Foxp3(+) T cells play a critical role for the maintenance of immune tolerance. Here we show that in mice, Foxp3(+) T cells contributed to diversification of gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous bacteria by Foxp3(+) T cells involved regulatory functions both outside and inside germinal centers (GCs), consisting of suppression of inflammation and regulation of immunoglobulin A (IgA) selection in Peyer's patches, respectively. Diversified and selected IgAs contributed to maintenance of diversified and balanced microbiota, which in turn facilitated the expansion of Foxp3(+) T cells, induction of GCs, and IgA responses in the gut through a symbiotic regulatory loop. Thus, the adaptive immune system, through cellular and molecular components that are required for immune tolerance and through the diversification as well as selection of antibody repertoire, mediates host-microbial symbiosis by controlling the richness and balance of bacterial communities required for homeostasis.

Immunoglobulin A (IgA) is essential to maintain the symbiotic balance between gut bacterial communities and the host immune system. Here we provide evidence that the inhibitory co-receptor programmed cell death-1 (PD-1) regulates the gut microbiota through appropriate selection of IgA plasma cell repertoires. PD-1 deficiency generates an excess number of T follicular helper (T(FH)) cells with altered phenotypes, which results in dysregulated selection of IgA precursor cells in the germinal center of Peyer's patches. Consequently, the IgAs produced in PD-1-deficient mice have reduced bacteria-binding capacity, which causes alterations of microbial communities in the gut. Thus, PD-1 plays a critical role in regulation of antibody diversification required for the maintenance of intact mucosal barrier.

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5 to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the S region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.camptothecin ͉ non-B DNA ͉ translation suppression

Activation-induced cytidine deaminase (AID) is an essential factor for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. CSR and SHM are initiated by AID-induced DNA breaks in the S and V regions, respectively. Because truncation or frame-shift mutations at the carboxyl (C)-terminus of AID abolishes CSR but not SHM, the C-terminal region of AID likely is required for the targeting of DNA breaks in the S region. To test this hypothesis, we determined the precise location and relative amounts of AID-induced DNA cleavage using an in situ DNA end-labeling method. We established CH12F3-2 cell transfectants expressing the estrogen receptor (ER) fused with wild-type (WT) AID or a deletion mutant lacking the C-terminal 16 aa, JP8Bdel. We found that AID-ER, but not JP8Bdel-ER, caused a CSR to IgA from the addition of 4-hydroxy tamoxifen. In contrast, both WT AID and JP8Bdel induced DNA breaks in both the V and S regions. In addition, JP8Bdel enhanced c-myc/IgH translocations. Our findings indicate that the C-terminal domain of AID is not required for S-region DNA breaks but is required for S-region recombination after DNA cleavage. Therefore, AID does not distinguish between the V and S regions for cleavage, but carries another function specific to CSR.C-terminal deletion mutant ͉ chromosomal translocation ͉ synapse formation ͉ target specificity A ctivation-induced cytidine deaminase (AID) is essential for the generation of antibody memory, which depends on 2 genetic alterations of Ig genes: somatic hypermutation (SHM) and class switch recombination (CSR) (1, 2). AID induces DNA cleavage in the V and S regions to trigger SHM and CSR, respectively (3, 4). The molecular mechanism of DNA cleavage by AID has been debated extensively. The RNA-editing hypothesis assumes that AID edits unknown mRNAs to generate a novel mRNA encoding endonucleases or its cofactor that cleave target DNA (5). In contrast, the DNA deamination hypothesis assumes that AID directly targets DNA and deaminates C to U, generating U-G mismatches that are subsequently recognized by the base excision repair pathway (6). By this hypothesis, the repair mechanism would create phosphodiester bond cleavages as intermediates that are proposed to trigger SHM or CSR. Lines of evidence support each hypothesis, but neither has been proven.The other question concerning AID function is how, with only 198 residues, it can distinguish between the V and S regions to initiate SHM or CSR appropriately. The RNA-editing hypothesis assumes that AID edits 2 different mRNAs, each specific for 1 of the 2 regions. The evidence supporting this hypothesis comes from studies on AID mutants in which alterations in the carboxyl (C)-terminal region cause a specific loss of CSR (7,8). In addition, point mutations in the amino (N)-terminal region of AID drastically reduce SHM, and also compromise CSR to some extent (9). The DNA deamination hypothesis does not provide a formal explanation for target specificity but assumes that some cofactors are required t...