Particulate Ag-CpG promotes B-cell proliferation and differentiation to form short-lived EF PCs in vivo. (A-D) CFSE-labeled MD4 B cells were adoptively transferred into C57BL/6 mice and challenged with 10 μL particulates containing either HEL^RD alone, CpG alone, or HEL^RD-CpG. (A) Four days after transfer, flow cytometry was used to measure CFSE dilution (top left panels) and CD138 up-regulation (bottom left panels) in HEL-binding cells in the spleen of recipient mice. The percentage of MD4 PCs (HEL intracellular^hi, CD138⁺) present (top right panel) is shown as a proportion of total splenocytes, and serum HEL-specific IgM^a (bottom right panel) was measured by ELISA. (B) Flow cytometry was used to measure CFSE dilution in HEL-binding cells in the spleens of recipient mice in stimulated (black line) and unstimulated (filled gray) MD4 cells at the indicated times after challenge. (C) ELISPOTs (left panel) were used to detect splenic HEL-specific ASCs, and ELISAs (right panel) were used to measure serum HEL-specific IgM^a. (D) After 5 days, immunofluorescence microscopy (Zeiss LSM 510 meter; Carl Zeiss MicroImaging, Gottingen, Germany) was used to detect HEL-binding cells (HEL–Alexa 488) and splenic follicular B cells (anti-B220–Alexa 543) in cryosections mounted in Fluoromount-G (Southern Biotech). Images were acquired using Zeiss LSM 510 version 3.2 software and processed in ImageJ version 1.41 (http://rsb.info.nih.gov/ij/). (E) CFSE-labeled MD4 B cells or MD4 TLR9^−/− B cells were adoptively transferred into C57BL/6 mice, and challenged with 10 μL HEL^RD-CpG particulates. Four days after the transfer flow cytometry was used to measure CFSE dilution (top left panels) and CD138 (bottom left panels) in HEL-binding cells in the spleen of recipient mice. The percentage of MD4 PCs (HEL intracellular^hi, CD138⁺) present (middle panel) is shown as a proportion of total splenocytes; serum HEL-specific IgM^a (right panel) was measured by ELISA.