Anti-Mouse IgG2b polyclonal antibody [DyLight® 488]

Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline I-H-C-H transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS-mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam(3)CSK(4)) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-gamma or TGF-beta, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline I-H-S-C-H transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing effective T-independent antibody responses to microbial pathogens, allergens and vaccines.

Quorum Sensing and type III secretion system play an important role in the virulence of Pseudomonas (P.) aeruginosa in burn wound infections. We aimed to explore the feasibility of using 3-oxo-C-12-HSL-r-PcrV conjugate as a candidate vaccine against P aeruginosa caused infections. 3-oxo-C-12-HSL-r-PcrV conjugate was prepared and used for immunization of mice (lo pig, subcutaneous, three times, at 2-week intervals). Mice were divided into five groups: I: PcrV; II: 3-oxo-C-12-HSL-r-PcrV 1.tg); III: 3-oxo-C-12-HSL-r-PcrV (20 1.1g); IV: 3-oxo-C-12-HSL; and V: PBS receiving groups. After each shot of immunization, total and isotype antibody responses against corresponding antigen were measured to determine the immunization efficacy. One month after the last immunization, all groups were burned and challenged subeschar with P aeruginosa PAO1. Survival rate and bacterial quantity in the skin and internal organs (liver and spleen) were evaluated 25-hr after burn infection. Immunization with 3-oxo-C-12-HSL-r-PcrV significantly increased total IgG and specific subclass antibodies (IgGi, IgG2a, IgG2b, and IgM) in the serum of the groups II and III compared to the control group (p