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B cells contribute to autoimmunity both as secretors of pathogenic antibodies and through the activation of autoreactive T cells. B cells and antibodies acquire higher affinity to self-antigen through a process known as immunoglobulin hypermutation or SHM. The contribution of SHM to pathogenic antibody development in lupus has been established in various autoimmune mouse models and by examining antibodies from patients. However, its role in the antibody-independent contribution of B cells to autoimmunity has not been examined. Herein, we generate lupus-prone MRL/lpr mice with a limited IgM-only B cell repertoire, no secreted antibodies and no SHM. This enabled us to isolate the role of somatic hypermutation in B cell-mediated autoimmunity. We found that SHM-deficiency correlated with a reduction in autoreactive B cells, a decrease in T cell activation and a decrease in kidney lymphocytic infiltration. These data establish AID as an important contributor to the antibody-independent role of B cells in autoimmunity. [ABSTRACT FROM AUTHOR]

We previously reported that some of the rare broadly reactive, HIV-1 neutralizing antibodies are polyreactive, leading to the hypothesis that induction of these types of neutralizing antibody may be limited by immunologic tolerance. However, the notion that such antibodies are sufficiently autoreactive to trigger B cell tolerance is controversial. To test directly whether rare neutralizing HIV-1 antibodies can activate immunologic tolerance mechanisms, we generated a knock-in mouse in which the Ig heavy chain (HC) variable region rearrangement (VHDJH) from the polyreactive and broadly neutralizing human monoclonal antibody 2F5 was targeted into the mouse Igh locus. In vitro, this insertion resulted in chimeric human/mouse 2F5 antibodies that were functionally similar to the human 2F5 antibody, including comparable reactivity to human and murine self-antigens. In vivo, the 2F5 VHDJH insertion supported development of large- and small pre-B cells that expressed the chimeric human/mouse lgμ chain but not the production of immature B cells expressing membrane IgM. The developmental arrest exhibited in 2F5 VHDJH knock-in mice is characteristic of other knock-in strains that express the Ig HC variable region of autoreactive antibodies and is consistent with the loss of immature B cells bearing 2F5 chimeric antibodies to central tolerance mechanisms. Moreover, homozygous 2F5 VHDJH knock-in mice support reduced numbers of residual splenic B cells with low surface 1gM density, severely diminished serum 1gM levels, but normal to elevated quantities of serum lgGs that did not react with autoantigens. These features are consistent with elimination of 2F5 HC autoreactivity by additional negative selection mechanism(s) in the periphery. [ABSTRACT FROM AUTHOR]

We previously reported that activation-induced deaminase (AID) heterozygous MRL/lpr mice have substantially lower levels of serum anti-dsDNA autoantibodies than AID wild-type littermates. Given the known functions of AID, here we examined whether this decrease in pathogenic autoantibodies in the heterozygotes was the result of a defect in class switch recombination, somatic hypermutation, or both. We report significant impairment of switch recombination to most isotypes except immunoglobulin G3 (IgG3) in vitro. However, serum levels of IgG were similar to AID wild-type levels even in very young mice. Mutation accumulation in the B cells from Peyer’s patches also revealed reduced somatic hypermutation in the heterozygotes. Unlike the switch defect, the hypermutation defect probably resulted in an in vivo effect because the serum IgG antibodies from the heterozygotes were of strikingly lower affinity to dsDNA than serum IgG antibodies from wild-type littermates. This suggests that the somatic hypermutation defect resulted in impaired affinity maturation of autoantibodies in these mice and explains the low levels of specific anti-dsDNA antibodies in the heterozygotes. This correlated with a delay in the development of kidney damage. These results imply that AID levels impact the class switch recombination and somatic hypermutation mechanisms and directly implicate affinity maturation of autoantibodies in autoimmunity. [ABSTRACT FROM AUTHOR]

Immunoglobulin genes undergo somatic hypermutation in activated B lymphocytes. The mutations are targeted to the variable region of the gene, apparently by co-transcriptional deposition of a mutation complex. The novel activation induced deaminase (AID) gene is required for this process and recent work indicates that it is the cytidine deaminase activity of the AID protein rather than an associated RNA editing function that is required [Curr. Biol. 12 (2002) 1748; Nature 419 (2002) 1; Nature 418 (2002) 99]. The authors review the implications of this work and argue that deamination of deoxy-cytidine to deoxy-uracil seems to initiate several pathways in which error-prone polymerases are postulated to create mutations. [Copyright &y& Elsevier]

The article presents study on lymphocytes, B cells, that lack a DNA-repair enzyme. It notes that the study challenges the dogma concerning the spatial separation of processes rearranging antibody genes and offer clues on the origin of B-cell cancers. It points out that vertebrate T and B lymphocytes have extraordinary diverse repertoire of surface receptors recognizing foreign antigens. Study shows that mature B-cells experienced frequent chromosome translocations at immunoglobulin genes due to the absence of DNA-repair enzyme. It was found out that chromosome translocations is attributed to incorrect repair of breaks through receptor editing and class switching.

