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

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

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

Suppressive capacity of CD19+CD24^hiCD38^hi B cells in healthy subjects.

<p>Total CD19+ or B cells depleted of CD19+CD24^hiCD38^hi cells (Breg-depleted CD19+ B cells), isolated from the peripheral blood of three healthy controls (HC), were established in culture, and stimulated with anti-IgM+IgG. Secretion of IFNγ was determined in culture supernatants by sandwich ELISA at 2, 5 or 7 days. Bar graphs represent the mean and SEM for IFNγ concentrations of 3 independent experiments.</p

Clinical characteristics of AS/nb patients.

<p>Clinical characteristics of AS/nb patients.</p

Numbers of circulating CD19+CD24^hiCD38^hi B cells in AS patients naïve for TNF blockers (AS/nb).

<p>PBMCs isolated from the peripheral blood of AS/nb patients (n = 42) and from age and gender-matched healthy controls (HC) (n = 42) were stained with fluorochrome-labeled antibodies against cell surface markers and examined by flow cytometry. AS/nb patients showed an increased proportion and absolute numbers of circulating CD19+CD24^hiCD38^hi B cells. A. Representative dot plots demonstrate CD24 and CD38 expression in cells gated for CD19. B, C. Frequency (B) and absolute numbers (C) of circulating CD19+CD24^hiCD38^hi B cells in HC and AS/nb patients. Bars represent the median and interquartile range. *p<0.01.</p

Treatment with anti-TNF agents is associated with a significant reduction of circulating CD19+CD24^hiCD38^hi B cell numbers in AS patients.

<p>A. Frequency of CD19+CD24^hiCD38^hi B cells in six AS patients before and six months after initiating treatment with anti-TNFα agents, and in their 6 age and gender-matched healthy controls at the basal and 6 month study. (*p<0.05) B. CRP, calprotectin and ASDAS-CRP values in these 6 AS/nb patients before and six months after initiating treatment with anti-TNFα. (*p<0.05)</p

Clinical data of patients providing surgical synovial samples.

<p>Clinical data of patients providing surgical synovial samples.</p