CXCL13 production by GC-Th cells is regulated by TCR activation and CD28 costimulation. (A) Regulation of CXCL13 production in GC-Th cells by PMA and/or ionomycin. (B) Efficient production of CXCL13 requires both TCR activation and CD28 costimulation. (C) GC-B cells support GC-Th cells in production of CXCL13. GC-B cells were cocultured with equal numbers of GC-Th cells in the presence of superantigen SEB (1 μg/mL) for 3 to 4 days. (D) High levels of CXCL13 mRNA were detected in GC-Th cells but not in B cells reisolated after culture. (E) High levels of CXCL13 messages were detected in GC-Th cells but not in cultured dendritic cells reisolated after culture. Expression of CXCL13 message was determined by quantitative real-time PCR assay (D-E). The CXCL13 message number of each sample was divided by the β-actin message number for normalization. Relative expression levels (when the normalized CXCL13 copy level in B cells or dendritic cells equals one) are shown. (F) GC-Th cells but not Th1 or Th2 cells produce CXCL13. GC-Th cells, Th1, or Th2 cells were in vitro generated and cultured with GC-B cells in the presence of SEB. CXCL13 production was examined by ELISA (A-C, F). Data shown are representatives of at least 3 separate experiments. The error bars are standard deviations of triplicate experiments.