We enrolled 60 patients with GPA, (12 with MPO-ANCA, 48 with PR3-ANCA, Table 1), mean age (± SD) 57 (± 13) years, and 14 age-matched healthy volunteers (healthy controls, HC), mean age 53 ± 5 years. Eighteen patients were in an active state of disease at presentation and forty-two were in remission (Table 1, 2). Informed patient consent and approval by the local ethics committee was obtained. Seven patients initially presenting with active disease were assessed again after remission was achieved. The diagnosis and classification of GPA was made according to criteria of the American College of Rheumatology, the Chapel Hill criteria and the algorithm published by Watts et al. 293031. Active disease was defined as clinical manifestation of new-onset or recurrent disease activity related to vasculitis requiring intensified immunosuppressive therapy 3233. All patients with active disease were either untreated or treated with maintenance therapy at the time of sampling. None of the active patients had received cyclophosphamide or high dose intravenous methylprednisolone at the time of sampling. Remission or quiescent disease was defined as absence of clinical disease activity reflecting a Birmingham Vasculitis Activity Score of zero 33. The cohort of patients in remission was divided for further analysis into two groups: patients in long-term stable remission, and patients with a relapsing disease course. Patients with a relapse-free disease course since the first onset of GPA and a minimum disease duration of 48 months, were defined as being in stable long-term remission (n = 13). Patients with a minimum disease duration of 48 months and at least one relapse within the first 48 months since diagnosis of GPA were defined as relapsers (n = 19).

Lymphocyte phenotypes were measured by five-colour surface staining. The following antibodies labeled with fluorescent dye were used: CD3 (mouse IgG1, HorV450), CD4 (mouse IgG1, PerCP; ITK/Biolegend, Uithoorn, The Netherlands), CD8 (mouse IgG1, APC-H7), CD25 (mouse IgG1, FITC), CD127 (mouse IgG1, PE). All antibodies except CD4 were purchased at BD Bioscience, Breda, The Netherlands. Appropriate isotype controls (BD Bioscience) were used. Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll density gradient centrifugation (Histopaque; Sigma Aldrich, Zwijndrecht, The Netherlands) and incubated with labeled monoclonal antibodies for 30 minutes in the dark at room temperature. Analysis was performed with a fluorescence-activated cell sorter (FACS) CANTO™ from BD Bioscience.

PBMC from patients and HC were separated by standard Ficoll density gradient centrifugation. The cells were resuspended in RPMI 1640 medium (Gibco Invitrogen, Breda, The Netherlands) supplemented with 10% heat-inactivated fetal calf serum (Greiner Bio-One, Alphen a/d Rijn, The Netherlands), 100 U/mL penicillin and 100 µg/mL Streptomycin (both Gibco Invitrogen). The cells were cultured in the absence or presence of PMA (50ng/mL) and Ionomycin (1µg/mL) (Sigma-Aldrich) for 4 hours. Cytokine secretion was inhibited by Brefeldin A (BD Bioscience). Surface staining was performed with CD3 HorV450 and CD8 APC-H7 (both BD Bioscience). Cells were fixed using a Cytofix/Cytoperm kit (BD Bioscience). Finally, the samples were stained intracellularly for CD69 (ITK/Biolegend), IFNγ (BD Bioscience), IL-4, IL-10 and IL-17A (all ITK/Biolegend). Unstimulated PBMC and appropriate isotype controls were used to confirm specificity of staining and to discriminate background staining. FSC-H vs. FSC-A gating allowed exclusion of cell doublets. As stimulation with PMA/ionomycin induced downregulation of CD4, CD4⁺ T cells were defined as CD3⁺CD8^neg, and an average of 30,000 events was acquired in this CD3⁺CD8^neg gate. CD69 expression was used as a control for the activation procedure but was not included in the gating strategy. CD69 expression did not differ between HC and patients with GPA after mitogen stimulation (94.97 ± 3.49% vs. 95.25 ± 3.54%, P > 0.05).

All values are expressed as mean ± SD. Significance for the differences between groups was determined using the Mann-Whitney U-test. Spearman's rank correlation test was applied to detect correlation between different study parameters.

