Figure 5. Amph-CpG and CD40 agonist improves Mtb control in vaccinated hosts.

(a) BMDC were cultured from B6 mice. After harvesting and resting overnight, BMDC were treated overnight with 1.24 nmol amph-CpG. CD103 expression on activated DCs was assessed by flow cytometry. (b–g) Mice were vaccinated with BCG s.c. and rested for 4 weeks. Vaccinated mice were infected with Mtb HN878 and treated with either Ag85B-Z-DC, amph-CpG (1.24 nmol), FGK4.5 (100 μg), or both amph-CpG and FGK4.5 along with Ag85B peptide (5 μg) on −1 and 4 dpi. Mice were collected at 8 dpi. (b) Lung bacterial burden was determined by plating. Flow cytometry was used to assess (c) numbers of CD4⁺CD44^hi cells in the lungs, and (d) IL-17 and (e) IFN-γ production by CD4⁺ T cells, (f) MHC-II expression on alveolar macrophages and fold change MFI relative to Vac was calculated. (g) Flow cytometry was used to calculate numbers of CD11c⁺CD103⁺ cells in the lungs. (a) n=4–5 technical replicates±s.d., (b–g) n=4–10 biological replicates. *P≤0.05, **P≤0.01, ***P≤0.001 by Student's t-test (a) or one-way analysis of variance (b–g). Dotted lines on bacterial burden plots represent the limit of detection by plating.