Figure 5

CD4⁺CD25^low/-GITR⁺but not CD4⁺CD25^highGITR^-cells from SLE patients exert a regulatory activity similar to HC. Regulatory activity is independent of cell contact. CFSE-labeled effectors, activated with cross-linked anti-CD3 Ab, were co-cultured with autologous unlabeled effectors (CD4⁺CD25^-GITR^-) (left, A, B), unlabeled CD25-depleted (including CD4⁺CD25^-GITR^- and CD4⁺CD25^low/-GITR⁺) (middle, A, B) or unlabeled GITR-depleted (including CD4⁺CD25^-GITR^- and CD4⁺CD25^highGITR^-) cells (right, A, B) at 1:3 cell ratio. Proliferation was evaluated after 4 days with flow cytometry. Graphs show inhibition of proliferation by CD4⁺CD25^low/-GITR⁺ and CD4⁺CD25^highGITR^-( histograms A, B) and the differences between the inhibition (plot on the right) in each subject (A, B). Experiments with cells from PBs of two representative HCs (A) and two representative SLE patients (B) are shown. (C, D) Histograms show mean percentage of inhibition of proliferation by CD4⁺CD25^low/-GITR⁺(C) and CD4⁺CD25^highGITR^-(D) cells from twelve HCs and six SLE patients. (E, F) Inhibition by CD4⁺CD25^low/-GITR⁺ and CD4⁺CD25^highGITR^- in an identical experiment as the described (classical) and in transwell experiments (unlabeled cells above and CFSE-labeled cells below the transwell). A representative SLE patient of 3 is shown. Graph in G shows differences in the inhibition of proliferation by CD4⁺CD25^low/-GITR⁺ in the classic and transwell experiments in the three patients. ***P <0.001 according to Mann-Whitney U test, comparing inhibition by CD4⁺CD25^highGITR^- cells from HC and SLE patients.