Fig. 5
Membrane-bound OVA of viable E. coli is highly efficient in activating antigen-specific CD4+ cells. a 5 × 103 adherent BMDCs were co-cultured with 5 × 106 ECCTRL, EC-ovaOM, EC-ovaSECR, EC-ovaCYTO1/2/3, or sOVA (10 µg) in 96-well plates in 200 µl medium. After 90 min, bacteria or sOVA were removed by washing three times. 200 µl medium containing 50 µg/ml gentamycin and 1 × 105 CFSE-labeled OTII T cells were added and co-cultured for 4 days. In the 90 min of BDMC/E. coli co-culture, bacterial numbers (intracellular + extracellular together) increased by approximately fourfold to 2 × 107 CFU (data not shown). At day 4, CFSE dilution in OTII cells was analyzed by FACS. Representative FACS plots of n = 5 experiments are shown. For gating strategy see Supplementary Figure 8B. b Dose-dependent effects on OTII proliferation (upper panel) and OTII cell death (HOECHST staining) of viable and heat-killed E. coli, bacterial lysates and outer membrane vesicles (OMVs). Numbers of viable E. coli correspond to the calculated number after 90 min of co-culture (input fourfold lower). Means ± SEM of two independent experiments performed in duplicates are shown. c Effects of bacterial culture supernatants (derived from OD595 ~0.5 cultures diluted 1:4 in cell culture medium) on OTII proliferation. d OTII proliferation induced by different concentration of sOVA ± 5 × 106 ECCTRL. Means ± SEM of two experiments performed in duplicates are shown
