The germinal center (GC) is the dymanic microenvironment where Ag-activated B cells rapidly expand and differentiate, generating plasma cells (PC) that produce high affinity antibodies. (3) CD9 expression was induced in CD9? GC-B cells under PC generating condition and gradually increased in the course of PC differentiation. Taken together, our data suggest that CD9 is a novel marker for a human GC-B cell subset that is committed to PC lineage. and CD9+ and CD9? GC-B cell populations were further separated using a MACS column (Figure 2A). Quantitative real-time PCR data showed CD9+ GC-B cells expressed higher levels of Blimp-1, a master transcription factor for PC differentiation [18], compared to CD9? GC-B MK-4305 cells (Figure 2B). Figure 2 CD9+ GC-B cells are more advanced cells than CD9- GC-B cells in the course of GC-B cell differentiation to PC At the same time, the expression of Pax-5 and Bcl-6, which are known to be switched off before PC differentiation was significantly lower in CD9+ GC-B cells (Figure 2B) [19, 20]. This data suggests that CD9+ population is a more differentiated population towards PC, compared to CD9? population, and corroborates a previous report that a MK-4305 subset of human GC-B cells express Blimp-1 [21]. To further confirm the differential expression in the transcription factors between CD9+ and CD9? populations functionally, we determined whether CD9+ GC-B cells generate PC faster than CD9? GC-B cells. CD9+ and CD9? GC-B cells were cultured with IL-2 and IL-10 in the presence of CD40L and an FDC line, HK cells [12] for 4 days to induce plasma cells [22] and at the end of the culture, cell surface phenotype and antibody production were examined. CD9+ GC-B cells generated a high percentage of CD20-CD38+ and CD27+CD38+ plasmablasts compared to CD9? GC-B cells (39.0% and 19.4% vs 22.8% and 10.4%, Figure 2B). Consistent with the phenotypic data, the numbers of CD20-CD38+ and CD27+CD38+ plasmablasts were significantly higher in the cultures of CD9+ GC-B cells compared to CD9? GC-B cells (Figure 2C). The amounts of the secreted IgG in the culture supernatants correlated with absolute numbers of plasmablasts generated (Figure 2D). This result is in agreement with a report using mouse B cells [8]. Although different target cells were used in the experiments, Won et al clearly demonstrated that CD9+ B1a cells could differentiate into CD138+ PC faster than CD9? B1a cells [8]. All together, the data suggest that CD9+ GC-B cells are more advanced cells than CD9? GC-B MK-4305 cells in the course of GC-B cell differentiation to PC. CD9 is induced during GC-B cell differentiation to PC Since CD9+ GC-B cells appear to be more differentiated towards PC, we examined whether CD9 is induced in the course of GC-B cell differentiation into PC. CD9? and CD9+ GC-B cells were cultured in the plasma cell generating culture condition for 4 days as described above or in the memory B cell generating culture condition by adding IL-2 plus IL-4 [22] in place of IL-2 plus IL-10, as a negative control MK-4305 and CD9 expression was quantified by FACS analysis. As shown in Figure 3A, both CD9? and CD9+ GC-B cells exhibited higher expression of CD9 when cultured with IL-2/IL-10 compared to IL-2/IL-4 (MFI 66.5 vs 25.4 for CD9?, MFI 183.3 vs 69.6 for CD9+). Furthermore, CD9 expression in CD20-CD38+ plasmablasts was higher than their precursors among the cells generated with IL-2/IL-10, suggesting that CD9 expression is upregulated during differentiation to PC (Figure 3B). MK-4305 Overall, CD9 expression is gradually increased in the course of GC-B cell differentiation to PC, confirming CD9 expression data obtained with ex vivo memory B cells and PC (Figure 1B). Figure 3 CD9 is induced during GC-B cell differentiation to PC Localization of CD9+ GC-B cells in vivo To localize CD9+ GC-B cells and experimental data presented above, supporting our conclusion that CD9 is a marker for PC precursors. Figure 4 Immunofluorescent staining for CD9 in the germinal centers of hCIT529I10 human tonsillar tissue sections ? Highlights Human tonsillar B cell subsets express CD9 differentially. Germinal center (GC) B cells contain CD9+ and CD9? populations. CD9+ GC-B cells are in more advanced stages of PC differentiation. CD9 expression is induced in the course of GC-B cell differentiation to PC. Acknowledgments This work was supported by NIH grant R01CA121039 to YSC. Footnotes Publisher’s Disclaimer: This is a.