FIG 3.

Leukocyte infiltration into the CNS is enhanced in WNV-infected Ccr7^−/− mice. (A) Dot plots of CD8⁺ T cells (lower right box) and CD4⁺ T cells (upper left box), after forward scatter/side scatter and UV-negative CD3⁺ gating, are shown at day 12 postinfection based on flow cytometry analysis from the CNS of WT and Ccr7^−/− mice. (B, C, and F to I) Following forward scatter/side scatter and UV-negative gating for live cells, total cell numbers of CD8⁺ T cells (CD3⁺ CD8⁺), CD4⁺ T cells (CD3⁺ CD4⁺), inflammatory monocytes (CD45^hi Ly6C^hi CD11b⁺), neutrophils (CD45^hi Ly6C^int CD11b⁺ Ly6G⁺; Ly6C^int indicates an intermediate expression level of Ly6C), activated CD8⁺ T cells (CD3⁺ CD8⁺ CD44⁺), and activated CD4⁺ T cells (CD3⁺ CD4⁺ CD44⁺) were assessed by flow cytometry from the CNS on days 0, 8, and 12 postinfection of WT and Ccr7^−/− mice. (D) Paraffin-embedded brain sections from WT and Ccr7^−/− mice on day 12 postinfection were stained for MAC2, myeloperoxidase (MPO), and CD3, and positive cells (shown in brown; indicated by black arrows) are shown within the parenchyma and meninges of WNV-infected mice. Representative images are shown at a magnification of ×20, with zoomed-in insets. (E) Dot plots of inflammatory monocytes (top box) and neutrophils (bottom box), after forward scatter/side scatter and UV-negative CD45^hi gating, are shown at day 12 postinfection based on flow cytometry analysis from the CNS of WT and Ccr7^−/− mice. (J and K) Protein levels of CCL19 and CCL21 from the CNS on days 0, 8, and 12 of WT and Ccr7^−/− mice were measured by an ELISA. (L and M) Following forward scatter/side scatter and UV-negative gating for live cells, total cell numbers of mDCs (MHC-II^hi CD11c⁺) and Treg cells (CD3⁺ CD4⁺ FoxP3⁺ CD25⁺) were assessed by flow cytometry from the CNS on day 8 postinfection in WT and Ccr7^−/− mice. Data are shown as means ± standard deviations for n = 3 to 9 mice per genotype and time point from two independent experiments. *, P < 0.05; ***, P < 0.001.