Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis
Fig 5
The effects of GSI on Th17 differentiation.
A-E. Effects of GSI on Th17 differentiation. Splenocytes were stimulated in vitro with anti-CD3 and anti-CD28 with TGFβ1 and IL-6 for 72 hours in the presence of either DMSO or GSI. Intracellular flow cytometry was used to measure IL-17 and RORγt expression. A. Representative flow cytometry plots showing IL-17 and RORγt expression. B. IL-17 expression was measured by intracellular flow cytometry. C. RORγt expression was measured by intracellular flow cytometry. D and E. Total cellular RNA was isolated from each culture and realtime PCR was performed to identify the expression of IL12Rβ1 and IL23R. D. Expression of IL12Rβ1. E. Expression of IL23R. F-H. Effects of GSI on encephalitogenic Th17 T cell responses. Splenocyte cultures from MBP1-11 TCR transgenic mice were activated in vitro with MBP ac1-11 peptide and polarized with TGFβ1, IL-6 and IL-23. Cells were cultured for 72 hours in the presence of DMSO or GSI. 8x106 T cells were transferred into B10.Pl recipients and mice were graded for the development of EAE. F. Mean EAE disease course. G. Peak clinical score. H. Cumulative EAE score. I-K. Effects of GSI on Th17 polarization with Splenocytes were stimulated in vitro with anti-CD3 and anti-CD28 for 96 hours in four Th17-inducing conditions: TGFβ1 and IL-6; TGFβ1, IL-6 and IL-23; TGFβ3 and IL-6; or IL-1β, IL-6 and IL-23. IL-17 and RORγt expression was measured by intracellular flow cytometry at 96 hours. I. Representative FACS plots for each polarizing condition. J. Percentages of IL-17 expressing CD4+ T cells for each polarizing condition. K. Percentages of T cells expressing RORγt for each polarizing condition. Results shown are representative of at least two experiments. Open circles indicate DMSO treatment. Filled squares indicate GSI. Symbols represent individual mice (panels B and C) or replicate cultures (panels E and F). Flow plots are gated on live CD4+ events. The numbers in FACS plots indicate cell percentages within each quadrant. Asterisks indicate statistically significant differences between groups (* p<0.05, ** p<0.01 and ***p <0.001).