IpaD binds to human CL-01 B lymphocytes and is internalized. (A) Confocal images of CL-01 B cells incubated with T3SA⁻ bacteria and IpaD or MxiH coupled to Alexa Fluor 488. An overview image (bar, 50 µm) is presented alongside single confocal slices of B cells (bars, 5 µm) after 2 h of incubation at 37°C. Cells were co-stained for GM1 (red, membrane) and DAPI (blue, nuclei). (B and C) Quantification of the AF488 mean fluorescence intensity (MFI) in a 15-µm radius around the center of the nuclei. (B) MFI of IpaD after co-incubation with T3SA⁻ for 2 h at 37°C. Values are compared with the MxiH control protein and to incubation at 4°C. (C) Time dependence of the MFI measured for IpaD. Cells were incubated with IpaD for different times at 37°C in the presence of T3SA⁻ bacteria. A minimum of 100 cells were analyzed for each condition for B and C. Data are presented as median ± interquartile range for one representative experiment and statistically significant differences were determined by Kruskal-Wallis test with Dunn’s post-test. ***, P < 0.0001. (D) IpaD internalization. Cells were incubated with fluorescent AF488-IpaD for 1 h at 4°C (time 0), and unbound AF488-IpaD was washed before incubation at 37°C for 30 min or 1 h. At each time point, IpaD bound to the cell surface was detected by flow cytometry using an anti-IpaD polyclonal serum, and a secondary antibody coupled to Alexa Fluor 647. Data are presented as mean ± SEM for three independent experiments and statistically significant differences to time 0 were determined by one-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001.