A RT I C L E in the C1 locus, with this strain crossed with the Rosa26- EYFP reporter mouse (Casola et al., 2006). In this mouse, every post-GC B cell expresses YFP, with this strain afford- ing the opportunity to track post-GC B cell subsets by YFP expression (Casola et al., 2006). Immunization of C1-cre/ Rosa26-EYFP mice with NP-keyhole limpet hemocyanin (KLH)/alum lead to the generation of NP-BMEM (Fig. S2 B; Casola et al., 2006). After negative enrichment, NP-BMEM were defined as B220+ NIP+IgG1/YFP+dumpCD38+, with post-sort analysis indicating purity (Fig. S2 B). Polyclonal BMEM cells. Polyclonal BMEM can be clearly enumerated in C1-cre/Rosa26-EYFP mice through ex- pression of YFP, loss of IgD and IgM, and expression of other BMEM markers. NP-immunized C1-cre/Rosa26-EYFP mice were negatively enriched for BMEM with polyclonal BMEM defined as B220+IgG1/YFP+CD38+dumpCD80+ and sorted to purity (Fig. 1 C). and NF B responded poorly or did not respond to agonists for TLR2, TLR3, and TLR5 (unpublished data). For each TLR agonist that NF B cells or poly-BMEM responded to, BMEM generated a sizable population of B22 0intCD138+ cells, whereas NF B cells maintained a B220+CD138 phenotype (Fig. 1 H). NF B cells also proliferated to a greater degree than BMEM (Fig. 1 I). Finally, it was observed that while e-n gagement of CD40 drove proliferation of both BMEM and NF B cells, BMEM did not differentiate into B220intCD138+ cells (Fig. 1, H–I). Collectively, these data show that BMEM are sensitive to multiple TLR agonists in vitro and do not re- quire BCR cross-linking to become activated and differenti- ate into ASCs. Furthermore, upon activation, BMEM have an enhanced intrinsic capacity to differentiate into CD138+ ASCs compared with NF B cells. Lastly, CD40 signaling a-p pears to drive BMEM proliferation, but to block differentiation in vitro. BMEM cells undergo rapid clonal expansion NF B cells. In parallel, NF B cells were defined as and differentiation into IgG+ ASCs in response B220+CD23+IgD+CD38+ and were additionally sorted to soluble antigen (Fig. S2 C). Having established that BMEM are responsive to bystander The response of PE-BMEM, NP-BMEM, poly-BMEM, and stimuli in vitro, we used the PE-antigen system to compare NF B cells to TLR agonists after 3 d of in vitro culture was BMEM responsiveness toward cognate antigen versus bystander analyzed (Fig. 1). PE-BMEM and corresponding NF B cells inflammation in vivo. We first performed a detailed analysis and IL-4–only controls were stained with CFSE, whereas of the secondary humoral immune response to soluble PE NP-BMEM and poly-BMEM with NF B cells and IL-4 only (sPE) to define the behavior and kinetics of BMEM activation controls were stained with DDAO to track proliferation. A-f in vivo. Unlike naive hosts, it is well-established that prev-i ter culture with the TLR4 agonist LPS, cells were analyzed ously immunized mice are responsive to low-doses of antigen for surface expression of B220 and CD138. The culture of in the absence of adjuvant (McHeyzer-Williams et al., 2006). all NF and BMEM with IL-4 resulted in a uniform B220+ pop- This heightened responsiveness results from the combined ulation after 3 d (unpublished data). Upon culture of NF B presence of antigen-reactive BMEM and CD4+ memory T cells and BMEM with LPS, NF B cells remained primarily cells. In concordance with this, we observed that injection of B220+CD138, whereas all three sources of BMEM generated naive mice with 1 µg sPE (or 10 µg; not depicted) failed to a B220intCD138+ population of cells (21–30% of total) rep- elicit PE-ASCs (Fig. 2, A and B), whereas PE-immunized resenting the phenotype of a ASC (Fig. 1, A and D). The mice produced high numbers of IgG+ PE-ASCs. A robust ASCs derived from the three sources of BMEM were of the IgM ASC response was only elicited when naive or PE-im- IgG isotype and, with the ASCs generated from PE-BMEM munized mice were injected with 1 µg of sPE along with the and NP-BMEM, specific for either PE or NP, respectively TLR9 agonist CpG ODN 1826 (CpG; Fig. 2 A). Thus, the (Fig. 1 G and not depicted). NF B cells generated a high fre- IgG+ PE-specific humoral response generated after sPE injec- quency of IgM+ ASCs (Fig. 1 G). The overall degree of pro- tion is likely the direct consequence of BMEM activation. liferation by the three sources of BMEM was moderately less These data demonstrate that a low level of sPE given in the than NF B cells, although proliferation did occur, as ind-i absence of adjuvant is capable of activating PE-BMEM to dif- cated by comparison to the IL-4–only cultured cells (Fig. 1, ferentiate into IgG+ ASCs. B and E). The population of B220intCD138+ cells generated The in vivo proliferation of PE-BMEM was measured by from poly-BMEM arose in the daughter peaks, showing that flow cytofluorimetric analysis of BrdU incorporation. PE- BMEM undergo cell division while differentiating into immunized mice were injected with sPE, and a 5-d BrdU pulse B220intCD138+ cells (Fig. 1 F). was performed starting on the day of sPE injection, followed The response of poly-BMEM and NF B cells was analyzed by a chase period. BrdU incorporation by BMEM was quantified to either Pam3CSK4 (TLR1/2 agonist), heat-killedListeria at various times during and after the pulse period. After preen- monocytogenes (TLR2 agonist), poly(I:C) (TLR3 agonist), richment, PE-BMEM were detected by excluding CD4/CD8/ LPS (TLR4 agonist), flagellin fromSalmonella typhimurium IgD/IgM+ cells via a dump gate and gating them on B220+PE+ (TLR5 agonist), FSL-1 (TLR6 agonist), S-27609 (TLR7 ag- cells, with these cells analyzed for BrdU incorporationFi(g. S3 A). onist), ODN 1826 (TLR9 agonist), and IL-4. Both NF B PE-immunized mice receiving BrdU, but no antigen, and cells and poly-BMEM were responsive to agonists for TLR1/2, immunized mice receiving neither antigen nor BrdU were TLR4, TLR6, TLR7, and TLR9 (Fig. 1 I). Both poly-BMEM included as negative controls (Fig. 2 C). 2 d after sPE JEM VOL. 206, August 31, 2009 2015