PWM-Induced B Cell Activation Figure 4. Detection of TLR ligands in PWM preparations. A: Human B cells were stimulated with different lots of PWM. Proliferation rates were quantified by 3H-thymidine incorporation (cpm = counts per minute). The diagram shows the mean values 6 SEM from n = 3 experiments. B: LAL- assay was performed to quantify the LPS content in PWM Lot. A–D. The diagram depicts the mean values 6 standard deviation obtained by testing in quadruplicates. C: TLR2 activity was assessed by measuring IL-8 concentrations in the supernatants of pTLR2-transfected or non-transfected HEK293 cells stimulated with different lots of PWM (left) or Pam3CSK4 (right) at the concentrations indicated. One representative experiment performed in triplicates of n = 2 is shown. doi:10.1371/journal.pone.0029806.g004 proliferation (Fig. 4A), thus, corroborating the concept that PWM- induced proliferation is driven by TLR ligands. (GlcNAc)3-binding lectins costimulate B cell proliferation induced by immune stimulatory DNA Earlier studies have described the (GlcNAc)3 carbohydrate specificity of Phytolacca americana (PWM) lectin [8]. It was further shown that the (GlcNAc)3 binding can be antagonized by N,N-di- acetylchitobiose and N,N,N-tri-acetylchitotriose. We, therefore, used these substances to assess the contribution of the lectin component within the PWM preparation. As shown in Figure 5A the effect of N,N-di-acetylchitobiose was negligible, but N,N,N-tri- acetylchitotriose inhibited PWM-triggered B cell proliferation, thus confirming the contribution of (GlcNAc)3 binding to mitogenicity. Based on these findings we hypothesized that the lectin component in PWM might act as a sensitizer for B cells towards microbial ligands as previously described for BCR ligation [7,22]. To test this hypothesis human B cells were stimulated with highly purified PWM (Lot. D) or a BCR stimulus (SpA or anti-Ig) in the presence and absence of GpC PTO, as introduced in Fig. 3B. The results indicated that, similarly to SpA and anti-Ig, PWM increases the proliferative activity of immune stimulatory DNA (Fig. 5B). Lastly, a non-related lectin with (GlcNAc)3 specificity from Lycopersicon esculentum (tomato) [23] was used to corroborate our findings. Albeit by themselves highly purified preparations of PWM and tomato lectins displayed very little activity, compar- ative analysis in CFSE dilution assays demonstrated that both lectins augmented B cell proliferation induced by GpC PTO (Fig. 5C). Lectin-typical carbohydrate binding may, thus, co- stimulate B cell activation by nucleic acid-sensing pattern recognition receptors. Discussion The results obtained in this study indicate that the mitogenic activity of PWM on human B cells is based on the synergistic action of the poke weed lectin and the co-purified bacterial TLR ligands specific for TLR2 and TLR9. DNA contained in the PWM preparation was found to be of bacterial origin, since ribosomal bacterial DNA was amplified with specific PCR primers when PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29806