Anti-CD40 antibody and LPS recruit and activate DCs in the draining lymph nodes and spleen. Eight- to 10-week-old C57BL/6 mice (n = 4 per cohorts) received intramuscular injection of 10¹¹ vg of AAV1-hFIX plus 100 μg of LPS plus 100 μg of anti-CD40 antibody (in the footpad) or 6 × 10¹⁰ vg of AAV2-hFIX plus 100 μg of LPS plus 100 μg of anti-CD40 antibody (in the footpad) on the same day. Control mice received PBS. Cells from the draining lymph nodes and spleen were collected after 24 hours and stained with the appropriate antibodies, and flow cytometry analysis was performed, as described. (A) Increase in DC numbers in draining lymph nodes and spleen after administration of LPS and anti-CD40 antibody in comparison with naive mice. The dot plots show the expression of CD11c on cells. Numbers indicate the percentage of CD11c⁺ cells among the total live cells in either the draining lymph nodes or the spleen. (B) Activation of DCs in draining lymph nodes and spleen after administration of LPS and anti-CD40 antibody. The dot plots show the expression of either MHCII and CD80 or MHCII and CD86 on cells gated with CD11c. Numbers indicate the percentage of MHCII⁺CD80⁺ or MHCII⁺CD86⁺ among the CD11c⁺ population. Data shown are mean ± SEM. A representative of 2 independent experiments is shown. (C) Cytokine profile of splenocytes 24 hours after administration of LPS and anti-CD40 antibody. The histograms show the expression of IL-12 (top panel), IL-6 (middle panel), and IL-10 (bottom panel) on cells gated with CD11c. Numbers indicate the percentage of CD11c⁺ cells that are expressing the particular cytokine. Data shown are mean ± SEM, n = 4 for each cohort. A representative of 2 independent experiments is shown.