After 7 days of culture, little difference was observed in CFSE profiles and per cent divided cells in SC-58125-treated B-cell cultures (Fig. 2b). Similar results were observed for B cells treated with NS-398, a different Cox-2 selective inhibitor (data not shown). The percentages of divided B cells following treatment with SC-58125 were averaged from three different donors (Fig. 2c). No significant change in the per cent divided B cells following

Cox-2 inhibitor treatment was detected, indicating that a decrease in proliferation does not account for the attenuation of antibody production. We next investigated whether attenuated antibody production was caused by a reduction in the Galunisertib differentiation of human B cells to antibody-secreting cells. Human plasma cell precursors, defined by multiple investigators as CD38+ antibody-secreting cells,17–19 can be generated in vitro. On day 7 of culture, B cells were stained for surface expression of CD38 and CD19, as well as for intracellular IgM or IgG. Intracellular antibody gates were determined based upon unpermeabilized stained controls. Multiple blood donors were assessed via this method with similar results. Freshly isolated B cells express a relatively low frequency of CD38+ antibody-secreting cells, which is significantly Alectinib order increased following 7 days of stimulation with CpG plus anti-IgM (Fig. 3a). A significant reduction in the

frequency of CD38+ antibody-secreting cells was observed following treatment with SC-58125 (Fig. 3a,c). In contrast there was no change in the frequency of CD38− Ig+-secreting cells (Fig. 3b). Generation of IgM-secreting, CD38+ B cells was significantly attenuated in a dose-dependent manner (Fig. 3c). These results mirrored the decrease in antibody production measured by ELISA (Fig. 1). Similarly, CD38+ IgG-secreting cells were also significantly decreased following treatment with the Cox-2 inhibitor (Fig. 3c). These new data demonstrate that the Cox-2 selective inhibitor, SC-58125 attenuated the ability of B cells to differentiate to CD38+ antibody-secreting plasma cell precursors. Cox-2 knockout

mice were next used to study the vital role of Cox-2 in B-cell differentiation to plasma cells. CD19+ B cells were N-acetylglucosamine-1-phosphate transferase isolated from the spleens of wild-type and Cox-2-deficient mice. Analysis of wild-type and Cox-2-deficient splenocytes revealed no significant differences in overall CD19+ cells or marginal zone B cells (CD19+ CD21+ CD23−), indicating that B-cell populations are similar. Following a 72-hr stimulation with LPS, Cox-2-deficient mice had a 60% reduction in the number of CD138+ plasma cells compared with wild-type controls (Fig. 4a,b). This indicates impairment in the differentiation of B cells to plasma cells in mice lacking Cox-2. Next, we tested whether expression of the essential plasma cell transcriptional regulator, Blimp-1, was regulated by Cox-2.