APMIS 123: 659–666
© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12399
Elevated circulating Th17 and follicular helper CD4+ T cells in patients with rheumatoid arthritis YAN ZHANG, YU LI, TING-TING LV, ZHEN-JIE YIN and XIN-BO WANG Department of Immunology and Rheumatology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China
Zhang Y, Li Y, Lv T-T, Yin Z-J, Wang X-B. Elevated circulating Th17 and follicular helper CD4+ T cells in patients with rheumatoid arthritis. APMIS 2015; 123: 659–666. It remains not fully elucidated the potential functions of Th17 cells and follicular helper T (Tfh) cells and secreting cytokines in the pathogenesis of rheumatoid arthritis (RA) and their association with disease activity. In this study, the frequencies of Th17 and Tfh cells were determined by flow cytometry, and the levels of interleukin (IL)-17, IL-21, and IL-22 were measured by ELISA in RA patients with different disease activities. The dynamic changes of cell subsets were also detected in response to disease-modify antirheumatic drugs (DMARDs) therapy. The percentages of CD3+CD4+IL-17A+ (Th17) cells and CD3+CD4+CXCR5+ICOShigh (Tfh) cells, as well as the concentrations of IL17, IL-21, and IL-22 were significantly elevated in RA patients than those in healthy individuals. Furthermore, Tfh cells, IL-21, and IL-22 in the serum was positively correlated with the values of disease activity score. Concentrations of IL-21 and IL-22 in the serum were remarkably reduced following the DMARDs therapies. Our data suggested that Th17 cells, Tfh cells as well as the secreting cytokines may be involved in the pathogenesis of RA. The frequency of circulating Tfh cells and the productions of IL-21 and IL-22 were associated with the disease activity of RA patients, and might be potential therapeutic targets for treatment of RA. Key words: Rheumatoid arthritis; Th17 cells; follicular helper T cells; interleukin-21; interleukin-22. Xin-Bo Wang, Department of Immunology and Rheumatology, Tangdu Hospital, Fourth Military Medical University, 569 Xinsi Rd, Xi’an, Shaanxi Province 710038, China. e-mail: [email protected]
Yan Zhang and Yu Li contributed equally to this work.
Rheumatoid arthritis (RA) is a long-term and systemic inflammatory disease which primarily affects small diarthrodial joints of hands and feet, with pain, stiffness, and fatigue (1, 2). It can be a disabling condition, which may lead to substantial loss of function and mobility if not adequately treated. The autoimmune disorder, which involves dysregulated-lymphocyte activation and circulating autoantibodies, was considered to play a crucial role in pathogenesis of RA although the etiology of RA is still not fully understood (3). Synovial joints are characterized by chronic inflammation and abnormally infiltrated by a variety of immune cells, including T cell, B cell, macrophage, granulocyte, and monocyte in RA patients (4). Moreover, the immunogenetics of RA suggested a key role for aberrant CD4+ T cell activation in the initiation and perpetuation of the disease (4–6). Received 3 December 2014. Accepted 3 April 2015
CD4+ T cells can differentiate into subsets of helper T (Th) cells once activated. Five different Th cell subsets, which are named as Th1, Th2, Th17, regulatory T, and follicular T (Tfh) are identified till now (7). Th cells are also critical in the activation of B cells and their differentiation into antibody-secreting cells. Th17 cells require transforming growth factor-b (TGF-b) and IL-6 for initiation and IL-23 to become an established population (8). Numerous studies have suggested that elevated T17 cells were involved in the pathogenesis of RA and might be negative predictors to the effect of anti-RA treatments (9–12). Recently, Tfh cells presented a new Th lineage to provide help to the B cells for the generation of germinal centers (GCs) and induce continuously protective humoral responses (13). Distinguishing features of Tfh cells are the expressions of the regulator transcription factor Bcl6, chemokine (C-X-C motif) receptor 5 (CXCR5), inducible costimulator (ICOS), interleukin-21 659
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(IL-21), and absence of Blimp-1 (14). Previous study has revealed that Tfh-producing IL-21 directly regulates B cells proliferation and class switching (15). Nurieva et al. also demonstrated that IL-21 could synergize IL-6 to induce Bcl-6 expression in CD4+ T cells, thus promoting Tfh cells differentiation (16). Importantly, generation of Th17 cells is also induced by IL-21 and IL-6 (17), with the evidence that elevated circulating CD4+ T cells-secreting IL-21 was correlated with increasing Th17 and memory B cells in patients with systemic lupus erythematosus (SLE) (18). This indicates that dysregulated functions of both Th17 and Tfh cells likely contributed to the pathogenesis of autoimmune diseases. An elegant study by Feng et al. (19) recently showed that frequencies of circulating Tfh cells, but not Th17 cells, were increased in SLE patients and correlated with disease activity, which prompted us to measure the circulating percentages of Th17 and Tfh cells, as well as their secreting cytokines in RA patients and the correlation with disease activity. In this study, the frequencies of IL-17-producing (Th17) and CXCR5+ICOShigh (Tfh) expressed CD4+ T cells were determined in RA patients with different disease activity, and the dynamic changes of those cell subsets were also detected in response to diseases-modified antirheumatic drugs therapy. MATERIALS AND METHODS Subjects Blood samples were collected from 54 patients with RA. Diagnoses were made with the diagnostic criteria of according to the criteria of the American College of Rheumatology (20). All patients were hospitalized or followed up in Tangdu Hospital, from June 2008 to March 2010. Patients with other rheumatic diseases, such as SLE, mixed connective tissue disease (MCTD) were excluded from this study. No patients had received steroids, chondrotin polysulphate, or hyaluronic for at least 2 months before sampling. For healthy controls (HCs), fresh blood samples were taken from 16 healthy individuals matched for sex ratio and mean age with the patient groups. The clinical data obtained for the enrolled subjects are listed in Table 1. The study protocol was granted by the ethics committee of Tangdu Hospital and informed consent was obtained from all participants.
