A Kinase-Dead Allele of Lyn Attenuates Autoimmune Disease Normally Associated with Lyn Deficiency

This information is current as Anne M. Verhagen, Morgan E. Wallace, Ankita Goradia, of October 2, 2021. Sarah A. Jones, Hayley A. Croom, Donald Metcalf, Janelle E. Collinge, Mhairi J. Maxwell, Margaret L. Hibbs, Warren S. Alexander, Douglas J. Hilton, Benjamin T. Kile and Robyn Starr J Immunol 2009; 182:2020-2029; ; doi: 10.4049/jimmunol.0803127 Downloaded from http://www.jimmunol.org/content/182/4/2020

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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

A Kinase-Dead Allele of Lyn Attenuates Autoimmune Disease Normally Associated with Lyn Deficiency1

Anne M. Verhagen,* Morgan E. Wallace,* Ankita Goradia,* Sarah A. Jones,* Hayley A. Croom,* Donald Metcalf,† Janelle E. Collinge,‡ Mhairi J. Maxwell,§ Margaret L. Hibbs,§ Warren S. Alexander,† Douglas J. Hilton,‡ Benjamin T. Kile,‡ and Robyn Starr2*

Lyn kinase, a member of the Src family of tyrosine kinases, functions as both a positive and negative regulator of activation. In the absence of Lyn, BCR signaling is unregulated, leading to perturbed B cell development, hyperactive B cells, and lethal Ab-mediated autoimmune disease. We have generated a mutant mouse pedigree, termed Mld4, harboring a novel mutation in the encoding Lyn, which renders the devoid of kinase activity. Despite similarities between the ؊ ؊ phenotypes of LynMld4/Mld4 and Lyn / mice, the spectrum of defects in LynMld4/Mld4 mice is less severe. In particular, Downloaded from although defects in the B cell compartment are similar, splenomegaly, myeloid expansion, and autoantibody production, -characteristic of Lyn؊/؊ mice, are absent or mild in LynMld4/Mld4 mice. Critically, immune complex deposition and comple ment activation in LynMld4/Mld4 glomeruli do not result in fulminant glomerulonephritis. Our data suggest that BCR hyper- ,sensitivity is insufficient for the development of autoimmune disease in Lyn؊/؊ mice and implicate other cell lineages particularly proinflammatory cells, in autoimmune disease progression. Furthermore, our results provide evidence for an additional role for Lyn kinase, distinct from its catalytic activity, in regulating intracellular signaling pathways. The http://www.jimmunol.org/ Journal of Immunology, 2009, 182: 2020–2029.

he Src family kinases (SFKs)3 are important mediators of signaling cascades that promote BCR-mediated signaling by phos- in hematopoietic cells, including sig- phorylating proximal signaling molecules, including CD19 and the T naling through immunoreceptors as well as cytokine and ITAMs of the Ig␣/Ig␤ BCR subunits. Lyn is not essential, how- growth factor receptors (1, 2). Src kinases share similar domain ever, for the initiation of BCR signaling, as this process still occurs structures that contain SH2, SH3, and kinase domains and a reg- in Lyn-deficient cells, suggesting that this function is shared with ulatory domain at the carboxyl terminus. A defining feature of

other SFKs such as Blk and Fyn (5). by guest on October 2, 2021 SFKs is an intramolecular interaction between the SH2 domain In contrast, Lyn plays a critical and nonredundant role in the neg- 527 and phosphorylated Tyr in the C-terminal regulatory domain ative regulation of BCR signaling. Upon BCR ligation, Lyn phos- that holds the protein in an inactive state. Dephosphorylation of phorylates ITIM-bearing coreceptors, including CD22 and Fc␥RIIB, 527 Tyr by CD45 permits an open, active conformation of Src. Full resulting in the recruitment and activation of the SH2 416 Src kinase activity requires autophosphorylation of Tyr in the domain-containing 1 (SHP-1) and SHIP-1. In turn, these activation loop of the catalytic domain (3). phosphatases dephosphorylate receptors, adaptors, and other signaling Lyn is the predominant SFK expressed in B lymphocytes and intermediates to switch off the activation pathways (6). The conse- acts as both a positive and negative regulator of BCR signaling Ϫ Ϫ quence of Lyn deficiency is profound, exemplified by Lyn / mice pathways (4). Lyn is activated following BCR ligation and initiates which have uncontrolled BCR responses and develop lethal Ab-me- diated glomerulonephritis (7, 8). *Signal Transduction Laboratory, St. Vincent’s Institute, Fitzroy, Victoria, Australia; The phenotype of Lyn-deficient mice is complex, reflecting the †Cancer and Haematology and ‡Molecular Medicine Divisions, The Walter and Eliza expression of Lyn in all hematopoietic cells except T cells (9). For Hall Institute, Parkville, Victoria, Australia; and §Signal Transduction Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia example, analogous to its role in B cells, Lyn phosphorylates Received for publication September 19, 2008. Accepted for publication December ITIM-containing receptors in mast cells and macrophages, limiting 5, 2008. signaling in response to Fc␧RI and Fc␥R ligation, respectively (10, The costs of publication of this article were defrayed in part by the payment of page 11). In addition, Lyn also regulates signaling in response to cyto- charges. This article must therefore be hereby marked advertisement in accordance kines and growth factors, including stem cell factor (12), G-CSF with 18 U.S.C. Section 1734 solely to indicate this fact. (13), GM-CSF and M-CSF, (14), IL-4 (15), erythropoietin (EPO) 1 This work was supported by Program Grant 461219 and fellowships (to M.J.M., M.L.H., W.S.A., and D.J.H.) from the Australian National Health and Medical Research (16), and thrombopoietin (17), as well as regulating cytokine pro- Council, fellowships from the Australian Research Council (to B.T.K.), the Cancer Coun- duction itself (18). In general, the precise role of Lyn in each of cil of Victoria (to D.M.), and the Sylvia and Charles Viertel Foundation (to R.S.). This study was supported in part by research funding from MuriGen Pty Ltd. these signaling pathways has not been determined. Although it has been proposed that Lyn directly phosphorylates STAT5 in re- 2 Address correspondence and reprint requests to Dr. Robyn Starr, St. Vincent’s Institute, 9 Princes Street, Fitzroy, Victoria 3065, Australia. E-mail address: [email protected] sponse to EPO (16), EPO-dependent signaling appeared normal in 3 Abbreviations used in this paper: SFK, ; SH, Src homology; studies of Lyn-deficient erythroblasts (19). Alternatively, it has SHP-1, SH2 domain-containing phosphatase 1; EPO, erythropoietin; ENU, N-ethyl- been suggested that there may be cross-talk between cytokine and N-nitrosourea; Syk, spleen ; IC, immune complex; wt/WT, wild type. signaling and signal transduction through Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 ITIM-containing receptors (20). www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803127 The Journal of Immunology 2021

Table I. Colony formation by cells from Lynϩ/ϩ, LynMld4/Mld4, and LynϪ/Ϫ mice

No. of Colonies.

