GA CD8⁺ T cells ameliorate demyelinating disease by a non-classical MHC class I-dependent mechanism requiring IFN-γ and perforin but not IL-10.

<p>(A–D) Donor wild-type C57BL/6 mice were immunized for 20 days with GA/IFA (2 mg) or OVAp/IFA (200 µg). Splenocytes and DLN cells were isolated and cultured <i>in vitro</i> for 3 days with antigen (20 µg/ml) and IL-2 (10 pg/ml). CD8⁺ T cells were purified by magnetic bead sorting and injected intravenously into wild-type (A), CD8^−/− (B), MHCI^−/− (β₂microglobulin^−/−) (C), and K^b−/−D^b−/− (D) recipient mice one day prior to disease induction. (E) <i>Ex vivo</i> GA CD8⁺ T cells were isolated from GA/IFA-immunized mice and stained with CFSE, followed by incubation with APC derived from naïve wild-type, β₂microglobulin^−/− (MHCI^−/−) or K^b−/−D^b−/− mice by depleting splenocytes of T cells using anti-CD3 magnetic beads. Replicate cultures were incubated with either vehicle or GA (20 µg/ml). Δ proliferation reflects difference in proportion of CFSE_low proliferating cells between GA and vehicle control cultures. * = p<0.05, “ns” = not statistically significant. (F-H) CD8⁺ T cells were obtained from GA/IFA or OVAp/IFA immunized wild-type (WT), IFN-γ^−/−, perforin^−/−, and IL-10^−/− mice as above and injected intravenously into wild-type recipient mice one day prior to EAE induction.