Development of functional antibodies against influenza B virus by activation-induced cytidine deaminase in hybridoma cells

Highlights
1. Class-switch recombination was mimicked in hybridomas through a controllable expression system of activation-induced cytidine deaminase.
2. IgG antibodies were generated through this system in an anti-Flu B IgM hybridoma 7G1.
3. IgG1 and IgG2a subtypes of 7G1 present improved antiviral activity in vitro and in vivo.     

The double-edged sword of activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is required for Ig class switch recombination, a process that introduces DNA double-strand breaks in B cells. We show in this study that AID associates with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) promoting cell survival, presumably by resolving DNA double-strand breaks. Wild-type cells expressing AID mutants that fail to associate with DNA-PKcs or cells deficient in DNA-PKcs or 53BP1 expressing wild-type AID accumulate gammaH2AX foci, indicative of heightened DNA damage response. Thus, AID has two independent functions. AID catalyzes cytidine deamination that originates DNA double-strand breaks needed for recombination, and it promotes DNA damage response and cell survival. Our results thus resolve the paradox of how B cells undergoing DNA cytidine deamination and recombination exhibit heightened survival and suggest a mechanism for hyperIgM type II syndrome associated with AID mutants deficient in DNA-PKcs binding.     

T-independent activation-induced cytidine deaminase expression, class-switch recombination, and antibody production by immature/transitional 1 B cells

Inflammation elicits a splenic lymphopoiesis of unknown physiologic significance but one that juxtaposes developing B cells and exogenous Ag. We show that immature and transitional 1 (immature/T1) B cells constitutively express activation-induced cytidine deaminase and B lymphocyte-induced maturation protein 1 in amounts that support accelerated plasmacytic differentiation and limited class-switch recombination. In vivo, activation of immature/T1 B cells by TLR ligands or bacterial vaccine rapidly induces T1 cells to divide, proliferate, and secrete IgM, IgG, or IgA Ab; in vitro, proliferation and differentiation are substantially enhanced by B cell-activating factor. We propose that inflammation-induced extramedullary lymphopoiesis represents a specialized mechanism for innate Ab responses to microbial pathogens.     

Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.

We have identified a novel gene referred to as activation-induced deaminase (AID) by subtraction of cDNAs derived from switch-induced and uninduced murine B lymphoma CH12F3-2 cells, more than 80% of which switch exclusively to IgA upon stimulation. The amino acid sequence encoded by AID cDNA is homologous to that of apolipoprotein B (apoB) mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC-1), a type of cytidine deaminase that constitutes a catalytic subunit for the apoB mRNA-editing complex. In vitro experiments using a glutathione S-transferase AID fusion protein revealed significant cytidine deaminase activity that is blocked by tetrahydrouridine and by zinc chelation. However, AID alone did neither demonstrate activity in C to U editing of apoB mRNA nor bind to AU-rich RNA targets. AID mRNA expression is induced in splenic B cells that were activated in vitro or by immunizations with sheep red blood cells. In situ hybridization of immunized spleen sections revealed the restricted expression of AID mRNA in developing germinal centers in which modulation of immunoglobulin gene information through somatic hypermutation and class switch recombination takes place. Taken together, these findings suggest that AID is a new member of the RNA-editing deaminase family and may play a role in genetic events in the germinal center B cell.     

Amino-terminal phosphorylation of activation-induced cytidine deaminase suppresses c-myc/IgH translocation

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates class switch recombination and somatic hypermutation of immunoglobulin genes (Ig) in B lymphocytes. However, AID also produces off-target DNA damage, including mutations in oncogenes and double-stranded breaks that can serve as substrates for oncogenic chromosomal translocations. AID is strictly regulated by a number of mechanisms, including phosphorylation at serine 38 and threonine 140, which increase activity. Here we show that phosphorylation can also suppress AID activity in vivo. Serine 3 is a novel phospho-acceptor which, when mutated to alanine, leads to increased class switching and c-myc/IgH translocations without affecting AID levels or catalytic activity. Conversely, increasing AID phosphorylation specifically on serine 3 by interfering with serine/threonine protein phosphatase 2A (PP2A) leads to decreased class switching. We conclude that AID activity and its oncogenic potential can be downregulated by phosphorylation of serine 3 and that this process is controlled by PP2A.     