Cells Cultured Stimulus Bl G GM M Eo Meg

Adult bone marrow (25,000) Lynϩ/ϩ GM-CSF 0 Ϯ 021Ϯ 34Ϯ 132Ϯ 11 3 Ϯ 10Ϯ 0 G-CSF 0 Ϯ 010Ϯ 10Ϯ 00Ϯ 00Ϯ 00Ϯ 0 M-CSF 0 Ϯ 02Ϯ 14Ϯ 148Ϯ 13 0 Ϯ 00Ϯ 0 IL-3 5 Ϯ 322Ϯ 311Ϯ 416Ϯ 83Ϯ 24Ϯ 3 SCF ϩ IL-3 ϩ EPO 15 Ϯ 623Ϯ 210Ϯ 122Ϯ 33Ϯ 219Ϯ 4 LynMld4/Mld4 GM-CSF 0 Ϯ 018Ϯ 511Ϯ 4* 34 Ϯ 10 3 Ϯ 10Ϯ 0 G-CSF 0 Ϯ 011Ϯ 30Ϯ 00Ϯ 00Ϯ 00Ϯ 0 M-CSF 0 Ϯ 03Ϯ 15Ϯ 161Ϯ 80Ϯ 00Ϯ 0 IL-3 9 Ϯ 317Ϯ 410Ϯ 524Ϯ 53Ϯ 14Ϯ 1 SCF ϩ IL-3 ϩ EPO 13 Ϯ 228Ϯ 912Ϯ 226Ϯ 83Ϯ 223Ϯ 3 LynϪ/Ϫ GM-CSF 1 Ϯ 128Ϯ 811Ϯ 646Ϯ 16 3 Ϯ 10Ϯ 0 G-CSF 0 Ϯ 016Ϯ 5* 0 Ϯ 00Ϯ 00Ϯ 00Ϯ 0 M-CSF 0 Ϯ 03Ϯ 18Ϯ 3* 81 Ϯ 20* 0 Ϯ 00Ϯ 0 IL-3 11 Ϯ 321Ϯ 311Ϯ 330Ϯ 3* 2 Ϯ 26Ϯ 1 SCF ϩ IL-3 ϩ EPO 17 Ϯ 827Ϯ 3* 13 Ϯ 334Ϯ 8* 2 Ϯ 222Ϯ 8 Adult spleen (50,000) Lynϩ/ϩ GM-CSF 0 Ϯ 01Ϯ 10Ϯ 10Ϯ 10Ϯ 10Ϯ 0 Downloaded from M-CSF 0 Ϯ 00Ϯ 00Ϯ 02Ϯ 20Ϯ 00Ϯ 0 SCF ϩ IL-3 ϩ EPO 1 Ϯ 11Ϯ 11Ϯ 11Ϯ 10Ϯ 16Ϯ 5 LynMld4/Mld4 GM-CSF 0 Ϯ 03Ϯ 1* 1 Ϯ 12Ϯ 21Ϯ 10Ϯ 0 M-CSF 0 Ϯ 00Ϯ 00Ϯ 18Ϯ 2** 0 Ϯ 00Ϯ 0 SCF ϩ IL-3 ϩ EPO 4 Ϯ 2* 2 Ϯ 1* 2 Ϯ 34Ϯ 50Ϯ 023Ϯ 6** LynϪ/Ϫ GM-CSF 1 Ϯ 16Ϯ 3* 2 Ϯ 1* 15 Ϯ 7** 0 Ϯ 00Ϯ 0

M-CSF 0 Ϯ 00Ϯ 01Ϯ 116Ϯ 12 0 Ϯ 00Ϯ 0 http://www.jimmunol.org/ SCF ϩ IL-3 ϩ EPO 8 Ϯ 86Ϯ 43Ϯ 37Ϯ 4* 1 Ϯ 120Ϯ 11

Cultures were scored after 7 days of incubation. Data are mean colony numbers in the entire culture Ϯ SDs counted in cultures stained with acetyl cholinesterase, Luxol Fast Blue, and hematoxylin. Bl, blast; G, granulocytic; GM, granulocyte-macrophage; M, macrophage; Eo, eosinophil; Meg, megakaryocyte colonies; SCF, stem cell factor. .p Ͻ 0.01, relative to Lynϩ/ϩ data ,ءء p Ͻ 0.05 and ,ء

In this study, we describe the characterization of mice express- Genetic mapping ing a kinase-dead allele of Lyn. We show that many of the abnor- Ϫ/Ϫ Mld4 Mld4 (m/m Mpl ϩ/ϩ by guest on October 2, 2021 Ϫ/Ϫ Male C57BL/6 / ) mice were mated to malities typical of Lyn mice are substantially ameliorated in ϩ/Ϫ Mpl 129/SV females. Random pairs of F1 mice were mated, a quarter mice expressing kinase-dead Lyn. In particular, despite similar de- of which produced affected m/m F2 mice, which were identified using flow fects in the B cell compartment, including the extent of B lym- cytometry of peripheral blood samples. DNA from 16 affected F2 mice and phopenia and strength of BCR signaling, few mutant mice develop 15 unaffected siblings was genotyped for 64 simple sequence length poly- morphisms spaced evenly genome-wide (23), upon which Mld4 was as- autoantibodies and autoimmune disease development is markedly signed to the proximal region of 4. The candidate interval was Ϫ/Ϫ reduced compared with Lyn mice. Our data indicate that B cell refined via analysis of additional MIT and in-house CA repeat markers in extrinsic factors play a critical role in the development of autoim- the region. mune disease in the absence of Lyn and also provide evidence for Sequencing and genotyping an additional role for Lyn kinase, distinct from its catalytic activ- ity, in regulating intracellular signaling pathways. DNA was prepared from tail biopsy and the coding exons and splice junc- tions of candidate were PCR amplified and products were treated with ExoSap-IT (GE Healthcare). Sequencing was conducted using Big- Materials and Methods Dye Terminator chemistry (Applied Biosystems) and reaction mixtures Generation and screening of mutant mice were resolved and analyzed on an Applied Biosystems 3730S Genetic An-

