Circulating T and B cell proportions in patients treated by fingolimod.

<p>PBMCs of MS patients before (t0, n = 16) or after three months of treatment by fingolimod (t3, n = 16) and healthy controls (HC, n = 10) were analysed <i>ex vivo</i> by flow cytometry. The percentage of (A) CD19⁺ B cells, (B) CD8⁺ and (C) CD4⁺ T cells within lymphoid cells is shown. The proportion of B cells and CD4⁺ T cells decreased after fingolimod while the proportion of CD8⁺ T cells was not significantly modified. The horizontal lines represent the median value in all subgroups. *, ** and *** indicate p-values of ≤0.05, ≤0.01 and ≤0.001 respectively.</p

Immune cell subpopulations in MS patients before (t0) or after three months of fingolimod treatment (t3) and healthy controls analysed by <i>ex vivo</i> flow cytometry.

<p>The median and range are shown. Lymphoid cells were gated according to their FSC/SSC profile.</p><p>Immune cell subpopulations in MS patients before (t0) or after three months of fingolimod treatment (t3) and healthy controls analysed by <i>ex vivo</i> flow cytometry.</p

<i>Ex vivo</i> mRNA expression in total PBMCs from fingolimod-treated patients.

<p>Scatter dot plots show relative mRNA expression levels in PBMCs from MS patients before (t0) or after 3 months (t3) of treatment by fingolimod, and healthy controls (HC) analysed by q-PCR. For each target, individual mRNA levels were expressed as relative values to the mean level of the control group. The mRNA expression levels of CD39 (p = 0.0033), as well as of AHR (p = 0.007) and CYP1B1, an AHR-induced gene (p<0.0001) were increased after fingolimod treatment. On the contrary, fingolimod reduced the mRNA levels of IL-17, IL-22 and FOXP3. Horizontal lines represent the median value in all subgroups. *, ** and *** indicate p-values of ≤0.05, ≤0.01 and ≤0.001 respectively.</p

Regulatory T cells in PBMCs of fingolimod-treated patients.

<p>Regulatory T cells of MS patients before (t0, n = 16) or after three months of treatment by fingolimod (t3, n = 16) and healthy controls (HC, n = 10) were analysed <i>ex vivo</i> by MethylS-qPCR (A) to quantify nTregs with demethylated <i>FOXP3i1</i>. The proportion of circulating CD4⁺CD25^hiFOXP3⁺ Tregs was also measured by flow cytometry and expressed as a percentage of lymphoid cells (B). The proportion of CD25^hiFOXP3⁺ cells was also expressed as a percentage of CD4⁺ T cells (C). nTreg proportion among total PBMCs was decreased by the treatment, as well as the proportion of Tregs within lymphoid cells. CD25^hiFOXP3⁺ Tregs were however enriched within the CD4⁺ cell population in 12 out of 16 patients. The horizontal lines represent the median value in all subgroups. ** indicates a p-value of ≤0.01.</p

IL-22, GM-CSF and IL-17 in peripheral CD4⁺ T cell subpopulations during multiple sclerosis relapses and remission. Impact of corticosteroid therapy

<div><p>Multiple sclerosis (MS) is thought to be a Th17-mediated dysimmune disease of the central nervous system. However, recent publications have questioned the pathogenicity of IL-17 per se and rather suggest the implication of other Th17-related inflammatory mediators. Therefore, we studied the expression of GM-CSF, IL-22, IL-24, IL-26 and CD39 in peripheral blood mononuclear cells (PBMCs) from MS patients during relapses, remission and following corticosteroid treatment. We performed qPCR to measure mRNA levels from <i>ex vivo</i> or <i>in vitro</i>-stimulated PBMCs. Cytokine levels were determined by ELISA. We used flow cytometry to assess GM-CSF⁺, IL-22⁺ and CD39⁺ cells in relationship to IL-17⁺ CD4⁺ T cells. Our results showed that IL-22 mRNA and IL-22⁺CD4⁺ lymphocytes are increased in circulating cells of relapsing MS patients compared to remitting patients while GM-CSF was unchanged. We have further shown that 12.9, 39 and 12.4% of Th17 cells from MS patients during relapses expressed IL-22, GM-CSF and CD39 respectively. No changes in these proportions were found in stable MS patients. However, the majority of GM-CSF⁺ or IL-22⁺ T cells did not co-express IL-17. GM-CSF mRNA, but not IL-22 mRNA, was dramatically decreased <i>ex vivo</i> by ivMP. Our results contribute to a better characterisation of Th17, Th22 and ThGM-CSF cells in the setting of MS and according to disease activity.</p></div

IL-22 mRNA and IL-22-producing cells in PBMCs from relapsing and stable MS patients and from healthy controls (HC).

<p>Quantitative-PCR to measure IL-22 mRNA expression (A) <i>ex vivo</i> (Relapsing MS: n = 56, Stable MS: n = 16, HC: n = 37) and (B) after 4h of stimulation by PMA/ionomycin (Relapsing MS: n = 14, Stable MS: n = 10, HC: n = 12). (C) PBMCs were stained for IL-22, CD3 and CD4 after 4h stimulation by PMA/ionomycin in the presence of a protein transport inhibitor and analysed by flow cytometry (Relapsing MS: n = 15, Stable MS: n = 12, HC: n = 14). Scatter dot plot shows the percentage of IL-22⁺CD4⁺ T cells. The horizontal lines of scatter plots represent the median value in all subgroups. * and *** indicate respectively p-values ≤ 0.05 and ≤ 0.001.</p

GM-CSF and IL-22 before and after intravenous methylprednisolone (ivMP) treatment in paired samples of relapsing MS patients.

<p>Quantitative-PCR to measure GM-CSF and IL-22 mRNA expression <i>ex vivo</i> (A, n = 13 and D, n = 17) and (B, n = 8 and E, n = 8) after 4h stimulation by PMA/ionomycin. Results are expressed relative to the mean of the relapsing patients set at 1. PBMCs were stained for GM-CSF (C, n = 8) or IL-22 (F, n = 15), CD3 and CD4 after 4h stimulation by PMA/ionomycin in the presence of a protein transport inhibitor and analysed by flow cytometry. The horizontal lines of scatter plots represent the median value in all subgroups. (G) PBMCs of HC (n = 5) were treated with methylprednisolone (MP) for 24h and stimulated 2h with PHA. Error bars represent the standard error of the mean. * and *** indicate respectively p-values ≤ 0.05 and ≤ 0.001.</p

FACS analysis of GM-CSF, IL-22 and CD39 coexpression with IL-17 in CD4⁺ T cells.

<p>PBMCs were fixed and stained after 4h stimulation by PMA/ionomycin in the presence of a protein transport inhibitor. CD4⁺ cells were gated among the CD3⁺ population. Representative dot plots of (A) GM-CSF, (B) IL-22 and (C) CD39 costaining with IL-17. Numbers in each quadrant represent the average percentage of positive CD4⁺ T cells in relapsing MS patients.</p

Main demographic features of MS patients and healthy control groups.

<p>Main demographic features of MS patients and healthy control groups.</p

GM-CSF expression analysis in PBMCs of MS patients and HC.

<p>GM-CSF expression analysis in PBMCs of MS patients and HC.</p