Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

Abstract

Multiple sclerosis (MS) is a T cell-mediated autoimmune disease. Fingolimod, a highly effective disease-modifying drug for MS, retains CCR7⁺ central memory T cells in which autoaggressive T cells putatively exist, in secondary lymphoid organs, although relapse may still occur in some patients. Here, we analyzed the T cell phenotypes of fingolimod-treated, fingolimod-untreated patients, and healthy subjects. The frequency of CD56⁺ T cells and granzyme B-, perforin-, and Fas ligand-positive T cells significantly increased during fingolimod treatment. Each T cell subpopulation further increased during relapse. Interestingly, T cells from fingolimod-treated patients exhibited interferon-γ biased production, and more myelin basic protein-reactive cells was noted in CD56⁺ than in CD56^- T cells. It is likely that the altered T cell phenotypes play a role in MS relapse in fingolimod-treated patients. Further clinical studies are necessary to investigate whether altered T cell phenotypes are a biomarker for relapse under fingolimod therapy.

Figure 1. Frequency of CD56⁺ T cells in healthy subjects (HS) and patients with multiple sclerosis (MS).

(a) CD56 expression on CD3-gated T cells. Representative FACS dot plots of HS, MS patients without disease-modifying drug (MS without DMD), MS patients with IFN-β therapy (MS with IFN-β), and those with fingolimod therapy (MS with fingolimod) are shown. (b) The frequency of CD56⁺ cells in CD3⁺ T cells in the indicated groups. (c) The frequency of CD56⁺ cells in CD4⁺ T cells in the indicated groups (left), and that of CD56⁺ cells in CD8⁺ T cells in the indicated groups (right). (d) The frequency of CD56⁺ T cells before and after fingolimod treatment in MS patients. The frequency of CD56⁺ T cells within total T cells, and CD4⁺ and CD8⁺ T cell subsets is shown. nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. Association of CD56 and CCR7 expression on T cells in healthy subjects (HS) and multiple sclerosis (MS) patients.

(a) The frequency of CCR7⁺ T cells within CD3⁺ T cells in F-MS patients both in remission and at relapse. (b) The frequency of central memory T cells (CCR7⁺CD45RA⁻ T cells) within CD3⁺, CD4⁺ or CD8⁺ T cells in F-MS patients both in remission and at relapse. (c) The frequency of CCR7⁻ T cells within CD56⁺ T cells in HS and each patient group. (d) The frequency of CD56⁺ cells within CCR7⁻ and CCR7⁺ T cells. (e) The frequency of CD56⁺ cells within naïve (CCR7⁺CD45RA⁺), central memory (CCR7⁺CD45RA⁻) (TCM), effector memory (CCR7⁻CD45RA⁻) (TEM) and CD45RA⁺ TEM (TEMRA) subsets in CD4⁺ and CD8⁺ T cells. nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. Effect of fingolimod, fingolimod-phosphate and the serums of patients under fingolimod therapy on CD56 expression in vitro.

(a) Kinetic change of CD56⁺ T cells co-cultured with fingolimod (F) or fingolimod-phosphate (F-p) for 5 days in healthy subjects (HS) and multiple sclerosis patients without disease-modifying drug (MS without DMD). (b) CD56 expression on T cells stimulated by PHA in the presence or absence of fingolimod or fingolimod phosphate at a concentration of 0.1 and 1 μM. Representative graphs of CD56 expression on T cells cultured in the presence of PHA, PHA with 0.1 μM fingolimod and PHA with 0.1 μM fingolimod-phosphate (left panel) (c) CD56 expression on T cells of MS without DMD co-cultured with serum of the same patient after fingolimod therapy. Each column represents mean ± SEM. p-values: *p < 0.05.

Figure 4. Expression of cytotoxic molecules in each T cell subset of healthy subjects (HS) and multiple sclerosis (MS) patients.