Ϫ/Ϫ alyzer at the Micromon DNA Sequencing Facility (Monash University, Male Mpl C57BL/6 mice (21) were treated with N-ethyl-N-nitrosourea Clayton, Victoria, Australia). Residue numbering corresponding to chicken (ENU) as previously described (22). ENU-treated mice were mated with c-Src is used. Genotyping of the Mld4 mutation was performed by PCR isogenic females to yield first generation (G1) progeny. G1 mice were in- amplification of exon 12 of Lyn, followed by digestion with Hpy8I (Fer- tercrossed to yield G2 progeny, which were brother/sister mated to produce mentas). The Mld4 mutation destroys an Hpy8I site, resulting in a 350-bp third generation (G3) mice. G3 mice were bled at 7 wk of age and periph- band instead of two bands of ϳ180 bp. eral blood cell values were determined using an Advia 120 automated hematological analyzer (Bayer). The percentage of B and T lymphocytes in Abs and flow cytometry peripheral blood was determined using flow cytometry. Upon isolation in ϩ/ϩ aG3 pedigree, multilineage defect 4 (Mld4) was bred to Mpl C57BL/6 Splenocyte suspensions were prepared by dissociating total spleens and mice. All animals used in the studies described were on a Mplϩ/ϩ C57BL/6 erythrocytes were lysed by incubation in 5 ml of warmed 156 mM ammo- genetic background, unless otherwise noted. LynϪ/Ϫ mice, backcrossed nium chloride, pH 7.3 (red cell removal buffer) at 37°C for 3 min. Leu- onto the C57BL/6 background for 20 generations, were as described pre- kocytes were enriched in blood samples by lysing erythrocytes twice in red viously (7). Mice were routinely housed in clean, conventional facilities cell removal buffer. Cells were pretreated with a blocking Ab against Fc␥R, and were used for experiments at 7–10 wk of age, unless otherwise de- 2.4G2, then stained with Abs specific for CD45R/B220 (clone RA3-6B2), scribed. Mice were housed side-by-side in identical conditions, with the CD90.2/Thy-1.2 (53-2.1), CD71 (C2), Ly-76 (Ter-119), Ly-6G/Gr-1 exception of mice used for the colony assays (described in Table I and see (RB6-8C5), CD11b/Mac-1 (M1/70), CD21/CD35 (7G6), CD23 (B3B4), Fig. 3, A and B) and the B cell analyses (see Fig. 2, A–D and F–H). The IgM (II/41), CD40 (3/23), CD19 (clone 1D3), MHC class II (AF6–120.1), B cell phenotyping data were confirmed subsequently in mice housed in CD5 (53-7.3), and CD43 (S7; BD Biosciences) and analyzed on a identical conditions (data not shown). All experiments using mice were FACSCalibur flow cytometer (BD Biosciences). Dead cells were excluded approved by St. Vincent’s Hospital Animal Ethics Committee. based on propidium iodide staining. 2022 AUTOIMMUNITY IS REDUCED IN Lyn KINASE-DEAD MUTANTS

ELISA marrow or 50,000 spleen cells and were stimulated by final concentrations of one or more of the following purified recombinant mouse pro- Anti-dsDNA Abs (IgG) were quantitated in mouse serum using an ELISA duced in-house (The Walter and Eliza Hall Institute) or purchased from (Alpha Diagnostic International) according to the manufacturer’s in- PeproTech: 10 ng/ml GM-CSF, 10 ng/ml M-CSF, 10 ng/ml IL-3, 100 structions. IgE and IgM were quantitated in mouse serum using a BD ng/ml stem cell factor, 2 IU/ml EPO, and 10 ng/ml human G-CSF. Culture Biosciences OptEIA IgE ELISA set or anti-mouse IgM Abs (capture: clone conditions and analysis were as described previously (26). II/41, detection: clone R6-60.2; BD Biosciences). Statistical analyses Histology and immunohistochemistry Significant differences were determined by Student’s two-tailed t tests for Kidneys were fixed in 10% buffered formalin, sectioned, and stained with independent events. H&E. Frozen kidney sections were fixed in acetone for 5 min at room temperature, followed by staining with FITC-conjugated goat anti-mouse IgG (Millipore) or rabbit anti-mouse C3c complement (Abcam). Results Generation of the Mld4 pedigree Immunoblotting To identify genes important for immune regulation, we conducted B cells were purified from mouse spleen by negative selection using a forward genetic screen for recessive mutations using the chem- MACS beads. Briefly, following RBC lysis of splenocyte suspensions, ical mutagen ENU. The mutant pedigrees used in this study had non-B cells were stained with biotinylated Abs specific for Mac-1, Gr-1, originally been generated in a genetic screen for mutations that Ly-76 (TER-119), CD5 (clone 53-7.3), and CD43 (S7), washed, incubated Ϫ Ϫ with streptavidin microbeads (Miltenyi Biotec) and depleted using a attenuate thrombocytopenia using Mpl / mice, which lack the MACS column (Miltenyi Biotec). The purity of the B cell preparations was receptor for thrombopoietin (27). We isolated a pedigree in which Ͼ ␮ typically 90%. Purified B cells were stimulated with 30 g/ml goat anti- multiple animals showed reduced numbers of white blood cells,

