![Figure 3. The induction of B-cell proliferation and Ig secretion by pDC is contact-dependent and independent of IFN and residual CpG-B. (A) pDC/B-cell coculture was established with pDCs sequestered in the upper transwell chamber and B cells in the lower chamber. B cells were labeled with CFSE before culture, and proliferation was assessed by measuring CFSE dilution on day 5. The shaded histogram represents B cells cultured with CD40L; dashed histogram, B cells cultured with resting pDCs; and solid histogram, B cells cultured with CpG-B-pDCs. Data shown indicate 1 donor representative of 3. Supernatant was collected on day 14, and IgG and IgM concentrations were determined by ELISA. The mean plus or minus SEM of duplicate results from 3 individual donors is shown. (B) pDC /B-cell coculture was carried out as described previously, except that a cocktail of antagonist antibodies against IFN-α, IFN-β, and IFN-α/β/R was added. Proliferation was assessed by measuring [3H]-thymidine incorporation on day 3, and IgG and IgM levels in supernatant were measured by ELISA on day 14. Mean results ± SEM from 1 experiment representative of 3 are shown. (C) B cells were labeled with CFSE and cultured with CD40L and 1μM CpG-B, resting pDCs, or CpG-B-pDCs. Chloroquine was added to the B cells at the indicated concentrations 30 minutes before the addition of CpG-B or pDCs. Proliferation was assessed by measuring CFSE dilution on day 5. Control experiments: Results are shown in the top panel; shaded histogram represents B cells cultured with CD40L; and dotted histogram, B cells cultured with CD40L plus 1μM CpG-B. pDC/B-cell coculture experiments: Results are shown in the bottom panel; shaded histogram represents B cells cultured with CD40L; dashed histogram, B cells cultured with resting pDCs; and solid histogram, B cells cultured with CpG-B-pDCs. Supernatant was collected on day 14, and IgG and IgM concentrations were determined by ELISA. Chloroquine was titrated to determine the optimal concentration. Fixed numbers of B cells and pDCs were added. Results of 1 experiment representative of 3 are shown. Data were analyzed by unpaired t test: *P < .05 and **P < .01.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/115/15/10.1182_blood-2009-08-239145/4/zh89991050970003.jpeg?Expires=1724239510&Signature=ZxGheQz9E4UK-gzmoO7GboLZ9~O6lMx0kU6MNiURfjvGgt47k2NsuHn8YHTc29uDkGojbfuKEvru7ha0WwRYP34la0xqKN-SvBqrRSaMvKr31ozWuKM4CM0w2Ot4YLlA1o5OsGv8zH9cx6vYeSOcO4wS9GMuXtouMxaosVy0rXxn4UkoquUfUDvJdVVj-umfsaIDpo44cl-FC0AOhHduoibJR78vYzL7O79vU~Pc0w4ks2nj-se5cV56jTfBxJSDeXDDN6WG4rsYrnvAnJvOFPHpl9hJcYCmYMMxeIzpyVThbNHvtngRNDt5bkwLvBZ0Xinn0sxiRIRreRxcQiAKfA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The induction of B-cell proliferation and Ig secretion by pDC is contact-dependent and independent of IFN and residual CpG-B. (A) pDC/B-cell coculture was established with pDCs sequestered in the upper transwell chamber and B cells in the lower chamber. B cells were labeled with CFSE before culture, and proliferation was assessed by measuring CFSE dilution on day 5. The shaded histogram represents B cells cultured with CD40L; dashed histogram, B cells cultured with resting pDCs; and solid histogram, B cells cultured with CpG-B-pDCs. Data shown indicate 1 donor representative of 3. Supernatant was collected on day 14, and IgG and IgM concentrations were determined by ELISA. The mean plus or minus SEM of duplicate results from 3 individual donors is shown. (B) pDC /B-cell coculture was carried out as described previously, except that a cocktail of antagonist antibodies against IFN-α, IFN-β, and IFN-α/β/R was added. Proliferation was assessed by measuring [3H]-thymidine incorporation on day 3, and IgG and IgM levels in supernatant were measured by ELISA on day 14. Mean results ± SEM from 1 experiment representative of 3 are shown. (C) B cells were labeled with CFSE and cultured with CD40L and 1μM CpG-B, resting pDCs, or CpG-B-pDCs. Chloroquine was added to the B cells at the indicated concentrations 30 minutes before the addition of CpG-B or pDCs. Proliferation was assessed by measuring CFSE dilution on day 5. Control experiments: Results are shown in the top panel; shaded histogram represents B cells cultured with CD40L; and dotted histogram, B cells cultured with CD40L plus 1μM CpG-B. pDC/B-cell coculture experiments: Results are shown in the bottom panel; shaded histogram represents B cells cultured with CD40L; dashed histogram, B cells cultured with resting pDCs; and solid histogram, B cells cultured with CpG-B-pDCs. Supernatant was collected on day 14, and IgG and IgM concentrations were determined by ELISA. Chloroquine was titrated to determine the optimal concentration. Fixed numbers of B cells and pDCs were added. Results of 1 experiment representative of 3 are shown. Data were analyzed by unpaired t test: *P < .05 and **P < .01.
