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ANA_21944_SupplementalFigure1.tif23488KSupplementary Figure 1: Effects of FoxP3 siRNA on FoxP3 expression levels of CD8+CD25+ TCCs, assessed by flow cytometry. As control, FoxP3 expression levels of natural occurring CD4+CD25 high+ T cells are shown.
ANA_21944_SupplementalFigure2.tif23581KSupplementary Figure 2: IFN-γ (A) and IL-17 (B) secretion by CD4+ TCCs under different conditions of stimulation from five representative patients. Three MS patients were studied during exacerbations and 2 during remission. MBP 83-102, MBP 143-168, and MOG 63-87 specific CD4+ TCCs were tested for cytokine secretion 7-10 days after their last stimulation with feeder cells. Non-specific cytokine secretion was tested by plating cloned T cells at a density of 5 × 104 cells/well and stimulated by 1 ?gr/ml of bound anti-CD3 mAb. To induce Ag-specific cytokine secretion 5 × 104 peptide specific T cells were co-cultured with 5 × 103 adherent irradiated autologous PBMC as APC in the presence of optimal peptide concentration (10-20 μg/ml). APC with Ag but without cloned cells were included as controls. Following 72h supernatants were harvested, and amounts of IFN-γ, and IL-17 were determined using commercial ELISA kits.
ANA_21944_SupplementalFigure3.tif23862KSupplementary Figure 3: Inhibition patterns of 3 representative CD8+CD25+FoxP3+ TCCs derived from 2 MS patients (KS and VD) and 1 healthy control (JC). Three thousand cloned CD4+ MBP 83-102 or MOG 63-87 autoreactive T cells per well were cultured with autologous CD8+CD25+FoxP3+ T cells at a 1:3 ratio, in the presence of optimal concentrations of the cognate Ag (10-20 μg/ml), and 3 × 104 /well irradiated PBMC depleted of CD3+ T cells as a source of APC. To establish specificity of the inhibition patterns, cloned CD4+ T cells specific for MBP1-20, tetanus toxoid, and influenza-heamglutinin 307-319- peptide were included. Proliferation (A) was determined after 72h, using a [3H] thymidine incorporation assay. To measure IFN-γ production (B) supernatants were removed from each well before [3H] thymidine addition, and analyzed using commercially available ELISA kits. CD8+CD25+FoxP3+ TCCs JC-6.1, KS-10.3, and VD-11.8 showed strong inhibitory activity towards MBP 143-168, MBP 83-102,and MOG 63-87-specific CD4+ inducer TCCs, but not against autologous CD4+ TCCs specific for MBP 1-20, tetanus toxoid (TT), or influenza hemaglutinin 307-319 peptide. The results shown represent the mean values + SEM of triplicate cultures. Bckg: Background.

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