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.

ATAD5 deficiency decreases B cell division and Igh recombination.

Mammalian ATPase family AAA domain-containing protein 5 (ATAD5) and its yeast homolog enhanced level of genomic instability 1 are responsible for unloading proliferating cell nuclear antigen from newly synthesized DNA. Prior work in HeLa and yeast cells showed that a decrease in ATAD5 protein levels resulted in accumulation of chromatin-bound proliferating cell nuclear antigen, slowed cell division, and increased genomic instability. In this study, B cells from heterozygous (Atad5(+/m)) mice were used to examine the effects of decreased cell proliferation on Ab diversity. ATAD5 haploinsufficiency did not change the frequency or spectrum of somatic hypermutation in Ab genes, indicating that DNA repair and error-prone DNA polymerase η usage were unaffected. However, immunized Atad5(+/m) mice had decreased serum IgG1 Abs, demonstrating a functional effect on class switch recombination. The mechanism of this altered immune response was then examined following ex vivo stimulation of splenic B cells, where Atad5(+/m) cells accumulated in the S phase of the cell cycle and had reduced proliferation compared with wild-type cells. These haploinsufficient cells underwent a significant decline in activation-induced deaminase expression, resulting in decreased switch region DNA double-strand breaks and interchromosomal translocations in the Igh locus. Class switch recombination to several isotypes was also reduced in Atad5(+/m) cells, although the types of end-joining pathways were not affected. These results describe a defect in DNA replication that affects Igh recombination via reduced cell division.

JH6 downstream intronic sequence is dispensable for RNA polymerase II accumulation and somatic hypermutation of the variable gene in Ramos cells.

Activation-induced deaminase (AID) introduces nucleotide substitutions within the variable region of immunoglobulin genes to promote antibody diversity. This activity, which is limited to 1.5 kb downstream of the variable gene promoter, mutates both the coding exon and downstream intronic sequences. We recently reported that RNA polymerase II accumulates in these regions during transcription in mice. This build-up directly correlates with the area that is accessible to AID, and manipulation of RNA polymerase II levels alters the mutation frequency. To address whether the intronic DNA sequence by itself can regulate RNA polymerase II accumulation and promote mutagenesis, we deleted 613 bp of DNA downstream of the JH6 intron in the human Ramos B cell line. The loss of this sequence did not alter polymerase abundance or mutagenesis in the variable gene, suggesting that most of the intronic sequence is dispensable for somatic hypermutation.

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.