CD28 Abs for five days, with or without addition of IFN-β. A) Concentrations of GM-CSF, IL-10, IL-17A and IFN-γ in cell culture supernatants from PBMCs (n=15) assayed by ELISA. B) Percentages of GM-CSF⁺ and IFN-γ⁺ CD4⁺ and CD8⁺ cells in total CD4⁺ and CD8⁺ T cells from above PBMC cultures (n=15). C) To evaluate direct effect of IFN-β on CD4⁺ T cells, purified CD45RA⁻CD4⁺ T cells and PBMCs from the same donors (n=8) were cultured in the presence of IFN-β. Percentages of GM-CSF⁺, GM-CSF⁺IFN-γ⁺ and GM-CSF⁺IFN-γ⁻ CD4⁺ cells in total CD4⁺ cells. GM-CSF concentrations measured by ELISA in cell culture supernatants. * p<0.05, ** p<0.01, *** p<0.001, NS: not significant (Paired, two-tailed Student t-test).

Figure 4. IFN-β reduces GM-CSF production by T cells from MS patients

PBMCs of IFN-β-treated and untreated MS patients were cultured with anti-CD3 and anti-CD28 Abs for five days with or without addition of IFN-β. A) GM-CSF, IL-10, IL-17A, and IFN-γ concentrations in above cell culture supernatants. B) Percentages of GM-CSF⁺ and IFN-γ⁺ CD4⁺ and CD8⁺ cells in total CD4⁺ and CD8⁺ T cells from above PBMC cultures. * p<0.05, ** p<0.01, *** p<0.001, NS: not significant (Paired, two-tailed Student t-test).

Figure 5. IFN-β reduces numbers of GM-CSF only CD4⁺ and CD8⁺ T cells

PBMCs from healthy donors (n=15) and MS patients (untreated MS n=13; IFN-β treated n=9) were stimulated with anti-CD3/anti-CD28 Abs in the presence or absence of IFN-β. Percentages of GM-CSF⁺IFN-γ⁺ and GM-CSF only CD4⁺ and CD8⁺ T cells in total CD4⁺ and CD8⁺ cells are shown. * p<0.05, ** p<0.01, *** p<0.001, NS: not significant (Paired, two-tailed Student t-test).

Figure 6. CD4⁺ and CD8⁺ T cells co-express GM-CSF and IL-17A in MS brain lesions

Serial sections from MS brain were stained with A) H&E and LFB to demonstrate the infiltration of T cells and demyelination in the MS lesion. B) CD4⁺ and C) CD8⁺ T cells were stained for GM-CSF and IL-17A and analyzed by confocal microscopy. Merged images show co-localization of GM-CSF and IL-17A in CD4⁺ and CD8⁺ T cells. Negative controls are virtually devoid of staining. Scale bars= 10μm.

Figure 7. CD4⁺ and CD8⁺ T cells co-express GM-CSF and INF-γ in MS brain lesions

Stained sections from three MS patients were analyzed by confocal microscopy. A) CD4⁺ and B) CD8⁺ T cells in brain lesions were stained for GM-CSF and INF-γ. Merged images show co-localization of GM-CSF and INF-γ in CD4⁺ and CD8⁺ T cells. Negative controls are virtually devoid of staining. C) Quantification of CD4⁺ and CD8⁺ T cells performed by counting average of 200 cells per group from three MS brains at 20X magnification. Scale bars = 10 μm.