Table 1. Characteristics of enrolled subjects Characteristics Rheumatoid arthritis Total High disease activity Case (n) 54 16 Age (years) 42.8 15.8 47.8 13.5 Gender 40/14 11/5 (female/male) DAS28 4.33 1.48 6.28 0.92
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Biochemical and immunological assessments C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and anti-cyclic citrullinated peptide (CCP) were routinely analyzed in the clinical laboratory of Tangdu Hospital. All participants underwent radiographs of their knees and wrists in the Department of Radiology of Tangdu Hospital. Disease activity score 28 (DAS28) was calculated according to Prevoo methods (21). Briefly, DAS28 = 0.56 9 numbers of tender joints + 0.28 9 numbers of swollen joints + 0.7 9 Ln (ESR) + 0.16. DAS28 > 5.1 referred to high disease activity, DAS28 < 3.2 referred to low disease activity, and DAS28 < 2.6 referred to remission.
Peripheral blood mononuclear cells and synovial fluid preparation Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation. Cells were incubated in RPMI 1640 (Hyclone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen GIBCO, Grand Island, NY, USA), penicillin (100 IU/mL), and streptomycin (100 lg/mL). PBMCs were cultured (106/well) in 24-well plates with or without PMA (50 ng/mL) and ionomycin (1 lg/mL), and monensin (10 lg/mL) at 37 °C under a 5% CO2 environment for 5 h. Synovial fluids were obtained from 16 patients undergoing knee joint cavity paracentesis. Synovial fluid samples were also centrifuged for 10 min at 1000 g to remove cells and joint debris. All samples were then frozen and stored at 80 °C until required for assay.
Flow cytometry The contents of the wells were transferred to 5-mL sterile tubes with 2 mL of phosphate-buffered saline (PBS) containing 1% FBS. PBMCs were harvested at 1000 g for 10 min at 4 °C. Anti-human CD3 APC, -CD4 FITC (eBioscience, San Diego, CA, USA) were used for surface staining, and anti-human IL-17A PE (eBioscience) was used for intracellular staining for detection of Th17 cells. Anti-human CD3 APC, -CD4 FITC, -CXCR5 PerCP Cy5.5, -ICOS(CD278) PE (eBioscience) were used for surface staining for detection of Tfh cells. A minimum of 10 000 events were collected for each analysis. All data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). All data were analyzed using FlowJo software version 8.6 (Tree Star Inc., Ashland, OR, USA).
Healthy controls Moderate disease activity 20 41.2 13.8 15/5
Low disease activity 18 40.2 19.4 14/4
16 34.2 6.3 10/6
3.95 0.65
3.00 0.12
N.A.
© 2015 APMIS. Published by John Wiley & Sons Ltd
TH17 AND TFH CELLS IN RA
Cytokine measurements Serum and synovial fluids were evaluated for cytokine production by enzyme-linked immunosorbent assay (ELISA). IL-17, IL-21, and IL-22 concentrations were measured by the use of commercial ELISA kits (eBioscience) according to the manufacturer’s instruction.
Statistical analyses Statistical significance was determined by Mann–Whitney U test, Dunn’s multiple comparison test, or Spearman correlation analysis using SPSS version 12.0 for Windows (SPSS, Chicago, IL, USA). Values of p < 0.05 were considered as a significant difference.
RESULTS Increased frequency of circulating Th17 and Tfh cells in patients with RA
For each tested PBMCs, 10 000 events were acquired in a stored live lymphocyte gates. The typical flow cytometry determination of the proportions of Th17
cells and Tfh cells are shown in Fig. 1. We gated the CD3+CD4+IL-17A+ cells as the Th17 cells challenged with mitogens (Fig. 1A). On the basis of intracellular cytokine staining (ICS) analyses, little IL-17A production can be found in CD4+ T cells without stimulation. After challenged with PMA and ionomycin, the percentage of Th17 cells in stimulated CD4+ cells from patients with RA were significantly higher than those from controls (2.28 1.02% vs 1.41 0.52%, p = 0.0016, Fig. 1B). However, Th17 cell frequencies revealed no significant difference among patients with high disease activity (2.42 1.21%), moderate disease activity (2.33 0.91%), and low disease activity (2.11 0.97%) (p > 0.05, Fig. 1C). We gated CD3+CD4+CXCR5+ICOShigh cells as the Tfh cells (Fig. 1D). Circulating Tfh cells in CD4+ T cells were also remarkably higher in RA patients than in healthy controls (1.01 0.63% vs 0.64 0.26%, p = 0.023, Fig. 1E). Furthermore, Tfh cells in both high DAS (1.20 0.77%) and moderate DAS (1.13 0.59%) were notably increased than low
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Fig. 1. Frequency of circulating Th17 and Thf cells was increased in patients with rheumatoid arthritis (RA). (A) Representative dot plots of IL-17+ expression in peripheral CD4+ T cells. (B) The percentage of Th17 cells in HCs (n = 16) and RA patients (n = 54). (C) The percentage of Th17 in high (n = 16), moderate (n = 20), and low (n = 18) disease activity score (DAS). (D) Representative dot plots of CXCR5+ and ICOShigh expression in peripheral CD4+ T cells. (E) The percentage of Tfh cells in HCs (n = 16) and RA patients (n = 54). (F) The percentage of Tfh cells in high (n = 16), moderate (n = 20), and low (n = 18) disease activity score (DAS). © 2015 APMIS. Published by John Wiley & Sons Ltd
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DAS patients (0.72 0.41%) (p = 0.025 and 0.019 respectively, Fig. 1F). It is also well understood that anti-CCP-positive RA with tissue types containing the shared epitope is pathogenetically distinct from anti-CCP-negative RA. Thus, we further analyzed the differences in Th17 and Tfh frequencies between anti-CCP positive and negative RA patients. Thirtytwo of anti-CCP positive and 22 of anti-CCP negative RNA patients were enrolled. However, there were no significant differences in either Th17 cells (2.32 0.99% vs 2.26 1.08%, p = 0.843) or Tfh cells (1.14 0.66% vs 0.91 0.58%, p = 0.282) between the two groups. IL-17, IL-21, and IL-22 expression in serum and synovial fluids
