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ABSTRACT: The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.
Methods in molecular biology (Clifton, N.J.) 01/2013; 971:93-111.
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Carolin M Oefner,
André Winkler,
Constanze Hess,
Alexandra K Lorenz,
Vivien Holecska,
Melanie Huxdorf,
Tim Schommartz,
Dominique Petzold,
Josephine Bitterling,
Anna-Lena Schoen, [......],
Peter König,
Arnd Petersen,
Elke O Luger,
Mattias Collin,
Jörg Köhl,
Andreas Hutloff,
Eckard Hamelmann,
Markus Berger, Hedda Wardemann,
Marc Ehlers
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ABSTRACT: Under inflammatory conditions, T cell-dependent (TD) protein antigens induce proinflammatory T- and B-cell responses. In contrast, tolerance induction by TD antigens without costimulation triggers the development of regulatory T cells. Under both conditions, IgG antibodies are generated, but whether they have different immunoregulatory functions remains elusive.
It was shown recently that proinflammatory or anti-inflammatory effector functions of IgG molecules are determined by different Fc N-linked glycosylation patterns. We sought to examine the Fc glycosylation and anti-inflammatory quality of IgG molecules formed on TD tolerance induction.
We administered chicken ovalbumin (OVA) with or without costimulus to mice and analyzed OVA-reactive IgG Fc glycosylation. The anti-inflammatory function of differentially glycosylated anti-OVA IgGs was further investigated in studies with dendritic cell cultures and in an in vivo model of allergic airway disease. Additionally, we analyzed the Fc glycosylation pattern of birch pollen-reactive serum IgGs after successful allergen-specific immunotherapy in patients.
Stimulation with TD antigens under inflammatory conditions induces plasma cells expressing low levels of α2,6-sialyltransferase and producing desialylated IgGs. In contrast, plasma cells induced on tolerance induction did not downregulate α2,6-sialyltransferase expression and secreted immunosuppressive sialylated IgGs that were sufficient to block antigen-specific T- and B-cell responses, dendritic cell maturation, and allergic airway inflammation. Importantly, successful specific immunotherapy in allergic patients also induced sialylated allergen-specific IgGs.
Our data show a novel antigen-specific immunoregulatory mechanism mediated by anti-inflammatory sialylated IgGs that are formed on TD tolerance induction. These findings might help to develop novel antigen-specific therapies for the treatment of allergy and autoimmunity.
The Journal of allergy and clinical immunology 04/2012; 129(6):1647-55.e13. · 9.17 Impact Factor
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ABSTRACT: Long-term humoral immunity is maintained by the formation of high-affinity class-switched memory B cells and long-lived antibody-secreting plasma cells. In healthy humans, a substantial fraction of IgG-positive memory B cells express self-reactive and polyreactive IgG antibodies that frequently develop by somatic mutations. Whether self- and polyreactive IgG-secreting B cells are also tolerated in the long-lived plasma cell pool is not known. To address this question, we cloned and expressed the Ig genes from 177 IgG-producing bone marrow plasma cells of four healthy donors. All antibodies were highly mutated but the frequency of self- and polyreactive IgG antibodies was significantly lower than that found in circulating memory B cells. The data suggest that in contrast to the development of memory B cells, entry into the bone marrow plasma cell compartment requires previously unappreciated selective regulation by mechanisms that limit the production of self- and polyreactive serum IgG antibodies.
Proceedings of the National Academy of Sciences 11/2011; 108(44):18044-8. · 9.68 Impact Factor
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Alexander D Stoehr,
Carolin T Schoen,
Maria M M Mertes,
Susanne Eiglmeier,
Vivien Holecska,
Alexandra K Lorenz,
Tim Schommartz,
Anna-Lena Schoen,
Constanze Hess,
André Winkler, Hedda Wardemann,
Marc Ehlers
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ABSTRACT: The role of TLR9 in the development of the autoimmune disease systemic lupus erythematosus is controversial. In different mouse models of the disease, loss of TLR9 abolishes the generation of anti-nucleosome IgG autoantibodies but at the same time exacerbates lupus disease. However, the TLR9-dependent tolerance mechanism is unknown. In this study, we show that loss of TLR9 is associated with low peritoneal B-1b cell numbers and low levels of protective self-reactive IgM serum autoantibodies in lupus-prone FcγRIIB-deficient mice leading to the uncontrolled accumulation of proinflammatory CD4(+) cells and exacerbated autoimmunity. TLR7 signaling was not able to compensate for the loss of TLR9 signaling in peritoneal B-1b cells to induce IgM Abs. Transfer of TLR9-expressing peritoneal B-1b cells from FcγRIIB-deficient mice or of recombinant monoclonal self-reactive IgM Abs was sufficient to reduce the frequency of proinflammatory Th17 cells and lupus disease in FcγRIIB/TLR9 double-deficient mice. Taken together, these data provide evidence for a TLR9-dependent tolerance mechanism of peritoneal B-1b cells generating protective self-reactive IgM in lupus-prone mice to control Th17 cell development and severe autoimmunity.