(a) Representative analysis of CD56 and granzyme B expression, gated on CD3⁺ T cells from left to right: a healthy subject, an MS patient not on fingolimod in remission (nF-MS rem) and one at relapse (nF-MS rel), a relapse-free MS patient with fingolimod therapy (rel-free F-MS) and one at relapse (F-MS rel). (b) The expression of granzyme B (GrzB), perforin, and Fas-Ligand (FasL) in or on CD3⁺ T cells are shown. (c) The frequency of cells expressing each cytotoxic molecule was compared between CD56⁺ and CD56⁻ T cells. (d) The frequency of cells expressing each cytotoxic molecule in CD4⁺CD56⁺ T cells, CD4⁺CD56⁻ T cells, CD8⁺CD56⁺ T cells, and CD8⁺CD56⁻ T cells. Bars represent median values. Each column represents mean ± SEM. p-values: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. The frequency of proinflammatory cytokine-producing cells in response to PMA/ionomycin stimulation.

(a) Representative data of FACS dot plots analyzing IFN-γ and IL-17A production gated on CD3⁺ T cells. Depicted as follows from left to right, a healthy subject (HS), a multiple sclerosis (MS) patient not on fingolimod in remission (nF-MS rem) and one at relapse (nF-MS rel), and a relapse-free patient with fingolimod therapy (rel-free F-MS) and one at relapse (F-MS rel). (b) The frequency of IL-17A-producing cells in CD3⁺ T cells (left) and that of IFN-γ-producing cells in CD3⁺ T cells (right). (c) The frequency of IFN-γ producing cells was compared between CD56⁺ and CD56⁻ T cells. (d) The frequency of IFN-γ-producing cells in CD4⁺CD56⁺ T cells and CD4⁺CD56⁻ T cells, CD8⁺CD56⁺ T cells and CD8⁺CD56⁻ T cells. Bars represent median values. Each column represents mean ± SEM. P-values: *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6. Myelin basic protein (MBP)-reactive proinflammatory cytokine production by CD56⁺ T cells from multiple sclerosis (MS) patients on fingolimod in remission.

CD56⁺ and CD56⁻ T cells were co-cultured with antigen-presenting cells (APC) in the presence or absence of MBP peptide mixture, and intracellular cytokines were analyzed by flow cytometry. (a) Representative FACS dot plots of IFN-γ-producing cells in CD56⁺ and CD56⁻ T cells when the cells were co-cultured with APC in the presence or absence of MBP. (b) The delta frequency of cells that produced IFN-γ (upper row) and IL-17A (bottom row) within CD56⁺ and CD56⁻ T cells. The delta frequency was calculated by subtracting the frequency of cytokine-producing cells in the absence of MBP from that in the presence of MBP. Diamonds are data from relapse-experienced MS patients with fingolimod therapy in remission, and circles represent relapse-free MS patients while on fingolimod treatment. Bars represent median values. p-values: **p < 0.01.

Figure 7. Longitudinal analysis of CD56 expression, granzyme B (GrzB) expression, and frequency of IFN-γ-producing cells in relapse-experienced multiple sclerosis (MS) patients on fingolimod.

(a) Temporal changes in the frequency of CD56-expressing cells within the CD3⁺ T cell fraction. (b) The frequency of GrzB-expressing cells and IFN-γ-producing cells in CD56⁺ and CD56⁻ T cell subsets decreased 3 months after relapse. (c) The frequency of CD56 expression in relapse-experienced MS patients with fingolimod therapy in remission remained higher than in patients who did not have any relapse while on fingolimod treatment. Each relapse-experienced patient is indicated by different-shaped dots. The frequencies at relapse are presented as red dots, and those in remission are presented as black dots. Bars represent median values. p-values: **p < 0.01.

Figure 8. The frequency of Foxp3+ regulatory T cells in multiple sclerosis patients and healthy subjects.

The frequency of regulatory T cells (Treg cells: CD3⁺CD4⁺CD25⁺Foxp3⁺ cells) in CD3⁺ T cells (upper) and CD4⁺ T cells (bottom). HS: healthy subjects, nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p < 0.05; **p < 0.01.