Serum IL-17, IL-21, and IL-22 were detected in 54 patients with RA and 16 healthy controls. As shown in Fig. 2A–C, the concentrations of IL-17 (controls: 6.64 2.17 pg/mL, patients: 17.52 9.76 pg/mL, p < 0.0001), IL-21 (controls: 17.59 6.96 pg/mL, patients: 33.82 12.76 pg/mL, p < 0.0001), and IL-22 (controls: 13.56 5.43 pg/mL, patients: 21.89 7.54 pg/mL, p = 0.0002) in the serum showed significantly higher levels in RA patients. However, there were no remarkable differences among the patients with different disease activities
(high DAS: 18.13 15.33 pg/mL, moderate DAS: 18.65 9.01 pg/mL, low DAS: 16.00 5.45 pg/ mL, p = 0.800, Fig. 2D) in IL-17. IL-21 concentration in RA patients with high DAS (37.33 12.72 pg/mL) was notably elevated compared with concentration in moderate and low DASs (31.29 11.87 pg/mL and 29.01 12.33 pg/mL, p = 0.039 and 0.049, respectively, Fig. 2E). Moreover, the serum levels of IL-22 were significantly higher in both patients with high (24.39 9.66 pg/ mL) and moderate DAS (23.75 7.15 pg/mL) compared to low DAS (18.35 4.26 pg/mL, p = 0.045 and 0.023, respectively, Fig. 2F). There were also no remarkable differences in these cytokines between anti-CCP positive and negative RA patients (data not shown). Furthermore, we measured the concentrations of IL-17, IL-21, and IL-22 in synovial fluids from patients with RA. Synovial fluids were obtained via knee joint cavity paracentesis from seven patients with high DAS, five patients with moderate DAS, and four patients with low DAS. IL-17 was highly expressed in synovial fluids from all patients and there was no remarkable significance among the different disease activities (high DAS: 159.8 83.22 pg/mL, moderate DAS: 152.6 121.8 pg/mL, low DAS: 140.8 60.77 pg/mL, p = 0.762, Fig. 3A). Interestingly, IL-22 levels revealed a simi-
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Fig. 2. Serum levels of interleukin (IL)-17, IL-21, and IL-22 in HC and rheumatoid arthritis (RA) patients. (A) IL-17, (B) IL-21, and (C) IL-22 expressions were measured in the serum HCs and RA patients. (D) IL-17, (E) IL-21, and (F) IL-22 concentrations in high, moderate, and low disease activity score (DAS) were also analyzed in the serum.
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Fig. 3. Interleukin (IL)-17 and IL-22 concentrations in synovial fluids. (A) IL-17 and (B) IL-22 concentrations in synovial fluids were measured by ELISA in high (n = 7), moderate (n = 5), and low (n = 4) disease activity score (DAS) of rheumatoid arthritis patients.
lar trend as the expression in the serum. Elevated IL22 was found in the synovial fluids from patients with high (449.5 147.5 pg/mL) and moderate DAS (414.9 100.4 pg/mL) than in low DAS RA patients (231.2 105.0 pg/mL, p = 0.024 and 0.05, respectively, Fig. 3B). However, synovial IL-21 levels were below the limit of detection in most patients and IL-21 was only tested in two patients with RA. Factors related to Th17 and Tfh cells in RA patients
To investigate whether the circulating Th17 and Tfh cells as well as the production of related cytokines were correlated with the disease progress of rheumatoid arthritis, we measured the ESR, CRF, and RF of the serum from all tested RA patients, and the DAS28 was calculated. Bivariate correlation showed that the percentage of Tfh cells within CD4+ T cells, IL-21, and IL-22 expression in the serum was directly associated with DAS28 with statistical significance in RA patients (r = 0.382, p = 0.0043; r = 0.296, p = 0.029; r = 0.273, p = 0.046, respectively, Fig. 4A–C). But there was no correlation between the Th17 cell frequency and DAS28 (r = 0.162, p = 0.615). Furthermore, synovial IL-22 concentration positively correlated with DAS28 scores (r = 0.506, p = 0.046, Fig. 4D). However, neither the frequencies of Th17 and Tfh cells nor the concentrations of the secreting cytokines were solely associated with the ESR, CRF, and RF levels (p > 0.05). Dynamic changes of Tfh cells, IL-21, and IL-22 in response to diseases-modify antirheumatic drugs
As disease activity is associated with a high frequency of circulating Tfh cells, we sought to determine whether this subset and related cytokines secretion would be downregulated as the remission © 2015 APMIS. Published by John Wiley & Sons Ltd
of disease activity by using diseases-modify antirheumatic drugs (DMARDs) (mostly methotrexate monotherapy except one received sulfasalazine monotherapy). To do so, we followed up with nine RA patients with high disease activity who received the DMARD treatments. Before the initiation of therapy, high levels of both serum IL-21 and IL-22 were detected in those subjects, while 24 weeks of DMARD therapy resulted in a dramatic decline in disease activity, with a simultaneous decrease in the concentrations of IL-21 and IL-22 (Fig. 5B,C). However, there was no significant change in the frequency of Tfh in response to DMARD therapy (Fig. 5A).
DISCUSSION CD4+ T cells play an important role in the disease pathogenesis of RA (6). The imbalance of Th1 and Th2 differentiation contributed to sustained rheumatoid inflammation in the patients with RA, since the elevated IFN-c production by Th1 cells and impaired IL-4 secretion by Th2 cells were found in a very early stage of the disease (22). The numbers of regulatory T cells (Tregs) were increased, however, the suppression function of these cells did not reveal activated in RA patients (23). Recently, two newly identified CD4+ T cells subsets, Th17 and Tfh, were proved to contribute to the development of autoimmune diseases. Th17-producing IL-17 was detected in RA synovial cell cultures and perivascular T cell rich zones (24), which could further promote the expression of many kinds of proinflammatory cytokines and chemokines (25). Tfh cells played an important role in the formation of follicular structures with germinal centers, which were pivotal for T–B cells cooperation and the local generation of specific auto-antibodies to establish autoimmune
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Fig. 4. The relationship between Tfh cells, interleukin (IL)-21, IL-22, and disease activity score (DAS) 28. (A) The frequency of Tfh cells, (B) serum concentration of IL-21, (C) serum concentration of IL-22 and (D) IL-22 expression in synovial fluids was positively correlated with DAS28.
