Figure 1.
MOG-specific Th1 and Th17 cell differentiation.
A. T cells from 2D2 mice were activated under Th1 and Th17 polarizing conditions. Vα3.2 and Vβ11 transgenic TCR chains, as well as intracellular IL-17 and IFN-γ cytokine expression was assessed by FACS. Data shown are gated in the CD4+ population. B. IFN-γ and IL-17 cytokines from culture supernatants of Th1 and Th17 polarized cells were quantified by ELISA. C. IFN-γ, Tbet, IL-17 and RORγt gene expression was quantified by real-time PCR of Th1 and Th17 polarized cells. Data shown are representative (A) or a mean of a minimum of 5 experiments. Error bars indicate SEM (B and C).
Figure 2.
Comparative characterization of MOG-specific Th1, Th2 and Th17 cells.
T cells from 2D2 mice were activated under Th1 and Th17 polarizing conditions. A. IL-4, IL-5, IL-10 and GM-CSF cytokines from culture supernatants of Th1, Th2 and Th17 polarized cells were quantified by ELISA (A and B). B. GM-CSF gene expression of Th1 and Th17 polarized cells was quantified by real-time PCR. C. CD25 and CD62L surface expression was assessed by FACS and represent the mean percentage of positive cells in the CD4+ population; **p<0.01; ***p<0.001. D. Antigen specific proliferation of differentiated Th1 and Th17 cells with titrated concentrations of rMOG was measured by quantification of radioactive 3H-thymidine uptake. *p<0.05; **p<0.01. Data shown are expressed as mean ± SEM of 3 (A, B), 9 (C) or representative of 3 (D) independent experiments.
Figure 3.
Adoptive transfer EAE with polarized Th1 and Th17 cells.
Th1 and Th17 polarized cells were adoptively transferred to Rag2−/− recipient mice, either alone or in combination or with anti-IFN-γ treatment as indicated. Recipient mice were scored for EAE disease. A. Shown is the percentage of EAE incidence in the different recipient mice. ***p<0.0001; Th1+Th17 vs. all other conditions. B. EAE clinical scores in the different recipient mice. Data is represented as mean ± SD. C. Incidence of classical and atypical EAE phenotype in different T cell transfers. Shown is the percentage of classical vs. atypical EAE incidence in the different recipient mice. Th1: n = 29; Th17: n = 40; Th1+Th17: n = 16; Th17+ anti-IFN-γ: n = 11.
Table 1.
Quantification of inflammation and demyelination in spinal cord, brain and PNS.
Figure 4.
Plasticity of Th17 cells in adoptive transfer EAE.
Th1, Th17, Th1+Th17 cells or Th17 cells and anti-IFN-γ antibodies were adoptively transferred into Rag2−/− mice. A. EAE sick mice with classical EAE, minimum score of 3 were sacrificed, and brain and spleen were removed. Immune cells were isolated and characterized for IFN-γ and IL-17 expression by intracellular FACS staining. Data shown is gated in the CD4+ population and is representative of a minimum of 5 animals analyzed per group. B. Expression of IFN-γ and IL-17 in the brain from EAE sick mice was analyzed by real time PCR. Rag2−/− control: n = 7; Th1: n = 7; Th17: n = 10; Th1+Th17: n = 5; Th17+anti-IFN-γ: n = 6. Data are represented as mean ± SEM. *p<0.05; ***p<0.0001.
Figure 5.
Cytotoxic molecules expression by Th1 and Th17 cells.
T cells from 2D2 mice were activated under Th1 and Th17 polarizing conditions. Expression of granzyme B, Fas-L and perforin in naive T cells (n = 4), Th1 (n = 6) and Th17 cells (n = 7) were measured by real time PCR. The data is represented as mean ± SEM.
Figure 6.
Cytotoxic potential of Th1 and Th17 cells towards astrocytes.
2D2 MOG-specific T cells were polarized in Th1 and Th17 conditions and co-cultured in duplicates with activated astrocytes as described in methods. A. MHC class II expression on astrocytes was analysed by FACS. B. GFP-labeled astrocytes were co-cultured with Th1 or Th17 cells in the presence of isotype control or anti-MHC class II antibodies. Cells were tracked every 30 minutes by fluorescent time-lapse microscopy. Shown is the snap-shot fluorescent picture after 48 h co-culture. Magnification: 10×. C. Quantification of the fluorescent area (surviving cells) every 6 hours in the conditions shown in B. The values were normalized to that of control (astrocytes only). D. IL-17 and IFN-γ levels were measured in the supernatants at 48 h after the co-culture by ELISA. Data shown are representative of minimum 3 experiments. *p<0.05; ns-not significant.
Figure 7.
Effect of blockade of cytotoxic molecules on Th1 mediated astrocyte cytotoxicity.
2D2 MOG-specific Th1 cells were co-cultured in duplicates for 48 hours with astrocytes in the presence or absence of the Th1 culture supernatant (A), anti-IFN-γ antibody, anti-FasL antibody, Granzyme B (GzmB) inhibitor and the pan-caspase inhibitor ZVAD (B). Cells were tracked every 30 minutes by fluorescent time-lapse microscopy. Shown is the snap-shot fluorescent picture at the end of the culture period and is representative of minimum 2 experiments.
Figure 8.
Cytotoxic potential of Th1 and Th17 cells towards FT7.1 cells.
2D2 MOG-specific T cells were polarized in Th1 and Th17 conditions and co-cultured in duplicates with FT7.1 cells as described in methods. A. MHC class II expression on FT7.1 cells was analysed by FACS. B. GFP-labeled FT7.1 cells were co-cultured with Th1 or Th17 cells in the presence of isotype control or anti-MHC class II antibodies. Cells were tracked every 30 minutes by fluorescent time-lapse microscopy. Shown is the snap-shot fluorescent picture after 48 h co-culture. Magnification: 10×. C. Quantification of the fluorescent area (surviving cells) every 6 hours in the conditions shown in B. The values were normalized to that of control (FT7.1 cells only). D. IL-17 and IFN-γ levels were measured in the supernatants at 48 h after the co-culture by ELISA. Data shown are representative of minimum 3 experiments. ns-not significant.