Fig. 4

Interleukin (IL-10) expression in T-helper type 1 (Th1) switching cells is dependent on intact cholesterol pathway fitness. Purified human CD4⁺ T cells stimulated in vitro with plate-bound α-CD3 (2 μgml⁻¹) + α-CD46 (5 μgml⁻¹) and recombinant human interleukin-2 (rhIL-2) (50 Uml⁻¹) were cultured for 36 h in the presence of 25-hydroxycholesterol (25-HC). a Normalised LDLr, HMGCS1, FDFT1 and DHCR7 messenger RNA (mRNA) levels (n = 6–7). b Representative flow cytometric analysis of intracellular interferon-γ (IFNγ) and IL-10 staining. c Normalised frequency of IFNγ⁻IL-10⁺- (left), IFNγ⁺IL-10⁺- (centre) and IFNγ⁺IL-10⁻- (right) producing cells (n = 16). d Normalised concentrations of secreted IL-10 (n = 9). e Normalised frequency of IFNγ⁻IL-10⁺- (left), IFNγ⁺IL-10⁺- (centre) and IFNγ⁺IL-10⁻- (right) producing cells cultured under lipid-free medium (LFM), fully supplemented medium (M) and cholesterol (500×) (Ch) in the presence or absence of 25-HC (2μM) for 36 h (n = 7). Graphs show independent donors (dots) normalised to untreated cells; bars represent median values. *<0.05, **<0.01 and ***<0.001 denote a significant difference compared to untreated cells by Friedman test with post hoc Dunn’s correction (a, c: IFNγ⁻IL-10⁺, e) or by repeated-measures one-way analysis of variance (ANOVA) test with post hoc Dunnett’s correction (c: IFNγ⁺IL-10⁺ and IFNγ⁺IL-10⁻, d)