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Antigens Break B Cell Anergy Cutting Edge: Complement Cutting Edge: Complement (C3d)-Linked Antigens Break B Cell Anergy Taras Lyubchenko, Joseph M. Dal Porto, V. Michael Holers and John C. Cambier This information is current as of September 29, 2021. J Immunol 2007; 179:2695-2699; ; doi: 10.4049/jimmunol.179.5.2695 http://www.jimmunol.org/content/179/5/2695 Downloaded from References This article cites 30 articles, 7 of which you can access for free at: http://www.jimmunol.org/content/179/5/2695.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE JOURNAL OF IMMUNOLOGY CUTTING EDGE Cutting Edge: Complement (C3d)-Linked Antigens Break B Cell Anergy Taras Lyubchenko,1* Joseph M. Dal Porto,1† V. Michael Holers,*† and John C. Cambier2† C3dg adducts of Ag can coligate complement receptor type anergic (8). These cells, although short lived, represent a signif- 2 (CR2; CD21) and the B cell Ag receptor. This interac- icant hazard of autoimmunity because they may bind cross-re- tion significantly amplifies BCR-mediated signals in Ag- active antigenic epitopes on infectious agents that stimulate B naive wild-type mice, lowering the threshold for B cell ac- cells via innate immune ligands. tivation and the generation of humoral immune responses The onset of autoimmunity often coincides with infection by as much as 1000-fold. In this study we demonstrate that pathogenic organisms (9, 10). Although the activation of self- CR2-mediated complementation of BCR signals can also reactive T cells has been attributed to superantigens, epitope- Downloaded from overcome B cell anergy. Unlike Ag alone, BCR/CR2 co- mimicking, epitope spreading, and other mechanisms, little is stimulation (Ars-CCG/C3dg complexes) of anergic known regarding the events leading to the failure of B cell tol- ؉ Ars/A1 B cells led to Ca2 mobilization in vitro and the erance. One major innate immunity factor known to influence production of autoantibodies in vivo. Interestingly, the in B cell responses is complement, particularly the C3dg protein vivo immune response of anergic cells occurs without the that is one of the cleavage products of the C3 component. In- vasive pathogenic organisms spontaneously bind and fix com- http://www.jimmunol.org/ formation of germinal centers. These results suggest that 3 the Ag unresponsiveness of anergic B cells can be overcome plement. C3dg is a ligand for complement receptor 2 (CR2) (CD21) on the B cell surface, and CR2-C3dg interaction by cross-reactive (self-mimicking) Ags that have been com- greatly amplifies signals elicited by the binding of a C3dg-at- plement-opsonized. This mechanism may place individu- tached Ag with its specific BCR (11, 12). It was recently shown als exposed to complement-fixing bacteria at risk for that C3dg association with collagen enhances its ability to in- autoimmunity. The Journal of Immunology, 2007, 179: duce arthritis and that CD21 expression by either B cells or 2695–2699. dendritic cells is sufficient to support this response (13). In this study we explored whether B cell anergy is susceptible to sub- by guest on September 29, 2021 reater than 60% of newly produced B cells are auto- version by components of the innate immune system, in this reactive and must be silenced to prevent autoimmu- case complement. G nity (1, 2). Silencing occurs by one of three primary The Ars/A1 murine model of B cell anergy offers an optimal mechanisms. Upon interaction with high avidity Ags, imma- setting in which to study the effects of complement-mediated ture B cells undergo receptor editing involving additional signaling in anergic B cells (14). Ars/A1 mice express a trans- V(D)J recombinations and the expression of a receptor with al- genic (Tg) Ig (␮␦/␬) that encodes a canonical anti- tered specificity (3). If this receptor is innocuous, cells enter the p-azophenylarsonate (Ars) BCR. The Ars-specific transgene-en- peripheral pool and can participate in immune responses. If ed- coded BCR cross-reacts with an undefined self-Ag in the bone iting does not eliminate autoreactivity, the cell may be elimi- marrow and periphery that induces anergy. Similar to other nated by clonal deletion that is thought to involve death by apo- well-established murine models of B cell anergy such as HyHEL ptosis (4). Twenty-five to 30% of mature peripheral B cells (15), Ars/A1 B cells that reach later transitional stages are capa- show signs of receptor editing and thus have escaped autoreac- ble of binding Ag; however, their receptors fail to