The Journal of Immunology 08/2011; 187(6):2953-65. · 5.79 Impact Factor
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ABSTRACT: Mucosal antibody responses play a major role in mediating homeostasis with the intestinal flora. It has been suggested that imbalance in the IgA+ and IgG+ intestinal B cell repertoire may be associated with the development of diseases such as inflammatory bowel disease. Despite this, little is known about the antibody specificity of human intestinal plasmablasts. Here, we have determined the reactivity profile of single isolated IgA+ and IgG+ plasmablasts from human terminal ileum using antibody cloning and in vitro expression. We found that approximately 25% of intestinal IgA and IgG plasmablast antibodies were polyreactive; the majority were antigen-specific. Antigen specificity was not only directed against enteropathogenic microbes but also against commensal microbes and self antigens. Regardless of their reactivity, all intestinal antibodies were somatically mutated and showed signs of antigen-mediated selection, suggesting that they developed from antigen-specific B cell responses. Together, our data indicate that antigen-specific immune responses to intestinal microbes are largely responsible for the maintenance of intestinal homeostasis and thus provide a basis for understanding the deregulated immune responses observed in patients with inflammatory bowel disease.
The Journal of clinical investigation 05/2011; 121(5):1946-55. · 15.39 Impact Factor
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ABSTRACT: Abnormalities in expression levels of the IgG inhibitory Fc gamma receptor IIB (FcγRIIB) are associated with the development of immunoglobulin (Ig) G serum autoantibodies and systemic autoimmunity in mice and humans. We used Ig gene cloning from single isolated B cells to examine the checkpoints that regulate development of autoreactive germinal center (GC) B cells and plasma cells in FcγRIIB-deficient mice. We found that loss of FcγRIIB was associated with an increase in poly- and autoreactive IgG(+) GC B cells, including hallmark anti-nuclear antibody-expressing cells that possess characteristic Ig gene features and cells producing kidney-reactive autoantibodies. In the absence of FcγRIIB, autoreactive B cells actively participated in GC reactions and somatic mutations contributed to the generation of highly autoreactive IgG antibodies. In contrast, the frequency of autoreactive IgG(+) B cells was much lower in spleen and bone marrow plasma cells, suggesting the existence of an FcγRIIB-independent checkpoint for autoreactivity between the GC and the plasma cell compartment.
Journal of Experimental Medicine 11/2010; 207(12):2767-78. · 13.85 Impact Factor
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Hugo Mouquet,
Johannes F Scheid,
Markus J Zoller,
Michelle Krogsgaard,
Rene G Ott,
Shetha Shukair,
Maxim N Artyomov,
John Pietzsch,
Mark Connors,
Florencia Pereyra,
Bruce D Walker,
David D Ho,
Patrick C Wilson,
Michael S Seaman,
Herman N Eisen,
Arup K Chakraborty,
Thomas J Hope,
Jeffrey V Ravetch, Hedda Wardemann,
Michel C Nussenzweig
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ABSTRACT: During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.
Nature 09/2010; 467(7315):591-5. · 36.28 Impact Factor
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Barbara Cassani,
Pietro Luigi Poliani,
Veronica Marrella,
Francesca Schena,
Aisha V Sauer,
Maria Ravanini,
Dario Strina,
Christian E Busse,
Stephan Regenass, Hedda Wardemann,
Alberto Martini,
Fabio Facchetti,
Mirjam van der Burg,
Antonius G Rolink,
Paolo Vezzoni,
Fabio Grassi,
Elisabetta Traggiai,
Anna Villa
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ABSTRACT: Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.
