Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma-globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.

The activation-induced cytidine deaminase (AID) gene, specifically expressed in germinal center B cells in mice, is a member of the cytidine deaminase family. We herein report mutations in the human counterpart of AID in patients with the autosomal recessive form of hyper-IgM syndrome (HIGM2). Three major abnormalities characterize AID deficiency: (1) the absence of immunoglobulin class switch recombination, (2) the lack of immunoglobulin somatic hypermutations, and (3) lymph node hyperplasia caused by the presence of giant germinal centers. The phenotype observed in HIGM2 patients (and in AID-/- mice) demonstrates the absolute requirement for AID in several crucial steps of B cell terminal differentiation necessary for efficient antibody responses.

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.

Activation-induced cytidine deaminase (AID) plays an essential role in class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes. We report here that deficiency in AID results in the development of hyperplasia of isolated lymphoid follicles (ILFs) associated with a 100-fold expansion of anaerobic flora in the small intestine. Reduction of bacterial flora by antibiotic treatment of AID-/- mice abolished ILF hyperplasia as well as the germinal center enlargement seen in secondary lymphoid tissues. Because an inability to switch to immunoglobulin A on its own does not lead to a similar phenotype, these results suggest that SHM of ILF B cells plays a critical role in regulating intestinal microflora.

The mechanism to maintain homeostasis of the gut microbiota remains largely unknown despite its critical role in the body defense. In the intestines of mice with deficiency of activationinduced cytidine deaminase (AID), the absence of hypermutated IgA is partially compensated for by the presence of large amounts of unmutated IgM and normal expression levels of defensins and angiogenins. We show here a predominant and persistent expansion of segmented filamentous bacteria throughout the small intestine of AID ؊/؊ mice. Reconstitution of lamina propria IgA production in AID ؊/؊ mice recovered the normal composition of gut flora and abolished the local and systemic activation of the immune system. The results indicate that secretions of IgAs rather than innate defense peptides are critical to regulation of commensal bacterial flora and that the segmented filamentous bacteria antigens are strong stimuli of the mucosal immune system. M ucosal epithelial surfaces represent points of continuous and intimate interactions between the immune system and the outside environment. Under a constant antigenic pressure from Ͼ400 species of commensal bacteria, the gut immune system has developed highly sophisticated and efficient defensive as well as symbiotic mechanisms (1, 2). Secretion of antibiotic peptides by epithelial cells represents an important component of the innate immune system in the gut. Bacteria or bacterial antigens are capable of stimulating secretion of large amounts of antimicrobial peptides by crypt Paneth cells (3, 4). Also, transgenic (Tg) mice expressing a human intestinal defensin are protected against enteric salmonellosis (5), whereas mice deficient in the metalloproteinase matrilysin MMP-7 and thus lacking mature cryptdins show decreased resistance to some intestinal infections (6). Therefore, antimicrobial peptides appear to be involved in the maintenance of the symbiotic environment in the gut and protection of crypt stem cells from infections.Another front line body defense mechanism that provides protection against microbial agents at mucosal surfaces is production and secretion of IgA (7). Indeed, IgA is the most abundant Ig isotype in mucosal secretions, and at least 80% of all plasma cells in mice are located in the intestinal lamina propria (LP) (8). The gut IgA responses are initiated primarily in organized lymphoid structures present in the intestine, namely Peyer's patches (PP) (9) and the isolated lymphoid follicles (ILFs), which have architecture similar to PP (10, 11). These structures contain a large number of conventional B2 cells, which are derived from precursor cells generated in the bone marrow (BM) (9, 10). In addition, peritoneal cavity B1 cells contribute to intestinal IgA plasma cells. B1 cells are shown to generate large amounts of IgAs independent of T cells and germinal centers (GC) (12, 13). Both B1 and conventional B2 cells are most likely to switch in situ from IgM to IgA in the LP with the help of dendritic cells and factors secreted by stromal cells (14). However, it is...

