Figure 3. dNK kill Lm in 1° trophoblasts in vitro and in 3D villous cultures in situ.
(A) Intracellular CFU in HLA-G+ EVT (left) and EVT survival (right) after co-culture with dNK, pNK or medium for 3 h.
(B) Representative immunofluorescence images of three consecutive 5 μm cryosections of a placental villous tree infected with Lm and stained for DAPI, Lm, SDC-1 (ST marker, left), HLA-G (EVT marker, middle) and E-cadherin (CDH-1, CT and EVT marker, right), acquired with an Axio Observer spinning disk confocal microscope. Cell types were identified by staining, nuclear size and localization. Scale bar, 50 μm.
(C) Distribution of Lm-infected cells amongst trophoblast cell types 72 h after infection of villous explants.
(D) Percentage of infected EVT, CT and ST in villi cultured with or without autologous dNK or medium for 72 h. Percentages in (C,D) were calculated in 10 imaging fields (217x magnification) from a representative donor.
(E) Lm CFU in villous cultures incubated for 72 h with dNK or medium. For each donor sample CFU were normalized to the tissue size estimated by DNA quantification and CFU in dNK samples were normalized to samples without added NK.
Shown are median ± interquartile range (A) or mean ± s.e.m. (C-E). *p<0.05, ** p<0.01, ***p<0.001; by unpaired non-parametric one-way ANOVA (Kruskall-Wallis test followed by Dunn’s post-test (A left, comparing cells co-cultured with and without NK), Wilcoxon rank test (A right), one-way ANOVA followed by Tukey’s post-test comparing each cell type with each other (C) and paired t-test (D-E).