The frequency of Treg subsets distinguishes disease activity in ANCA vasculitis

Abstract

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.

Keywords: ANCA vasculitis; Tregs; autoimmune disease.

Figure 1

Mass cytometry gating strategy: Tregs were defined as live CD3⁺CD4⁺FOXP3⁺ after single and live cell discrimination.

Figure 2

The expansion of Tregs in anti‐neutrophil cytoplasmic autoantibody (ANCA) vasculitis is associated with remission and the administration of immunosuppressive drugs. (a) Frequency of Tregs from CD4⁺ T cells in the peripheral circulation of HCs; closed circle (n = 8) and patients; open circle (n = 25). (b) Frequency of Tregs from CD4⁺ T cells in the peripheral circulation of HCs; closed circle (n = 8) and patients in remission; closed square (n = 9) and with active disease; closed triangle (n = 14). (c) Correlation between the frequency of Tregs and Birmingham Vasculitis Activity Score (BVAS). (d) Frequency of Tregs from CD4⁺ T cells in the peripheral circulation of HCs (n = 8), patients that did not receive rituximab (no Ritux; n = 14); closed diamond and patients that received rituximab; closed star (Ritux; n = 11) at the time of sample collection or 6 months prior. (e) Frequency of Tregs from CD4⁺ T cells in the peripheral circulation of HCs (n = 8), patients that did not receive prednisone; closed diamond (no Pred; n = 15) and patients that received prednisone; closed star (Pred; n = 10) at the time of sample collection or 6 months prior. (f) Normalised FOXP3 protein expression levels in peripheral Tregs from HCs (n = 8) and patients (n = 25). Bars shown are median and interquartile range.

Figure 3

FOXP3Δ2 CD4⁺ T cells are not conventional (CD25^highCD127^−/low) Tregs and cannot distinguish disease activity in anti‐neutrophil cytoplasmic autoantibody (ANCA) vasculitis. (a) Representative mass cytometry gate for the detection of FOXP3Δ2 CD4⁺ T cells from total FOXP3⁺CD4⁺ T cells. (b) Frequency of FOXP3⁺CD4⁺ T cells from CD4⁺ T cells in the peripheral circulation of HCs (n = 8) and patients (n = 25). (c) Frequency of peripheral FoxP3Δ2 CD4⁺ T cells from FOXP3⁺CD4⁺ T cells in HCs (n = 8) and patients in remission (n = 9) and with active disease (n = 13). (d) VisNE analysis of FOXP3⁺CD4⁺ T revealing the expression levels of FOXP3 exon 2, CD127, CD25 and Helios (from left to right) in FoxP3Δ2 CD4⁺ T cells. Bars shown are median and interquartile range.

Figure 4

Changes in the frequency of CD103⁺, CCR7⁺ and CXCR3⁺ Tregs distinguish disease activity in anti‐neutrophil cytoplasmic autoantibody (ANCA) vasculitis. (a, d, g) Frequency of CD103⁺, CCR7⁺ and CXCR3⁺ Tregs in the peripheral circulation of HCs (n = 8) and patients (n = 23). (b, e, h) Frequency of CD103⁺, CCR7⁺ and CXCR3⁺ Tregs from CD4⁺ T cells in the peripheral circulation in HCs (n = 8) and patients in remission (n = 10) and with active disease (n = 13). (c, f, i) Correlation between the frequency of CD103⁺, CCR7⁺ and CXCR3⁺ Tregs and Birmingham Vasculitis Activity Score (BVAS). Bars shown are median and interquartile range. Patients with active disease with renal involvement are depicted in closed squares. Correlation's significance was determined using the non‐parametric Spearman's correlation test.

Figure 5

A combination of the frequencies of CD103⁺, CCR7⁺ and CXCR3⁺ Tregs segregates active patients from patients in remission and healthy controls. (a) VisNE analysis revealing clusters of CD103⁺, CCR7⁺ and CXCR3⁺ Tregs (left to right). (b) Three‐dimensional plot showing the segregation of active patients (blue) from patients in remission (yellow) and healthy controls (orange) when the frequencies of CD103⁺, CCR7⁺ and CXCR3⁺ Tregs are combined.