Ј Downloaded from mouse IgM F(ab )2 (Jackson Immunoresearch Laboratories) at 37°C and Ϫ/Ϫ lysed for1hinTriton X-100 lysis buffer (1% Triton X-100, 1% glycerol, RBC, and platelets compared with control Mpl mice in their 150 mM NaCl, 20 mM Tris (pH 7.5), and 2 mM EDTA) supplemented with peripheral blood (Fig. 1A). Flow cytometric analysis of the blood protease and phosphatase inhibitors (Complete protease inhibitor and from mice of this pedigree, named multilineage-deficient 4 (Mld4), PhosSTOP; Roche). The nuclear and cellular debris were removed by cen- showed that the lymphopenic phenotype was due predominantly to trifugation. SDS-PAGE of total cell lysates and immunoblots using Abs a loss of B, rather than T, lymphocytes (Fig. 1B). specific for ERK1/2 (K-23/sc-153; Santa Cruz Biotechnology), Lyn (Lyn- 01; Abcam), phosphorylated forms of CD19 (Tyr513; catalog no. 3571), The Mld4 mutation was mapped to a 6.48-Mb region of chro- Akt (Ser473; catalog no. 9271), Stress-activated /JNK mosome 4 using a standard positional cloning approach (Fig. 1C). http://www.jimmunol.org/ (Thr183/Tyr185; catalog no. 9251), SHIP-1 (Tyr1020; catalog no. 3941), The gene encoding Lyn kinase, located within this region, was 323 202 spleen tyrosine kinase (Syk; Tyr ; catalog no. 2715), ERK1/2 (Thr / considered a strong candidate because LynϪ/Ϫ mice are profoundly Tyr204; catalog no. 9106), and c-Src Tyr416 (catalog no. 2101), all from Cell Signaling Technology, phosphotyrosine (4G10; Millipore), or phospho- B lymphopenic (7, 8). Sequence analysis of the exons and intron/ SHP-1 (Tyr536; catalog no. SP1571; ECM Biosciences) were performed exon boundaries of Lyn revealed a C-to-A conversion of base 25 in Ϫ Ϫ essentially as described previously (24). Proteins were visualized by ECL exon 12 which was not found in either parental Mpl / nor (Amersham Biosciences) following incubation of membranes with HRP- C57BL/6 wild-type (wt) mice (Fig. 1D). This mutation is predicted coupled secondary Abs (sheep anti-rabbit IgG; Chemicon International, or to cause the substitution of a lysine for a threonine codon at residue anti-mouse IgG; Amersham Biosciences). 429 in the highly conserved Src activation loop (Fig. 1E). by guest on October 2, 2021 Calcium flux B cell lymphopenia is similar in LynMld4/Mld4 and LynϪ/Ϫ mice Calcium flux was performed essentially as previously described (25). Ϫ Ϫ Briefly, splenocytes (1 ϫ 107) were loaded with Indo-1 AM (Molecular B lymphopenia is a hallmark of Lyn / mice (5, 7, 8). To deter- Probes) and stained with Abs specific for IgM and CD23. The ratio of mine the effect of the Mld4 mutation on B cell development, we blue:green fluorescence (FL5/FL4) was measured on an LSR flow cytom- ␮ analyzed splenic B cell subsets by flow cytometry. Similar to eter (BD Biosciences) before and after stimulation of cells with 4 g/ml Ϫ/Ϫ Ј Lyn mice, there was a profound reduction in the proportion of goat anti-mouse IgM F(ab )2. Flux was measured for at least 5 min. transitional stage 2, marginal zone, and CD23ϩ (mainly follicular) In vitro kinase assays B cells in LynMld4/Mld4 spleen (Fig. 2A). This B lymphopenia was Mld4/Mld4 Cell lysates from purified splenic B cells (107 cells/100 ␮l) were precleared reflected in reduced total cellularity in both Lyn and Ϫ Ϫ with protein G-Sepharose (Fast Flow 4; GE Healthcare) for1hat4°C. Lyn Lyn / spleens and a significant decrease in follicular and mar- was immunoprecipitated from precleared lysates (100 ␮l/immunoprecipi- ginal zone B cell number (Fig. 2, B and C). ␮ tation) for2hat4°Cusing 1 g of anti-Lyn Ab (sc-15; Santa Cruz Bio- Previous studies have characterized differences in the expres- technology) and 20 ␮l of protein G-Sepharose. Control immunoprecipita- sion of cell surface molecules on B cells between wt and tions were performed with protein G-Sepharose alone. Immunoprecipitates Ϫ Ϫ were washed three times in Triton X-100 lysis buffer and twice in kinase Lyn / mice, which are thought to reflect compensatory adjust- buffer (50 mM Tris (pH 6.8), 50 mM NaCl, 0.1% Triton X-100, 10 mM ments in response to uncontrolled BCR signaling (28). Surpris- Mld4/Mld4 MgCl2, 5 mM MnCl2, and 0.1 mM sodium vanadate) and resuspended in ingly, receptor density on Lyn follicular B cells dif- 20 ␮l of kinase buffer supplemented with 0.5 mM DTT, 5 ␮M Src optimal Ϫ/Ϫ peptide (60222-1; AnaSpec), and 0.5 ␮M[␥-32P]ATP (3000 Ci/mmol; fered from that of Lyn cells. As reported previously (28), Ϫ/Ϫ PerkinElmer) at 30°C. At regular intervals, 4-␮l aliquots of supernatant Lyn B cells expressed lower levels of CD21 compared with were transferred onto 2 ϫ 2-cm squares of Whatman P81 chromatography wt, but this difference was not seen in LynMld4/Mld4 B cells (Fig. paper, which were washed, shaking, for1hin0.4% orthophosphoric acid, 2D). In contrast, surface IgM and CD19 expression were in- rinsed briefly in 100% ethanol, air dried, immersed in scintillation fluid variably increased in LynMld4/Mld4 B cells, as was CD23 expres- (Ultima Gold; PerkinElmer), and counted on a beta counter (Tri-Carb 2500 sion (Fig. 2, E–G). A small but significant increase in CD19 and TR; PerkinElmer). Kinase reactions were also performed using lysates (100 Ϫ Ϫ ␮g of precleared extract/immunoprecipitation) from macrophages derived CD23 expression was also seen in Lyn / cells compared with wt, from the bone marrow of mice by culture in L cell-conditioned medium for but this difference was less pronounced than for LynMld4/Mld4 cells 6–7 days. Equivalent Lyn protein input for B cell and macrophage in vitro (Fig. 2, F and G). No difference in surface IgM expression on kinase reactions was established by immunoblotting lysates with anti-Lyn Ϫ/Ϫ Ab (Lyn-01; Abcam). Lyn cells was observed (Fig. 2E). B cells were activated in both LynϪ/Ϫ and LynMld4/Mld4 mice, with significant increases in MHC Colony assays class II expression (Fig. 2H). Elevated levels of circulating IgE and Ϫ/Ϫ Clonal cultures were prepared in 35-mm petri dishes containing 1 ml of IgM, characteristic of Lyn mice (7, 8, 15), were also seen in DMEM with 20% FCS and 0.3% agar. Cultures contained 25,000 bone LynMld4/Mld4 mice (Fig. 2I). The Journal of Immunology 2023

FIGURE 1. Mld4 is a missense mutation in Lyn. A, Analysis of peripheral blood pa- rameters in 7-wk-old control (MplϪ/Ϫ, n ϭ 138) or Mld4 (n ϭ 42) mice. Values shown -p Ͻ 0.001. B, B lym ,ءءء ,are mean ϩ SEM phopenia in peripheral blood of Mld4 mice. C, Mapping haplotypes for Mld4. Markers used are indicated and defining recombina- tion events are shaded gray. Black squares indicate C57BL/6 homozygosity and divided squares indicate C57BL/6:129/Sv heterozy- gosity. The candidate interval was refined to 6.5 Mb between JCCA197 and D4Mit101. D, C to A transversion in exon 12 of Lyn in Mld4 genomic DNA. E, Alignment of amino acid sequences corresponding to the Src ac- tivation loop in the indicated species using chicken c-Src numbering. Arrows indicate Downloaded from Y416 (Lyn Y397), which increases kinase activity when phosphorylated, and T429 (Lyn T410), which is mutated to K429 in Mld4 mice. http://www.jimmunol.org/

