Expression of IL-17A by RA CD4+ T cells.

<p>CD4+ T cells were isolated from the peripheral blood of healthy controls (HCPB) (n = 53), the peripheral blood of early RA patients (eRAPB) (n = 33), the peripheral blood of established RA patients (RAPB) (n = 20) and the synovial fluid of established RA patients (RASF) (n = 20), and stimulated with PMA/Ionomycin for 16 h or with anti-CD3/CD28/CD49d for 4 days. A, B. Percentage of CD4+ T cells expressing IL-17A (Th17 cells) after a 16 h stimulation, as determined by flow cytometry. Because the CD4 molecule is downregulated upon stimulation with PMA, shown is CD3 expression on isolated CD4+ T cells. C. Representative flow cytometry dot-plots showing expression of CD45RO, CD45RA and CCR6 versus IL-17A in isolated CD4+ T cells. D. Secretion of IL-17A to the culture medium of CD4+ T cells stimulated for 16 h with PMA/ionomycin or for 4 days with anti-CD3/CD28/CD49d. Box-plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values. *p<0.05 vs HCPB; † p<0.05 vs RAPB; ¥ p<0.05 vs eRAPB.</p

Relation of Th17 and Th17/Th1 frequencies with anti-CCP antibodies and with erosions in eRA.

<p>A. Percentage of Th17 and Th17/Th1 cells among CD4+ T cells isolated from the peripheral blood of healthy controls (HCPB) (n = 53), the peripheral blood of anti-CCP+ eRA patients (ACCP+) (n = 15) and the peripheral blood of anti-CCP- eRA patients (ACCP-) (n = 18). *p<0.05 vs HCPB. B. Linear correlation between the percentage of circulating Th17 or Th17/Th1 cells and the anti-CCP antibody titre in anti-CCP+ eRA patients. C. Frequencies of Th17 and Th17/Th1 cells in the peripheral blood of eRA patients with erosive (n = 9) or non-erosive disease (n = 24) at initial presentation. *p<0.05 vs patients with erosive disease. D. Left panel: Percentage of patients with erosive disease at initial presentation among anti-CCP+ and anti-CCP- eRA subjects; Right panel: anti-CCP antibody titres in anti-CCP+ eRA patients with erosive or non-erosive disease at initial presentation. *p<0.05 vs patients with erosive disease. Box-plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values.</p

In vivo effect of treatment on the circulating Th17 and Th17/Th1 frequencies.

<p>Eight patients with anti-CCP+ eRA donated blood for a second time, one year after the first visit, and while receiving treatment with oral MTX with or without low-dose prednisone. Four out of these patients had achieved remission and 4 of them demonstrated persistent disease activity. Shown are the frequencies of circulating Th17 and Th17/Th1 cells in patients who achieved remission (ACCP+ remission) (n = 4), in patients who did not achieve remission (ACCP-non-remission) (n = 4) and in their age and sex-matched controls (HCPB) (n = 8), observed at first evaluation (“before treatment”) and at the one-year follow-up visit (“after treatment”). Box plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values. *p<0.05 vs HCPB.</p

Proportion of Th17/Th1 cells in RA.

<p>CD4+ T cells were isolated from the peripheral blood of healthy controls (HCPB) (n = 53), the peripheral blood of early RA patients (eRAPB) (n = 33), the peripheral blood of established RA patients (RAPB) (n = 20) and the synovial fluid of established RA patients (RASF) (n = 20). A, B. Percentage of CD4+ T cells expressing both IL-17A and IFN-γ (Th17/Th1 cells) after a 16 h stimulation with PMA/ionomycin, as determined by cytometry. Box-plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values. *p<0.05 vs HCPB; † p<0.05 vs RAPB; ¥ p<0.05 vs eRAPB.</p

Expression of IFN-γ, TNF-α and IL-10 by RA CD4+ T cells.

<p>CD4+ T cells were isolated from the peripheral blood of healthy controls (HCPB) (n = 33), the peripheral blood of early RA patients (eRAPB) (n = 33) and the synovial fluid of established RA patients (RASF) (n = 20), and stimulated with PMA/Ionomycin for 16 h or with anti-CD3/CD28/CD49d for 4 days. A, B. Percentage of CD4+ T cells expressing IFN-γ (Th1 cells) after a 16 h stimulation, as determined by flow cytometry. Because the CD4 molecule is downregulated upon stimulation with PMA, shown is CD3 expression on CD4+ T cells. C. Secretion of IFN-γ by CD4+ T cells stimulated for 16 h with PMA/ionomycin or for 4 days with anti-CD3/CD28/CD49d. D. Secretion of TNF-α and of IL-10 by CD4+ T cells stimulated for 16 h with PMA/ionomycin. Box-plots represent the median and interquartile range of all studied subjects, whiskers represent the maximum and minimum values. *p<0.05 vs HCPB; † p<0.05 vs eRAPB.</p

Functional capacity of circulating CD4+CXCR5+ T cells.

