Figure 4: Z-DC transfer induces early genes associated with APC activation.

B6 mice were vaccinated with BCG s.c. followed by mucosal boost with Ag85B_240–254 peptide in mucosal adjuvant, rested for 4 weeks, and Mtb HN878-infected (∼100 c.f.u.) and treated with Ag85B-Z-DC on −1 and 4 dpi. RNA-seq was performed on lungs at 8 dpi. (a) Expression values of each biological replicate (n=5 for Vac, n=4 for Vac+Ag85B-Z-DC) for each group of a subset of selected genes of interest (all selected genes had a differential expression of at least twofold). These genes were differentially expressed using CuffDiff output with a P<0.05, and Benjamini–Hochberg false discovery rate (FDR) of 5%. The scaled expression of each replicate, denoted as the row Z-score, is plotted in red–blue colour scale with red indicating high expression and blue indicating low expression. q values for each gene of interest are shown in Supplementary Table 1 for the top 100 upregulated genes. (b) Expression of the top 100 upregulated genes shown in Supplementary Table 1 was considered in the published transcriptional data following expression in lungs of mice infected (red curves) with Mtb, or uninfected controls (blue curves) along the timecourse of 14, 21, 28 and 42 dpi³³. (c) Gene Set Enrichment Analysis was used to analyse the expression of the top 100 upregulated genes shown in Supplementary Table 1 in the context of the infection model of Cd40l^−/− mice with P. murina. Genes differentially expressed between wild type (WT) and Cd40l^−/− mice on 32 days post-P. murina infection were compared and the top 1,000 expressed genes shown in the heat map, ranked by the level of differential expression between the two genotypes⁷⁰. Orange lines indicate positions of 100 genes from Supplementary Table 1, P<10⁻³. (d) Numbers of CD11c⁺CD103⁺ cells in lungs of Mtb-infected-vaccinated Z-DC-recipient mice was determined using flow cytometry. n=4–5 biological replicates±s.d. ***P≤0.001 by Student’s t-test.