Assessment of macrophages isolated from the West Nile virus (WNV) infected brain. (A, B) Macrophages isolated from day 7 p.i. brain tissue infected with West Nile virus expressed major histocompatability complex (MHC)-II and CD86. (C) More than 70% of Ly6Chi macrophages in the WNV-infected brain were positive for fluorescent agent (ProSense; VisEn Medical, Bedford, MA, USA), indicative of cathepsin activity. (D) The ability of macrophages (CD45+/CD11b+/Ly6G−/GFP+/CD11c−/Ly6Chi) and resident microglia (CD45lo/int/CD11b+/GFP−/CD11c−/Ly6Chi), isolated from WNV-infected colony-stimulating factor 1 receptor (cFMS)-enhanced green fluorescent protein (EGFP) chimeric mice
 to stimulate naïve and activated CD3+/CD4+ T cells from sham-infected and WNV-infected mice, respectively, was measured. Except at the highest antigen-presenting cell (APC):CD4 ratios, neither population was capable of stimulating naive T-cell proliferation, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (E) Notwithstanding the apparent APC capabilities, depletion of CD8+ or CD4+ or CD4+ and CD8+ had no effect on overall survival in this model. All experiments were performed at least twice with four to five mice per group. Cells were sorted from 10 pooled cFMS-EGFP chimeric mice, and data represent the mean of triplicate wells and the standard deviation of absorbance. T-cell depletion was performed at least twice with eight to ten mice per group. Statistical analysis was conducted using one-way ANOVA with a Tukey-Kramer post hoc test. * Indicates significance of comparison of CD4+ T-cell proliferation between wells with macrophages and wells with CD4+ T cells only. * P < 0.05; **P < 0.001; ***P < 0.001. Mice infected with 6 ×104 plaque-forming units were used to obtain APC populations.