Journal of Experimental Medicine 07/2010; 207(7):1525-40. · 13.85 Impact Factor
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ABSTRACT: We have established a highly efficient 96-well format based strategy to characterize the expressed murine antibody repertoire by combining immunoglobulin (Ig) gene cloning with antibody expression and reactivity profiling at the single cell level. Individual mouse B lineage cells are isolated based on defined surface marker expression patterns by fluorescence-activated cell sorting (FACS) and corresponding full-length Ig heavy (H) and Ig light (L) chain variable (V) region gene transcripts are amplified by RT-PCR. Cloning of the amplified products into eukaryotic expression vectors enables the in vitro production of monoclonal antibodies with antigen specificities identical to the initial B cell antigen receptors. IgH and IgL chain gene sequence information is obtained as part of the cloning procedure and can be directly linked to reactivity profiles of the recombinant antibodies. In summary, our RT-PCR based strategy to generate recombinant monoclonal antibodies from single mouse B cells allows the highly efficient and unbiased characterization of the expressed murine antibody repertoire by sequence analysis and parallel antibody reactivity testing.
Journal of immunological methods 09/2009; 350(1-2):183-93. · 2.35 Impact Factor
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Johannes F Scheid,
Hugo Mouquet,
Niklas Feldhahn,
Michael S Seaman,
Klara Velinzon,
John Pietzsch,
Rene G Ott,
Robert M Anthony,
Henry Zebroski,
Arlene Hurley, [......],
Yuxing Li,
Mark Connors,
Florencia Pereyra,
Bruce D Walker, Hedda Wardemann,
David Ho,
Richard T Wyatt,
John R Mascola,
Jeffrey V Ravetch,
Michel C Nussenzweig
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ABSTRACT: Antibodies to conserved epitopes on the human immunodeficiency virus (HIV) surface protein gp140 can protect against infection in non-human primates, and some infected individuals show high titres of broadly neutralizing immunoglobulin (Ig)G antibodies in their serum. However, little is known about the specificity and activity of these antibodies. To characterize the memory antibody responses to HIV, we cloned 502 antibodies from HIV envelope-binding memory B cells from six HIV-infected patients with broadly neutralizing antibodies and low to intermediate viral loads. We show that in these patients, the B-cell memory response to gp140 is composed of up to 50 independent clones expressing high affinity neutralizing antibodies to the gp120 variable loops, the CD4-binding site, the co-receptor-binding site, and to a new neutralizing epitope that is in the same region of gp120 as the CD4-binding site. Thus, the IgG memory B-cell compartment in the selected group of patients with broad serum neutralizing activity to HIV is comprised of multiple clonal responses with neutralizing activity directed against several epitopes on gp120.
Nature 05/2009; 458(7238):636-40. · 36.28 Impact Factor
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ABSTRACT: The majority of early immature B cells express autoreactive B cell receptors (BCRs) that are, according to the current view, negatively selected to avoid the production of self-reactive antibodies. Here, we show that polyreactive BCRs, which recognize multiple self-antigens, induced autonomous signaling and selective expansion of B cell precursors in a manner comparable to the pre-BCR. We found that the pre-BCR was capable of recognizing multiple self-antigens and that a signaling-deficient pre-BCR lacking the non-Ig region of the surrogate-light-chain component lambda5 was rescued by the complementarity-determining region 3 derived from heavy chains of polyreactive receptors. Importantly, bone marrow B cells from mice carrying Ig transgenes for an autoreactive BCR showed increased cell-cycle activity, which could not be detected in cells lacking the transgenic BCR. Together, the pre-BCR has evolved to ensure self-recognition because autoreactivity is required for positive selection of B cell precursors.
