Figure 6: Pharmacological inhibition of Tiam1/Rac1 signaling regulates human Th17 Cells.

(a) Tiam1 and Rac1 expression is induced in human Th17 cells. Naïve CD4⁺ T cells were isolated from fresh PBMC obtained from healthy subjects (n=7) by MACS negative selection and cells were cultured under Th1 and Th17 cell polarizing conditions in the presence or absence of NSC23766 (94 μM) for 7 days. Tiam1 and Rac1 expression was analyzed by Taqman PCR. RORγt and IL-17A mRNA levels are shown as controls for polarization. (b) Cytokine expression of NSC23766-treated Th17 cells by flow cytometry. Naive CD4⁺ T cells were polarized under Th0, Th1, and Th17 conditions in the presence or absence of NSC23766 for 7 days followed by activation for 4 h with PMA and ionomycin. Cells were fixed, permeabilized then incubated with fluorochrome-conjugated anti-IL-17A (x axis) and anti-IFNγ (y axis) antibodies. Numbers in outlined areas indicate per cent of positive cells. (c) Mean of the frequency of IL-17A⁺IFNγ⁻ and IL-17A⁻IFNγ⁺ in Th17 cells starting from naïve CD4⁺ T cells isolated from healthy subjects (n=5) and differentiated in the presence or absence of NSC23766 is shown. (d) Cytokine profile of NSC23766-treated Th17 cells by Luminex. Supernatants were collected from Th17 cell cultures at the end of the differentiation and were analyzed by bead-based Luminex assay according to the manufacturer’s instructions. Data represent mean±s.e.m. of a representative experiment each performed in triplicate. (e) Tiam1 and Rac1 expression in MS patients. Gene expression profiling of CD4⁺ T cells from MS patients (n=8) and healthy controls (HC) (n=4) analyzed by Affymetrix gene array and deposited on GEO (GSE32988). *P<0.05; **P<0.01; ***P<0.001 by Student t-test. Each dot represents an individual. NS, non-significant.