Activation-induced cytosine deaminase (AID) is a cytosine deaminase that is critical to immunoglobulin hypermutation, class switch recombination, and gene conversion. In the context of hypermutating B cells, AID deaminates cytosine in the DNA of immunoglobulin genes, leading to the accumulation of mutations in the variable regions. However, when AID is expressed ectopically, it is a generalized mutator of G:C base pairs. Therefore, we asked whether AID may be partially regulated by an active system of nuclear export. We found that removal of a highly conserved nuclear export signal in the C terminus of AID causes accumulation of AID in the nucleus. However, a putative nuclear localization signal in the N terminus does not appear to be functional. Finally, we found that agents that induce DNA breaks caused retention of AID in the nucleus, suggesting that DNA breaks or the repair patches initiated as a result are a substrate for AID binding. [ABSTRACT FROM AUTHOR]

We recently identified a novel germinal center GTPase, SLIPGC, that localizes to replication factories in B cells and that, when reduced, induces DNA breaks in lymphoma B cell lines in an activation-induced deaminase (AID)-dependent manner. Herein, we generated mice deficient in SLIP-GC and examined the impact of SLIP-GC deficiency in immunoglobulin hypermutation and class switch recombination, both AID-dependent mechanisms. SLIP-GC-deficient mice experienced a substantial increase in mutations at G:C base pairs at the region downstream of JH4 in the immunoglobulin heavy chain locus. This change was reflected in the overall mutation frequency, and it was associated with an increase in transitions from G:C base pairs, a hallmark of AID-mediated deamination during replication. In addition, G:C transitions at non-immunoglobulin loci also increased in these mice. Given the intracellular localization of SLIP-GC to sites of replicating DNA, these results suggest that SLIP-GC protects replicating DNA from AID-mediated deamination of cytosines in both strands. [ABSTRACT FROM AUTHOR]

The HIV-1 broadly neutralizing Ab (bnAb) 2F5 has been shown to be poly-/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 VH knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. In this study, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 VH × VL KI mice, and find an even higher degree of tolerance control than observed in the 2F5 VH KI strain. Although B cell development was severely impaired in 2F5 VH × VL KI animals, we demonstrate rescue of their B cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, ? editing and anergy are additional safeguards preventing 2F5 VH/VL expression by immature/transitional B cells. Importantly, 7% of rescued B cells retained 2F5 VH/VL expression and secreted Env-specific mAbs with HIV-1-neutralizing activity. This partial rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B cells in 2F5 VH × VL KI × Eμ-Bcl-2 transgenic mice and significant (yet modest) enrichment of Env-specific B cells and serum Igs. The rescued 2F5 mAb-producing B cell clones in this study are the first examples, to our knowledge, of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs and provide a starting point for design of strategies aimed at rescuing such B cells. [ABSTRACT FROM AUTHOR]

We identified a novel GTPase, SLIP-GC, with expression limited to a few tissues, in particular germinal center B cells. It lacks homology to any known proteins, indicating that it may belong to a novel family of GTPases. SLIP-GC is expressed in germinal center B cells and in lymphomas derived from germinal center B cells such as large diffuse B cell lymphomas. In cell lines, SLIP-GC is expressed in lymphomas that express activation-in- duced deaminase (AID) and that likely undergo somatic hypermutation. SLIP-GC is a nuclear protein, and it localizes to replication factories. Reduction of SLLP-GC levels in the Burkitt lymphoma cell line Raji and in non-Hodgkin lymphoma cell lines resulted in an increase in DNA breaks and apoptosis that was AID-dependent, as simultaneous reduction of AID abrogated the deleterious effects of SLIP-GC reduction. These results strongly suggest that SLIP-GC is a replication-related protein in germinal center B cells whose reduction is toxic to cells through an AID-dependent mechanism. [ABSTRACT FROM AUTHOR]

Abstract: The basis for mutations at A:T base pairs in immunoglobulin hypermutation and defining how AID interacts with the DNA of the immunoglobulin locus are major aspects of the immunoglobulin mutator mechanism where questions remain unanswered. Here, we examined the pattern of mutations generated in mice deficient in various DNA repair proteins implicated in A:T mutation and found a previously unappreciated bias at G:C base pairs in spectra from mice simultaneously deficient in DNA mismatch repair and uracil DNA glycosylase. This suggests a strand-biased DNA transaction for AID delivery which is then masked by the mechanism that introduces A:T mutations. Additionally, we asked if any of the known components of the A:T mutation machinery underscore the basis for the paucity of A:T mutations in the Burkitt lymphoma cell lines, Ramos and BL2. Ramos and BL2 cells were proficient in MSH2/MSH6-mediated mismatch repair, and express high levels of wild-type, full-length DNA polymerase η. In addition, Ramos cells have high levels of uracil DNA glycosylase protein and are proficient in base excision repair. These results suggest that Burkitt lymphoma cell lines may be deficient in an unidentified factor that recruits the machinery necessary for A:T mutation or that AID-mediated cytosine deamination in these cells may be processed by conventional base excision repair truncating somatic hypermutation at the G:C phase. Either scenario suggests that cytosine deamination by AID is not enough to trigger A:T mutation, and that additional unidentified factors are required for full spectrum hypermutation in vivo. [Copyright &y& Elsevier]