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Fig. 5. Dynamic changes of Tfh cells, interleukin (IL)-21, and IL-22 in response to diseases-modify antirheumatic drug (DMARD) therapy. (A) Tfh cell proportion, (B) IL-21, and (C) IL-22 expression at baseline, 12 and 24 weeks after treatment.
disease (14). Over-activation of Tfh cell immune responses have the potential to mount unwarranted germinal centers, composed of aberrantly mutated B cells that can drive antibody-mediated autoimmune diseases (26). Elevated levels of Tfh-like cells can be detected in the blood of a subset of human patients with SLE and Sjogren’s syndrome (27). However, evidence suggesting Tfh cells can definitively cause autoimmunity in humans remains incomplete. In this study, we investigated the percentage of circulating Th17 and Tfh cells in patients with RA, and demonstrated that the elevated CD3+CD4+IL17A+ Th17 cells and CD3+CD4+CXCR5+ICOShigh Tfh cells were found in RA patients when compared with health controls. Furthermore, the expressions of Th17-secreting cytokines IL-17 and 664
IL-22, as well as Tfh-secreting cytokine IL-21 were all significantly increased in RA patients. Thus, in consistence with the previously published data (28, 29), our results suggested that the circulating Th17 and Tfh cells may be involved in the pathogenesis of RA. Clinical assessment and diagnosis of RA are based on the history of joint pain and stiffness, symmetric polyarticular joint swelling, radiographs, and biomarkers tested in the bloods (30). RF and anti-CCP are two important auto-antibodies in the diagnostic measures of RA, while ESR and CRP reflect the degree of inflammation in the bloods (31). Furthermore, DAS28 is a composite score derived from both clinical presentation and laboratory test for the monitoring and assessment of RA. © 2015 APMIS. Published by John Wiley & Sons Ltd
TH17 AND TFH CELLS IN RA
In this study, we found that frequency of Tfh cells as well as the expression of IL-21 and IL-22 was significantly increased in patients with high DAS28, compared with moderate and low DAS28 patients. Moreover, the DAS28 score correlated positively with the frequency of circulating Tfh cells, serum IL-21, and IL-22 expressions in both serum and synovial fluids. However, Th17 and IL-17 did not reveal remarkable correlations with DAS28. Th17 cells have been demonstrated to take part in the immunopathogenesis of RA in many previous studies (32, 33). Kim et al. (34) also showed that the percentage of Th17 cells correlated with that of DAS28. Unfortunately, however, this study did not reveal the correlation between Th17 cells and DAS28. This was further confirmed by the finding that the frequency of Th17 cells did not change significantly during treatments. Thus, we thought that Th17 cells may not contribute to the severity of the diseases. In contrast, Tfh cells proportion and IL21 level was positively correlated with DAS28. Moreover, IL-21 concentration was also remarkably reduced in response to DMARD therapy. Although we did not find the similar trend of Tfh cells during therapy, previous studies have demonstrated that Tfh cells secrete IL-21, and IL-21 could also promote the differentiation and survival of Tfh cells (35). Together, Tfh cells were thought to be in relation to pathogenesis and progression of RA. IL-17, IL-21, and IL-22 concentrations were also measured in the serum and synovial fluids of RA patients. The levels of IL-17, IL-21, and IL-22 were all significantly elevated in the serum of RA patients. However, IL-17 did not show correlation with severity of the diseases, which manifested as similar IL-17 levels in serum and synovial fluids in high, moderate, and low DAS28 patients, as well as no correlation between IL-17 and DAS28. This result suggested that Th17-secreting IL-17 may not be involved in the progression of RA. Furthermore, another Th17-secreting cytokine, IL-22 concentration in both serum and synovial fluids demonstrated a positive correlation with DAS28. More importantly, IL-22 levels were reduced in response to therapy. This is consistent with the previous studies, which revealed that the elevated IL-22 and CD4+IL-22+ T cells were associated with disease activity and radiographic progression (12, 36, 37). IL-22 was proved to play either a protective or a pathological role in several tissues and organs, and the precise beneficial or detrimental effects may vary among different disease state (38). Geboes et al. (33) showed that IL-22 plays a proinflammatory role in collagen-induced arthritis in the animal model. Thus, IL-22 may promote the disease progression in RA patients and be a potential
© 2015 APMIS. Published by John Wiley & Sons Ltd
therapeutic targeting for intervention of RA. Moreover, the Tfh-secreting IL-21 was also associated with DAS28 and decreased during DMARD treatment. However, little production of IL-21 was found in the synovial fluids of RA patients. Wang et al. (39) also found that IL-21 was significantly higher in RA patients, while Sglunda et al. (40) showed a decrease in serum IL-21 in response to treatment in recent-onset RA patients. Thus, the present results indicated that IL-21 may also be a biomarker for predicting the disease progression in patients with RA. In conclusion, the current results suggested that Th17 cells, Tfh cells as well as the secreting cytokines may be involved in the pathogenesis of RA. The frequency of circulating Tfh cells and the productions of IL-21 and IL-22 were associated with the disease activity of RA patients, and might become potential therapeutic targets for treatment of RA.
We thank all the volunteers who participated in the study.
