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Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance

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Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance This information is current as Andrew J. Gross, Julia R. Lyandres, Anil K. Panigrahi, Eline of October 1, 2021. T. Luning Prak and Anthony L. DeFranco J Immunol 2009; 182:5382-5392; ; doi: 10.4049/jimmunol.0803941 http://www.jimmunol.org/content/182/9/5382 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2009/04/20/182.9.5382.DC1 Material References This article cites 48 articles, 26 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/182/9/5382.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021 • Fast Publication! 4 weeks from acceptance to publication *average 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance1 Andrew J. Gross,* Julia R. Lyandres,† Anil K. Panigrahi,§ Eline T. Luning Prak,§ and Anthony L. DeFranco2‡ To better understand whether autoimmunity in Lyn-deficient mice arises from compromised central or peripheral B cell tolerance, we examined BCR signaling properties of wild-type and Lyn-deficient B cells at different stages of development. Wild-type mature follicular B cells were less sensitive to BCR stimulation than were immature transitional stage 1 B cells with regard to BCR- induced calcium elevation and ERK MAPK activation. In the absence of Lyn, mature B cell signaling was greatly enhanced, whereas immature B cell signaling was minimally affected. Correspondingly, Lyn deficiency substantially enhanced the sensitivity of mature B cells to activation via the BCR, but minimally affected events associated with tolerance induction at the immature stage. The effects of CD22 deficiency on BCR signaling were very similar in B cells at different stages of maturation. These results Downloaded from indicate that the Lyn-CD22-Src homology region 2 domain-containing phosphatase-1 inhibitory pathway largely becomes oper- ational as B cell mature, and sets a threshold for activation that appears to be critical for the maintenance of tolerance in the B cell compartment. The Journal of Immunology, 2009, 182: 5382–5392. cell receptor hyperresponsiveness is associated with a would lead to a loss of tolerance in lynϪ/Ϫ mice is not entirely breakdown in tolerance and the development of autoan- self-evident, because whereas BCR signaling drives activation of B tibodies in several genetically modified mouse strains (1, mature B cells, it also promotes tolerance-inducing mechanisms in http://www.jimmunol.org/ 2). A good example of this is the Lyn-deficient mouse (3). Lyn is immature B cells. For example, binding of Ag by immature B cells a Src family kinase (SFK)3 that, like two other SFKs expressed by in the bone marrow (BM) induces expression of RAG 1 and 2, B cells, Blk and Fyn, phosphorylates ITAMs on BCR Ig␣/Ig␤ which can generate L chain rearrangement and thereby change the chains following Ag binding (4–6). Lyn also functions to phos- specificity of the BCR (receptor editing) (16–18). Continued rec- phorylate ITIMs on inhibitory receptors that negatively regulate ognition of Ag by the BCR may induce apoptosis of self-reactive BCR signaling, including CD22 (5, 7–9), Fc␥RIIb (7, 8), and per- B cells (clonal deletion) (16). Therefore, genetic alterations that haps others (10–13). In this way, Lyn facilitates recruitment of the enhance BCR signaling should increase the sensitivity of immature Src homology region 2 domain-containing phosphatase-1 (SHP-1) B cells to self Ags, causing more thorough removal of autoreactive by guest on October 1, 2021 and SHIP phosphatases to the plasma membrane, which down- BCR specificities from the newly formed B cell repertoire. For this modulate BCR signaling (5, 8, 9). In the absence of Lyn, BCR reason, it seems paradoxical that autoimmunity develops in the signaling is supported by Blk and Fyn, but inhibitory receptors are lynϪ/Ϫ mouse and in other mice with genetic alterations increasing ineffective at down-regulating BCR signaling, thereby leading to the strength of BCR signaling. BCR hyperresponsiveness (3). To account for the break in B cell tolerance observed in Lyn- Lyn-deficient mice exhibit increased plasma cell numbers and deficient mice, we hypothesized that the level of Lyn-mediated serum Ig levels. Surprisingly, these mice also produce autoanti- inhibitory signaling might change during the course of B cell de- bodies to nuclear Ags (4, 14, 15). Why elevated BCR signaling velopment. Interestingly, we found that B cells become less