Figure 2.

Expression of NID at the surface of E. coli binds to IgD and activates B cells. (A) NID is expressed at the surface of E. coli as revealed by flow cytometry and rabbit anti-MID pAbs and FITC-conjugated secondary anti-rabbit pAbs. The E. coli-ctrl (control) was transformed with an empty vector. (B) NID-expressing E. coli binds IgD. Soluble recombinant IgD⁵ was detected with FITC-conjugated anti-IgD pAbs. (C) Peripheral blood lymphocytes (PBLs) were incubated with FITC-labeled E. coli-NID or E. coli–ctrl. PBLs were isolated from human blood using a Lymphoprep one-step gradient (Nycomed) and incubated with an RPE-conjugated anti-CD19 monoclonal antibody, washed and further incubated with the FITC-labeled bacteria. In (A) and (B), background fluorescence was determined by incubating bacteria with the secondary detection pAbs only (non-shaded). In (C), dot plots from the flow cytometry are shown. (D) E. coli-NID stimulates B cells to proliferate. PBLs were analyzed by measuring [³H]-thymidine uptake at 72 h. PBLs were stimulated with different concentrations of formaldehyde-killed E. coli either containing NID or control bacteria with the empty vector only. (E) E. coli OMVs were isolated from the E. coli-NID and E. coli (control) as described.¹⁹ OMVs were probed with anti-MID pAbs and FITC-conjugated secondary anti-rabbit pAbs. The control (ctrl) designates the secondary pAbs only. The y-axis denotes the fold change with the anti-MID pAbs. Data were normalized to the secondary detection-pAbs that were set as 1.0.