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27. Craft JE. Follicular helper T cells in immunity and systemic autoimmunity. Nat Rev Rheumatol 2012;8:337–47. 28. Ma J, Zhu C, Ma B, Tian J, Baidoo SE, Mao C, et al. Increased frequency of circulating follicular helper T cells in patients with rheumatoid arthritis. Clin Dev Immunol 2012;2012:827480. 29. van Hamburg JP, Asmawidjaja PS, Davelaar N, Mus AM, Colin EM, Hazes JM, et al. Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin-17A production. Arthritis Rheum 2011;63:73–83. 30. Farheen K, Agarwal SK. Assessment of disease activity and treatment outcomes in rheumatoid arthritis. J Manag Care Pharm 2011;17:S09–13. 31. Agrawal S, Misra R, Aggarwal A. Autoantibodies in rheumatoid arthritis: association with severity of disease in established RA. Clin Rheumatol 2007;26:201– 4. 32. Yamada H, Nakashima Y, Okazaki K, Mawatari T, Fukushi JI, Kaibara N, et al. Th1 but not Th17 cells predominate in the joints of patients with rheumatoid arthritis. Ann Rheum Dis 2008;67:1299–304. 33. Geboes L, Dumoutier L, Kelchtermans H, Schurgers E, Mitera T, Renauld JC, et al. Proinflammatory role of the Th17 cytokine interleukin-22 in collageninduced arthritis in C57BL/6 mice. Arthritis Rheum 2009;60:390–5. 34. Kim J, Kang S, Kwon G, Koo S. Elevated levels of T helper 17 cells are associated with disease activity in patients with rheumatoid arthritis. Ann Lab Med 2013;33:52–9. 35. Linterman MA, Beaton L, Yu D, Ramiscal RR, Srivastava M, Hogan JJ, et al. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J Exp Med 2010;207:353–63. 36. daRocha LF Jr., Duarte AL, Dantas AT, Mariz HA, Pitta Ida R, Galdino SL, et al. Increased serum interleukin 22 in patients with rheumatoid arthritis and correlation with disease activity. J Rheumatol 2012;39:1320–5. 37. Leipe J, Schramm MA, Grunke M, Baeuerle M, Dechant C, Nigg AP, et al. Interleukin 22 serum levels are associated with radiographic progression in rheumatoid arthritis. Ann Rheum Dis 2011;70:1453–7. 38. Cobleigh MA, Robek MD. Protective and pathological properties of IL-22 in liver disease: implications for viral hepatitis. Am J Pathol 2013;182:21–8. 39. Wang J, Shan Y, Jiang Z, Feng J, Li C, Ma L, et al. High frequencies of activated B cells and T follicular helper cells are correlated with disease activity in patients with new-onset rheumatoid arthritis. Clin Exp Immunol 2013;174:212–20. 40. Sglunda O, Mann HF, Hulejova H, Pecha O, Plestilova L, Ruzickova O, et al. Decrease in serum interleukin 21 levels is associated with disease activity improvement in patients with recent-onset rheumatoid arthritis. Physiol Res 2014;63:475–81.
© 2015 APMIS. Published by John Wiley & Sons Ltd
© 2015 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12399
Elevated circulating Th17 and follicular helper CD4+ T cells in patients with rheumatoid arthritis YAN ZHANG, YU LI, TING-TING LV, ZHEN-JIE YIN and XIN-BO WANG Department of Immunology and Rheumatology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China
Zhang Y, Li Y, Lv T-T, Yin Z-J, Wang X-B. Elevated circulating Th17 and follicular helper CD4+ T cells in patients with rheumatoid arthritis. APMIS 2015; 123: 659–666. It remains not fully elucidated the potential functions of Th17 cells and follicular helper T (Tfh) cells and secreting cytokines in the pathogenesis of rheumatoid arthritis (RA) and their association with disease activity. In this study, the frequencies of Th17 and Tfh cells were determined by flow cytometry, and the levels of interleukin (IL)-17, IL-21, and IL-22 were measured by ELISA in RA patients with different disease activities. The dynamic changes of cell subsets were also detected in response to disease-modify antirheumatic drugs (DMARDs) therapy. The percentages of CD3+CD4+IL-17A+ (Th17) cells and CD3+CD4+CXCR5+ICOShigh (Tfh) cells, as well as the concentrations of IL17, IL-21, and IL-22 were significantly elevated in RA patients than those in healthy individuals. Furthermore, Tfh cells, IL-21, and IL-22 in the serum was positively correlated with the values of disease activity score. Concentrations of IL-21 and IL-22 in the serum were remarkably reduced following the DMARDs therapies. Our data suggested that Th17 cells, Tfh cells as well as the secreting cytokines may be involved in the pathogenesis of RA. The frequency of circulating Tfh cells and the productions of IL-21 and IL-22 were associated with the disease activity of RA patients, and might be potential therapeutic targets for treatment of RA. Key words: Rheumatoid arthritis; Th17 cells; follicular helper T cells; interleukin-21; interleukin-22. Xin-Bo Wang, Department of Immunology and Rheumatology, Tangdu Hospital, Fourth Military Medical University, 569 Xinsi Rd, Xi’an, Shaanxi Province 710038, China. e-mail: [email protected]
Yan Zhang and Yu Li contributed equally to this work.
Rheumatoid arthritis (RA) is a long-term and systemic inflammatory disease which primarily affects small diarthrodial joints of hands and feet, with pain, stiffness, and fatigue (1, 2). It can be a disabling condition, which may lead to substantial loss of function and mobility if not adequately treated. The autoimmune disorder, which involves dysregulated-lymphocyte activation and circulating autoantibodies, was considered to play a crucial role in pathogenesis of RA although the etiology of RA is still not fully understood (3). Synovial joints are characterized by chronic inflammation and abnormally infiltrated by a variety of immune cells, including T cell, B cell, macrophage, granulocyte, and monocyte in RA patients (4). Moreover, the immunogenetics of RA suggested a key role for aberrant CD4+ T cell activation in the initiation and perpetuation of the disease (4–6). Received 3 December 2014. Accepted 3 April 2015
CD4+ T cells can differentiate into subsets of helper T (Th) cells once activated. Five different Th cell subsets, which are named as Th1, Th2, Th17, regulatory T, and follicular T (Tfh) are identified till now (7). Th cells are also critical in the activation of B cells and their differentiation into antibody-secreting cells. Th17 cells require transforming growth factor-b (TGF-b) and IL-6 for initiation and IL-23 to become an established population (8). Numerous studies have suggested that elevated T17 cells were involved in the pathogenesis of RA and might be negative predictors to the effect of anti-RA treatments (9–12). Recently, Tfh cells presented a new Th lineage to provide help to the B cells for the generation of germinal centers (GCs) and induce continuously protective humoral responses (13). Distinguishing features of Tfh cells are the expressions of the regulator transcription factor Bcl6, chemokine (C-X-C motif) receptor 5 (CXCR5), inducible costimulator (ICOS), interleukin-21 659
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(IL-21), and absence of Blimp-1 (14). Previous study has revealed that Tfh-producing IL-21 directly regulates B cells proliferation and class switching (15). Nurieva et al. also demonstrated that IL-21 could synergize IL-6 to induce Bcl-6 expression in CD4+ T cells, thus promoting Tfh cells differentiation (16). Importantly, generation of Th17 cells is also induced by IL-21 and IL-6 (17), with the evidence that elevated circulating CD4+ T cells-secreting IL-21 was correlated with increasing Th17 and memory B cells in patients with systemic lupus erythematosus (SLE) (18). This indicates that dysregulated functions of both Th17 and Tfh cells likely contributed to the pathogenesis of autoimmune diseases. An elegant study by Feng et al. (19) recently showed that frequencies of circulating Tfh cells, but not Th17 cells, were increased in SLE patients and correlated with disease activity, which prompted us to measure the circulating percentages of Th17 and Tfh cells, as well as their secreting cytokines in RA patients and the correlation with disease activity. In this study, the frequencies of IL-17-producing (Th17) and CXCR5+ICOShigh (Tfh) expressed CD4+ T cells were determined in RA patients with different disease activity, and the dynamic changes of those cell subsets were also detected in response to diseases-modified antirheumatic drugs therapy. MATERIALS AND METHODS Subjects Blood samples were collected from 54 patients with RA. Diagnoses were made with the diagnostic criteria of according to the criteria of the American College of Rheumatology (20). All patients were hospitalized or followed up in Tangdu Hospital, from June 2008 to March 2010. Patients with other rheumatic diseases, such as SLE, mixed connective tissue disease (MCTD) were excluded from this study. No patients had received steroids, chondrotin polysulphate, or hyaluronic for at least 2 months before sampling. For healthy controls (HCs), fresh blood samples were taken from 16 healthy individuals matched for sex ratio and mean age with the patient groups. The clinical data obtained for the enrolled subjects are listed in Table 1. The study protocol was granted by the ethics committee of Tangdu Hospital and informed consent was obtained from all participants.
