Fingolimod modulates T cell phenotype and regulatory T cell plasticity in vivo

Abstract

Fingolimod is an approved therapeutic option for patients with relapsing-remitting multiple sclerosis that primarily functions by sequestering T cells in lymph nodes inhibiting their egress to the central nervous system. However, recent data suggests that Fingolimod may also directly affect the immune cell function. Here we examined the in vivo effects of Fingolimod in modulating the phenotype and function of T cell and Foxp3 regulatory T cell populations in patients with multiple sclerosis under Fingolimod treatment. Besides decreasing the cell numbers in peripheral blood and sera levels of pro-inflammatory cytokines, Fingolimod inhibited the expression of Th1 and Th17 cytokines on CD4⁺ T cells and increased the expression of exhaustion markers. Furthermore, treatment increased the frequency of regulatory T cells in blood and inhibited the Th1-like phenotype that is characteristic of patients with multiple sclerosis, augmenting the expression of markers associated with increased suppressive function. Overall, our data suggest that Fingolimod performs other important immunomodulatory functions besides altering T cell migratory capacities, with consequences for other autoimmune pathologies characterized by excessive Th1/Th17 responses and Th1-like regulatory T cell effector phenotypes.

Keywords: Fingolimod; Foxp3; Gilenya; Multiple sclerosis; Regulatory T cells; Th1-like Tregs; Treg plasticity.

Figure 1.. Fingolimod treatment decreased both B and T cells and increased Tregs in the peripheral blood.

Blood was collected from 17 MS patients at baseline and after 3, 6 and 12 months of fingolimod treatment. Proportion and absolute numbers of PBMC populations were measured in freshly collected whole blood by flow cytometry using trucount tubes. (A) Representative example of percentage of different populations of white blood cells in whole blood of one MS patient before and at different times after fingolimod treatment. (B) Absolute counts of PBMC populations in peripheral blood (n = 17). *p < 0.0001, paired t test (compared to baseline, 0 months). (C) Proportion of Tregs in CD4⁺ population before and after fingolimod treatment.

Figure 2.. Fingolimod treatment decreases the amounts of TNFα and IL27 in the plasma of MS patients.

Concentrations of inflammatory cytokines were measured in the plasma of MS patients at baseline (0), and after 3, 6 and 12 months of fingolimod treatment using Luminex based assay. Data are shown as mean±SEM (n=17). *p<0.03, paired t test (compared to baseline levels).

Figure 3.. Effect of fingolimod on gene and protein expression in T effector cells.

PBMC were isolated from MS patients at baseline and 3 months after fingolimod treatment, labeled with T cell regulatory and effector marker antibodies. (A) T effector cells were sorted (Supplementary Fig. 2) and stimulated with PMA and ionomycin, for 4 hours. RNA expression was analyzed by real-time PCR. *p<0.05, paired t test (compared to baseline levels, n=9). (B) IL-17 and (C) IL-10 production was measured by flow cytometry in CD4⁺ effector cells from patients after stimulation with PMA and ionomycin. (D) Protein expression of CCR7, PD-1, Tim-3 and CD25 was measured by mass cytometry on CD4⁺ effector cells of 9 patients, before and 3, 6, 12 months after fingolimod treatment.

Figure 4.. Increased secretion of Granzyme B by CD8⁺ T cells after fingolimod treatment.

PBMC from patients were stained for CD8 and granzyme B at baseline and 3 months after fingolimod treatment. (A) Granzyme B was measured intracellularly by flow cytometry after gating on live CD3⁺ cells. (B) Statistical analysis of the proportion of CD8⁺ cells producing granzyme B after gating on CD8⁺ cells. (C) Flow plots of granzyme B production in a representative patient before and at 3, 6 and 12 months after fingolimod treatment.

Figure 5.. Effect of fingolimod on gene and protein expression in Tregs.

PBMC were isolated from MS patients at baseline and 3 months after fingolimod treatment, labeled with T cell regulatory and effector marker antibodies. (A) Tregs were sorted (supplementary figure 2) and stimulated with PMA+ionomycin, for 4 hours. RNA expression was analyzed in batches by qRT-PCR. *p<0.05, paired t tests (compared to baseline levels), n=9. (B) Tim-3 expression was measured by flow cytometry in CD4 from patients before and 3 months after fingolimod treatment. (C) Protein expression of CCR7, PD-1, Tim-3 and CD25 was measured by mass cytometry on Tregs of 9 patients, before and at 3, 6, 12 months after fingolimod treatment.