RESUMO
Objectives: T regulatory cells (Tregs) are a heterogeneous group of immunoregulatory cells that dampen self-harming immune responses and prevent the development of autoimmune diseases. In anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis, Tregs possess diminished suppressive capacity, which has been attributed to the expression of a FOXP3 splice-variant lacking exon 2 in T cells (FOXP3Δ2 CD4+ T cells). However, the suppressive capacity of Tregs varies between subsets. We evaluated the frequency of Treg subsets in ANCA vasculitis as a potential explanation for diminished suppressive capacity. Methods: We developed a custom mass cytometry panel and performed deep immune profiling of Tregs in healthy controls, patients with active disease and in remission. Using these data, we performed multidimensional reduction and discriminant analysis to identify associations between Treg subsets and disease activity. Results: Total Tregs were expanded in ANCA vasculitis, which was associated with remission and the administration of rituximab and/or prednisone. The frequency of FOXP3Δ2 CD4+ T cells did not distinguish disease activity and this population had high expression levels of CD127 and lacked both CD25 and Helios, suggesting that they are not conventional Tregs. The frequency of CXCR3+, CD103+ and CCR7+ Tregs distinguished disease activity, and the combination of the frequency of these three Treg subsets segregated active patients from patients in remission and healthy controls. From these three subsets, the frequency of CXCR3+ Tregs distinguished patients with active disease with renal involvement. Conclusion: Treg heterogeneity can discriminate disease activity and should be explored as a biomarker of disease activity in ANCA vasculitis.
RESUMO
OBJECTIVE: There is accumulating evidence that complement activation is important in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) pathogenesis. This study was undertaken to investigate complement activation in AAV with myeloperoxidase (MPO) positivity and AAV with proteinase 3 (PR3) positivity after determining optimal methods for measuring activated complement factors in circulation. METHODS: Participants included 98 patients with AAV (45 MPO-ANCA positive, 53 PR3-ANCA positive) and 35 healthy controls. Plasma was obtained from blood collected using EDTA tubes, with or without 100 µg/ml Futhan. Levels of Bb, C3a, C5a, soluble C5b-9 (sC5b-9), properdin, and C4d were measured by enzyme-linked immunosorbent assay. Group comparisons were made using Wilcoxon's 2-sample test. Paired data were analyzed using a matched pairs signed rank test. RESULTS: Compared to healthy controls, certain complement analyte levels were high in patients with active AAV with MPO positivity, including C3a (P < 0.0001), C5a (P = 0.0004), and sC5b-9 (P = 0.0007). During remission, levels of Bb (P = 0.001), C3a (P < 0.0001), and sC5b-9 (P = 0.003) were higher. Compared to healthy controls, C3a (P < 0.0001), C5a (P = 0.002), sC5b-9 (P = 0.0001), and C4d (P = 0.005) levels were higher in patients with active AAV with PR3 positivity; levels of C3a (P < 0.0001) and C4d (P = 0.007) were also higher duriing remission. There were no significant differences in any complement analyte for either ANCA serotype between patients with active disease and those with disease in remission. Among patients with paired samples, sC5-9 levels were significantly lower during disease remission compared to active disease. C5a was significantly lower among patients with disease in long-term remission who were not receiving therapy. For Bb, C5a, and sC5b-9, median levels and individual values were considerably higher in control and patient samples processed without Futhan compared to those processed with Futhan. CONCLUSION: Complement activation occurs in both MPO-positive AAV and PR3-positive AAV. The complement activation profile differs according to disease activity and possibly ANCA serotype. Futhan reduces in vitro complement activation and provides a more accurate measurement.