Table 1. Characteristics of enrolled subjects Characteristics Rheumatoid arthritis Total High disease activity Case (n) 54 16 Age (years) 42.8 15.8 47.8 13.5 Gender 40/14 11/5 (female/male) DAS28 4.33 1.48 6.28 0.92
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Biochemical and immunological assessments C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and anti-cyclic citrullinated peptide (CCP) were routinely analyzed in the clinical laboratory of Tangdu Hospital. All participants underwent radiographs of their knees and wrists in the Department of Radiology of Tangdu Hospital. Disease activity score 28 (DAS28) was calculated according to Prevoo methods (21). Briefly, DAS28 = 0.56 9 numbers of tender joints + 0.28 9 numbers of swollen joints + 0.7 9 Ln (ESR) + 0.16. DAS28 > 5.1 referred to high disease activity, DAS28 < 3.2 referred to low disease activity, and DAS28 < 2.6 referred to remission.
Peripheral blood mononuclear cells and synovial fluid preparation Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation. Cells were incubated in RPMI 1640 (Hyclone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen GIBCO, Grand Island, NY, USA), penicillin (100 IU/mL), and streptomycin (100 lg/mL). PBMCs were cultured (106/well) in 24-well plates with or without PMA (50 ng/mL) and ionomycin (1 lg/mL), and monensin (10 lg/mL) at 37 °C under a 5% CO2 environment for 5 h. Synovial fluids were obtained from 16 patients undergoing knee joint cavity paracentesis. Synovial fluid samples were also centrifuged for 10 min at 1000 g to remove cells and joint debris. All samples were then frozen and stored at 80 °C until required for assay.
Flow cytometry The contents of the wells were transferred to 5-mL sterile tubes with 2 mL of phosphate-buffered saline (PBS) containing 1% FBS. PBMCs were harvested at 1000 g for 10 min at 4 °C. Anti-human CD3 APC, -CD4 FITC (eBioscience, San Diego, CA, USA) were used for surface staining, and anti-human IL-17A PE (eBioscience) was used for intracellular staining for detection of Th17 cells. Anti-human CD3 APC, -CD4 FITC, -CXCR5 PerCP Cy5.5, -ICOS(CD278) PE (eBioscience) were used for surface staining for detection of Tfh cells. A minimum of 10 000 events were collected for each analysis. All data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). All data were analyzed using FlowJo software version 8.6 (Tree Star Inc., Ashland, OR, USA).
Healthy controls Moderate disease activity 20 41.2 13.8 15/5
Low disease activity 18 40.2 19.4 14/4
16 34.2 6.3 10/6
3.95 0.65
3.00 0.12
N.A.
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Cytokine measurements Serum and synovial fluids were evaluated for cytokine production by enzyme-linked immunosorbent assay (ELISA). IL-17, IL-21, and IL-22 concentrations were measured by the use of commercial ELISA kits (eBioscience) according to the manufacturer’s instruction.
Statistical analyses Statistical significance was determined by Mann–Whitney U test, Dunn’s multiple comparison test, or Spearman correlation analysis using SPSS version 12.0 for Windows (SPSS, Chicago, IL, USA). Values of p < 0.05 were considered as a significant difference.
