The Common Key to Class-Switch Recombination and Somatic Hypermutation: Discovery of AID and Its Role in Antibody Gene Diversification

The mechanisms by which lymphocytes engineer their genomes, allowing the adaptive immune system to respond to an enormous spectrum of Ags, have been one of the most exciting topics in immunology. By the late 1990s, a detailed understanding of V(D)J recombination mechanisms, found to be controlled by the RAG enzymes (1–3), was emerging. Additional processes by which mature B lymphocytes diversified their Ig genes, namely class-switch recombination (CSR) and somatic hypermutation (SHM), also had been characterized at the genetic level. Both of these processes take place in the germinal center (GC), the anatomic niche where B cells undergo the final steps of their maturation to become activated plasma cells (4, 5). The process of CSR, by which B cells respond to various Ags through changing the effector function of the Abs they express (6), and the Igh constant gene locus where CSR occurs, was dissected (7–11). Furthermore, V(D)J-recombined DNA was found to undergo physiologic SHM, driving GC B cells through a unique evolutionary process until a high-affinity Ag receptor emerged (12, 13). However, identification of the cellular machinery responsible for a B cell’s unique ability to perform both CSR and SHM remained a central goal in immunology.

The establishment of the CH12 cell line, a murine lymphoma clone that would switch from IgM- to IgA-producing cells upon stimulation, yielded a model system to evaluate the determinants of CSR (14). In 1999, Muramatsu et al. (15) identified a novel gene expressed in stimulated CH12 cells and mouse splenic B cells: activation-induced cytidine deaminase (AID), an enzyme related to the RNA-editing enzyme APOBEC-1. Intriguingly, AID was found to be preferentially expressed in GC B cells and, in culture, to be expressed in splenic B cells that had been stimulated to undergo CSR.

Soon afterward, in an important paper, truly a pillar of immunology, Muramatsu et al. (16) investigated the role of AID as an initiator of SHM and CSR in vivo. What emerged was a clear demonstration of B cell reliance on AID, not for survival or activation, but for the genomic alterations responsible for Ab diversification in the GC. AID, when overexpressed through various means, increased CSR by 2–5-fold in stimulated CH12 cells. Although large GCs still formed in AID-deficient mice, analysis of their serum Ig levels revealed a complete lack of IgG3, IgG2, and IgA and almost null levels of IgG1 and IgG2a (with residual levels reflecting Igs from maternal serum). IgM levels, however, were elevated two to three times the levels observed in animals with intact AID. Even after stimulation with sheep RBCs, B cells in AID-deficient animals were found to express only IgD or IgM. In vitro, stimulated splenic B lymphocytes from AID-deficient mice expressed IgM only, compared with B cells from AID-intact mice that, upon stimulation, could also express IgG, IgE, and IgA. Additionally, expressed rearrangements of the I_μ exon with C_γ1, C_γ2a, C_γ2b, C_γ3, C_ε, and C_α were not detectable in AID^−/− splenic B cells. In other words, AID^−/− mice were unable to perform CSR. Importantly, sequencing of the VH186.2 transcripts from mesenteric lymph nodes of (4-hydroxy-3-nitrophely) acetyl–immunized mice revealed that B cells lacking AID had no detectable mutations in the complementarity-determining regions compared with the SHM evident in AID-intact B cells. Remarkably, therefore, in this single paper, the authors identified the key element that enabled B cells to undergo both mechanisms of diversification/maturation taking place within the GC: CSR and SHM.

Concurrently, Revy et al. (17) showed that the human immunodeficiency, hyper-IgM syndrome, is observed in patients that carry mutations in AICDA. These mutations were later proven to result in loss of AID function (18, 19). AID-mediated DNA mutation and DNA double-strand break activity was found on template DNA, as well as nontemplate ssDNA in a noncoding RNA expression–dependent fashion (20, 21). In addition, the base excision repair and mismatch repair pathways were shown to collaborate with AID, introducing breaks and mutations in DNA (22–26). Honjo and colleagues (27, 28) also demonstrated that nonlymphoid cells (i.e., fibroblasts) could undergo some steps of both CSR and SHM, the key genetic alterations thought to be unique to B lymphocytes, once expression of AID was introduced. AID, it seems, can confer this essence of B cell function to any cell type, as was shown in several additional studies (29).

The identification of AID as the cellular component required for physiologic mutagenesis in B cells provided an explanation for their unique ability to evolve and adapt in the face of a pathogen. This finding was leveraged quickly to show a role for AID in tumorigenesis, including its ability to mutate oncogenes like BCL6 (30, 31). Additional non-Ig proto-oncogene targets of AID, including PIM1, CMYC, RhoH/TTF, and PAX5 were later identified in human diffuse large B cell lymphoma (32–34). Furthermore, AID-transgenic mice were found to develop T cell lymphomas (35), which demonstrated evidence of non-Ig SHM (36). Furthermore, AID was found to be important for the IGH/CMYC rearrangements present in some B lineage neoplasms (murine plasmacytomas, human Burkitt lymphoma, and other lymphomas) (37). More recent studies have identified numerous genes harboring oncogenic mutations that can be traced backed to AID because of their unique mutational signatures (38–42). Overall, these findings have clarified the natural history of a substantial subset of lymphomas. Furthermore, a role for AID and AID-mediated mutagenesis has been proposed in several nonlymphoid neoplasms (43), involving mechanisms that are yet to be well understood. Finally, a distinct role for AID in antiviral host defense, through processes that overlap with, but differ from, those observed in Ab gene diversification, is beginning to emerge (44).

In summary, the importance of the findings detailed in this landmark paper to our understanding of the B cell arm of immunity cannot be overstated. The revelation that a single mutagenic enzyme endowed B cells with their unique ability to adapt and evolve through CSR and SHM was startling. The implications of this work have been far reaching and crucial for our understanding of adaptive immunity, lymphocyte genetics, and tumorigenesis.