FIGURE 2.

The monocyte response to cffDNA activates bystander PBMCs and T cells to secrete IFN-γ and GzmB. (A) Maternal monocytes were stimulated with Effectene alone or Effectene with cffDNA overnight. CM were harvested from both groups, and autologous maternal PBMCs were cultured in same-donor monocyte supernatants (CM or cffDNA CM) for 18 h before cytokine levels were measured using Multiplex analysis (n = 1 donor). (B) Third trimester maternal monocytes were stimulated with cffDNA. After overnight stimulations, CM and cffDNA CM were harvested and added to maternal PBMCs from the same donor. Levels of IFN-γ and GzmB (n = 4–6 donors) were measured in duplicate by ELISA. Data are presented as median and IQR. The p values were calculated using Mann–Whitney U test: ***p < 0.0005, ****p < 0.0001. (C) As in (B), monocytes were stimulated overnight with either Effectene alone or Effectene with cffDNA. Supernatants from these cultures (termed CM and cffDNA CM, respectively) were used as media for same-donor PBMCs with and without neutralizing Abs specific for IL-1β and CXCL10. After overnight incubation, levels of IFN-γ and GzmB (n = 6 donors) were measured in duplicate by ELISA. Data are displayed as medians and IQRs. The p values were compared by Kruskal–Wallis test, with correction using false discovery rate for multiple comparison testing and two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. Significant comparisons (p ≤ 0.05) are indicated by CM versus cffDNA CM (≠), cffDNA CM versus IL-1β block (Δ), cffDNA CM versus CXCL10 block (ϕ), and cffDNA CM versus IL-1β and CXCL10 block (Ω). (D) Maternal T cells were cultured overnight in CM from same-donor monocytes treated with either Effectene reagent alone (CM) or Effectene and cffDNA (cffDNA CM). Levels of IFN-γ and GzmB were measured in duplicate T cell supernatants after overnight incubation (n = 5 donors). Data are presented as median and IQR, with p values calculated by Mann–Whitney U test: ****p < 0.0001.