RESULTS Increased frequency of circulating Th17 and Tfh cells in patients with RA
For each tested PBMCs, 10 000 events were acquired in a stored live lymphocyte gates. The typical flow cytometry determination of the proportions of Th17
cells and Tfh cells are shown in Fig. 1. We gated the CD3+CD4+IL-17A+ cells as the Th17 cells challenged with mitogens (Fig. 1A). On the basis of intracellular cytokine staining (ICS) analyses, little IL-17A production can be found in CD4+ T cells without stimulation. After challenged with PMA and ionomycin, the percentage of Th17 cells in stimulated CD4+ cells from patients with RA were significantly higher than those from controls (2.28 1.02% vs 1.41 0.52%, p = 0.0016, Fig. 1B). However, Th17 cell frequencies revealed no significant difference among patients with high disease activity (2.42 1.21%), moderate disease activity (2.33 0.91%), and low disease activity (2.11 0.97%) (p > 0.05, Fig. 1C). We gated CD3+CD4+CXCR5+ICOShigh cells as the Tfh cells (Fig. 1D). Circulating Tfh cells in CD4+ T cells were also remarkably higher in RA patients than in healthy controls (1.01 0.63% vs 0.64 0.26%, p = 0.023, Fig. 1E). Furthermore, Tfh cells in both high DAS (1.20 0.77%) and moderate DAS (1.13 0.59%) were notably increased than low
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Fig. 1. Frequency of circulating Th17 and Thf cells was increased in patients with rheumatoid arthritis (RA). (A) Representative dot plots of IL-17+ expression in peripheral CD4+ T cells. (B) The percentage of Th17 cells in HCs (n = 16) and RA patients (n = 54). (C) The percentage of Th17 in high (n = 16), moderate (n = 20), and low (n = 18) disease activity score (DAS). (D) Representative dot plots of CXCR5+ and ICOShigh expression in peripheral CD4+ T cells. (E) The percentage of Tfh cells in HCs (n = 16) and RA patients (n = 54). (F) The percentage of Tfh cells in high (n = 16), moderate (n = 20), and low (n = 18) disease activity score (DAS). © 2015 APMIS. Published by John Wiley & Sons Ltd
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DAS patients (0.72 0.41%) (p = 0.025 and 0.019 respectively, Fig. 1F). It is also well understood that anti-CCP-positive RA with tissue types containing the shared epitope is pathogenetically distinct from anti-CCP-negative RA. Thus, we further analyzed the differences in Th17 and Tfh frequencies between anti-CCP positive and negative RA patients. Thirtytwo of anti-CCP positive and 22 of anti-CCP negative RNA patients were enrolled. However, there were no significant differences in either Th17 cells (2.32 0.99% vs 2.26 1.08%, p = 0.843) or Tfh cells (1.14 0.66% vs 0.91 0.58%, p = 0.282) between the two groups. IL-17, IL-21, and IL-22 expression in serum and synovial fluids
Serum IL-17, IL-21, and IL-22 were detected in 54 patients with RA and 16 healthy controls. As shown in Fig. 2A–C, the concentrations of IL-17 (controls: 6.64 2.17 pg/mL, patients: 17.52 9.76 pg/mL, p < 0.0001), IL-21 (controls: 17.59 6.96 pg/mL, patients: 33.82 12.76 pg/mL, p < 0.0001), and IL-22 (controls: 13.56 5.43 pg/mL, patients: 21.89 7.54 pg/mL, p = 0.0002) in the serum showed significantly higher levels in RA patients. However, there were no remarkable differences among the patients with different disease activities
(high DAS: 18.13 15.33 pg/mL, moderate DAS: 18.65 9.01 pg/mL, low DAS: 16.00 5.45 pg/ mL, p = 0.800, Fig. 2D) in IL-17. IL-21 concentration in RA patients with high DAS (37.33 12.72 pg/mL) was notably elevated compared with concentration in moderate and low DASs (31.29 11.87 pg/mL and 29.01 12.33 pg/mL, p = 0.039 and 0.049, respectively, Fig. 2E). Moreover, the serum levels of IL-22 were significantly higher in both patients with high (24.39 9.66 pg/ mL) and moderate DAS (23.75 7.15 pg/mL) compared to low DAS (18.35 4.26 pg/mL, p = 0.045 and 0.023, respectively, Fig. 2F). There were also no remarkable differences in these cytokines between anti-CCP positive and negative RA patients (data not shown). Furthermore, we measured the concentrations of IL-17, IL-21, and IL-22 in synovial fluids from patients with RA. Synovial fluids were obtained via knee joint cavity paracentesis from seven patients with high DAS, five patients with moderate DAS, and four patients with low DAS. IL-17 was highly expressed in synovial fluids from all patients and there was no remarkable significance among the different disease activities (high DAS: 159.8 83.22 pg/mL, moderate DAS: 152.6 121.8 pg/mL, low DAS: 140.8 60.77 pg/mL, p = 0.762, Fig. 3A). Interestingly, IL-22 levels revealed a simi-
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Fig. 2. Serum levels of interleukin (IL)-17, IL-21, and IL-22 in HC and rheumatoid arthritis (RA) patients. (A) IL-17, (B) IL-21, and (C) IL-22 expressions were measured in the serum HCs and RA patients. (D) IL-17, (E) IL-21, and (F) IL-22 concentrations in high, moderate, and low disease activity score (DAS) were also analyzed in the serum.
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Fig. 3. Interleukin (IL)-17 and IL-22 concentrations in synovial fluids. (A) IL-17 and (B) IL-22 concentrations in synovial fluids were measured by ELISA in high (n = 7), moderate (n = 5), and low (n = 4) disease activity score (DAS) of rheumatoid arthritis patients.
lar trend as the expression in the serum. Elevated IL22 was found in the synovial fluids from patients with high (449.5 147.5 pg/mL) and moderate DAS (414.9 100.4 pg/mL) than in low DAS RA patients (231.2 105.0 pg/mL, p = 0.024 and 0.05, respectively, Fig. 3B). However, synovial IL-21 levels were below the limit of detection in most patients and IL-21 was only tested in two patients with RA. Factors related to Th17 and Tfh cells in RA patients
To investigate whether the circulating Th17 and Tfh cells as well as the production of related cytokines were correlated with the disease progress of rheumatoid arthritis, we measured the ESR, CRF, and RF of the serum from all tested RA patients, and the DAS28 was calculated. Bivariate correlation showed that the percentage of Tfh cells within CD4+ T cells, IL-21, and IL-22 expression in the serum was directly associated with DAS28 with statistical significance in RA patients (r = 0.382, p = 0.0043; r = 0.296, p = 0.029; r = 0.273, p = 0.046, respectively, Fig. 4A–C). But there was no correlation between the Th17 cell frequency and DAS28 (r = 0.162, p = 0.615). Furthermore, synovial IL-22 concentration positively correlated with DAS28 scores (r = 0.506, p = 0.046, Fig. 4D). However, neither the frequencies of Th17 and Tfh cells nor the concentrations of the secreting cytokines were solely associated with the ESR, CRF, and RF levels (p > 0.05). Dynamic changes of Tfh cells, IL-21, and IL-22 in response to diseases-modify antirheumatic drugs
As disease activity is associated with a high frequency of circulating Tfh cells, we sought to determine whether this subset and related cytokines secretion would be downregulated as the remission © 2015 APMIS. Published by John Wiley & Sons Ltd
of disease activity by using diseases-modify antirheumatic drugs (DMARDs) (mostly methotrexate monotherapy except one received sulfasalazine monotherapy). To do so, we followed up with nine RA patients with high disease activity who received the DMARD treatments. Before the initiation of therapy, high levels of both serum IL-21 and IL-22 were detected in those subjects, while 24 weeks of DMARD therapy resulted in a dramatic decline in disease activity, with a simultaneous decrease in the concentrations of IL-21 and IL-22 (Fig. 5B,C). However, there was no significant change in the frequency of Tfh in response to DMARD therapy (Fig. 5A).
