Figure 7. PIKFYVE deletion in CD4 T... | Rockefeller University Press

Figure 7.

$PIKFYVE deletion in CD4+T cells attenuates EAE pathogenesis. PIKFYVEf/f or CD4crePIKFYVEf/f mice were immunized with 200 µg of MOG35–55 peptide containing CFA. On days 0 and 2, mice received 200 ng/mouse of pertussis toxin. (A and B) (A) Clinical score was measured daily and (B) its linear regression was computed with six mice per group. Draining lymph nodes were collected 7 days after EAE induction and cells were restimulated with MOG35–55 for 96 h. Cells were stained with viability probe and antibodies for CD3 and CD4. (C and D) A fraction of the cells were stained with antibodies for (C) IL-17A and Foxp3 or (D) GM-CSF and IFNγ. Spinal cords were harvested on day 15 after EAE induction. Isolated cells were stimulated, stained with multiple antibody panels, and analyzed by flow cytometry. (E) The level of CD4+ T cell infiltration, CD4+CD45hi cells, was determined. (F) High dimensional flow cytometry and tSNE analysis were utilized to measure other immune cell populations that infiltrated the spinal cord in the PIKFYVEf/f or CD4crePIKFYVEf/f mice. (G and H) The immune infiltrate in the spinal cord was analyzed for (G) Th17 cells (CD3+CD4+CD45hiIL17+) and Tregs (CD3+CD4+CD45hiFoxP3+) or (H) GM-CSF (CD3+CD4+CD45hiGM-CSF+) and IFNγ (CD3+CD4+CD45hiIFNγ+) on day 15 after EAE induction with flow cytometry. (I) Histological analysis of the leukocyte infiltration and demyelination was performed on spinal cord sections collected on day 30 after EAE induction from PIKFYVEf/f and CD4crePIKFYVEf/f mice. The black scale bar represents 100 μM. Data shown depict the mean ± SEM (n = 6 biological replicates). HE, hematoxylin and eosin; LFB, Luxol Fast Blue. P values were calculated by two-way ANOVA test followed by Sidak test in panels A and B and by Student’s t test in panels C–E, G, and H.$

PIKFYVE deletion in CD4 ⁺ T cells attenuates EAE pathogenesis. PIKFYVE^f/f or CD4^crePIKFYVE^f/f mice were immunized with 200 µg of MOG_35–55 peptide containing CFA. On days 0 and 2, mice received 200 ng/mouse of pertussis toxin. (A and B) (A) Clinical score was measured daily and (B) its linear regression was computed with six mice per group. Draining lymph nodes were collected 7 days after EAE induction and cells were restimulated with MOG_35–55 for 96 h. Cells were stained with viability probe and antibodies for CD3 and CD4. (C and D) A fraction of the cells were stained with antibodies for (C) IL-17A and Foxp3 or (D) GM-CSF and IFNγ. Spinal cords were harvested on day 15 after EAE induction. Isolated cells were stimulated, stained with multiple antibody panels, and analyzed by flow cytometry. (E) The level of CD4⁺ T cell infiltration, CD4⁺CD45^hi cells, was determined. (F) High dimensional flow cytometry and tSNE analysis were utilized to measure other immune cell populations that infiltrated the spinal cord in the PIKFYVE^f/f or CD4^crePIKFYVE^f/f mice. (G and H) The immune infiltrate in the spinal cord was analyzed for (G) Th17 cells (CD3⁺CD4⁺CD45^hiIL17⁺) and Tregs (CD3⁺CD4⁺CD45^hiFoxP3⁺) or (H) GM-CSF (CD3⁺CD4⁺CD45^hiGM-CSF⁺) and IFNγ (CD3⁺CD4⁺CD45^hiIFNγ⁺) on day 15 after EAE induction with flow cytometry. (I) Histological analysis of the leukocyte infiltration and demyelination was performed on spinal cord sections collected on day 30 after EAE induction from PIKFYVE^f/f and CD4^crePIKFYVE^f/f mice. The black scale bar represents 100 μM. Data shown depict the mean ± SEM (n = 6 biological replicates). HE, hematoxylin and eosin; LFB, Luxol Fast Blue. P values were calculated by two-way ANOVA test followed by Sidak test in panels A and B and by Student’s t test in panels C–E, G, and H.