PWM-Induced B Cell Activation Figure 1. TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation. A: B cell proliferation. Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 mg/ml PWM in the presence or absence of 5 mg/ml Staphylococcus aureus protein A (SpA) or 5 mg/ml anti-human Ig F(ab9)2 fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. B: TNF- induction. Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam3CSK4 (P3), phosphorothioate- modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided 6 SEM. C: Antagonization with Polymyxin B. CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. D: MyD88- dependency of B cell stimulation. B220+ B cells were isolated from the spleens of MyD882/2 mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with 3H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments 6 SEM. E: TLR-dependency. Wild type, TLR22/2 and TLR92/2 B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam3CSK4 (P3) and FSL-1R (FSL), PWM (10 mg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value 6 SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation. doi:10.1371/journal.pone.0029806.g001 Results Poke weed mitogen activity is modulated by B cell receptor ligation Despite the mechanism of action remains unclear, researchers and clinicians frequently use PWM as a B cell-specific stimulatory reagent with the aim of inducing polyclonal B cell proliferation and immunoglobulin (Ig) secretion. As shown in Figure 1A PWM triggers a long lasting B cell expansion visualized by CFSE dilution and concomitant terminal differentiation into antibody-secreting cells (ASC) (reflected by loss of CD20 expression). It should, however, be noted that high concentrations of PWM ranging from 1–10 mg/ml are required to elicit this response. Next, we assessed the co-stimulatory potential of the PWM preparation. To this end, we combined PWM with two distinct B cell receptor (BCR) stimuli, i.e. Staphylocoocus aureus protein A (SpA) and anti-human IgM+IgG+IgA F(ab9)2 fragments (anti-Ig). The results showed that the percentage of PWM-induced proliferating cells and the overall B cell survival were almost unaffected by SpA (Fig. 1A). In marked contrast, BCR crosslinking with anti-Ig resulted in an increased percentage of proliferating B cells and a concomitant decrease in B cell survival (Fig. 1A). Moreover, stimulation with PWM or PWM with SpA not only supported prolonged B cell division cycles but also promoted plasma blast differentiation coinciding with loss of CD20 surface expression in the cells with the highest CFSE dilution (Fig. 1A). In contrast, combination of PWM with anti-Ig did not support terminal differentiation and prevented repeated cell division ($2 genera- tions) despite the increased cellular turnover (Fig. 1A). BCR crosslinking with anti-Ig, thus, interferes with PWM-triggered effector functions. PWM induces TNF in monocytes and PWM-induced B cell expansion is blocked by TLR4 antagonist polymyxin B The finding that PWM displayed co-stimulatory activity when combined with a BCR stimulus prompted us to ask whether the PWM preparations contained TLR ligands, e.g. microbial substanc- es well-known to synergize with BCR signaling [7,17,18]. This assumption was further supported by the finding that PWM activated human monocytes (Fig. 1B). PWM-induced TNF expression was observed in a flow cytometric assay routinely used to detect minute amounts of microbial contaminants in cell culture reagents [16]. We concluded that low concentrations of microbial molecules such as LPS could account for this finding. Furthermore, PWM-induced B cell proliferation could be inhibited in the presence of polymyxin B, a cationic detergent frequently employed to antagonize LPS (Fig. 1C). PWM activates B cells in a MyD88-dependent manner In line with this observation PWM-triggered murine B cell proliferation was found to depend on MyD88, the key adaptor molecule in TLR signaling: in contrast to the wild type B cells MyD882/2 B cells failed to proliferate in response to PWM albeit proliferation could be induced by PMA and ionomycine (Fig. 1D). However, MyD88-deficiency is associated with attenuated B cell responses [19] and MyD882/2 B cells were, thus, also less responsive to stimulation with PMA and ionomycine than wild type B cells. We, therefore, limited our conclusions to the statement that a role for Toll-like receptor (TLR) ligands in PWM-driven B cell proliferation could not be excluded. Experiments using B cells deficient in single TLRs, i.e. purified from TLR2- or TLR9-deficient mice, did not display any relevant deficit in their response to PWM albeit selective unresponsiveness to TLR2- or TLR9 ligands, respectively, was confirmed (Fig. 1E). This finding was compatible with previously described endotoxin (LPS) contamination in PWM preparations [10] or a redundant contribution of multiple TLR ligands to B cell proliferation. Proliferation of human peripheral blood B cells is triggered via TLR2 or TLR9 but not by TLR4 ligand lipopolysaccharide To assess a possible contribution of TLR-stimulatory substances in PWM-induced human B cell activation, we stimulated human B cells with ligands for TLR2 (MALP-2), TLR4 (LPS) and TLR9 (CpG ODN) in the presence or absence of BCR crosslinking with SpA or anti-Ig. After 5 days B cell survival and proliferation were detectable after exposure of B cells to TLR2 and TLR9 ligands and were more pronounced when the TLR agonists were combined with SpA and/or anti-Ig (Fig. 2A). In contrast, survival and proliferation above the levels observed in unstimulated B cells were not observed when B cells were treated with LPS at varying concentrations (Fig. 2A). This indicated that LPS contamination cannot account for human B cell activity of PWM preparations, although it could contribute to murine B cell activation as suggested in [10]. PWM preparations contain TLR2 ligands This prompted us to ask whether PWM contained other TLR ligands. To test for TLR2 activity we transfected HEK293 cells with or without a human TLR2 expression plasmid (Fig. 2B), and challenged these cells with PWM or a synthetic triacylated lipopeptide, namely Pam3CSK4. TLR2-dependency of lipopeptide activity indicated specificity of the assay (Fig. 2C). Interestingly, PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e29806