The B lymphopenia was mainly restricted to mature B cell pop- megaly in LynϪ/Ϫ mice (average spleen weight of 0.61 g in 1-year- ulations. No defect in early B cell development in the bone marrow old mice) (14). In contrast, no difference was observed between the was seen, while the proportion of recirculating mature B cells in spleen weight of wt and LynMld4/Mld4 mice (Fig. 3D). Similar to the bone marrow was reduced in both LynMld4/Mld4 and LynϪ/Ϫ LynϪ/Ϫ mice, erythropoiesis was enhanced in LynMld4/Mld4 mice, mice as expected (Fig. 2, J and K). The effect of Lyn deficiency in with a large increase in the proportion of CD71ϩTer-119ϩ eryth- by guest on October 2, 2021 the B1 cell compartment has been contentious, with some studies roblasts in the spleens of LynMld4/Mld4 mice (Fig. 3E). indicating an increase in B1 B cell number in LynϪ/Ϫ mice (5, 8) Mld4 and others reporting little or no change (7, 29–31). We found no Lyn is a kinase-dead allele of Lyn consistent change in the number of B1a or B1b cells in the peritoneal To address how the Mld4 mutation affects Lyn function, we mea- cavity of either LynMld4/Mld4 or LynϪ/Ϫ mice (Fig. 2, L and M). The sured the catalytic activity of Lyn using an in vitro transkinase proportion and number of peritoneal T cells, however, was increased assay. Lyn immunoprecipitated from purified wt splenic B cell in both LynMld4/Mld4 and LynϪ/Ϫ mice, which may be a reflection of lysates effectively phosphorylated Src optimal peptide over a time an ongoing immune response in these mice (Fig. 2, L and M). course (Fig. 4A). In contrast, despite normal levels of Lyn in LynMld4/Mld4 B cells, mutant Lyn immunoprecipitated from these Intermediate enhancement of myelopoiesis and erythropoiesis in cells completely lacked kinase activity. Similar results were seen Mld4/Mld4 Lyn mice using macrophage lysates (Fig. 4B). In addition, Lyn immunopre- Hypersensitivity to myeloid growth factors in the absence of Lyn cipitated from LynMld4/Mld4 macrophage lysates was completely has been shown to result in an age-dependent increase in myeloid, devoid of activity in an autokinase assay (data not shown). Al- erythroid, and primitive hematopoietic progenitor numbers and the though our results strongly suggest that the LynMld4/Mld4 mutant development of splenomegaly and macrophage tumors (14, 19). protein is catalytically inactive, we cannot completely exclude the To assess the effect of the Mld4 mutation on myelopoiesis, we possibility that some activity remains that is below the threshold determined the myeloid progenitor number in 8-wk-old Lynϩ/ϩ, for detection, but given the sensitivity of these assays, any residual LynMld4/Mld4, and LynϪ/Ϫ mice using colony assays. As reported activity would be expected to be minimal. Surprisingly, mutant previously, there was a significant increase in the number of col- Lyn expressed in LynMld4/Mld4 B cells was phosphorylated on onies generated in response to GM-CSF, M-CSF, or IL-3 in both Tyr416 in the catalytic domain in response to BCR ligation, albeit the bone marrow and spleen of LynϪ/Ϫ mice (Table I and Fig. 3, to a lesser extent than in wt samples (Fig. 4C). Although this A and B) (14, 19). The number of LynMld4/Mld4 myeloid progenitors critical tyrosine residue is generally considered to be an autophos- was also enhanced relative to Lynϩ/ϩ cells, but in general was phorylation site, these results indicate that it can be phosphorylated intermediate between that of Lynϩ/ϩ and LynϪ/Ϫ cells (Table I and in trans by other kinases as well. Ϫ/Ϫ Fig. 3, A and B). Unlike Lyn mice, which develop a mye- Ϫ/Ϫ Mld4/Mld4 loproliferative syndrome, the increase in myelopoiesis in BCR signaling defects are similar in Lyn and Lyn LynMld4/Mld4 mice resulted in only minor increases in the propor- B cells tion of neutrophils and macrophages in peripheral lymphoid tis- Following BCR ligation, Lyn is rapidly activated and phosphory- sues, even in 1-year-old mice (Fig. 3C) (14). The expanded splenic lates substrates involved in both the positive and negative regula- myeloid compartment contributes to the development of spleno- tion of BCR signaling (5, 32, 33). We compared the strength of 2024 AUTOIMMUNITY IS REDUCED IN Lyn KINASE-DEAD MUTANTS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. B lymphopenia in LynMld4/Mld4 mice is similar to that of LynϪ/Ϫ mice. Splenocytes from 8-wk-old Lynϩ/ϩ (ϩ/ϩ), LynMld4/Mld4 (m/m), and LynϪ/Ϫ (Ϫ/Ϫ) mice were stained with the indicated Abs and analyzed by flow cytometry. A, Dot plots gated on CD23Ϫ and CD23ϩ populations show the proportions of marginal zone (MZ) and transitional stage 1 (T1) (middle), and transitional stage 2 (T2) and follicular (Fo) (lower) B cells. Total splenocytes (B) and numbers of follicular and marginal zone/B-1 B cells (C) per spleen are graphed at the right. Splenocytes were stained with Abs specific for CD21 (D), surface IgM (sIgM; E), CD19 (F), CD23 (G), or MHC class II (H) and analyzed by flow cytometry. The median fluorescence intensity (MFI) ϩ SEM of data gated on CD23ϩ (follicular, T2) B cells is shown. I, Levels of IgE and IgM in the serum of resting 27- to 30-wk-old Lynϩ/ϩ and LynMld4/Mld4 mice (n ϭ 6–12). Horizontal bar indicates mean level. J, Bone marrow from Lynϩ/ϩ, LynMld4/Mld4, and LynϪ/Ϫ mice was stained with the indicated Abs and The Journal of Immunology 2025

FIGURE 3. Enhanced myelopoie- sis and erythropoiesis in LynMld4/Mld4 mice. Progenitors responsive to GM- CSF, IL-3, or M-CSF in the bone marrow (A) and spleen (B) of mice of the indicated genotypes were assessed by semisolid agar assays in vitro. Val- ues shown are mean ϩ SD of n ϭ 4 mice/genotype (n ϭ 2 mice/genotype p Ͻ ,ء .(for IL-3 stimulation of spleen p Ͻ ,ءءء p Ͻ 0.01; and ,ءء ;0.05 0.001, relative to Lynϩ/ϩ data. C, Limited expansion of myeloid cells in LynMld4/Mld4 mice. Flow cytometry of spleen populations from Lynϩ/ϩ (43 wk) and LynMld4/Mld4 (51 wk) mice. Spleen weights for these mice were 0.086 g (Lynϩ/ϩ) and 0.135 g (LynMld4/Mld4). D, Spleen weight of Downloaded from mice of the indicated age and genotype, on a MplϪ/Ϫ genetic background, is shown. Horizontal bar indicates mean weight. E, Flow cytometry of spleen populations from 8-wk-old Lynϩ/ϩ and LynMld4/Mld4 mice showing the expan- http://www.jimmunol.org/ sion of Ter-119ϩCD71ϩ erythroblasts in LynMld4/Mld4 spleen. Mature RBC were excluded from the analysis. Spleen weights for these mice were 0.095 g (Lynϩ/ϩ) and 0.081 g (LynMld4/Mld4). by guest on October 2, 2021

