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. 2021 Feb 22;10:e57417. doi: 10.7554/eLife.57417

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© 2021, Zhou et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 7. — (A and B) EAE clinical score and FFPE-processed spinal cords sectioned and stained with H and E and LFB from mice challenged with myelin oligodendrocyte glycoprotein (MOG) peptide with and without combined IL-2/IL-4 and subclinically effective doses of the individual cytokines (For statistical analyses, see Figure 7—figure supplement 1). Histological images of H and E and Luxol Fast Blue (LFB) staining represent a mouse of the mean disease score of each condition. N = 10–12 for EAE mice; N = 4 for vehicle control mice. (C) EAE clinical score and FFPE-processed spinal cords sectioned and stained with H and E and LFB from mice challenged with MOG peptide with and without the combined cytokines according to the indicated dosing schedule (for statistical analyses, see Figure 7—figure supplement 2A and B). N = 10–12 for EAE mice; N = 4 for vehicle control mice. (D) Analysis of MOG-loaded MHCII tetramer positive Tconv cells in the CNS as determined by flow cytometry. The color legend is indicated for panels D–H. N = 3 (E) Quantification of IL-17A secreted by CNS leukocytes isolated by Percoll gradient and incubated overnight in media, as quantified by ELISA. The conditions are described by the key in 7C. N = 3. (F and G) Analysis of Treg abundance and IL-10-expression in Tregs isolated from the CNS or spleen of Foxp3^RFP/Il10^GFP mice in the EAE trials by flow cytometry. The conditions are described by the key in 7C. N = 3. (H) Analysis of IL-10 expression of F4/80⁺ CNS macrophages from Foxp3^RFP/Il10^GFP mice in the EAE trials by flow cytometry. The conditions are described by the key in 7C. N = 3. (I and J) Assessment of EAE severity in IL-10 cKO mice quantified by daily clinical scoring (For statistical analyses, see Figure 7—figure supplement 3B) and FFPE-processing of spinal cords stained with H and E and LFB. Images represent a mouse of the mean disease score of each condition. N = 10–12 for EAE mice; N = 4 for negative control mice for disease. N = 10–12 for EAE mice; N = 4 for vehicle control mice. (K) Assessment of EAE severity in Il10^-/- mice as quantified by daily clinical scoring. N = 7–8 for EAE mice; N = 4 for negative control mice for disease (see also Figure 7—figure supplement 3C). (L) Quantification of IL-17A secreted by CNS leukocytes of Il10^-/- mice isolated by Percoll gradient and incubated overnight in media, as quantified by ELISA. N = 3. For all panels, mean ± SEM are indicated. *p<0.05, **p<0.01, ****p<0.0001.

Figure 7—figure supplement 1. — (A and B) EAE clinical score and FFPE-processed spinal cords sectioned and stained with H and E and LFB from mice challenged with myelin oligodendrocyte glycoprotein (MOG) peptide with and without combined IL-2/IL-4 and subclinically effective doses of the individual cytokines (For statistical analyses, see Figure 7—figure supplement 1). Histological images of H and E and Luxol Fast Blue (LFB) staining represent a mouse of the mean disease score of each condition. N = 10–12 for EAE mice; N = 4 for vehicle control mice. (C) EAE clinical score and FFPE-processed spinal cords sectioned and stained with H and E and LFB from mice challenged with MOG peptide with and without the combined cytokines according to the indicated dosing schedule (for statistical analyses, see Figure 7—figure supplement 2A and B). N = 10–12 for EAE mice; N = 4 for vehicle control mice. (D) Analysis of MOG-loaded MHCII tetramer positive Tconv cells in the CNS as determined by flow cytometry. The color legend is indicated for panels D–H. N = 3 (E) Quantification of IL-17A secreted by CNS leukocytes isolated by Percoll gradient and incubated overnight in media, as quantified by ELISA. The conditions are described by the key in 7C. N = 3. (F and G) Analysis of Treg abundance and IL-10-expression in Tregs isolated from the CNS or spleen of Foxp3^RFP/Il10^GFP mice in the EAE trials by flow cytometry. The conditions are described by the key in 7C. N = 3. (H) Analysis of IL-10 expression of F4/80⁺ CNS macrophages from Foxp3^RFP/Il10^GFP mice in the EAE trials by flow cytometry. The conditions are described by the key in 7C. N = 3. (I and J) Assessment of EAE severity in IL-10 cKO mice quantified by daily clinical scoring (For statistical analyses, see Figure 7—figure supplement 3B) and FFPE-processing of spinal cords stained with H and E and LFB. Images represent a mouse of the mean disease score of each condition. N = 10–12 for EAE mice; N = 4 for negative control mice for disease. N = 10–12 for EAE mice; N = 4 for vehicle control mice. (K) Assessment of EAE severity in Il10^-/- mice as quantified by daily clinical scoring. N = 7–8 for EAE mice; N = 4 for negative control mice for disease (see also Figure 7—figure supplement 3C). (L) Quantification of IL-17A secreted by CNS leukocytes of Il10^-/- mice isolated by Percoll gradient and incubated overnight in media, as quantified by ELISA. N = 3. For all panels, mean ± SEM are indicated. *p<0.05, **p<0.01, ****p<0.0001.