Increased circulating FOXP3+ T-cells in patients with granulomatosis with polyangiitis are attributed to an increase in the non-suppressor FOXP3LOW CD45RO+ treg cell subpopulation

Background/Purpose: Human FoxP3+T-cells are functionally heterogeneous, and can be classified into three phenotypically distinct subpopulations based on the expression levels of FoxP3 and the memory T-cell marker CD45RO. These three subpopulations can be defined as: activated suppressor TRegs (FoxP3HighCD45RO+; ASTReg), resting suppressor TRegs (FoxP3Low CD45RO-; RSTReg), and cytokine-secreting non-suppressor TRegs (FoxP3Low CD45RO+; NONTReg). This study aimed to evaluate the identity of the elevated frequency of FoxP3+T-cells in patients with granulomatosis with polyangiitis (GPA). Methods: Peripheral blood mononuclear cells were isolated from 46 GPApatients (27 ANCA-positief and 19 ANCA-negative at the time of inclusion) in remission and from 22 age- and sex-matched HCs. Expression of CD4 CD45RO, and FoxP3 was determined by flow cytometric analysis. The expression levels of FoxP3 and CD45RO were used for distinction between ASTReg, RSTReg, and NONTRegFoxP3+T-cells. Next, frequencies of intracellular production of IL-17 within the FoxP3+ cell subpopulations were analyzed by flow cytometry in peripheral blood of ANCA-positive (n=10) and ANCA-negatieve (n=9) GPApatients and matched HCs (n=12) upon ex vivo stimulation with phorbolmyristate- acetate and calcium ionophore in the presence of brefeldin A. Results: A significant increase in the frequency of FoxP3+TRegcells was observed in GPA-patients as compared with HCs. No differences were detected in RSTReg- and ASTRegcells between GPA-patients and HCs, whereas the NONTRegcells were significantly increased in GPA-patients. The distribution of RSTReg- and NONTRegcells did not differ between ANCA-negative and ANCApositive patients, whereas lower percentages of ASTRegcells were observed in ANCA-positive patients as compared to ANCA-negative patients and HCs. In addition, frequencies of IL-17+ T-cells were significantly increased within the NONTRegsubset from ANCA-positive patients in comparison with ANCA-negative patients and HCs, whereas no such difference was found between ANCA-negative patients and HCs. Conclusion: Increased FoxP3 expression in CD4+T-cells from GPA-patients is related to an increase in a subset of non-suppressive T-cells that produce higher levels of IL-17 and display low expression of FoxP3. Plasticity of CD4+T-cells in GPA points towards FoxP3+IL-17+ effector cells and decrease in suppressive TRegcells in relation to ANCA production.