Figure 2: Z-DC transfer-mediated superior Mtb control is CD4⁺ T-cell dependent.

(a–d) Unvaccinated B6 mice, or B6 mice vaccinated with BCG s.c. followed by mucosal boost with Ag85B_240–254 peptide in mucosal adjuvant, were rested for 4 weeks, and Mtb HN878-infected (∼100 c.f.u.) and treated with Ag85B-Z-DC on −1 and 4 dpi, and (a) bacterial burden was assessed by plating at 20 dpi. Flow cytometry was used to determine production of (b) IL-17 and (c) IFN-γ by Ag85B-specific CD4⁺CD44^hi T cells. (d) B-cell lymphoid follicle formation was determined by CD3 (red) and B220 (green) staining on formalin-fixed, paraffin-embedded sections by immunofluorescence staining on 20 dpi. The average size of B-cell follicles per lobe was quantified using the morphometric tool of the Zeiss Axioplan. (e–g) B6 mice were vaccinated as above, and infected with Mtb HN878 (∼100 c.f.u.) via aerosol. Mice received Ag85B-Z-DC transfer on −1 and 4 dpi; and were treated with 300 μg GK1.5 (anti-CD4) or isotype control delivered i.p. on 0 and 7 dpi. Lungs were collected at 20 dpi. (e) Lung bacterial burden was determined by plating. Flow cytometry was used to assess (f) IL-17 and (g) IFN-γ production by Ag85B-specific CD4⁺CD44^hi T cells. (a–d) n=8–10 biological replicates±s.d., (e–g) n=5–10 biological replicates±s.d., *P≤0.05, **P≤0.01, ***P≤0.001 by one-way analysis of variance or Kruskall–Wallis test (e) or Student’s t-test (d) (UnVac+Ag85B-Z-DC compared with Vac+Ag85B-Z-DC). Dotted lines on bacterial burden plots represent the limit of detection by plating.