B cells were purified as a CD43 negative fraction using anti-CD43 beads (Miltenyi) and cultured at 5 106 cells per mL for 48 h or 7 d as indicated

B cells were purified as a CD43 negative fraction using anti-CD43 beads (Miltenyi) and cultured at 5 106 cells per mL for 48 h or 7 d as indicated. BCR and TLR7. Splenocytes from IFNR1?/? mice were incubated with TLR7 agonist and/or anti-BCR, and B-cell expression of T-bet was analyzed. The absence of IFNR did not affect the levels of T-bet induced in response to TLR7 agonist alone but considerably reduced the amount of T-bet induced in B cells in CPI-268456 response to the combination of TLR7 agonist and anti-BCR (compare Fig. 1 and and and and and and = 3 mice per group. * 0.05, ** 0.001, *** 0.0001 (test). Data are representative of three independent experiments. To find out what kind of B cells is induced by the procedures described here, we assessed the phenotype of the MD4 B cells in vivo after immunization with HEL and a TLR7 Rabbit Polyclonal to ETV6 agonist. Twenty-four hours after immunization, there was a significant increase of MD4, but not WT B cells expressing CD11c and CD11b (Fig. 2and Fig. S6). This phenomenon occurred only in animals immunized with a mixture of HEL and TLR7 ligand (Fig. 2and Fig. S6). The joint expression by these cells of T-bet, CD11b, and CD11c is CPI-268456 reminiscent of another population of B cells, so-called ABCs, which CPI-268456 we and others have recently reported (19, 21), a result we consider in and and Fig. S6) even though T-bet levels were increased in all of the MD4 B cells (Fig. 2and and and = 5 mice per group. (= 5 mice per group. Data are representative of two independent experiments. Overall, these data demonstrate that elevated levels of T-bet expression in B cells are sufficient to drive CD11b and CD11c expression on B cells. This fact leads to the hypothesis that synergistic stimulation via TLR7, BCR, and IFN leads to the induction of T-bet in B cells, which, as a transcription factor, directly or indirectly induces CD11b and CD11c. T-bet/CD11b/CD11c Positive B Cells Appear at the Peak of Antiviral Response and Produce Viral Specific IgG2a Antibodies. So far, we have shown that TLR7, BCR, and IFN synergistically drive T-bet and consequent CD11b and CD11c expression in B cells. The next question we asked was why this pathway of B-cell activation has evolved. Does it play any role in protective immune responses to pathogens? Because it has been previously reported CPI-268456 that T-bet drives IgG2a isotype switching (5, 25), we decided to test whether synergistic activation of B cells via TLR7, BCR, and IFN also leads to IgG2a production. To address this question, purified B cells were incubated for 7 d in the presence of different stimuli, and the levels of different IgG isotypes in supernatants were then evaluated. Synergistic activation of B cells with TLR7, BCR, and IFN drove the highest levels of IgG2a and IgG2b production, compared with other stimulation conditions (Fig. 4). Thus, one outcome of synergistic induction of T-bet in B cells is an isotype switch to IgG2a production. Open in a separate window Fig. 4. T-bet induction in B cells by TLR7, BCR, and IFNR leads to IgG2a production. Purified B cells from young C57BL/6 mice were cultured under the indicated conditions, and supernatants were analyzed for the presence of different IgG isotypes by ELISA at day 7. Bars represent the means SEM of = 3 samples per group. Data are representative of three independent experiments. IgG2a is well known to be the most potent isotype for antibody-dependent cellular cytotoxicity (ADCC) and is the major isotype produced during antiviral responses (1, 2, 25, 26). To address whether T-bet expression in B cells plays a role in antiviral responses, we examined the phenotype of spleen B cells at the peak of the antibody response (10 or 15 dpi) (26) to murine gammaherpesvirus 68 (gHV68), lymphocytic choriomeningitis virus (LCMV), and vaccinia infection. As shown in Fig. 5 and = 5 mice per group. (= 3 samples per group). (and = 5 mice per group. (= 0.02 (test). All data are representative of three independent experiments. To assess the function of this B subset in responses to viruses, we isolated CD11c+ B cells and FO B cells from gHV68-infected mice at.