To reconcile conflicting reports on the role of CD40 signaling in germinal center (GC) formation, we examined the earliest stages of murine GC B cell differentiation. staining. DOI: http://dx.doi.org/10.7554/eLife.19552.005 We examined the expression levels of RelB, IRF4 and BCL6 in GFP+ NP-specific B cells during the early stages of GC?B cell differentiation using the adoptive transfer model described above (Figure 1A). Two days p.i., GFP+ NP-specific B cells were found predominantly in the IF zone and at the T / Tubacin tyrosianse inhibitor B border and had been RelB+ and IRF4+, but indicated undetectable degrees of BCL6 (Shape 1B). BCL6 manifestation had not been seen in NP-specific B cells after Compact disc40 blockage, corroborating the specificity of BCL6 staining (Shape 1figure health supplement 3). As of this accurate time, all RelB+ responding B cells indicated raised degrees dJ857M17.1.2 of IRF4 almost, even though the reverse had not been true. In keeping with our prior research, manifestation of BCL6 had not been obvious among NP-specific B cells until d3 p.we., a point over time when they continued to be largely constrained towards the IF area (Kerfoot et al., 2011) (Shape 1B). Strikingly, we discovered that all BCL6 expressing B cells as of this correct period stage harbored nuclear RelB and IRF4, even though the BCL6 expression degrees of such cells was significantly less than observed in completely differentiated GC?B cells (Shape 1B and data not shown; discrimination of nuclear RelB through the cytoplasmic form can be demonstrated in Shape 1figure health supplement 2). Just a half day time later on (d3.5), GFP+ B cells expressing higher degrees of BCL6 with diminished levels of RelB and IRF4 began to emerge (Figure 1B,D). Image analysis comparing BCL6+ RelB+ cells to BCL6+ RelB- cells revealed that the newly formed BCL6hi RelB- cells were located much deeper within follicles, whereas BCL6int RelB+ cells resided mainly outside of follicles or close to follicular borders (Figure 1C,D). Thus, intermediate levels of BCL6 are first observed in RelB+ B cells, suggesting that ongoing CD40 signals are important to this differentiation step. The BCL6int RelB+ IRF4+ population is transient and has an incomplete GC phenotype Flow cytometry results support the conclusion that BCL6int RelB+ IRF4+ B cells temporally precede follicular BCL6hi GC?B cells (Figure 2A). Consistent with the histology data, the expression of RelB in BCL6int IRF4+ cells is significantly higher in BCL6hi IRF4lo GC?B cells (Figure 2B). The BCL6int population evidenced an early and transient pattern: it emerged by 3 days pi., before the appearance of intrafollicular GC?B cells, peaked at day Tubacin tyrosianse inhibitor 3.5 and rapidly declined by day 8 when GC?B cells were abundant (Figure 2C,D). The BCL6int RelB+ IRF4+ nascent GC?B cell precursors displayed a partial GC phenotype (Figure 2E). They expressed lower levels of PNA binding and Fas and less repression of the BCL6 target gene CD38 compared to their BCL6hi GC?B cell counterparts (Figure 2E). Interestingly, significantly higher levels of CD86 were observed among the BCL6int RelB+ IRF4+ GC precursors. It is important to note that these markers are not exclusive to GCs during the early stages of the response, and that other activated B cell subsets not expressing BCL6 can also show elevated levels of Fas and PNA binding (Figure 3). Together these results implicate BCL6int RelB+ IRF4+ B cells as a GC precursor population that immediately precedes BCL6hi RelBlo IRF4lo GC?B cells. From here on, we refer to BCL6int RelB+ IRF4+ B cells as Tubacin tyrosianse inhibitor the GC precursors (or pre-GC) and BCL6hi RelBlo IRF4lo cells as GC?B cells. Open in a separate window Figure 2. BCL6int RelB+ IRF4+ Ag-specific B cells emerge early during immune responses and.