BCG stimulates CD16^lo natural killer (NK) cells to produce interferon (IFN)-γ in a TLR2-dependent manner.

<p>Human neonatal NK cells were isolated from umbilical cord blood, stimulated with Bacille Calmette Guérin (BCG) overnight (in the presence of Brefeldin-A for the last 4 hours), stained with a vital dye (L/D Aqua), and intracellularly stained for CD3, CD16, CD56, and interferon (IFN)-γ for flow cytometry analysis. In Fig 4a-e, one representative experiment is shown. (a) gating strategy, (b) unstimulated control, (c) BCG stimulated, (d) BCG stimulated and pre-incubated with an isotype control antibody, (e) BCG stimulated and pre-incubated with a Toll-like receptor (TLR)2 blocking antibody. (f) Summary data for <i>n</i> = 5 independent experiments. Bars are mean values and error bars are S.D. ^† p = 0.06, BCG+TLR2 blocking IgG vs. BCG+isotype control IgG.</p

The Bacille Calmette Guérin (BCG) vaccine contains extracellular mycobacterial (BCG) DNA which induces Type I interferon (IFN) production in human neonatal plasmacytoid dendritic cells (pDCs).

<p>(a) The cell-free supernatants of reconstituted BCG vaccine vials from two different manufacturers contain BCG DNA. PCR for 2 mycobacterial (BCG-specific) genes on the cell-free supernatants was performed as described in the Methods section, and the PCR products were run on an agarose gel and stained with ethidium bromide. Isolated human umbilical cord blood pDCs were stimulated with BCG DNA (100 μg/ml) for 8 hours and the relative expression levels of (b) IFN-α1 mRNA and (c) IFN-α2 mRNA were determined by qRT-PCR. ^†† p = 0.07, <i>n</i> = 3 independent experiments, bars are median values and error bars are S.D.</p

Bacille Calmette Guérin (BCG) does not induce IL-12 p35 mRNA production from human neonatal monocyte-derived dendritic cells (mdDCs), but induces IL-12 p40 mRNA production in a Toll-like receptor (TLR)2-dependent manner.

<p>Human umbilical cord blood CD14+ monocytes were differentiated into mdDCs using rIL-4 and rGM-CSF, stimulated with BCG x 18–24 hours, and then cellular mRNA was isolated for qRT-PCR. (a) relative expression of IL-12 p35 mRNA levels upon BCG stimulation compared to unstimulated control. Bars are median values, ^‡ p = 0.3, <i>n</i> = 6 independent experiments; (b) relative expression of IL-12 p40 mRNA levels upon BCG stimulation compared to unstimulated control, in the presence or absence of a TLR2 blocking antibody. Bars are median values, * p<0.05 compared to BCG stimulation with isotype control antibody pre-incubation, <i>n</i> = 6 independent experiments.</p

Optimization of interleukin (IL)-12 p35 and p40 mRNA expression in human neonatal monocyte-derived dendritic cells (mdDCs).

<p>Neonatal mdDCs were primed with recombinant (r) interferon-β (IFN-β) (Type I IFN), and rIFN-γ (Type II IFN) for 12 hours prior to stimulating them with a synthetic TLR2 agonist, Pam3CSK4. The combination of Pam3CSK4, rIFN-β, and rIFN-γ induced the maximal levels of (a) interleukin (IL)-12 p35 mRNA and (b) IL-12 p40 mRNA expression in human neonatal monocyte-derived dendritic cells (mdDCs). * p<0.05 compared to unstimulated control. Data points represent individual donors, the number of individual donors for each condition is shown in the figure, and bars are mean values.</p

Illustrative model demonstrating how the Bacille Calmette Guérin (BCG) vaccine might drive interleukin (IL)-12 p35 and p40 (IL-12 p70) production and CD4+ T-cell T-helper 1 (Th1) polarization in neonates.

<p>IFN = interferon; cDC = conventional dendritic cell; NK = natural killer; pDC = plasmacytoid dendritic cell; TLR = Toll-like receptor.</p