<p>CD4+CXCR5+ T cells isolated from peripheral blood of HC are more efficient than cells from AS/nb patients at inducing maturation of cocultured autologous naïve B cells. A. CD19+CD20-CD38^high plasmablasts in 9-day cocultures of naïve B cells from AS/nb patients or HC with autologous CXCR5+ or CXCR5- CD4+ T cells. B, C. Concentrations of IgG, A and M at different time points (B) or at 13 days (C) in cocultures of naïve B cells from AS/nb patients or HC with autologous CXCR5+ or CXCR5- CD4+ T cells. D. Recovered viable B and T cells in 9-day cocultures of naïve B cells from AS/nb patients or HC with autologous CXCR5+ or CXCR5- CD4+ T cells. Line and bar graphs represent the mean and SD of 3 independent experiments.</p

Numbers of circulating plasmablasts in patients with AS.

<p>A, B. AS/nb but not AS/b patients demonstrate a decreased frequency of circulating plasmablasts. Shown are representative dot plots of CD20 and CD38 expression on CD19+ cells (A). C. Decreased absolute numbers of circulating plasmablasts in AS/nb patients. B and C represent box and whiskers plots from 25 AS/nb patients, 25 AS/b patients and 50 HC; * p <0.005 vs HC. D. The frequency of circulating plasmablasts in AS/nb patients is positively correlated with the frequency of circulating Tfh counterparts, with the frequency of Tfh(Th2+Th17) and with the ratio [%Tfh(Th2+Th17)]/%Tfh(Th1) cells, and negatively correlated with the frequency of Tfh-Th1 cells.</p

Numbers of circulating Tfh counterparts (cTfh) in patients with AS.

<p>AS/nb but not AS/b patients demonstrate decreased frequencies and absolute numbers of cTfh. A, B. Frequency of cTfh in HC, AS/nb and AS/b patients. Representative dot plots demonstrate ICOS and CXCR5 expression (A) or ICOS and PD-1 expression (B) in cells gated for CD3, CD4 and CXCR5. C. Absolute numbers of circulating Tfh counterparts (cTfh) in HC, AS/nb and AS/b patients. Box and whiskers plots represent the median, interquartile range, maximum and minimum values calculated from 25 AS/nb patients, 25 AS/b patients and 50 HC. *p<0.0001 vs HC; † p<0.01 vs AS/nb patients.</p

Frequency of circulating Tfh subsets in patients with AS.

<p>A. AS/nb, but not AS/b patients, demonstrate an increased frequency of circulating CD4+CXCR5+CXCR3+CCR6- (Tfh-Th1) cells and a decreased frequency of CD4+CXCR5+CXCR3-CCR6+ (Tfh-Th17) cells as compared with HC, whereas the frequency of CD4+CXCR5+CXCR3-CCR6- (Tfh-Th2) cells is not different among the three groups. Representative dot plots demonstrate CXCR3 and CCR6 expression in cells gated for CD3, CD4 and CXCR5. B. Underrepresentation of cTfh subsets with a B cell helper phenotype (Tfh-Th17 plus Tfh-Th2 cells) in AS/nb but not in AS/b patients. Box and whiskers plots represent the median, interquartile range, maximum and minimum values calculated from 25 AS/nb patients, 25 AS/b patients and 50 HC. *p<0.05 vs HC, ^†p<0.005 vs AS/nb.</p

Serum levels of IgG, IgA and IgM in patients with AS.

<p>A. IgG, IgA and IgM were determined by nephelometry in the serum of AS/nb and AS/b patients. For AS/b patients, determinations were not only done in sera collected on the day when phenotypical studies were done, but also on sera that had been taken just before initiation of treatment with TNF blockers. B. Relation of serum Ig concentrations with cTfh, circulating plasmablasts and disease activity. Shown are cTfh proportions, cTfh subset ratio and circulating plasmablast proportions together with serum IgG, IgA and IgM concentrations in HC, AS patients with inactive disease or moderate disease activity (ASDAS-CRP < 2.1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107086#pone.0107086-Machado1" target="_blank">[38]</a> and AS patients with high or very high disease activity (ASDAS-CRP > 2.1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107086#pone.0107086-Machado1" target="_blank">[38]</a>. Normal or elevated serum Ig levels are observed despite the presence of decreased or normal cTfh and plasmablasts, respectively. Note that serum Ig concentrations vary with disease activity whereas cTfh and plasmablast numbers do not. Box and whiskers plots represent the median, interquartile range, maximum and minimum values. *p<0.05 vs HC, ^†p<0.05 vs AS/nb.</p