Patients with active GPA (n = 18) showed a significantly increased percentage of circulating IL-17A⁺ T cells compared to HC (n = 14) (% of CD4⁺ T cells = 1.7 ± 1.4% vs. 0.7 ±0.3%, P = 0.006 (Figure 1A, B), while there was no difference in IFNγ (14.5 ± 9.9% vs. 17.7 ± 7.9%, P = 0.17), or IL-4-producing CD4⁺ T cells (5.6 ± 3.3% vs. 4.9 ± 2.1%, P = 0.7). There were no significant differences between active patients with or without maintenance therapy at the time of sampling.

Th17 expansion was similar between active GPA and quiescent GPA (n = 42, 1.7 ± 1.4% vs. 1.9 ± 1.5%, P = 0.37 (Figure 1B). Seven patients first assessed during the active state of disease were sampled again during remission (median time between visits 118 days, range 71 to 567 days). The percentage of Th17 cells did not decline during remission (Figure 1C).

Accordingly, circulating IL-17A⁺ T cells were elevated in GPA patients in remission compared to HC (% of CD4⁺ T cells = 1.9 ± 1.5% vs. 0.7 ± 0.3%, P < 0.0001) (Figure 1A, B) whereas IFNγ (22.1 ± 10.8% vs. 17.7 ± 7.9%, P = 0.20), and IL-4 producing-T-helper-cells (6.1 ± 2.5% vs. 4.9 ± 2.1%, P = 0.11) did not differ between patients with GPA and the HC. Of note, IFNγ⁺ T-cells were significantly reduced in patients with active GPA compared to those with quiescent disease (14.5 ± 9.9% vs. 22.1 ± 10.8%, P = 0.002).

CD3⁺CD4^negCD8^neg (DN) T-cells were previously described as producers of IL-17 34. However, there was no difference between HC and patients with GPA in the percentage of DN T cells (% of CD3⁺ T-cells in the HC vs. quiescent GPA = 2.9 ± 2.0% vs. 3.1 ± 2.9%, P > 0.05, and in the HC vs. active GPA = 2.9 ± 2.0% vs. 3.4 ± 4.1%, P > 0.05). Moreover, DN T cells did not correlate with Th17 cells (r = -0.4, P = 0.2 in the HC; r = -0.3, P = 0.3 in patients with GPA).

ANCA-status, that is, PR3/MPO-ANCA negative (n = 12) vs. positive (n = 30) at the time of sampling, did not have impact on the percentage of Th17 cells (data not shown). Patients with (n = 16) or without maintenance (n = 26) therapy both had a significant increase of Th17 cells compared to the HC (1.33 ± 0.94% vs. 0.7 ± 0.3%, P = 0.03 and 2.32 ± 1.61% vs. 0.7 ± 0.3%, P < 0.0001). However, patients on maintenance therapy harbored a significantly smaller fraction of Th17 cells than untreated patients (1.33 ± 0.94% vs. 2.32 ± 1.61%, P = 0.01).

In addition, administration of steroids had an impact on Th17 cells as there was a negative correlation of steroid dosage and percentage of Th17 cells (r = -0.46, P = 0.002). A similar effect was observed for Th1 cells and steroid dosage (r = -0.32, P = 0.04), and so a separate analysis accounting for maintenance therapy was also performed. To assess the impact of Th17 cells on the clinical course of the disease, we stratified the patient cohort into relapse-free and relapsing patients, and identified 13 relapse-free patients and 19 relapsing patients. Two relapse-free patients and nine relapsing patients received maintenance treatment at the time of sampling. As shown before, treatment seemed to have impact on the percentage of Th17 cells. Therefore, we compared untreated relapsing patients in remission (n = 10) with untreated relapse-free patients in remission (n = 11). Th17 cells were similar between both groups (% Th17 cells = 2.3 ± 1.0% vs. 2.6 ± 2.2%, P = 0.7).