BCR signaling in LynMld4/Mld4 and LynϪ/Ϫ B cells to determine and CD22, enabling recruitment, phosphorylation, and activation whether expression of a kinase-dead Lyn allele could lead to par- of the inositol phosphatase SHIP-1 and tyrosine phosphatase tial regulation of these pathways. Although BCR-induced tyrosine SHP-1 (25, 28, 32–34). BCR ligation failed to induce phosphory- phosphorylation of cellular proteins was evident for all three ge- lation of SHIP-1 in LynMld4/Mld4 and LynϪ/Ϫ lysates, whereas li- notypes, phosphorylation of several proteins was absent or sub- gand-induced phosphorylation of SHP-1 was reduced (Fig. 5B). stantially reduced in LynMld4/Mld4 and LynϪ/Ϫ lysates relative to The tyrosine kinase Syk also becomes phosphorylated on several Lynϩ/ϩ (Fig. 5A). The phosphorylation of these proteins was sites following BCR ligation, resulting in Ca2ϩ mobilization. One equally reduced in LynMld4/Mld4 and LynϪ/Ϫ lysates. A 130-kDa of these sites, Tyr323, has been shown to be a negative regulatory protein, inducibly phosphorylated in Lynϩ/ϩ but to a lesser extent site that is phosphorylated by Lyn, resulting in suppression of the in LynMld4/Mld4 and LynϪ/Ϫ cells, was subsequently confirmed to Ca2ϩ signal (35). Consistent with this, phosphorylation of Tyr323 be CD22, an inhibitory coreceptor that is directly phosphory- on Syk was substantially reduced in the absence of active Lyn (Fig. lated by Lyn (Fig. 5A and data not shown) (28, 32, 34). Other 5B). Thus, negative regulatory actions attributable to Lyn appeared proteins of low molecular mass were more highly phosphory- to be perturbed to a similar extent in LynMld4/Mld4 and LynϪ/Ϫ lated in LynMld4/Mld4 and LynϪ/Ϫ lysates than in wt, suggesting cells. increased activity of another tyrosine kinase or decreased ac- Lyn also has a key role in the initiation of pathways that pro- tivity of a tyrosine phosphatase in cells lacking catalytically mote B cell activation, although this role is likely to be shared with active Lyn (Fig. 5A). other SFKs (5). The requirement for Lyn in CD19 phosphorylation Further detailed biochemical analyses of cellular lysates were is controversial, with reports in the literature either supporting or undertaken with a panel of Abs that detect phosphorylated proteins refuting an essential role for Lyn in this process (36, 37). In our involved in B cell signaling. BCR ligation results in Lyn-induced experiments, CD19 phosphorylation did not appear to depend on phosphorylation of ITIMs on the inhibitory coreceptors Fc␥RIIB Lyn activity, as CD19 phosphorylation was induced with similar

analyzed by flow cytometry. The percentage of pro- and pre-B cells (B220lowIgMϪ), immature B cells (B220lowIgMϩ), and recirculating mature B cells (B220highIgMϩ) is indicated. K, Total bone marrow cell count/femur from mice of the indicated genotypes is shown. L, Flow cytometry analysis of peritoneal cells showing the percentage of T cells (CD5highIgMϪ), B1a (CD5lowIgMϩ), and B2/B1b (CD5ϪIgMϩ) B cells. M, Absolute numbers of T, B1a, B1b, and conventional B2 cells from the peritoneal cavity. Cells were stained with IgM, CD5, and CD43 to distinguish B1b (IgMϩCD5ϪCD43ϩ) from B2 ,ءءء p Ͻ 0.01; and ,ءء ;p Ͻ 0.05 ,ء .IgMϩCD5ϪCD43Ϫ) cells (data not shown). Data in bar graphs represent mean ϩ SEM of n ϭ 4–8 mice/genotype) p Ͻ 0.001, relative to Lynϩ/ϩ data; ††, p Ͻ 0.01 and †††, p Ͻ 0.001, relative to LynMld4/Mld4. 2026 AUTOIMMUNITY IS REDUCED IN Lyn KINASE-DEAD MUTANTS

FIGURE 4. LynMld4 is a kinase-dead allele of Lyn. In vitro kinase activity of Lyn immu- noprecipitated from purified splenic B cells (A) or bone marrow-derived macrophages (B) from mice of the indicated genotypes was as- sayed using the Src optimal peptide substrate. Insets show that lysates used for the immuno- precipitations contained similar amounts of Lyn. Control samples refer to wt lysates im- munoprecipitated with protein G-Sepharose in the absence of Lyn Ab. Lyn kinase activity from B cell or macrophage lysates is represen- tative of two or three independent experiments, respectively. C, The regulatory tyrosine Tyr416 of c-Src is phosphorylated in trans in LynMld4/Mld4 B cells. Purified splenic B cells were stimulated with 30 ␮g/ml anti-IgM Ј F(ab )2 for the indicated times and immuno- blotted with Abs specific for phospho-Tyr416 of Src (upper panel), Lyn (middle panel), and ERK2 as a loading control (lower Downloaded from panel).Notethatimmunoblottingwithananti- ERK Ab indicates equal protein loading in C. This is not apparent with the anti-Lyn Ab, as this Ab does not recognize the phosphor- ylated form of Lyn. http://www.jimmunol.org/ kinetics and intensity in wt, LynMld4/Mld4, and LynϪ/Ϫ splenic B Development of autoimmune disease is attenuated in cells (Fig. 5C). Conversely, BCR ligand-induced activation of LynMld4/Mld4 mice ERK1/2, JNK, and Akt was grossly elevated in both LynMld4/Mld4 Ϫ/Ϫ and LynϪ/Ϫ lysates relative to wt (Fig. 5C). Further, calcium flux Previous studies have shown that Lyn mice develop Ab-medi- was amplified to a similar extent in LynMld4/Mld4 and LynϪ/Ϫ cells ated autoimmune disease as they age, with autoantibodies detect- (Fig. 5D). able in virtually all LynϪ/Ϫ mice by 4 mo of age (5, 7, 8). We by guest on October 2, 2021

FIGURE 5. BCR hyperresponsiveness is similar in LynϪ/Ϫ and LynMld4/Mld4 B cells. A–C, Purified splenic B cells were stimu- ␮ Ј lated with 30 g/ml anti-IgM F(ab )2 for the indicated times and immunoblotted with the indicated Abs. A, Proteins that are hypo- phosphorylated in LynMld4/Mld4 (Mld4) and LynϪ/Ϫ B cells in response to BCR ligation are indicated by an arrow (the top arrow in- dicates CD22) and the asterisk indicates those that are hyperphosphorylated. Arrow- head indicates the doublet corresponding to the two isoforms of Lyn. An Ab specific for ERK1/2 was used as a loading control. Note that the loading control for B is the same as for C because the same filter was immuno- blotted multiple times. D, Time course of calcium flux in splenic IgMϩCD23ϩ B cells from mice of the indicated genotypes stim- ␮ Ј ulated with 4 g/ml F(ab )2 anti-IgM. The Journal of Immunology 2027

