Compromised CD4+ CD25(high) regulatory T-cell function in patients with relapsing-remitting multiple sclerosis is correlated with a reduced frequency of FOXP3-positive cells and reduced FOXP3 expression at the single-cell level

Abstract

CD4+ CD25(high) regulatory T cells (Tregs) of patients with relapsing-remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single-cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4+ CD25(high) FOXP3+ T cells and lower FOXP3 protein expression per cell in RR-MS patients than in SP-MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)-beta-treated RR-MS patients showed restored numbers of FOXP3+ Tregs. Furthermore, a higher percentage of CD4+ CD25(high) FOXP3+ Tregs in RR-MS patients, as compared with controls and SP-MS patients, expressed CD103 and CD49d, adhesion molecules involved in T-cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27+ CD25(high) CD4+ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR-MS patients. Taken together, these data show aberrant FOXP3 expression at the single-cell level correlated with Treg dysfunction in RR-MS patients. Our results also suggest that Tregs accumulate in the CSF of RR-MS patients, in an attempt to down-regulate local inflammation in the central nervous system.

Figure 1

Intracellular forkhead box P3 (FOXP3) expression in peripheral blood mononuclear cells (PBMC) isolated from healthy controls (HCs) and multiple sclerosis (MS) patients. PBMC of HCs and MS patients were surface-stained with anti-CD4 and anti-CD25 antibodies. After permeabilization, cells were labelled with anti-human FOXP3 antibody. Cells were analysed by means of flow cytometry. The CD25 signal was subdivided into negative, intermediate (int) and high levels. The CD25^high CD4⁺ T-cell population was defined as the top 2% of CD4+ T cells showing the brightest CD25 expression. The figure shows costainings of FOXP3 and CD4 (dot plots) or FOXP3 expression of CD4⁺ CD25^–, CD4⁺ CD25^int and CD4⁺ CD25^high T cells (histograms). The cut-offs for positive and negative signals were set based on isotype-matched control antibody staining. Numbers in each histogram indicate the percentage of FOXP3-positive cells. Data are shown for one HC, one relapsing-remitting multiple sclerosis (RR-MS) patient and one secondary progressive multiple sclerosis (SP-MS) patient, and are illustrative for all study subjects, as indicated in Figure 2.

Figure 2

Frequency of CD4⁺ CD25⁺ FOXP3⁺ T cells and forkhead box P3 (FOXP3) expression at the cellular level in multiple sclerosis (MS) patients and control subjects. Peripheral blood mononuclear cells (PBMC) of 40 healthy controls (HCs), 18 patients with other neurological diseases (ONDs), 55 patients with relapsing-remitting multiple sclerosis (RR-MS) (40 untreated and 15 IFN-β-treated), 15 patients with secondary progressive multiple sclerosis (SP-MS), 10 patients with rheumatoid arthritis (RA) and five patients with systemic lupus erythematosis (SLE) were stained for FOXP3, CD25 and CD4 as described in Fig. 1. (a) The percentage of FOXP3⁺ CD25⁺ CD4⁺ T cells relative to the total number of CD4⁺ T cells. (b) Percentage of FOXP3⁺ cells within the CD4⁺ CD25^–, CD4⁺ CD25^int and CD4⁺ CD25^high T-cell populations. (c) For five untreated RR-MS patients and three HCs, FOXP3⁺ CD25⁺ CD4⁺ T-cell frequencies were measured at different time-points (0, 6 and 12 months after initial measurement). (d) Mean fluorescence intensity (MFI) of FOXP3 from CD4⁺ CD25^high FOXP3⁺ T cells in patients and controls. Indicated values (shown as box and whiskers) represent MFI signals obtained from FOXP3 stainings. MFI values of cells stained by isotype-matched control antibody (background MFI) were subtracted from FOXP3 MFI values. *P < 0·05; **P < 0·01 as compared with the indicated study populations. (e) FOXP3 MFI of CD4⁺ CD25^high FOXP3⁺ T cells was plotted against percentages of FOXP3⁺ cells within the CD4⁺ CD25^high T-cell populations of 55 MS patients, 18 OND patients and 40 HCs. r and P values were calculated using Spearman's correlation test.

Figure 3

The suppressive capacity of CD4⁺ CD25^high T cells in function of forkhead box P3 (FOXP3) expression. The suppressive capacity of CD4⁺ CD25^high T cells from 10 patients with relapsing-remitting multiple sclerosis (RR-MS; ), 10 patients with secondary progressive multiple sclerosis (SP-MS; ♦) and 10 healthy controls (HCs; ○) were plotted against the percentage of CD4⁺ CD25^high T cells positive for FOXP3, and FOXP3 mean fluorescence intensity (MFI). Suppression values represent percentages of inhibition of proliferation (5,6-carboxy fluorescein diacetate succinimidyl ester (CFSE)) of CD4⁺ CD25^– T cells (Tresp) by CD4⁺ CD25^high T cells (Tregs) as measured in coculture experiments at a 1 : 1 (Tresp:Treg) ratio. r and P values were calculated using Spearman's correlation tests.

Figure 4

Expression of adhesion molecules on regulatory T cells (Tregs) of multiple sclerosis (MS) patients and controls. CD4⁺ CD25^high and CD4⁺ CD25^– T cells within the peripheral blood mononuclear cell (PBMC) population of 20 healthy controls (HCs), 15 patients with other neurological diseases (ONDs), 15 untreated and 10 interferon (IFN)-β-treated relapsing-remitting multiple sclerosis (RR-MS) patients and 12 secondary progressive multiple sclerosis (SP-MS) patients were analysed for expression of the indicated markers [percentage positive cells or mean fluorescence intensity (MFI)] by means of flow cytometry. *P < 0·05; **P < 0·01 compared with the corresponding population.

Figure 5

Analysis of regulatory T cell (Treg) frequency in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and patients with other neurological diseases (ONDs). (a) Flow cytometric analysis of forkhead box P3 (FOXP3), CD25 and CD4 on cells isolated from the CSF of relapsing-remitting multiple sclerosis (RR-MS) patients. The dot plots shown are representative for data obtained from two RR-MS patients. (b) Percentages of CD27⁺ CD25^high and FOXP3⁺ CD25^high CD4⁺ T cells (within the total CD4⁺ T-cell population) were determined by flow cytometric analysis of peripheral blood mononuclear cells (PBMC) isolated from 11 OND and 12 RR-MS patients. The cut-off value for high staining of CD25 was set at a constant value (10²) to allow comparison between the T-cell subsets. The percentages of CD27⁺ CD25^high and FOXP3⁺ CD25^high CD4⁺ T cells were significantly correlated (P < 0·0001) in the PBMC of the same individuals as measured by linear regression analysis (r² = 0·92). (c) Frequency of CD4⁺ CD27⁺ CD25^high T cells in paired peripheral blood and CSF samples of 12 OND, eight RR-MS and three SP-MS patients.