Fig 2: Characterization of the PeptiENV Priorix complexes(A) SPR analysis of the interaction between the CPP-OVA and Priorix vaccine and OVA peptide without the attachment moiety and Priorix vaccine. (B) Mouse DC line JawsII was pulsed with PeptiENV-OVA Priorix, Priorix mixed with OVA peptide (a non-interacting control peptide), Priorix, CPP-containing SIINFEKL peptide alone or left unpulsed (Mock). Cross-presentation was determined by flow cytometry using APC-conjugated anti-H-2Kb bound to SIINFEKL. CD86 expression and changes in morphology of the cells (as measures of DC maturation and activation) were determined by flow cytometry. Each bar is the mean ± standard error of the mean of biological triplicates. Statistical analysis was performed with one-way ANOVA. ∗∗∗∗p < 0.0001.

Fig 3: Effects of BEA on BMDCs. (A, B) 5x105 BMDCs from IL-12p40/GFP reporter mice were stimulated with indicated concentration of BEA with or without LPS (1 ng/ml) and CpG2216 (0.5 μM) for 16 hours. IL-12p40/GFP (A) and MFI of CD86 expression (B) by BMDCs were detected by flow cytometry. Analysis of IL-12p40/GFP and CD86 expression were gated on MHC IIhigh CD11c+ cells. Results shown are representative of three independent experiments using cells from 3 mice per group (n=3). (C) 106 BMDCs were stimulated with 5 μM BEA for the indicated time. IL-12p40, IFNβ, TNF and IL-6 were analyzed by Real time PCR. Results shown are representative of two independent experiments using cells from 3 mice per group (n=3). Data in A, B and C are presented as mean ± SEM and analyzed by one-way ANOVA with Dunnett’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ns, not significant.

Fig 4: Myeloid immune cell subsets differ significantly between the different versions of the MMTV-PyMT breast tumor model. Tumors from the autochthonous MMTV-PyMT model were harvested and single-cell suspensions were generated. Cells (1E6, 1E5, or 1E4) were injected into the mammary fat pad of FVB/NJ wild-type mice. When tumors reached 100 mm3, tumors were obtained and processed into a single-cell suspension for immunophenotyping by flow cytometry. a–d Analysis of cells subsets as a percent of myeloid cells (CD11b+) include a myeloid-derived suppressor cells (MDSC (GR1+)), b PD-L1+F4/80+, c PD-L1– F4/80+, and d other CD11b+ populations; e Analysis of the myeloid cells subsets as a percent of viable cells include f analysis of PD-L1 expression on myeloid cells (CD11b+) and g cancer cells represented by CD45-. h Analysis of the ratio of lymphocytes expressing PD-1. i Analysis of M1-like macrophage markers, CD40+, CD80+, and CD86+ and an M2-like macrophage marker, CD206+, as a ratio of total myeloid cells (CD11b+, top), and mature macrophages (F4/80+, bottom). Graphs show mean ± SEM from at least two independent experiments with at least three mice per group. Each data point represents an individual mouse. Unpaired two-tailed t test. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.001

Fig 5: AGK2 enhances the BCG-induced innate and adaptive immune cell activation(A–I) Gating strategy used to screen the antigen-presenting cells in the lungs and the spleen of infected animals. The cells were ex vivo stimulated with M.tb CSA for 24 h and stained with anti-CD11b (APCCy7), anti-CD11c (APC), anti-CD40 (PE), anti-CD80 (FITC), anti-CD86 (PerCP). Representative dot plots are shown. Percentage of (B) CD11b+, (C) CD11b+CD40+, (D) CD11b+CD80+, and (E) CD11b+CD86+ in the lungs of infected mice. Percentage of (F) CD11c+, (G) CD11c+CD40+, (H) CD11c+CD80+, and (I) CD11c+CD86+ in the lungs of infected mice.(J–N) Representative dot plots to demonstrate the gating strategy used. The single-cell suspensions obtained from the lungs of infected animals were stimulated with M.tb CSA for 24 h before staining with anti-CD3 (Pacific Blue), anti-CD4 (PreCPCy5.5), anti-CD8 (APCCy7), and anti-CD69 (FITC). Percentage of (K) CD4+, (L) CD8+, (M) CD4+CD69+, and (N) CD8+CD69+ T cells. Data are representative of two independent experiments with 5 mice in each group. The graphs represent mean ± SD. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005.