mice assayed (LynMld4/Mld4, n ϭ 7; LynϪ/Ϫ, n ϭ 6), along with the deposition of complement C3 (86% of LynMld4/Mld4 (n ϭ 7) and 83% LynϪ/Ϫ (n ϭ 6) kidneys positive for C3; Fig. 6B, lower pan- els and data not shown). Neither IgG nor C3 aggregates were seen in kidney sections from wt mice of similar ages (n ϭ 4; Fig. 6B). These results suggest that despite being undetectable in many mice by ELISA, autoantibodies are present in young LynMld4/Mld4 and LynϪ/Ϫ mice at sufficient concentrations to aggregate in glomeruli. Alternatively, autoantibodies specific for Ags other than dsDNA or nuclear Ags may be present that would not be detected by this assay. More than 90% of LynϪ/Ϫ mice aged 6 wk or older show his- tological signs of autoimmune disease (7). Despite the appearance of autoantibodies and IC deposition in the glomeruli, there was no evidence of either hematopoietic cellular infiltration in the kidney, pancreas, or thyroid of LynMld4/Mld4 mice or major glomerular damage by histological staining (n ϭ 6 mice, 6–12 mo of age; Fig. 6C and data not shown). Histological changes in LynMld4/Mld4 kid- neys were minimal and were limited to mild hypercellularity ap- parent in one of the mice analyzed (12 mo of age; data not shown). Downloaded from This is in contrast to the severe glomerulonephritic changes char- acteristic of LynϪ/Ϫ mice, including global sclerosis, crescent for- mation and lobularity (7, 8). At 1 year of age, 90% of LynMld4/Mld4 mice were healthy (n ϭ 10; data not shown), whereas 40% of LynϪ/Ϫ mice had died by this age (25). These results suggest that the presence of kinase-dead Lyn affords protection against the fatal http://www.jimmunol.org/ glomerulonephritis characteristic of Lyn-deficient mice.

Discussion We describe here the characterization of a mutant mouse strain that FIGURE 6. Mice expressing a Lyn kinase-dead allele have attenuated expresses a kinase-dead allele of Lyn kinase. Despite displaying a autoimmune disease. A, Quantitation of anti-dsDNA IgG Abs in the serum Ϫ/Ϫ ϩ/ϩ ϩ ϩ ϭ ϩ/Ϫ ϩ Ϫ ϭ ϩ/Mld4 similar spectrum of defects to Lyn mice, the overall phenotypic of Lyn ( / ; n 2–5/age group), Lyn ( / ; n 6), Lyn Mld4/Mld4 (ϩ/m; n ϭ 3–4), LynMld4/Mld4 (m/m; n ϭ 9–23), LynϪMld4 (-/m; n ϭ 2–7), severity of Lyn mice is substantially reduced. In particu- Ϫ/Ϫ lar, the extent of splenomegaly, myeloid expansion, and autoanti- and Lyn (Ϫ/Ϫ; n ϭ 2–18) mice of the indicated ages. Horizontal bar by guest on October 2, 2021 Mld4/Mld4 p Ͻ 0.001, relative to Lynϩ/ϩ body production in Lyn mice is reduced compared with ,ءءء p Ͻ 0.05 and ,ء .indicates mean value Ϫ/Ϫ data of the same age group. B, IC and complement deposition are similar Lyn mice, and they do not develop the fulminant glomerulo- Ϫ Ϫ in LynMld4/Mld4 and LynϪ/Ϫ kidneys. Immunohistochemical analysis of kid- nephritis characteristic of Lyn / mice. neys from mice of the indicated genotypes (8 wk old; original magnifica- The Mld4 mutation is predicted to cause the substitution of a tion, ϫ400) stained with FITC-conjugated Abs specific for IgG (upper lysine for a threonine codon in the Src activation loop. This non- panels) and complement C3 (lower panels). C, Glomerulonephritis is ame- conservative substitution, resulting in a change of charge, is pre- Mld4/Mld4 liorated in Lyn mice. Representative renal glomeruli of 1-year-old dicted to affect Lyn structure in this region, but it is unclear how mice of the indicated genotypes (original magnification, ϫ400) showing catalytic activity is affected by this change. The tyrosine residue in normal pathology of LynMld4/Mld4 glomeruli. the activation loop critical for catalytic activity, Tyr416, is still accessible to kinases, but phosphorylation of this residue is clearly quantitated serum autoantibodies (IgG isotype) recognizing insufficient for kinase activity in the presence of the Mld4 muta- dsDNA from mice at various ages by ELISA. In young mice (7–9 tion. Although there is indisputable evidence that Tyr416 is an au- wk), autoantibodies were detectable in only 10% of LynMld4/Mld4 tophosphorylation site in all SFK members (3), our data indicate sera (mean anti-dsDNA Abs ϭ 29.0 ␮g/ml, n ϭ 10; Fig. 6A). that other tyrosine kinases, most likely other SFKs, can also phos- Serum levels of these Abs increased moderately in older mice phorylate this site. Phosphorylation of Tyr416, located between the (mean, 111 ␮g/ml at 42–54 wk, n ϭ 9; Fig. 6A). Slightly higher two lobes of the kinase domain, is thought to trigger a conforma- titers of anti-dsDNA Abs were measured in the sera of young tional reorganization of the activation loop, allowing displacement LynϪ/Ϫ mice (mean, 352 ␮g/ml at 7–9 wk, n ϭ 18; Fig. 6A), but of the C-terminal helix into the (38). It is conceivable dramatic increases in autoantibody concentration were evident as that this displacement may be affected by the Mld4 mutation. LynϪ/Ϫ mice aged (mean, 5.6 mg/ml at 27–30 wk, n ϭ 2; Fig. 6A). Given that there are multiple mutations present in the genome of The titer of anti-dsDNA Abs in the sera of compound hetero- LynMld4/Mld4 mice, there is a small possibility that unlinked muta- zygotes (LynϪMld4; Ϫ/m) was intermediate between that of tions segregating in these mice may affect the autoimmune phe- LynMld4/Mld4 and LynϪ/Ϫ mice. Similar results were obtained by notype. We consider this possibility to be unlikely, however, since measuring Abs to nuclear Ags (IgA, IgG, and IgM isotypes; data unlinked mutations would be segregating randomly and would not not shown). be present in all mice with the LynMld4/Mld4 genotype, leading to an Despite low or undetectable serum autoantibody levels in inconsistency in the phenotype of the LynMld4/Mld4 mice that is younger mice, glomerular immune complex (IC) deposition was contrary to our observation. The most likely explanation is that the evident in kidney sections from both LynMld4/Mld4 and LynϪ/Ϫ mutant Lyn protein retains some function in regulating signaling, mice (Fig. 6B, upper panels). These ICs, which contained both IgG at least in some lineages. Although catalytically inactive, intact and IgM aggregates, were evident in the kidneys of all 8-wk-old interaction motifs such as the SH2 and SH3 domains, as well as 2028 AUTOIMMUNITY IS REDUCED IN Lyn KINASE-DEAD MUTANTS multiple potential tyrosine phosphorylation sites, may influence alytically inactive Lyn to limit the activation of the proinflam- downstream signaling events by competing for interaction sites matory response to some degree. that might otherwise be occupied by other signaling molecules. For Collectively, our data indicate that autoimmune and inflamma- instance, it is conceivable that mutant Lyn could limit positive tory disease associated with Lyn deficiency is attenuated in mice signaling events in both B and myeloid cells, emanating from the expressing a kinase-dead allele of Lyn. Future studies will address surface receptors BCR/CD19 and Fc␥RI/III respectively, by block- whether signaling cascades differ in intensity in LynMld4/Mld4 ing the recruitment and activation of other Src kinases. Although phagocytes and will characterize the effect of the Mld4 mutation on we did not observe signaling differences between LynMld4/Mld4 and Lyn function in dendritic cells and other myeloid cells. LynϪ/Ϫ B cells, subtle differences may occur in vivo when cells are exposed to physiological levels of stimuli or in other signaling Acknowledgments pathways not examined in this study. Indeed, significant differ- We thank J. Corbin, L. DiRago, and S. Mifsud for first-class technical ences in autoantibody titers and B cell surface receptor expression assistance; Drs. S. Nutt and P. Lock for the generous provision of flow Mld4/Mld4 Ϫ/Ϫ suggest that Lyn and Lyn B cells are not functionally cytometry and Western blot Abs; Dr. D. Tarlinton for the provision of mice equivalent. The most dramatic differences we observed, however, and advice; Drs. K. Graham, M. Fuchsberger, and K. Greig for assistance occurred in the myeloid compartment where myeloid expansion is with immunohistochemistry and calcium flux experiments; and M. Rowe, observed in LynϪ/Ϫ but not LynMld4/Mld4 mice, strongly suggesting R. Branch, C. Van Puyenbroek, K. Hibbins, S. Ross, and S. Guzzardi for a role for proinflammatory cell types in the development of expert animal husbandry. autoimmunity. Several mouse models have been generated which rescue the

