Internalized MID-expressing <i>M</i>. <i>catarrhalis</i> do not survive in tonsillar B cells.

<p>(A) A representative dot plot displaying forward and side scatters of the purified tonsillar B cells used for live-cell gating. Each preparation of purified tonsillar B lymphocytes were analysed as routine control by flow cytometry for IgD (B), CD19 (C), and CD3 (D) expression. White profiles correspond to the isotype control Abs used as negative controls. Representative flow cytometry plots are shown. (E) <i>M</i>. <i>catarrhalis</i> wild type (BBH18 wt) or an isogenic MID-deficient mutant (BBH18 Δ<i>mid</i>) were incubated with purified tonsillar B lymphocytes for 1 h. Thereafter, B cells were washed and treated with gentamicin to kill extracellular bacteria followed by thorough washes. At the indicated times, infected cells were lysed mechanically and plated on agar plates. Colony forming units (cfu) were counted after incubation for 24 h at 37°C. The mean values of three separate experiments with different donors are demonstrated. Error bars indicate SEM. Insert shows IgD binding to <i>M</i>. <i>catarrhalis</i> BBH18 wt and the MID-deficient mutant BBH18 Δ<i>mid</i>. Bacteria were grown on solid medium overnight. After incubation with a human IgD standard serum followed by a FITC-conjugated anti-human IgD pAb and several washings, bacteria were analyzed by flow cytometry. (F) The viability of B cells infected with <i>M</i>. <i>catarrhalis</i> BBH18 wt and BBH18 Δ<i>mid</i> was assessed at the indicated times by trypan blue exclusion staining. The mean values of three independent experiments are demonstrated. Error bars indicate SEM. (G) The capacity of purified human IgD to block <i>M</i>. <i>catarrhalis</i> binding to B cells was analyzed by flow cytometry. FITC-labelled <i>M</i>. <i>catarrhalis</i> wild type were treated with increased concentrations of purified human IgD, washed and incubated with human B cells for 30 min at 37°C. After several washes, bacterial binding to B cells were analyzed by flow cytometry. The mean values of three independent experiments are demonstrated. Error bars indicate SEM. Insert shows IgD-binding to FITC-labelled <i>M</i>. <i>catarrhalis</i>. Bacteria were incubated with a human IgG standard (50 µg/ml) (left panel) or purified IgD (50 µg/ml) (right panel). After several washes, <i>Moraxella</i> were incubated with an RPE-conjugated anti-human IgD mAb and analyzed by flow cytometry.