Increased presence of CD4⁺CD25⁺ and CD25^highFoxP3^highCD127^- cells in the circulation of patients with systemic sclerosis (SSc).

<p>Flow cytometry analysis of and CD4⁺CD25⁺ and CD25^highFoxP3^highCD127^- cells was performed in healthy controls (n = 26) and patients (n = 68) with different phenotypes of SSc. Peripheral blood mononuclear cells (PBMC's) were stained with anti-CD4, anti-CD25, anti-CD127 and anti-FoxP3, and analyzed by flow cytometry. (a) One representative individual from each group is shown. (b) Percentage of CD4+CD25+ and CD25+FoxP3+ cells are presented for each group, consisting of healthy controls (n = 26), lSSc (n = 20), ldSSc (n = 24) and edSSc (n = 24) patients. (c) Based upon CD25 expression, the top 10% (CD25^bright) and top 2% (CD25^verybright) were gated and FoxP3 expression analyzed as the percentage positive cells. (d) Based upon FoxP3 expression, the top 10% (FoxP3^bright) and top 2% (FoxP3^verybright) were gated and CD127 expression analyzed as the percentage positive cells. Data is presented as mean±sem.</p

SSc patients express high levels of IL-17-positive T cells and the co-presence of IL-17 with either IFNγ, IFNα or TGFβ reflects SSc phenotype.

<p>Panel A presents the percentage of IL-23R positive cells in the CD4+ positive pool of T cells (left panel) and CD45Ro and CD45Ra positive cells (right panel) from healthy controls (n = 14), and SSc patients with the lcSSc (n = 12), ldcSSc (n = 11) and edcSSc (n = 13) phenotype. Panel B shows the mean intracellular expression level of IL-17, IFNγ and TGFβ in CD4+ cells from each a representative individual from each tested group (left side). On the right side, the mean percentage of CD45Ro-positive or CD45Ra-positive cells that express IL-17, IFNγ or TGFβ and the mean intensity thereof are presented for the whole group of healthy controls (n = 14), and SSc patients with the lcSSc (n = 12), ldcSSc (n = 11) and edcSSc (n = 13) phenotype. Panel C depicts the percentage of CD25^high cells that co-express IL-17 in healthy controls and different SSc phenotypes (* represents a p-value<0.01). Panel D represents the level of cytokines (IL-17 and IFNγ) spontaneously secreted by CD3+ T cells from healthy controls and SSc patients after 24 hrs of incubation. * <i>P</i>-value<0.0001, ** <i>P</i>-value<0.002, *** <i>P</i>-value<0.004.</p

Impaired suppressive function by Tregs from SSc patients correlates with surface expression of CD69 and intracellular expression of TGFβ.

<p>Unsorted CD3+ (MACS bead isolated) were stimulated with PHA (5 µg/ml) and consecutively incubated with CD25^highCD127^- or CD25^lowCD127^high cells for 5 days. Thereafter, CD3+ cells were incubated with 3^H-thymidine for 24 more hours after which 3^H-thymidine incorporation was measured. Panel (a) reflects the suppressive capacity of Tregs from healthy donors and SSc patients. Proliferation of CD3+ effector cells was effectively inhibited by T regulatory cells from healthy controls, whereas a clearly diminished suppressive activity was observed in the experiments with Tregs from SSc patients. Suppressive effect of Treg (CD25^highCD127^-) and non-Tregs (CD25^lowCD127^high) is presented in black and white bars, respectively. Results are the mean and SEM of 6 separate experiments using cells from healthy donors (n = 9), lSSc (n = 7), ldSSc (n = 9) and edSSc (n = 7). Panel (b) represents the correlation of CD69 expression and Treg suppressive capacity in Tregs from the various groups under investigation. The percentage of CD69 positive regulatory T cells (CD25^highCD127^-) correlates well with the percentage of inhibition of CD3+ cells in healthy controls (triangles), lSSc (diamonds), ldSSc (circles) and edSSc (squares). Panel (c) reflects the expression of intracellular TGFβ in Tregs from healthy controls and SSc patients as measured using intracellular flow cytometry. CD25^highCD127^- cells from all SSc patients express lower TGFβ levels compared to controls. Left panel reflects an representative individual from each group whereas the right panel displays the mean of each group comprising 6 individuals (per group) coming forth from 4 independent experiments.</p

Phenotypical characterization of T regs reveals diminished expression of CD62L and CD69 in SSc patients.

