Fig. 3

Astrocyte IFNγ signaling is protective and upregulates PD-L1 during chronic EAE. A EAE was induced in Ifngr1^fl/fl Aldh1l1-Cre^ERT2+ mice (n = 7) and Ifngr1^fl/fl littermate controls (n = 8) and EAE clinical course was blindly monitored. On day 16 ± 1 mice were injected i.p. with tamoxifen for 5 consecutive days to induce recombination (black arrows). Graph is representative of two combined independent experiments. 35 days post-immunization, mice were perfused and the CNS was removed and cryopreserved for IHC analysis. Ventral white matter tracts of the lumbar spinal cord were imaged using confocal microscopy. B Ifngr1^fl/fl and C Ifngr1^fl/fl Aldh1l1-Cre^ERT2+ tissue sections were labeled for GFAP, PD-L1, and nuclei were counterstained with DAPI. D Total PD-L1 area and E PD-L1 colocalized with GFAP were analyzed using ImageJ. F, G Tissue sections were labeled for GFAP, CD3, and PD-1 and H–K for Iba1, CD45, PD-1. Nuclei were counterstained with DAPI and imaged at (H, I) 20 × and (J, K) 63 × magnification. L Total PD-1 area, (M) CD3⁺ cells and (N) Iba1⁺ and CD45⁺ cells per high powered field were quantified. O Colocalization of PD-1 was assessed for CD3⁺, Iba1⁺, and GFAP⁺ cells using ImageJ. Data in panel A represent the mean ± SEM and were analyzed using a Mann–Whitney U test for nonparametric data. Data represent the mean ± SEM and were analyzed using a two-tailed Student’s t test (D, E, L, M) or two-way ANOVA (N, O). Data are combined from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001