DISCUSSION CD4+ T cells play an important role in the disease pathogenesis of RA (6). The imbalance of Th1 and Th2 differentiation contributed to sustained rheumatoid inflammation in the patients with RA, since the elevated IFN-c production by Th1 cells and impaired IL-4 secretion by Th2 cells were found in a very early stage of the disease (22). The numbers of regulatory T cells (Tregs) were increased, however, the suppression function of these cells did not reveal activated in RA patients (23). Recently, two newly identified CD4+ T cells subsets, Th17 and Tfh, were proved to contribute to the development of autoimmune diseases. Th17-producing IL-17 was detected in RA synovial cell cultures and perivascular T cell rich zones (24), which could further promote the expression of many kinds of proinflammatory cytokines and chemokines (25). Tfh cells played an important role in the formation of follicular structures with germinal centers, which were pivotal for T–B cells cooperation and the local generation of specific auto-antibodies to establish autoimmune
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Fig. 4. The relationship between Tfh cells, interleukin (IL)-21, IL-22, and disease activity score (DAS) 28. (A) The frequency of Tfh cells, (B) serum concentration of IL-21, (C) serum concentration of IL-22 and (D) IL-22 expression in synovial fluids was positively correlated with DAS28.
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Fig. 5. Dynamic changes of Tfh cells, interleukin (IL)-21, and IL-22 in response to diseases-modify antirheumatic drug (DMARD) therapy. (A) Tfh cell proportion, (B) IL-21, and (C) IL-22 expression at baseline, 12 and 24 weeks after treatment.
disease (14). Over-activation of Tfh cell immune responses have the potential to mount unwarranted germinal centers, composed of aberrantly mutated B cells that can drive antibody-mediated autoimmune diseases (26). Elevated levels of Tfh-like cells can be detected in the blood of a subset of human patients with SLE and Sjogren’s syndrome (27). However, evidence suggesting Tfh cells can definitively cause autoimmunity in humans remains incomplete. In this study, we investigated the percentage of circulating Th17 and Tfh cells in patients with RA, and demonstrated that the elevated CD3+CD4+IL17A+ Th17 cells and CD3+CD4+CXCR5+ICOShigh Tfh cells were found in RA patients when compared with health controls. Furthermore, the expressions of Th17-secreting cytokines IL-17 and 664
IL-22, as well as Tfh-secreting cytokine IL-21 were all significantly increased in RA patients. Thus, in consistence with the previously published data (28, 29), our results suggested that the circulating Th17 and Tfh cells may be involved in the pathogenesis of RA. Clinical assessment and diagnosis of RA are based on the history of joint pain and stiffness, symmetric polyarticular joint swelling, radiographs, and biomarkers tested in the bloods (30). RF and anti-CCP are two important auto-antibodies in the diagnostic measures of RA, while ESR and CRP reflect the degree of inflammation in the bloods (31). Furthermore, DAS28 is a composite score derived from both clinical presentation and laboratory test for the monitoring and assessment of RA. © 2015 APMIS. Published by John Wiley & Sons Ltd
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In this study, we found that frequency of Tfh cells as well as the expression of IL-21 and IL-22 was significantly increased in patients with high DAS28, compared with moderate and low DAS28 patients. Moreover, the DAS28 score correlated positively with the frequency of circulating Tfh cells, serum IL-21, and IL-22 expressions in both serum and synovial fluids. However, Th17 and IL-17 did not reveal remarkable correlations with DAS28. Th17 cells have been demonstrated to take part in the immunopathogenesis of RA in many previous studies (32, 33). Kim et al. (34) also showed that the percentage of Th17 cells correlated with that of DAS28. Unfortunately, however, this study did not reveal the correlation between Th17 cells and DAS28. This was further confirmed by the finding that the frequency of Th17 cells did not change significantly during treatments. Thus, we thought that Th17 cells may not contribute to the severity of the diseases. In contrast, Tfh cells proportion and IL21 level was positively correlated with DAS28. Moreover, IL-21 concentration was also remarkably reduced in response to DMARD therapy. Although we did not find the similar trend of Tfh cells during therapy, previous studies have demonstrated that Tfh cells secrete IL-21, and IL-21 could also promote the differentiation and survival of Tfh cells (35). Together, Tfh cells were thought to be in relation to pathogenesis and progression of RA. IL-17, IL-21, and IL-22 concentrations were also measured in the serum and synovial fluids of RA patients. The levels of IL-17, IL-21, and IL-22 were all significantly elevated in the serum of RA patients. However, IL-17 did not show correlation with severity of the diseases, which manifested as similar IL-17 levels in serum and synovial fluids in high, moderate, and low DAS28 patients, as well as no correlation between IL-17 and DAS28. This result suggested that Th17-secreting IL-17 may not be involved in the progression of RA. Furthermore, another Th17-secreting cytokine, IL-22 concentration in both serum and synovial fluids demonstrated a positive correlation with DAS28. More importantly, IL-22 levels were reduced in response to therapy. This is consistent with the previous studies, which revealed that the elevated IL-22 and CD4+IL-22+ T cells were associated with disease activity and radiographic progression (12, 36, 37). IL-22 was proved to play either a protective or a pathological role in several tissues and organs, and the precise beneficial or detrimental effects may vary among different disease state (38). Geboes et al. (33) showed that IL-22 plays a proinflammatory role in collagen-induced arthritis in the animal model. Thus, IL-22 may promote the disease progression in RA patients and be a potential
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therapeutic targeting for intervention of RA. Moreover, the Tfh-secreting IL-21 was also associated with DAS28 and decreased during DMARD treatment. However, little production of IL-21 was found in the synovial fluids of RA patients. Wang et al. (39) also found that IL-21 was significantly higher in RA patients, while Sglunda et al. (40) showed a decrease in serum IL-21 in response to treatment in recent-onset RA patients. Thus, the present results indicated that IL-21 may also be a biomarker for predicting the disease progression in patients with RA. In conclusion, the current results suggested that Th17 cells, Tfh cells as well as the secreting cytokines may be involved in the pathogenesis of RA. The frequency of circulating Tfh cells and the productions of IL-21 and IL-22 were associated with the disease activity of RA patients, and might become potential therapeutic targets for treatment of RA.
We thank all the volunteers who participated in the study.
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