Fig 1.
Gamma-secretase inhibitors alter Th1 and Th17 responses but do not inhibit EAE.
B6 mice were immunized with MOG35-55 to induce active EAE. Beginning on the day after immunization, the mice were randomized and treated with DMSO or GSI every other day for 18 days. A. Clinical course of EAE. B. Peak clinical score of DMSO and GSI-treated mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or FoxP3 (I) among CNS-infiltrating CD4+ T cells. J-L, Presence of cytokine expression in the spleens of EAE mice on day 17 post-immunization. Distribution of the percentages of cells expressing IFNγ (J), IL-17 (K), GM-CSF (L). Symbols indicate the percentage of cells from individual mice. Also plotted are the mean and SEM for each treatment group. Open circles indicate DMSO treatment, filled squares indicate treatment with GSI. Error bars represent SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.01 and ***p <0.001).
Fig 2.
GSI treatment reduces T effector numbers and effector differentiation in vivo.
A. 2D2 transfer model. T cells from 2D2 TCR transgenic, CD90.1 congenic mice were isolated and transferred on day -1. Mice were immunized with MOG35-55/CFA on day 0. Mice were treated with vehicle (DMSO) or GSI beginning on day -1 as indicated (arrows) and cell responses were detected on day 7 post-immunization. B. Total splenic CD4+ T cell counts. C. Percentages of donor (CD90.1+) among CD4 T cells. D. Total numbers of donor CD4 T cells present in the spleen. E. Percentage of donor T cells that had diluted CFSE. F-K Impact of GSI on Th1 differentiation in vivo. F. Histogram overlay of Tbet expression. G. Comparison of Tbet expression (MFI) in donor T cells. H. Numbers of donor cells expressing Tbet. I. the total number of IFNγ+ donor T cells in the spleen. J. Numbers of host-derived CD4+ cells expressing Tbet. K. the total number of IFNγ+ donor T cells in the spleen. L-Q GSI treatment alters Th17 responses L. Histogram overlay of RORγt expression. M. Comparison of RORγt expression (MFI) in donor T cells. N. Numbers of donor cells expressing RORγt. O. the total number of IL-17+ donor T cells in the spleen. P. number of host-derived T cells expressing RORγt. Q. Number of IL-17+ host-derived T cells. Symbols indicate results from individual mice. Open circles indicate treatment with DMSO, filled squares indicate GSI treatment. Error bars indicate standard deviation. Asterisks indicate significant differences (* p<0.05).
Fig 3.
GSI treatment inhibits effector differentiation, activation and proliferation in vitro.
A-E. Effects of GSI on Th1 differentiation. Splenocytes were stimulated in vitro with anti-CD3 and anti-CD28 for 72 hours in the presence of anti-IL-4 and either DMSO or GSI. A-C. IFNγ and Tbet expression were determined by intracellular staining and flow cytometry. A. Representative flow cytometry plots showing IFNγ and Tbet expression. B. IFNγ expression was measured by intracellular flow cytometry. C. Tbet expression was measured by intracellular flow cytometry. D and E. Expression analysis of IL12Rβ1 and IL12Rβ2. Total cellular RNA was isolated from each culture and target gene expression determined by real time PCR. D. Expression of IL12Rβ1. E. Expression of IL12Rβ2. F-J. Effects of GSI on T cell activation. Splenocytes from 2D2 TCR transgenic mice were activated in vitro with MOG35-55 peptide in the presence of DMSO or GSI. At 72 hours of stimulation, flow cytometry was used to measure expression of the activation markers CD25, CD44 and CD69. F. Representative flow cytometry plots showing CD25 and CD44 expression. G. The percentage of T cells expressing CD25. H. Expression of CD44 by T cells. I. The percentages of T cells expressing CD69. J. Quantitation of CD25 expression on activated T cells (CD4 and CD44 gated events). K-N Effects of GSI on T cell proliferation. Splenocytes were labeled with Cell-trace and stimulated for 96 hours with antibodies to CD3 and CD28 in the presence of DMSO or DBZ. Th1 differentiation was promoted by the addition of anti-IL-4 alone (neutral), or in combination with IL-2. K. Proliferation and intracellular IFNγ staining were detected by flow cytometry. L and M. Division index and Proliferation index were calculated using gates to measure the percentages of T cells within each cell division. O. Expression of IFNγ by T cells that had undergone 4 or more cell divisions. P. Expression of CD25 by T cells cultured in the absence or presence of IL-2. Flow cytometry plots are gated on live CD4+ T cells. Results shown are representative of at least two experiments. The numbers in FACS plots indicate cell percentages within each quadrant. Individual symbols indicate results from replicate wells. Open circles indicate treatment with DMSO, filled squares indicate GSI treatment. Error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.01 and ***p <0.001).
