Multimericity Amplifies the Synergy of BCR and TLR4 for B Cell Activation and Antibody Class Switching

Abstract

Sustained signaling through the B cell antigen receptor (BCR) is thought to occur only when antigen(s) crosslink or disperse multiple BCR units, such as by multimeric antigens found on the surfaces of viruses or bacteria. B cell-intrinsic Toll-like receptor (TLR) signaling synergizes with the BCR to induce and shape antibody production, hallmarked by immunoglobulin (Ig) class switch recombination (CSR) of constant heavy chains from IgM/IgD to IgG, IgA or IgE isotypes, and somatic hypermutation (SHM) of variable heavy and light chains. Full B cell differentiation is essential for protective immunity, where class switched high affinity antibodies neutralize present pathogens, memory B cells are held in reserve for future encounters, and activated B cells also serve as semi-professional APCs for T cells. But the rules that fine-tune B cell differentiation remain partially understood, despite their being essential for naturally acquired immunity and for guiding vaccine development. To address this in part, we have developed a cell culture system using splenic B cells from naive mice stimulated with several biotinylated ligands and antibodies crosslinked by streptavidin reagents. In particular, biotinylated lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, and biotinylated anti-IgM were pre-assembled (multimerized) using streptavidin, or immobilized on nanoparticles coated with streptavidin, and used to active B cells in this precisely controlled, high throughput assay. Using B cell proliferation and Ig class switching as metrics for successful B cell activation, we show that the stimuli are both synergistic and dose-dependent. Crucially, the multimerized immunoconjugates are most active over a narrow concentration range. These data suggest that multimericity is an essential requirement for B cell BCR/TLRs ligands, and clarify basic rules for B cell activation. Such studies highlight the importance in determining the choice of single vs multimeric formats of antigen and PAMP agonists during vaccine design and development.

Lassa virus glycoprotein nanoparticles elicit neutralizing antibody responses and protection

“…Indeed, we observed an overall trend toward lower IgM levels in the rabbits that received GPC NPs, with IgM levels being significantly lower after the second immunization (median mean fluorescence intensity [MFI] of 67 versus 320, respectively; two-tailed Mann-Whitney U -test; p = 0.0022), consistent with observations that multivalent antigen presentation accelerates class-switching. 32 …”

Influenza viral matrix 1 protein aggravates viral pathogenicity by inducing TLR4-mediated reactive oxygen species production and apoptotic cell death

“…It has been reported that several viral proteins can activate TLR4 [ 13 ]. Recently, it was proposed that the multimeric or oligomeric structure of viral proteins is important for TLR4 activation [ 8 , 13 ].…”

“…It has been reported that several viral proteins can activate TLR4 [ 13 ]. Recently, it was proposed that the multimeric or oligomeric structure of viral proteins is important for TLR4 activation [ 8 , 13 ]. Although oligomeric M1 is abundant, monomeric M1 also exists at equilibrium in vitro and in vivo (Fig.…”

Trimeric, APC-Targeted Subunit Vaccines Protect Mice against Seasonal and Pandemic Influenza

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