Immunity 01/2009; 29(6):912-21. · 21.64 Impact Factor
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Johannes F Scheid,
Hugo Mouquet,
Niklas Feldhahn,
Bruce D Walker,
Florencia Pereyra,
Emily Cutrell,
Michael S Seaman,
John R Mascola,
Richard T Wyatt, Hedda Wardemann,
Michel C Nussenzweig
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ABSTRACT: Antibodies to HIV are potentially important reagents for basic and clinical studies. Historically, these reagents have been produced by random cloning of heavy and light chains in phage display libraries [Burton, D.R., Barbas, C.F. III, Persson, M.A.A., Koenig, S., Chanock, R.M., and Lerner, R.A., (1991), A large array of human monoclonal antibodies to type 1 immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. U. S. A. 88, 10134-10137.] and electrofusion techniques [Buchacher, A., Predl, R., Tauer, C., Purtscher, M., Gruber, G., Heider, R., Steindl, F., Trkola, A., Jungbauer, A., and Katinger, H., (1992), Human monoclonal antibodies against gp41 and gp120 as potential agent for passive immunization. Vaccines 92, 191-195]. Here we describe a method to identify and potentially enrich human memory B cells from HIV infected patients that show serum titers of neutralizing antibodies. When biotinylated gp140 is used to stain peripheral blood mononuclear cells it identifies a distinct population of gp140 binding B cells by flow cytometry.
Journal of immunological methods 01/2009; 343(2):65-7. · 2.35 Impact Factor
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ABSTRACT: The enormous diversity of the antibody repertoire is generated by two mechanisms: recombination of immunoglobulin (Ig) gene variable (V), diversity (D), and joining (J) gene segments during the early stages of B-cell development in the bone marrow and somatic hypermutation (SHM) of functional Ig genes from antigen-activated B cells within secondary lymphoid organs. Diversity by V(D)J recombination and SHM not only provides protective humoral immunity but also generates potentially harmful clones expressing autoantibodies. Under normal circumstances, several mechanisms regulate the removal of autoreactive B cells and defects in central and peripheral B cell tolerance checkpoints are associated with the development of autoimmunity in humans.
Current opinion in immunology 11/2008; 20(6):632-8. · 10.88 Impact Factor
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Brun Mietzner,
Makoto Tsuiji,
Johannes Scheid,
Klara Velinzon,
Thomas Tiller,
Klaus Abraham,
Jose B Gonzalez,
Virginia Pascual,
Dorothee Stichweh, Hedda Wardemann,
Michel C Nussenzweig
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ABSTRACT: Persistent autoantibody production in patients with systemic lupus erythematosus (SLE) suggests the existence of autoreactive humoral memory, but the frequency of self-reactive memory B cells in SLE has not been determined. Here, we report on the reactivity of 200 monoclonal antibodies from single IgG+ memory B cells of four SLE patients. The overall frequency of polyreactive and HEp-2 self-reactive antibodies in this compartment was similar to controls. We found 15% of IgG memory B cell antibodies highly reactive and specific for SLE-associated extractable nuclear antigens (ENA) Ro52 and La in one patient with serum autoantibody titers of the same specificity but not in the other three patients or healthy individuals. The germ-line forms of the ENA antibodies were non-self-reactive or polyreactive with low binding to Ro52, supporting the idea that somatic mutations contributed to autoantibody specificity and reactivity. Heterogeneity in the frequency of memory B cells expressing SLE-associated autoantibodies suggests that this variable may be important in the outcome of therapies that ablate this compartment.
Proceedings of the National Academy of Sciences 08/2008; 105(28):9727-32. · 9.68 Impact Factor
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ABSTRACT: We have developed an efficient strategy that combines immunoglobulin (Ig) gene repertoire analysis and Ig reactivity profiling at the single cell level. Based on surface marker expression individual cells at different stages of human B cell development are isolated by fluorescence-activated cell sorting. For each cell Ig heavy and corresponding Ig light chain gene transcripts are amplified by nested RT-PCR and cloned into eukaryotic expression vectors to produce monoclonal human antibodies of the same specificity in vitro. All reactions are performed in 96-well plates and allow cloning of large numbers of Ig genes. The recombinant antibodies are tested for reactivity with diverse self- and non-self antigens and the reactivity profile can be directly linked to the complete Ig heavy and Ig light chain gene sequence information that is obtained as part of the cloning strategy. In summary, our method to clone and express human monoclonal antibodies is unbiased, highly efficient, requires only small cell numbers and the recombinant antibodies allow direct conclusions on the frequency of specific human B cells in a diverse repertoire.