Disclosures Downloaded from Ϫ/Ϫ autoimmune phenotype of Lyn mice while retaining other as- We disclose that this work was supported in part by Murigen Pty Ltd for pects, including B lymphopenia. Mice deficient in both Lyn and whom W.S.A., B.T.K., and D.J.H. consult and/or have a financial interest. Ϫ Ϫ Ϫ Ϫ Btk, as well as Lyn / Cd19 / mice, do not develop autoimmu- The remaining authors have no financial conflict of interest. nity, most likely due to reduced BCR signaling in the absence of Btk or CD19 (29, 30, 39). Introduction of a transgene expressing Ϫ/Ϫ Ϫ/Ϫ References a low level of Btk into Lyn Btk mice restored B cell num- 1. Latour, S., and A. Veillette. 2001. Proximal protein tyrosine kinases in immu- bers and BCR signaling hypersensitivity but failed to restore au- noreceptor signaling. Curr. Opin. Immunol. 13: 299–306. http://www.jimmunol.org/ toimmunity, suggesting that BCR hyperresponsiveness is insuffi- 2. Gauld, S. B., and J. C. Cambier. 2004. Src-family kinases in B-cell development and signaling. Oncogene 23: 8001–8006. cient for the development of autoimmune disease, consistent with 3. Sun, G., L. Ramdas, W. Wang, J. Vinci, J. McMurray, and R. J. Budde. 2002. our data (31). Effect of autophosphorylation on the catalytic and regulatory properties of protein Autoimmune disease also failed to develop in mice lacking tyrosine kinase Src. Arch. Biochem. Biophys. 397: 11–17. 4. Xu, Y., K. W. Harder, N. D. Huntington, M. L. Hibbs, and D. M. Tarlinton. 2005. MyD88 and Lyn, indicating that TLR signaling contributes to the Lyn tyrosine kinase: accentuating the positive and the negative. Immunity 22: autoimmune phenotype and that inappropriate activation of other 9–18. cell lineages collaborates in autoimmune disease development 5. Chan, V. W., F. Meng, P. Soriano, A. L. DeFranco, and C. A. Lowell. 1997. Characterization of the B lymphocyte populations in Lyn-deficient mice and the (40). 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Colony-stimulating factors not only induce myeloid differ- cells and indication of autoimmune disease in lyn-deficient mice. Immunity 3: entiation, but also increase myeloid activity, including inflamma- 549–560. tory cytokine secretion and Ag-presenting activity, both of which 9. Lowell, C. A. 2004. Src-family kinases: rheostats of immune cell signaling. Mol. Immunol. 41: 631–643. may promote the progression of autoimmune disease. Indeed, renal 10. Parravicini, V., M. Gadina, M. Kovarova, S. Odom, C. Gonzalez-Espinosa, pathology was suppressed in autoimmune-prone MRL-Faslpr mice Y. Furumoto, S. Saitoh, L. E. Samelson, J. J. O’Shea, and J. Rivera. 2002. Fyn when generated on a M-CSF-deficient background (41). kinase initiates complementary signals required for IgE-dependent de- granulation. Nat. Immunol. 3: 741–748. Deposition of glomerular ICs results in complement activation 11. Strzelecka-Kiliszek, A., K. Kwiatkowska, and A. Sobota. 2002. Lyn and Syk and recruitment of phagocytic cells, initiating inflammation and kinases are sequentially engaged in phagocytosis mediated by Fc␥R. J. Immunol. renal injury. Phagocytosis of ICs is largely mediated by Fc␥R. 169: 6787–6794. 12. Linnekin, D., C. S. DeBerry, and S. Mou. 1997. Lyn associates with the jux- Innate immune effector cells such as macrophages and neutrophils tamembrane region of c-Kit and is activated by stem cell factor in hematopoietic express combinations of Fc␥R, comprising both activating (Fc␥RI, cell lines and normal progenitor cells. J. Biol. Chem. 272: 27450–27455. Fc␥RIII) and inhibitory (Fc␥RIIB) receptors, thereby ensuring ad- 13. Corey, S. J., A. L. Burkhardt, J. B. Bolen, R. L. Geahlen, L. S. Tkatch, and D. J. Tweardy. 1994. Granulocyte colony-stimulating factor receptor signaling equate regulation of the effector responses (42). Interestingly, dis- involves the formation of a three-component complex with Lyn and Syk protein- ruption of the gene encoding the ␥-chain of Fc␥R uncouples IC tyrosine kinases. Proc. Natl. Acad. Sci. USA 91: 4683–4687. 14. Harder, K. W., L. M. Parsons, J. Armes, N. Evans, N. Kountouri, R. Clark, formation and complement activation from renal injury in lupus- C. Quilici, D. Grail, G. S. Hodgson, A. R. Dunn, and M. L. Hibbs. 2001. Gain- Mld4/Mld4 prone mice, an analogous phenotype to Lyn mice (43). and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in FcR␥Ϫ/Ϫ mice do not express activation receptors but maintain the myeloid lineage. Immunity 15: 603–615. ␥ 15. Janas, M. L., P. Hodgkin, M. Hibbs, and D. Tarlinton. 1999. Genetic evidence for expression of the inhibitory receptor Fc RIIB, which is activated Lyn as a negative regulator of IL-4 signaling. J. Immunol. 163: 4192–4198. by Lyn. Furthermore, expression of intact FcR␥ in myeloid cells 16. Chin, H., A. 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