Increased frequency of circulating follicular helper T cells in patients with rheumatoid arthritis

Abstract

Follicular helper T (Tfh) cells are recognized as a distinct CD4(+) helper T-cell subset, which provides for B-cell activation and production of specific antibody responses, and play a critical role in the development of autoimmune disease. So far, only one study investigated the circulating Tfh cells increased in a subset of SLE patients. Since relatively little is known about the Tfh cells in rheumatoid arthritis (RA) patients, in this study, Tfh-cell frequency, related cytokine IL-21, and transcription factor Bcl-6 were investigated in 53 patients with RA and 31 health controls. Firstly, we found that the frequency of CD4(+)CXCR5(+)ICOS(high) Tfh cells was increased significantly in the peripheral blood of RA patients, compared with that in healthy controls. It is known that Tfh cells are critical for directing the development of an antibody response by germinal centers B cells; secondly, we observed that the Tfh-cell frequency is accompanied by the level of anti-CCP antibody in RA patients. Furthermore, expression of Bcl-6 mRNA and plasma IL-21 concentrations in RA patients was increased. Taken together, these findings have shown that the increased frequency of circulating Tfh cells is correlated with elevated levels of anti-CCP antibody, indicating the possible involvement of Tfh cells in the disease progression of RA.

Figure 1

Increased frequency of circulating follicular helper T (Tfh) cells in peripheral blood from rheumatoid arthritis (RA) patients. Peripheral blood mononuclear cells (PMBCs) from RA patients (n = 31) and healthy controls (n = 30) were stained with labelled antibodies as described in Methods. (a) Representative expression of ICOS versus CD4 expression by flow cytometry, values in each CD4⁺ gate (ICOS^high [black box], ICOS positive [ICOS intermediate plus ICOS^high [+]], and ICOS negative). (b) Representative flow cytometry plots of Tfh (CD4⁺CXCR5⁺) cells. (c) Representative flow cytometry plots of Tfh (CD4⁺CXCR5⁺ICOS^high) cells. (d) Percentage of CD4⁺ICOS⁺ T lymphocytes in RA patients and healthy controls (***P < 0.001). (e) Percentage of CD4⁺ICOS^high T lymphocytes in RA patients and healthy controls (***P < 0.001). (f) Percentage of CD4⁺CXCR5⁺ T lymphocytes in RA patients and healthy controls (**P < 0.01). (g) Percentage of CD4⁺CXCR5⁺ICOS^high T lymphocytes in RA patients and healthy controls (***P < 0.001). Each data point represents an individual subject, and horizontal lines show the median.

Figure 2

Correlation of circulating Tfh cells frequency and autoantibody in RA patients. (a) Relationship between the frequency of circulating Tfh cells and the positive anti-CCP antibody (r = 0.5429, P = 0.0163) (n = 19). (b) Relationship between the frequency of circulating Tfh cells and the negative anti-CCP antibody (r = −0.2000,  P = 0.6134) (n = 6). (c) Relationship between the frequency of circulating Tfh cells and the level of RF (r = −0.1356, P = 0.5577) (n = 21). (d) Increased frequency of circulating Tfh cells in RA patients according to anti-CCP antibody. Horizontal lines show the median.

Figure 3

Increased Bcl-6 mRNA, IL-21 mRNA expression, and plasma IL-21 concentration in RA patients. (a) Detection of Bcl-6 mRNA expression in RA patients and healthy controls. Bcl-6 mRNA was estimated by real-time RT-PCR. Data correspond to the mean ± SD of 7 RA patients and 7 healthy controls (**P < 0.01). (b) Detection of IL-21 mRNA expression in RA patients and healthy controls. IL-21 mRNA was estimated by real-time RT-PCR. Data correspond to the mean ± SD of 7 RA patients and 7 healthy controls (**P < 0.01). (c) Plasma IL-21 concentration in RA patients (n = 35) and healthy controls (n = 29) (*P < 0.05). (d) Relationship between plasma IL-21 concentration and the frequency of circulating Tfh cells in RA patients (r = −0.0320, P = 0.9100) (n = 15).