Figure 5.

Survival, infection and cytokine production by CD1c⁺ DCs is not affected by pDCs. (A) One representative gating strategy used to visualize DC subsets and apoptotic–necrotic CD1c⁺ DCs. After exclusion of doublets, pDCs were gated as CD123^high CD11c^– cells and CD1c⁺ DCs as CD123^low/–CD11c⁺ cells. CD1c⁺ DCs were then gated as early apoptotic cells (Annexin A⁺ PI^–), late apoptotic cells (Annexin A⁺ PI⁺), and necrotic cells (PI⁺). (B) Relative number of apoptotic and necrotic CD1c⁺ DCs at different time points after Mtb infection in the presence (Mtb + pDCs) or absence of pDCs (Mtb). The number was obtained by normalizing the percentages of apoptotic–necrotic CD1c⁺ DCs after infection to the percentages of the respective controls (CD1c⁺ DCs ± pDCs in the absence of Mtb). (C) Percentage of Mtb-infected (GFP⁺)-CD1c⁺ DCs in the presence or absence of pDCs. (D) Mean fluorescence intensity of HLA-DR, CD40, and CD83 in Mtb-infected mono- (white bars) or co-cultures (black bars) gated on CD1c⁺ DCs (top) or pDCs (bottom panel) (Mann–Whitney test). (E) Cytokine production by pDCs, CD1c⁺ DCs, or CD1c-pDC cultures 16 h post-infection. (F) Production of IL-12p70 by CD1c⁺ DC or CD1c⁺ DC-pDC cultures 16 h post-infection in the absence (Mtb) or in the presence (Mtb/R848) of TLR7/8 ligands or after stimulation with TLR4 and TLR7/8 ligands (LPS/R848). Control (Cntr) indicated unstimulated cells. Data are obtained from six donors in two independent experiments. One-way ANOVA; *p < 0.05, ***p < 0.001.