Previous estimates of the diversity of the mouse antibody repertoire have been based on fragmentary data owing to many technical limitations, in particular the many samples necessary to provide adequate coverage. Here, we used 5' coding end amplification of Igκ mRNAs from bone marrow, splenic and lymph node B cells of C57BL/6 mice combined with amplicon pyrosequencing to assess the functional and non-functional Vκ repertoire. To evaluate the potential effects of receptor editing, we also compared V/J associations and usage in bone marrows of mouse mutants under constitutive negative selection or an altered ability to undergo secondary recombination. In order to focus on preimmune B cells, our cell sorting strategy excluded memory B cells and plasma cells. Analysis of approximately 90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functionalVκ genes are used, but at frequencies ranging from ~.001% to ~10%.Usage of sevenVκgenes made up over 40% of the repertoire. A small class of transcripts from apparently nonfunctional Vκ genes was found, as were occasional transcripts from several apparently functional genes that carry aberrant recombination signals. Of 404 potential V-J combinations (101 Vκs X 4 Jκs), 398 (98.5%) were found at least once in our sample. For most Vκ transcripts, all Jκs were used, but V-J association biases were common.Usage patterns were remarkably stable in different selective conditions.Overall, the primary κ repertoire is highly skewedby preferred rearrangements, limiting antibody diversity, but potentially facilitating receptor editing.

Dsg3-specific naive lymphocytes in Dsg3(-/-) mice can be primed and activated by the endogenous Dsg3 in recipient mice to produce pathogenic anti-Dsg3 IgG without active immunization. This approach using naive lymphocytes provides a unique model to dissect immunological mechanisms of tolerance against peripheral autoimmune targets.

Broadly neutralizing antibodies (bNAbs) against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic (“knock-in”) mice expressing, in the physiological immunoglobulin heavy (H) and light (L) chain loci, two well-studied bNAbs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the accompanying paper. Here, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV Env gp140 trimers and to BCR ligands, but only weakly to HIV pseudovirions. Upon transfer to wild type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, while generating abundant precursors and antibodies with affinity for Env, were unable to rapidly generate neutralizing antibodies, highlighting the importance of developing antigen forms that better focus responses to neutralizing epitopes. b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding non-protective specificities.

To analyze B lymphocyte central tolerance in a polyclonal immune system, mice were engineered to express a superantigen reactive to IgM of allotype b (IgMb). IgMb/b mice carrying superantigen were severely B cell lymphopenic, butsmall numbers of Bcells matured. Their sera contained low levels of IgG andoccasionally high levels of IgA. In bone marrow, immature B cells were normal in number, but internalized IgM and had a unique gene expression profile, compared to thoseexpressing high levels of surface IgM, including elevated recombinase activator gene expression. A comparable B cell population was defined in wild-type bone marrows, with an abundance suggesting that at steady state ∼20% of normal developing B cells are constantly encountering autoantigens in situ. In superantigen-expressing mice, as well as in mice carrying the 3H9 anti-DNA Ig heavy chain transgene, or 3H9 H along with mutation in the murine kappa deleting element RS, IgM internalization was correlated with CD19 downmodulation. CD19low bone marrow cells from 3H9;RS−/− mice were enriched in light chains that promote DNA binding. Our results suggest that central tolerance and attendant light chain receptor editing affect a large fraction of normal developing B cells.IgHa/b mice carrying the superantigen had a ∼50% loss in follicular B cell numbers, suggesting that escape from central tolerance by receptor editing from one IgH allele to another was not a major mechanism. IgMb superantigen hosts reconstituted withexperimental bone marrow were demonstrated to be useful in revealing pathways involved in central tolerance.

Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by IgG autoantibodies against desmoglein 3 (Dsg3). We have recently developed an active disease mouse model for PV by adoptive transfer of splenocytes from Dsg3 À/À mice. The purpose of this study was to determine the role of CD40/CD154 interaction in the pathogenic antibody production and development of the disease in PV model mice. When anti-CD154 monoclonal antibody (mAb) was administered to recipient mice prior to adoptive transfer, anti-CD154 mAb almost completely blocked the anti-Dsg3 IgG production and prevented blister formation. The blockade of CD40/CD154 interaction induced tolerance against Dsg3 as the suppression of antibody production was observed through day 70, and it was maintained even after challenge by immunization with recombinant mouse Dsg3 or by adoptive transfer of immunized Dsg3 À/À splenocytes. Furthermore, the tolerance to Dsg3 was transferable because cotransfer of splenocytes from anti-CD154 mAb-treated mice and naı ¨ve Dsg3 À/À splenocytes significantly suppressed anti-Dsg3 IgG production in recipient mice. In contrast, when anti-CD154 mAb was injected after the mice had developed the PV phenotype, no significant suppression of the production of anti-Dsg3 IgG was observed. These findings indicate that the CD40/CD154 interaction is essential for the induction of pathogenic anti-Dsg3 IgG antibodies and that antigen-specific immune-regulatory cells induced by anti-CD154 mAb would hold a therapeutic option for autoimmune diseases.

Transitional B cells turn over rapidly in vivo and are sensitive to apoptosis upon BCR ligation in vitro. However, little direct evidence addresses their tolerance sensitivity in vivo. A key marker used to distinguish these cells is IgD, which, through alternative RNA splicing of H chain transcripts, begins to be coexpressed with IgM at this stage. IgD is also expressed at high levels on naive follicular (B-2) and at lower levels on marginal zone and B-1 B cells. In this study, mice were generated to ubiquitously express a membrane-bound IgD-superantigen. These mice supported virtually no B-2 development, a greatly reduced marginal zone B cell population, but a relatively normal B-1 compartment. B cell development in the spleen abruptly halted at the transitional B cell population 1 to 2 stage, a block that could not be rescued by either Bcl-2 or BAFF overexpression. The developmentally arrested B cells appeared less mature and turned over more rapidly than nontransgenic T2 cells, exhibiting neither conventional features of anergy nor appreciable receptor editing. Paradoxically, type-2 T-independent responses were more robust in the transgenic mice, although T-dependent responses were reduced and had skewed IgL and IgH isotype usages. Nevertheless, an augmented memory response to secondary challenge was evident. The transgenic mice also had increased serum IgM, but diminished IgG, levels mirrored by the increased numbers of IgM+ plasma cells. This model should facilitate studies of peripheral B cell tolerance, with the advantages of allowing analysis of polyclonal populations, and of B cells naturally lacking IgD.

IgE plays a pivotal role in allergic reactions and asthma through its ability to bind to the mast cell FcR for IgE (FcεRI). Current therapies to suppress such reactions include passive treatment with neutralizing Abs to IgE that block its binding to FcεRI. In theory, induction of immune tolerance in the B lymphocytes that carry IgE Ag receptors and give rise to IgE-secreting cells should provide longer term efficacy. However, recent data have suggested that such memory cells may lack cell surface IgE. Using a gene therapy approach, we show that a recombinant single-chain neutralizing anti-IgE could not only neutralize circulating IgE, but also reduce IgE+ B cell numbers and H chain transcripts. Therapeutic anti-IgE stimulated a calcium response in primary B cells or in a B cell line expressing membrane IgE and suppressed IgE secretion in vitro, suggesting that active signaling through membrane IgE likely promoted tolerance. Interestingly, upon subsequent challenge of anti-IgE-treated mice with an IgE cross-linking reagent capable of inducing activation of IgE-decorated mast cells, an anaphylaxis reaction was induced, apparently via a FcγRIII pathway involving recognition of anti-IgE Ab itself. These studies have important implications for the optimal design of safe and effective anti-IgE therapies and suggest that the IgE memory B cells may be targeted by such genetic Ab therapies.

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