A two-sided P-value of <0·05 was considered statistically significant. To determine the role of different differentiation stages of B cells and Tfh cells in the pathogenesis of RA, a total of 25 patients with new-onset RA and 15

gender- and age-matched HC were recruited. There was no significant difference in the distribution of age and gender and the numbers of white blood cells (WBC) and lymphocytes between the patients and HC (Table 1). As expected, the levels of serum RF, CRP and anti-CCP and the values of ESR in the patients were significantly higher than that in the HC. We characterized the frequency of different differentiation stages of B cells by flow cytometry analysis. As shown in Fig. 1, the percentages of IgD+CD27−CD19+ (naive B), CD86+CD19+, CD95+CD19+ B cells in those patients were significantly higher than that in the HC. In contrast, the frequency of IgD+CD27+CD19+ mTOR inhibitor preswitch Bcl-2 inhibitor memory B cells was significantly lower in the patients than that in the HC. There was no significant difference in the frequency of IgD−CD27+CD19+ post-switch memory B cells, IgD−CD27−CD19+ double-negative

B cells, CD38+CD19+ and TLR-9+CD19+ B cells between the RA patients and HC. Interestingly, the percentages of CD86+CD19+ B cells were correlated positively with the values of DAS28 in those patients (Fig. 1c). However, there was no significant correlation between the values of DAS28 and the frequency of other B cell subsets in this population (data

not shown). Given that CD86 and CD95 were up-regulated in B cells, our data indicated that the higher frequency of activated B cells contributed to the pathogenesis of RA in Chinese patients with new-onset RA. Tfh cells can promote B cell activation, expansion and differentiation. To investigate the potential role of Tfh cells in the development of RA, we characterized the percentages of peripheral blood CD3+CD4+CXCR5+ cells in total CD3+CD4+ T cells in patients and HC by flow cytometry analysis (Fig. 2a). We found that the percentages of CD3+CD4+CXCR5+cells, CD3+CD4+ICOS+CXCR5+, CD3+CD4+PD-1+CXCR5+ and CD3+CD4+ICOS+PD-1+CXCR5+ Tfh cells in CD3+CD4+CXCR5+ cells in the patients were significantly higher than those in the HC (Fig. 2b). Given that Tfh cells can secrete IL-21, which has been shown to regulate all B cell differentiation and proliferation [23-25], we examined the concentrations of serum IL-21 in those patients and HC by ELISA (Fig. 2c). We found that the levels of serum IL-21 in the patients were significantly higher than that in the HC. These data clearly indicated a higher frequency of activated Tfh cells and higher levels of serum IL-21 in patients with new-onset RA, and may contribute to the development of RA. Next, we examined the relationship between Tfh and B cells in RA patients and found that the percentages of CD3+CD4+CXCR5+ cells were correlated positively with the frequency of CD19+ B cells in those patients (Fig. 3a).