Fig 4.
Impact of GSI on Th1 effector differentiation.
A-C. Effects of GSI on the generation of encephalitogenic Th1 T cells. T cells from MBP1-11 TCR transgenic mice were activated in vitro with MBP ac1-11 peptide and IL-12. Cell cultures were stimulated 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. A. Mean EAE disease course. B. Peak clinical score. C. Cumulative EAE score. D-F. Effects of GSI on Th1 polarization with IL-12. Splenocytes were stimulated in vitro with anti-CD3 and anti-CD28 for 72 hours in the presence of anti-IL-4 alone with IL-12. IFNγ and Tbet expression was measured by intracellular flow cytometry at 72 hours using gates for live CD4+ T cell events. E. Percentages of IFNγ expressing CD4+ T cells. F. Percentages of T cells expressing Tbet. 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). 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).
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).
Fig 6.
PSEN1 cKO mice are susceptible to EAE.
WT and PSEN1 cKO mice were immunized with MOG35-55 to induce active EAE. A. Clinical course of EAE. B. Peak clinical score of EAE mice. C. Cumulative EAE scores for DMSO and GSI-treated animals. T cell cytokine expression in CNS-infiltrating CD4+ T cells from DMSO or GSI-treated mice following the Peak of EAE. On day 17 post-immunization, CNS cells were isolated and intracellular cytokine staining performed. D. Expression of IL-17 and IFNγ. E. Expression of GM-CSF and IFNγ. Distribution of the percentages of cells expressing IFNγ (F), IL-17 (G), GM-CSF (H) or FoxP3 (I) among CNS-infiltrating CD4+ T cells. Symbols indicate the percentage of cells from individual mice. Open circles indicate results from WT and filled squares indicate PSEN1 cKO mice. Error bars represent SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.01 and ***p <0.001).
Fig 7.
Altered Th1 and Th17 differentiation in the absence of PSEN1 expression.
A-C. Impact of PSEN1 on Th1 differentiation in vitro. Splenocytes isolated from WT and PSEN1 cKO donors were stimulated in vitro with anti-CD3 and anti-CD28 for 72 hours in the presence of anti-IL-4 alone or together with recombinant IL-12. IFNγ and Tbet expression were measured by intracellular flow cytometry at 72 hours. A. Representative FACS plots. B. Percentages of IFNγ expressing CD4+ T cells was determined in replicate cultures containing DMSO, GSI and either neutral, IL-2 or IL-12 polarizing conditions. C. Percentages of T cells expressing Tbet was determined under each culture condition. D-F. PSEN1 regulates Th17 polarization in vitro. Splenocytes isolated from WT and PSEN1 cKO donors 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. D. Representative FACS plots for each polarizing condition. E. Percentages of IL-17 expressing CD4+ T cells for each polarizing condition. F. Percentages of T cells expressing RORγt for each polarizing condition. Results shown are representative of at least two experiments. Open circles identify cells from WT donors, filled squares are from PSEN1 cKO donors. Symbols represent replicate cultures. 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).
Fig 8.
PSEN1 regulates T cell proliferation but not activation.
Panels A-D. Activation in PSEN1 cKO T cells. Splenocytes from WT and PSEN1 cKO mice were isolated and stimulated with soluble anti-CD3 and anti-CD28 antibodies. A. At 72 hours of culture T flow cytometry was performed to measure the expression of CD44 and CD25 on CD4+ T cells. B. Percentage of CD4+ T cells that express CD25. C. Quantitation of CD44 expression on CD4+ gated T cells D. CD25 surface expression level (MFI) of activated T cells that express both CD4 and CD44. Panels E-G. T cell proliferative responses. E. Cell Trace-labeled splenocytes were activated with anti-CD3 and anti-CD28 for 72 hours. Flow cytometry was performed to measure dye dilution by CD4+ gated events. F. The division index was calculated for WT and PSEN1 cKO T cells. G. The proliferation index was calculated for WT and PSEN1 cKO T cells. Mean expression data are plotted+/- SEM. Open circles and filled squares indicate WT and PSEN1 cKO T cells, respectively. Asterisks indicate significant differences (* p<0.05, ** p<0.01 and ***p <0.001).