Fig. 3

CNS restricted IFNγ promotes CD8 infiltration and MHC on OPCs. TRE/IFNγ × GFAP/tTA mice were fed CPZ for 6 weeks and either maintained on doxycycline (gray; n = 5) or removed from doxycycline (blue; n = 5) to induce IFNγ expression. Two-weeks after CPZ withdrawal the corpus callosum was dissected and qPCR analysis was performed (a). Significance for qPCR was determined by two-tailed, unpaired t-test between doxycycline and no doxycycline conditions. b Whole brains from TRE/IFNγ × GFAP/tTA mice under the same experimental paradigm were isolated for flow cytometric analysis. The OPC population was determined by CD11b negativity and Olig2, A2B5, and PDGFRα positivity. H2Kb expression is shown in the histogram plot in which the staining control FMO (white) is compared to mice kept on doxycycline (gray; n = 5) and mice removed from doxycycline (blue; n = 5). Significance for flow cytometry was determined by two-tailed, unpaired Student’s t-test between doxycycline and no doxycycline conditions (H2Kb⁻ mean diff = −31.3 ± 3.5; 95% CI = 39.3–(–)23.4, H2Kb⁺ mean diff = 17.0 ± 3.2, 95% CI = 9.9–24.2, H2Kb⁺⁺⁺ mean diff = 14.3 ± 2.4, 95% CI = 8.8–19.7). c The same gating strategy was used to determine MHC Class II (IA/IE) expression by H2Kb. Significance for flow cytometry was determined by two-tailed, unpaired t-test between doxycycline (gray; n = 6) and no doxycycline (blue; n = 6) conditions were compared (IAIE⁺ of H2Kb⁺ mean diff = 10.9 ± 2.1, 95% CI = 6.3–15.5). CD4⁺/CD8⁺ populations were also analyzed within the whole brain tissue as determined by flow cytometry. The CD4⁺ population remains unchanged between doxycycline (gray; n = 6) and doxycycline removal (blue; n = 6). However, the removal of doxycycline significantly increased the number of CD8⁺ cells in the brain. Significance for flow cytometry was determined by two-tailed, unpaired Student’s t-test between doxycycline and no doxycycline conditions were compared, (CD8⁺ T cell mean diff = 11.55 ± 1.399, 95% CI = 8.429–14.66). e Percent OPC numbers were also analyzed for PDGFRα, A2B5, and O4. PDGFRα⁺ (top), PDGFRα⁺/A2B5⁺ (middle), and PDGFRα⁺/O4⁺ (bottom) OPC percentages were all significantly decreased in animals in which doxycycline was removed. Significance for flow cytometry was determined by two-tailed, unpaired Student’s t-test between doxycycline and no doxycycline conditions were compared (PDGFRα⁺ mean diff = −10.5 ± 2.1, 95% CI = −15.3–(−)5.8, PDGFRα⁺/A2B5⁺ mean diff = −4.1 ± 1.2, 95% CI = −6.9–(−)1.4, PDGFRα⁺/O4⁺ mean diff = −3.6 ± 1.4, 95% CI = −6.7–(−)0.5). All error bars represent standard deviation. (P*≤0.05,**≤0.01,***≤0.001,****≤0.0001)