sen- sitive to BCR engagement as they proceed through development. This reduction in BCR sensitivity was mediated by Lyn and CD22, *Department of Medicine, †Department of Surgery, and ‡Department of Microbiol- because Lyn or CD22 deficiency strongly increased BCR signaling ogy and Immunology, University of California, San Francisco, CA 94143; and §De- partment of Pathology and Laboratory Medicine, University of Pennsylvania, Phila- in follicular B cells, but only weakly increased signaling in tran- delphia, PA 19104 sitional stage 1 (T1) B cells. Consistent with these findings, Lyn Received for publication December 1, 2008. Accepted for publication February deficiency had a modest effect on events associated with receptor 13, 2009. editing, whereas it more strongly enhanced the sensitivity of ma- The costs of publication of this article were defrayed in part by the payment of page ture B cells to BCR-induced proliferation. These results indicate charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that inhibition of BCR signaling by the Lyn-CD22-SHP-1 pathway is up-regulated as B cells mature in the spleen, and suggest that this 1 This work was supported by National Institutes of Health Grant K08 AI52249 (to A.J.G.), by the Rosalind Russell Medical Research Center for Arthritis, and by Na- developmental change is involved in the maintenance of peripheral tional Institutes of Health Grant R01 AI20038 (to A.L.D.). immune tolerance of B cells. 2 Address correspondence and reprint requests to Dr. Anthony L. DeFranco, Univer- sity of California, 400 Parnassus Avenue, Box 0414, San Francisco, CA 94143. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: SFK, Src family kinase; BM, bone marrow; Mice [Ca2ϩ] , intracellular Ca2ϩ concentration; HEL, hen egg lysozyme; mIg, membrane- i Mice aged 7–12 wk were used for most experiments. Four-week-old mice bound Ig; NF, immature newly formed; SHP1, Src homology region 2 domain-con- me-v taining phosphatase-1; T1, transitional stage 1; T2, transitional stage 2; T3, transi- were used in experiments using motheaten-viable mice (Ptpn6 ) back- Ϫ/Ϫ tional stage 3; WT, wild type; RS, recombining sequence. crossed onto C57BL/6 background (The Jackson Laboratory). The lyn mice were used as described (4), and backcrossed at least 15 generations Ϫ Ϫ Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 onto C57BL/6 background. The cd22 / mice (19) backcrossed at least 16 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803941 The Journal of Immunology 5383 generations onto C57BL/6 background were obtained from E. Clark (Uni- Proteins were separated on a NuPAGE bis-Tris gel (Invitrogen) with versity of Washington, Seattle, WA). MD4 transgenic IgHel mice were MOPS buffer and immunoblotted on Immobilon-FL transfer membrane obtained from J. Cyster (University of California, San Francisco, CA). All (Millipore). Abs were directed against phospho-Src family kinase Tyr416 animals were housed in a specific pathogen-free facility at University of (Cell Signaling Technology; 2101s), phospho-Lyn Tyr507 (Abcam California, according to University and National Institutes of Health guide- ab53122), Lyn (Abcam ab1890), and BAP31 (Abcam ab37120) as a load- lines. Animal use was approved by the University of California Institu- ing control. These Abs were detected with fluorescently labeled secondary tional Animal Care and Use Committee. Abs using the Odyssey Infrared Imaging System (LI-COR Biosciences). Abs, immunofluorescence analysis, B cell purification, and cell Real-time RT-PCR sorting Total RNA was isolated from sorted B cells using the RNeasy Micro kit Fluorophore-conjugated Abs directed against the following molecules were (Qiagen), and cDNA was transcribed using the iScript cDNA synthesis kit ␭ used: B220 (RA3-6B2), CD23 (B3B4), IgM (II/41), Ig 1–3 (R26-46), (Bio-Rad), according to the manufacturers’ instructions. Equivalent CD16/CD32 (2.4G2) CD22.2 (Cy34.1), and CD72 (K10.6) all from BD amounts of cDNA were used in quantitative PCR on an ABI Prism 7700 Pharmingen; CD24 (M1/69) from BioLegend; CD93 (AA4.1) and CD5 sequence detection instrument (Taqman; PerkinElmer Applied Biosys- ␬ ␭ (53-7.3) from eBioscience; Ig (187.1) and Ig (JC5-1) from Southern tems). Primer and probe sequences for RAG1 and RAG2 (20), and Biotechnology Associates; and IgM (goat polyclonal Fab monomer, GAPDH (21) were used as published. ␮-chain specific) (Jackson ImmunoResearch Laboratories). Cells were an- alyzed on an LSR-II or FACSCalibur (both from BD Pharmingen).
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