B cells from young and old mice switch isotypes with equal frequencies after ex vivo stimulation.

To determine whether old B cells have the same capacity to switch isotypes as young cells, we purified splenic follicular, marginal zone, and age-associated B cell subsets from C57BL/6 mice. Cells were stimulated in culture with interleukin 4 and either lipopolysaccharide or anti-CD40, and switching to IgG1 was measured by flow cytometry of surface immunoglobulin. The results show that switching was robust in follicular and marginal zone B cells from old mice and was comparable to their young counterparts. However, age-associated B cells from old mice switched poorly relative to the other subsets. Expression of activation-induced deaminase, which initiates switching, was quantified by qPCR of mRNA, and it was equal between young and old follicular B cells. Thus, in this ex vivo system, the follicular and marginal zone cells from young and old mice behaved similarly, showing that the molecular machinery to perform switching is intact in old B cells.

Age-Associated B Cells Express a Diverse Repertoire of VH and Vκ Genes with Somatic Hypermutation.

The origin and nature of age-associated B cells (ABCs) in mice are poorly understood. In this article, we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for 1 mo in the absence of external Ag. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors but not from MHC class II- or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what Igs ABCs express, we sequenced VH and Vκ rearranged genes from unimmunized 22-mo-old C57BL/6 mice and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting that ABCs have undergone mild stimulation from endogenous Ags over time. These observations show that quiescent ABCs are Ag-experienced cells that accumulate during T cell-dependent responses to diverse Ags during the life of an individual.

Exceptional Antibodies Produced by Successive Immunizations.

Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations.

Co-Stimulation of BCR and Toll-Like Receptor 7 Increases Somatic Hypermutation, Memory B Cell Formation, and Secondary Antibody Response to Protein Antigen.

The goal of immunization is to produce both a flood of antibodies to neutralize antigen and memory cells to accelerate the secondary response. To enhance the generation of memory B cells, we examined the effect of co-engaging BCR and toll-like receptor (TLR) 7 receptors by immunizing mice with a hapten-protein antigen, NP-CGG, and a ligand, R837 (imiquimod). During the early and late primary responses, there was no augmentation with R837 on the number of germinal center B cells or serum antibody. However, in the niche of germinal centers, R837 increased somatic hypermutation in the canonical VH1-72 gene that encodes NP-specific antibody. Increased mutation was not due to enhanced expression of activation-induced deaminase, but was likely a result of selection for high-affinity B cells with altered codons in the gene. This correlated with the appearance of antigen-specific B cells with a memory phenotype, which was intrinsic to TLR7 on B cells. To determine if these memory cells produced a recall response after a secondary challenge, spleen cells from mice that were immunized with NP-CGG and R837 were adoptively transferred into muMT recipients, and boosted with NP-CGG. Cells from mice that initially received both antigen and R837 generated a robust increase in germinal center B cells, plasmablasts, plasma cells, and serum antibody, compared with their cohorts who received antigen alone. These results support the use of co-immunization with TLR7 ligands to promote vigorous memory B cell responses to protein antigens.