Regulatory T-helper-cell subsets control and limit immune responses of pro-inflammatory T cells. In this study, we focused on two types of regulatory T cells (Tregs), which were defined according to literature: CD4⁺CD25^highCD127^low Tregs and IL-10⁺ Tregs 3536. CD4⁺CD25^highCD127^low Tregs were increased in quiescent GPA (8.3 ± 3.5% in untreated patients, n = 22; 6.0 ± 1.1% in treated patients, n = 13) and active GPA (7.0 ± 2.8%, n = 18) compared to the HC (5.1 ± 1.0%, n = 13)(P < 0.006, P = 0.06, and P = 0.02 respectively, for comparison with the HC) (Figure 2A). In contrast, IL-10⁺ Tregs did not differ between quiescent GPA (0.7 ± 0.4% in untreated patients, n = 26; 0.5 ± 0.2% in treated patients, n = 16) or active GPA (0.6 ± 0.4%, n = 17) compared to the HC (0.5 ± 0.3%, n = 14) (P = 0.13, P = 0.7, and P = 0.52 respectively, for comparison with the HC) (Figure 2A). As Th17 cells and Treg share developmental pathways, a reciprocal relationship has been described 37. Accordingly, Th17 expansion is associated with Treg depletion in human SLE 22. Therefore, a correlation analysis was performed to assess the Th17/Treg relationship in GPA. Th17 cells were positively correlated with CD4⁺CD25^highCD127^low or IL-10⁺ Tregs in quiescent GPA (r = 0.34, P < 0.05 and r = 0.38, P = 0.01) and active GPA (r = 0.36, P = 0.14 and r = 0.60, P = 0.01) but not in the HC (r = -0.04, P = 0.90 and r = 0.19, P = 0.52). Separate analysis of GPA patients in remission stratified by maintenance treatment showed only a significant positive correlation between IL-10⁺ Tregs and Th17 in patients on maintenance therapy (r = 0.68, P = 0.004). Thus, a reciprocal relationship or a negative association between Th17 cells and Tregs was not found.

Ratios were calculated to assess if the balance between Th17 cells and Tregs was preserved in GPA. Importantly, Th17 expansion was not balanced by Tregs in quiescent, untreated GPA and active GPA indicated by both a higher Th17/CD25^highCD127^low Treg ratio and Th17/IL-10⁺ Treg ratio compared to HC (Figure 2B). Interestingly, in quiescent, treated GPA, only the Th17/IL-10⁺ Treg ratio was significantly skewed towards Th17, whereas the Th17/ CD25^highCD127^low Treg ratio was not different from HC (Figure 2B). Relapsing and relapse-free patients had comparable Th17/Treg ratios, with 0.36 ± 0.32 vs. 0.39 ± 0.38 (P = 1.0) for Th17/CD25^highCD127^low Treg, and 4.9 ± 2.8 vs. 4.3 ± 3.8 (P = 0.6) for Th17/IL-10⁺ Treg.

IFNγ and IL-10 co-expression of Th17 cells was measured in a subset of GPA patients. Both IL-10⁺/IL-17A⁺ and IFNγ⁺/IL-17A⁺ CD4⁺ T cells could be detected in GPA and HC. The percentage of IL-10⁺/IL-17A⁺ CD4⁺ T cells was higher in untreated quiescent GPA (0.0501 ± 0.031%, n = 23), but not in treated quiescent GPA (0.0432 ± 0.053%, n = 15), than in the HC (0.0282 ± 0.016%, n = 14) (P = 0.007 and P = 0.91 respectively, for comparison with the HC) (Figure 3A).

In active GPA, the percentage of the IL-10⁺/IL-17A⁺population (0.0703 ± 0.076%, n = 12) did not differ from the HC (0.0282 ± 0.016%, n = 14, P > 0.05) (Figure 3A).

IFNγ⁺/IL-17A⁺CD4⁺ T cells were significantly increased in untreated, quiescent GPA (0.24 ± 0.18%, n = 24, P = 0.001), but not in treated, quiescent GPA (0.20 ± 0.18%, n = 15, P = 0.09), and in active GPA (0.19 ± 0.19%, n = 12, P < 0.05) compared to the HC (0.09 ± 0.07%, n = 14) (Figures 3B and 3C). IFNγ⁺/IL-10⁺ T cells did not differ between the patients and HC (data not shown). Thus, Th17 cells producing IL-10 or IFNγ were increased only in untreated, quiescent and active GPA.