Class switch recombination (CSR) is a region-specific DNA recombination reaction that replaces one immunoglobulin heavy-chain constant region (CH) gene with another. This enables a single variable (V) region gene to be used in conjunction with different downstream CH genes, each having a unique biological activity. The molecular mechanisms that mediate CSR have not been defined, but activation-induced cytidine deaminase (AID), a putative RNA-editing enzyme, is required for this reaction 1 . Here we report that the Nijmegen breakage syndrome protein (Nbs1) and phosphorylated H2A histone family member X (γ-H2AX, also known as γ-H2afx), which facilitate DNA double-strand break (DSB) repair 2-4 , form nuclear foci at the CH region in the G1 phase of the cell cycle in cells undergoing CSR, and that switching is impaired in H2AX -/-mice. Localization of Nbs1 and γ-H2AX to the IgH locus during CSR is dependent on AID. In addition, AID is required for induction of switch region (Sμ)-specific DNA lesions that precede CSR. These results place AID function upstream of the DNA modifications that initiate CSR.Correspondence and requests for materials should be addressed to A.N. (andre_nussenzweig@nih.gov) or M.C.N. (nussen@mail.rockefeller.edu).. Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Competing interests statementThe authors declare that they have no competing financial interests. To determine whether DNA repair factors associate with DSBs at the switch regions, we first examined the intracellular localization of γ-H2AX, Nbs1, Rad51 and Brca1 in activated B cells by immunofluorescence. Brca1 and Rad51 are required for homologous recombination, the Mre11-Rad50-Nbs1 complex has been implicated in both homologous recombination and non-homologous end-joining (NHEJ), and γ-H2AX is critical for recruiting these repair factors to DSBs 6 and facilitates NHEJ in Saccharomyces cerevisiae 4 . All four proteins showed diffuse nuclear staining in most of the resting B cells from C57BL/6 wild-type mice. High local concentrations of these factors (nuclear foci) were detected in a very small percentage of cells (<5%), which increased significantly when the cells were stimulated to undergo CSR in vitro with lipopolysaccharide (LPS) and interleukin (IL)-4 (Fig. 1a). After 3 days of stimulation, 37% of the B cells contained discrete Brca1 foci (12 ± 6 per cell) and 43% contained Rad51 foci (7 ± 3 per cell), consistent with previous results 7 ; the remaining cells exhibited a weak, diffuse pattern of nuclear staining (Fig. 1a). Many of the stimulated B cells also formed Nbs1 foci (32% contained, on average, 3 ± 2 per cell) and γ-H2AX foci (40% contained, on average, 4.5 ± 3 per cell). To determine which of these repair factors are co-localized in activated B cells, we performed two colour immunofluoresence experiments (Fig. 1b). Only 20% of the cells that contained Rad51 and Nbs1 (n = 687) or Brca1 and Nbs1 foci (n = 431) exhibited co-localization. In contrast, γ-H2AX foci co-localiz...

Class switch recombination (CSR) and somatic hypermutation (SHM) have been considered to be mediated by different molecular mechanisms because both target DNAs and DNA modification products are quite distinct. However, involvement of activation-induced cytidine deaminase (AID) in both CSR and SHM has revealed that the two genetic alteration mechanisms are surprisingly similar. Accumulating data led us to propose the following scenario: AID is likely to be an RNA editing enzyme that modifies an unknown pre-mRNA to generate mRNA encoding a nicking endonuclease specific to the stem-loop structure. Transcription of the S and V regions, which contain palindromic sequences, leads to transient denaturation, forming the stem-loop structure that is cleaved by the AID-regulated endonuclease. Cleaved single-strand tails will be processed by error-prone DNA polymerase-mediated gap-filling or exonuclease-mediated resection. Mismatched bases will be corrected or fixed by mismatch repair enzymes. CSR ends are then ligated by the NHEJ system while SHM nicks are repaired by another ligation system.

Many microbial pathogens employ antigenic variation as a strategy to evade the immune system, posing a challenge in vaccine development. To understand the requirements for immunity against such pathogens, we studied Borrelia hermsii, a relapsing fever bacterium. We found that mice deficient in T, follicular B, marginal zone B, or B1a lymphocytes resolved B. hersmii bacteremia and became resistant to reinfection. The resolution of bacteremia coincided with an expansion and persistence of B1b lymphocytes, and purified B1b lymphocytes from convalescent wild-type or TCR-betaxdelta-/- mice conferred immunity to Rag1-/- mice. The B1b lymphocytes in the reconstituted Rag1-/- mice provided long-lasting immunity by rapidly generating B. hermsii-specific IgM but not IgG upon bacterial challenge. Unmutated IgM is sufficient to eliminate B. hermsii, because AID-/- mice deficient in somatic hypermutation and class switch recombination efficiently resolved all bacteremic episodes. These data demonstrate that B1b lymphocytes can provide long-lasting T cell-independent IgM memory.