Fig. 7

Kv1.3 blockade inhibits LPS-induced MHCI-restricted antigen presentation to CD8 T cells. a Experimental plan for MHCI-restricted T cell proliferation studies: BV2 microglia were treated in vitro with PBS, ShK-223 (100 nM), LPS (100 ng/mL), or LPS+ShK-223 for 24 h (four replicates/group) and then loaded with 2 μg/mL Ova (257-264) peptide for 30 min at 37 °C. Washed microglia were co-cultured with CFSE-labeled splenic T cells from Ova (257-264)-specific OT-1 mice for 48 h (25,000 microglia:150,000 T cells/well). For in vivo experiments, adult wild-type mice were treated with four daily IP doses of saline, ShK-223, LPS, or LPS+ShK-223 (n = 6/group) and isolated brain mononuclear cells were used for in vitro T cell proliferation studies. After 48 h, CFSE dilution (leftward shift in CFSE staining) was assessed by flow cytometry as a measure of CD8⁺ T cell proliferation. b Comparison of proliferating CD8⁺ cells across various treatment groups from in vitro and in vivo studies. Right: Representative frequency histograms from in vivo experiments. No direct effects of LPS or Ova peptide on T cells were noted without the presence of microglia. c Flow cytometric detection of CD3⁺ CD11b⁻ T cells in brain mononuclear cells after thorough cardiac perfusion. Mice were injected (IP, once daily for 4 days) with PBS, LPS, ShK-223, or LPS+ShK-223. Panel a shows the gating strategy. After forward and side-scatter gating for live mononuclear cell population, CD11b⁻ CD3⁺ cells were gated and assessed for T cell markers (CD3, CD8) and activation/cytolytic marker CD95 (Fas). d Flow cytometric evidence for increased CD8⁺ T cell trafficking to the brain following LPS treatment. e Comparison of proportions of brain CD8⁺ and CD8⁻ T cell populations across the 4 treatment groups. f Comparison of CD95 (Fas) expression in brain CD8⁺ and CD8⁻ T cell populations across treatment groups (*p < 0.05, **p < 0.01, ***p < 0.005)