<p>Panel (a) of this figure depicts the expression of the T cell activation marker GITR on CD25+, CD25^bright and CD25^verybright cells from healthy controls (white bars, n = 24) and SSc patients having limited cutaneous SSc (light gray bars, n = 18), late diffuse SSc (dark gray bars, n = 22) and early diffuse SSc (black bars, n = 22) patients. In panel (b) the expression of CD62L on Tregs is investigated. CD25+ and CD25^bright cells from SSc and healthy controls express similar levels of CD62L, whereas CD25^verybright from SSc patient subsets exhibit lower levels of CD62L compared to those from healthy controls. Panel (c) reflects the expression of CD69 on Tregs from healthy donors and SSc patients. CD25+, CD25^bright and CD25^verybright cells from SSc patients express significant lower levels of CD69 than those from healthy donors. CD69 expression on CD25^bright and CD25^verybright cells from edSSc patients was significantly lower then that from ldSSc patients, and ldSSc expressed CD69 significantly lower than those from lSSc. In panel (d) the expression on CD3+ cells is shown for all investigated groups. In contrast with that observed on Tregs from SSc patients, CD69 expression on CD4+ cells was significantly higher in all SSc patient groups. Panel (e) reflects the potential association between CD69 expression on Tregs and disease duration. CD69 expression on T regs from patients with lSSc correlated with disease duration, whereas this was not the case either with ldSSc nor edSSc. In all figures the white bars represent healthy controls, whereas lSSc, ldSSc and edSSc patients are represented by light gray, dark gray and black bars, respectively.</p

Patients clinical characteristics.

*<p>P value 0.03.</p

Cytokine expression pattern in SSc clinical phenotypes.

<p>Cytokine expression pattern in SSc clinical phenotypes.</p

SSc patients have more and more activated CD4+ T cells compared to healthy controls.

<p>Panel A depicts the percentage of CD4+ and CD8+ cells in the whole T cell pool (CD3+ cells) from healthy controls (n = 14), and SSc patients with the lcSSc (n = 12), ldcSSc (n = 11) and edcSSc (n = 13) phenotype. The CD3+ cells were isolated using MACS bead isolation after which CD4 and CD8 positivity was analyzed using flow cytometry. Panel B (left) depicts the percentage of CD4+ and CD8+ cells that were double-positive for the T cell activation markers CD69 and GITR. For this aim a representative individual from each group was selected. In the right panel, the percentage of CD4-CD69, CD8-CD69, CD4-GITR, CD8-GITR, CD45Ro-CD69 and CD45Ra-CD69 double positive cells is presented over the whole group of healthy donors (n = 14) and/or SSc patients (n = 36).</p

Plasma from SSc patients abrogates T cell suppression and up regulates CD69.

<p>(a) During the co-cultures of unsorted CD3+ cells with either Tregs (CD25^highCD127^-) or non-Tregs (CD25^lowCD127^high) 10 or 25% plasma from an edSSc patient or healthy control was added to the culture. The graph represents data from 3 independent experiments using 3 healthy control cells, and plasma derived from two edSSc patients and two control individuals. (b) The effect of SSc plasma was evaluated by adding 10% to CD3+ cells for 24 hrs stimulated with PHA or unstimulated. As a control, CD69 expression was measured on CD3+ cells stimulated with PHA only. CD4 and CD25^high/FoxP3^high cells were gated based on the expression of these markers using flow cytometry. (c) CD69 expression and induction upon PHA mediated stimulation of CD4+ and CD25^high/FoxP3^high obtained from healthy donors, lSSc, ldSSc and edSSc patients was investigated using flow cytometry. One representative patient from each group is shown.</p

Clinical characteristics of patients included in in vitro assays.

*<p>0.03.</p

Increased level of Th17 inducing cytokines in the circulation of SSc patients.

<p>Panel A depicts the presence of IL-17 in the circulation of SSc patients and healthy controls. Panel B, C and D represents the levels of IL-6, IL-1α and IL-23, respectively. The circulating levels of IL-17, IL-1α and IL-23 was measured by ELISA whereas IL-6 was studied by Bioplex assays.</p