Figure 4. Tregs in sJIA patients maintain suppressive capacity and CNS2 hypomethylation.

(A) Representative histograms from a healthy control and patient with chronic sJIA. CellTrace Violet–labeled peripheral blood (PB) Teffs from a third-party control were cultured alone, with autologous PB Tregs, or with sJIA PB Tregs at the given ratios after stimulation with anti-CD2/CD3/D28 beads. Teff proliferation was measured by flow cytometry as dye dilution. (B) The percent suppression of PB Teff proliferation from controls (n = 3) by autologous Tregs (CD4⁺CD25⁺CD127^lo) at the given ratios (mean ± SEM). The percent suppression of PB Teff proliferation from a common third-party control by Tregs from acute sJIA (n = 3) and chronic sJIA (n = 4) are also depicted. (C) The percent suppression of PB Teff proliferation from controls (n = 3) by autologous Tregs (CD4⁺CD25⁺CD127^lo) at the given ratios (mean ± SEM). The percent suppression of PB Teffs from a common third-party control by Tregs (CD4⁺CD25⁺CD127^lo) from the SF of a chronic sJIA patient at the given ratios is also depicted. (D) Heatmap depicting methylation rates of CpG sites in CNS2 of FOXP3 in sorted Tregs (CD4⁺CD25⁺CD127^lo) and Teffs (CD4⁺CD25^–) from acute sJIA PB, chronic sJIA PB, chronic sJIA SF, and healthy control PB. Acute sJIA9, chronic sJIA 5, and HC Adult 4 are all males. (E) Mean percentage of methylation rates (mean ± SD) of all evaluated 9 CpG sites in CNS2 of FOXP3 in sorted Tregs (CD4⁺CD25⁺CD127^lo) and Teffs (CD4⁺CD25^–) from acute sJIA PB (n = 5), chronic sJIA PB (n = 4), and healthy control PB (n = 5) (t tests, ***P ≤ 0.001; ****P ≤ 0.0001). Teff, T effector cell; D0, % nondividing cells; sJIA, systemic juvenile idiopathic arthritis; SF, synovial fluid; CNS2, conserved noncoding sequences 2.