Journal of Immunological Methods 02/2008; 329(1-2):112-24. · 2.20 Impact Factor
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ABSTRACT: More than half of the nascent B cells in humans initially express autoreactive antibodies. However, most of these autoantibodies are removed from the repertoire at two checkpoints before maturation into naive B cells. A third checkpoint excludes remaining autoantibodies from the antigen-experienced IgM(+) memory B cell pool. Nevertheless, low-affinity self-reactive antibodies are frequently found in the serum of normal humans. To determine the source of these antibodies, we cloned and expressed antibodies from circulating human IgG(+) memory B cells. Surprisingly, we found that self-reactive antibodies including anti-nuclear antibodies were frequently expressed by IgG(+) memory B cells in healthy donors. Most of these antibodies were created de novo by somatic hypermutation during the transition between mature naive and IgG(+) memory B cells. We conclude that deregulation of self-reactive IgG(+) memory B cells may be associated with autoimmunity.
Immunity 03/2007; 26(2):205-13. · 21.64 Impact Factor
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ABSTRACT: Two mechanisms account for generation of the human antibody repertoire; V(D)J recombination during the early stages of B-cell development in the bone marrow and somatic mutation of immunoglobulin genes in mature B cells responding to antigen in the periphery. V(D)J recombination produces diversity by random joining of gene segments and somatic mutation by introducing random point mutations. Both are required to attain the degree of antigen receptor diversification that is necessary for immune protection: defects in either mechanism are associated with increased susceptibility to infection. However, the downside of producing enormous random diversity in the antibody repertoire is the generation of autoantibodies. To prevent autoimmunity B cells expressing autoantibodies are regulated by strict mechanisms that either modify the specificity of autoantibodies or the fate of cells expressing such antibodies. Abnormalities in B-cell self-tolerance are associated with a large number of autoimmune diseases, but the precise nature of the defects is less well defined. Here we summarize recent data on the self-reactive B-cell repertoire in healthy humans and in patients with autoimmunity.
Advances in Immunology 02/2007; 95:83-110. · 5.76 Impact Factor
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ABSTRACT: A majority of the antibodies expressed by nascent B cells in healthy humans are self-reactive, but most of these antibodies are removed from the repertoire during B cell development. In contrast, untreated systemic lupus erythematosus (SLE) patients fail to remove many of the self-reactive and polyreactive antibodies from the naive repertoire. Here, we report that SLE patients in clinical remission continue to produce elevated numbers of self-reactive and polyreactive antibodies in the mature naive B cell compartment, but the number of B cells expressing these antibodies is lower than in patients with active disease. Our finding that abnormal levels of self-reactive mature naive B cells persist in the majority of patients in clinical remission suggests that early checkpoint abnormalities are an integral feature of SLE.
Journal of Experimental Medicine 11/2006; 203(10):2255-61. · 13.85 Impact Factor
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ABSTRACT: Autoantibodies are removed from the repertoire at two checkpoints during B cell development in the bone marrow and the periphery. Despite these checkpoints, up to 20% of the antibodies expressed by mature naive B cells in healthy humans show low levels of self-reactivity. To determine whether self-reactive antibodies are also part of the antigen-experienced memory B cell compartment, we analyzed recombinant antibodies cloned from single circulating human IgM+ memory B cells. Cells expressing antibodies specific for individual bacterial polysaccharides were expanded in the IgM+ memory compartment. In contrast, B cells expressing self-reactive and broadly bacterially reactive antibodies were removed from the repertoire in the transition from naive to IgM+ memory B cell. Selection against self-reactive antibodies was implemented before the onset of somatic hypermutation. We conclude that a third checkpoint selects against self-reactivity during IgM+ memory B cell development in humans.
Journal of Experimental Medicine 03/2006; 203(2):393-400. · 13.85 Impact Factor
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ABSTRACT: Antibodies are protective effector molecules in the body's immune defense against pathogens. However, if directed against the body's own tissues or organs, antibodies can also play a role in the pathogenesis of autoimmune disease. Here, we discuss our data on how autoreactive antibodies are regulated in healthy humans and how a failure to establish self-tolerance during early B-cell development in patients with systemic lupus erythematosus may predispose to the development of this autoimmune disease.
Annals of the New York Academy of Sciences 01/2006; 1062:165-74. · 3.15 Impact Factor