Identification of a specific domain required for dimerization of activation-induced cytidine deaminase

Activation-induced cytidine deaminase (AID) is essential to all three genetic alterations required for generation of antigen-specific immunoglobulin: class switch recombination, somatic hypermutation, and gene conversion. Here we demonstrate that AID molecules form a homodimer autonomously in the absence of RNA, DNA, other cofactors, or post-translational modifications. Studies on serial deletion mutants revealed the minimum region between Thr27 and His56 responsible for dimerization. Analyses of point mutations within this region revealed that the residues between Gly47 and Gly54 are most important for the dimer formation. Functional analyses of these mutations indicate that all mutations impairing the dimer formation are inefficient for class switching, suggesting that dimer formation is required for class switching activity. Dimer formation and its requirement for the function of AID are features that AID shares with APOBEC-1, an RNA editing enzyme of apolipoprotein B100 mRNA.     

Characterization of Ig gene somatic hypermutation in the absence of activation-induced cytidine deaminase

Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W = A/T, R = A/G, Y = C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase eta (POL eta). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL eta-mediated mutations, 1470 H chain and 1313 kappa- and lambda-chain rearrangements from three AICDA(-/-) patients were analyzed. The Ig mutation frequency of all V(H) genes from AICDA(-/-) patients was 40-fold less than that of normal donors, whereas the mutation frequency of mutated V(H) sequences from AICDA(-/-) patients was 6.8-fold less than that of normal donors. AICDA(-/-) B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity-determining regions. A significantly reduced mutation frequency in WA motifs compared with normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase activity, suggest a role for AICDA in regulating POL eta and uracil DNA-glycosylase activity. Similar results were observed in V(L) rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes.     

Helicobacter pylori infection triggers aberrant expression of activation-induced cytidine deaminase in gastric epithelium

Infection with Helicobacter pylori (H. pylori) is a risk factor for the development of gastric cancer. Here we show that infection of gastric epithelial cells with 'cag' pathogenicity island (cagPAI)-positive H. pylori induced aberrant expression of activation-induced cytidine deaminase (AID), a member of the cytidine-deaminase family that acts as a DNA- and RNA-editing enzyme, via the IkappaB kinase-dependent nuclear factor-kappaB activation pathway. H. pylori-mediated upregulation of AID resulted in the accumulation of nucleotide alterations in the TP53 tumor suppressor gene in gastric cells in vitro. Our findings provide evidence that aberrant AID expression caused by H. pylori infection might be a mechanism of mutation accumulation in the gastric mucosa during H. pylori-associated gastric carcinogenesis.     

Phosphorylation directly regulates the intrinsic DNA cytidine deaminase activity of activation-induced deaminase and APOBEC3G protein

The beneficial effects of DNA cytidine deamination by activation-induced deaminase (AID; antibody gene diversification) and APOBEC3G (retrovirus restriction) are tempered by probable contributions to carcinogenesis. Multiple regulatory mechanisms serve to minimize this detrimental outcome. Here, we show that phosphorylation of a conserved threonine attenuates the intrinsic activity of activation-induced deaminase (Thr-27) and APOBEC3G (Thr-218). Phospho-null alanine mutants maintain intrinsic DNA deaminase activity, whereas phospho-mimetic glutamate mutants are inactive. The phospho-mimetic variants fail to mediate isotype switching in activated mouse splenic B lymphocytes or suppress HIV-1 replication in human T cells. Our data combine to suggest a model in which this critical threonine acts as a phospho-switch that fine-tunes the adaptive and innate immune responses and helps protect mammalian genomic DNA from procarcinogenic lesions.