transduce tivity by changing receptor specificity (5). The third known si- proximal biochemical signaling events as indicated by intracel- ϩ lencing mechanism is anergy, which is induced by low avidity lular Ca2 mobilization or tyrosine phosphorylation. Pheno- binding to self-Ags (6, 7). Anergic cells persist in the periphery typically, the Ars/A1 model is in many aspects similar to the for a few days and, despite expressing unoccupied Ag receptors, HyHEL model (16). Thus, using this system it is possible to are unresponsive to Ag stimulation. We have recently shown assess the effects of C3dg-associated cross-reactive Ags on B cell that ϳ50% of newly produced B cells are destined to become anergy. *Division of Rheumatology, Department of Medicine, University of Colorado School of 2 Address correspondence and reprint requests to Dr. John C. Cambier, Department of Medicine, Denver, CO 802602; and †Department of Immunology, University of Colo- Immunology, K803A, National Jewish Medical and Research Center, 1400 Jackson Street, rado School of Medicine and National Jewish Medical and Research Center, Denver, CO Denver, Colorado 80206. E-mail address: [email protected] 80206 3 Received for publication February 22, 2007. Accepted for publication July 11, 2007. Abbreviations used in this paper: CR2, complement receptor 2; AFC, Ab-forming cell; AM, acetoxymethyl ester; Ars, p-azophenylarsonate; CCG, chicken ␥-globulin; GC, ger- The costs of publication of this article were defrayed in part by the payment of page charges. minal center; PNA, peanut agglutinin; PtdInsP3, phosphatidylinositol 3,4,5-trisphos- This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. phate; Tg, transgenic. Section 1734 solely to indicate this fact. 1 T.L. and J.D. contributed equally to this work. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org 2696 CUTTING EDGE: C3d BREAKS ANERGY In this report we describe the ability of C3dg augmentation of BCR signals to overcome proximal inhibitory mechanisms responsible for the Ag unresponsiveness of anergic B cells. Materials and Methods Generation of anti-IgM/C3dg conjugates Biotin-conjugated C3dg and anti-IgM/C3dg/streptavidin conjugates were gen- Ј erated as described (12). Briefly, biotin-conjugated anti-mouse-IgM F(ab )2 Ab (anti-IgM) was titrated down from its optimal concentration (1 ␮g/ml) to the concentration at which splenic B cells no longer mobilized intracellular Ca2ϩ (suboptimal, 0.001 ␮g/ml; data not shown). Recombinant biotin-conjugated C3dg and streptavidin were titrated to concentrations capable of amplifying the suboptimal anti-IgM stimulus (0.2 and 0.04 ␮g/ml, respectively). Animals and reagents C57BL/6 mice were obtained from The Jackson Laboratory. Ars/A1 mice (B6.Cg-Tg(IghAb36–65)1Wys Tg(IgkAb36–71)1Wys) were generated and bred in our laboratory (14). Abs and their sources are as follows: biotin-conju- FIGURE 1. Intracellular Ca2ϩ influx in Ars/A1 B cells stimulated by anti- Ј gated goat-anti-mouse-IgM F(ab )2 (Southern Biotechnology Associates); un- IgM/C3dg complexes. Splenocytes isolated from C57BL/6 (solid line) or labeled or FITC-conjugated anti-mouse-CD21/CD35 mAb (7G6), FITC- Ars/A1 (dashed line) mice were labeled with Indo-1 AM and allophycocyanin- Downloaded from conjugated anti-mouse-CD19 mAb, FITC-conjugated anti-mouse-TCR mAb, ϩ ϩ conjugated anti-B220 mAb, and Ca2 influx in B220 B cells was analyzed by allophycocyanin-conjugated anti-mouse-B220 mAb, FITC-conjugated anti- flow cytometry (ratio units). After a 1-min baseline cells were stimulated with mouse-CD80, and anti-mouse-CD86 mAb (BD Pharmingen); Cy5-conju- ␮ ␮ gated donkey-anti-mouse IgM, normal rabbit serum (Jackson ImmunoRe- either 1 g/ml anti-IgM-biotin (A), anti-IgM-biotin (0.001 g/ml)/streptavi- ␮ search); IgMa (RS3.1; American Type Culture Collection), ␬ (187.1; American din (B), or anti-IgM-biotin (0.001 g/ml)/C3dg-biot/streptavidin conjugates Type Culture Collection), and anti-idiotypic Ab (17). Streptavidin was pur- (C). Expression of IgM (D), CD21 (E), and CD19 (F) in C57BL/6 and Ars/A1 chased from BioSource International, Indo-1 acetoxymethyl ester (AM) was B cells. Ars/A1 B cells bind specific Ag (H) and anti-Idiotypic (Id) Ab (G). Data ϩ from Molecular Probes, and LPS and PE-conjugated peanut agglutinin (PNA) presented in D–H was obtained by flow cytometric analysis of B220 spleno- http://www.jimmunol.org/ ␥ were from Sigma-Aldrich. Anti-Ars-chicken -globulin (CCG) and Ars-CCG- cytes costained with specific Abs as indicated.
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