Continuous signaling of CD 79b and CD 19 is required for the fitness of Burkitt lymphoma B cells

• DOI: 10.15252/embj.201797980
• Publication Date: 2018-04-18T12:50:28Z
• Authors: Xiaocui He; Kathrin Kläsener; Joseena M Iype; Martin Becker; Palash C Maity; Marco Cavallari; Peter J Nielsen; Jianying Yang; Michael Reth
• Keywords: 0301 basic medicine; CRISPR/Cas-Methode; B-Lymphocytes; Gene Expression Regulation, Leukemic; Antigens, CD19; 610; Immunoglobulins; Immunoreceptor Tyrosine-Based Activation Motif; Articles; Burkitt Lymphoma; Phosphatidylinositol 3-Kinases; 03 medical and health sciences; Burkitt-Lymphom; Humans; Genetic Fitness; CRISPR-Cas Systems; CD79 Antigens; Signal Transduction

Article18 April 2018Open Access Transparent process Continuous signaling of CD79b and CD19 is required for the fitness Burkitt lymphoma B cells Xiaocui He orcid.org/0000-0002-0106-057X BIOSS Centre For Biological Signaling Studies, Department Molecular Immunology, Biology III, Faculty Biology, University Freiburg, Germany Max Planck Institute Immunobiology Epigenetics, Search more papers by this author Kathrin Kläsener orcid.org/0000-0002-5969-2553 Joseena M Iype Martin Becker orcid.org/0000-0002-1751-1056 Palash C Maity Marco Cavallari Peter J Nielsen Jianying Yang orcid.org/0000-0001-8197-5413 Michael Reth Corresponding Author [email protected] orcid.org/0000-0002-1025-7198 Information He1,2, Kläsener1,2, Iype1,3, Becker1,4, Maity1,3, Cavallari1, Nielsen2, Yang1,2 *,1,2 1BIOSS 2Max 3Present address: Uni Hospital Ulm, 4Present Helmholtz Zentrum München, *Corresponding author. Tel: [phone]; E-mail: The EMBO Journal (2018)37:e97980https://doi.org/10.15252/embj.201797980 PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Expression B-cell antigen receptor (BCR) essential not only development but also maintenance mature cells. Similarly, many lymphomas, (BL), require continuous BCR their tumor growth. This growth driven immunoreceptor tyrosine-based activation motif (ITAM) PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 delete co-receptor genes in human BL cell line Ramos. We find that Ramos expression component Igβ (CD79b), CD19, competitive culture. Furthermore, show absence any other component, can be expressed on surface, where it found close proximity signals an ITAM-dependent manner. These data suggest are part alternative module use ITAM/PI3K promote survival normal Synopsis (CD79b) act as promotes via integrated A new strategy functional studies lines mediated deletion genes. requires CD19. Only Igα transported surface components. BCR-independent Introduction consists membrane-bound immunoglobulin (mIg) comprising two heavy (H) light (L) chains signal-transducing Igα/Igβ (CD79a/CD79b) heterodimer (Reth Wienands, 1997). cytoplasmic tails each contain with conserved tyrosines crucial (Reth, 1989; Torres et al, 1996; Kraus 2001, 2004; Reichlin 2001). Upon ligation opening BCR, spleen tyrosine (Syk) phosphorylates interacts ITAM (Rolli 2002). resulting ITAM/Syk complex amplifies signal connects several downstream pathways, leading activation, proliferation, differentiation (Johnson 1995; Kurosaki, 1999; Deane Fruman, Werner 2010). prominent substrate Syk SLP65/BLNK adaptor protein which, upon phosphorylation, organizes calcium signalosome thus mobilization activated (Jumaa Kulathu 2008). Another important hub molecule, which forms plasma membrane together tetraspanners, CD81 (TAPA-1) leu-13, complement CD21 (CR2; Fearon Carroll, 2000). long cytosolic tail contains nine most phosphorylated src-family Lyn activation. Once phosphorylated, these serve binding partners proteins Grb2 Vav, phosphoinositide-3 (PI3K), phospholipase C-γ (Fearon Signal transduction from involves PI3K cytoskeleton rearrangements. Mature co-express different classes namely IgM-BCR IgD-BCR, reside islands (Yang Reth, 2010; 2015). On resting cells, CD81-tetraspanin localized inside IgD-class-specific (Mattila 2013; however, open gains access (Klasener 2014). IgM/CD19/CD81/CD21 stabilized its co-ligation complement-bound antigens reduce threshold 10- 100-folds (Carter Fearon, 1992). mouse CD19-deficient have defects proliferation maturation peripheral lymph tissues spleen, defective T-cell dependent response (Rickert 1995). pre-BCR Mice one H chain, Igα, or display developmental block at pro-B-cell stage (Kitamura 1991; Gong Nussenzweig, Minegishi 2001; Meffre 2002; Pelanda chain gene mutation results apoptosis disappearance periphery few days (Lam 1997; 2004). proper was therefore suggested unengaged emits tonic signal. However, some loose after inducible survive mice than 20 days, suggesting (Levit-Zerdoun 2016). of, from, lymphomas (Gauld Kuppers, 2005; Lenz Staudt, Stevenson 2011). instance, chronic lymphocytic leukemia (B-CLL) carry auto-aggregated expansion (Duhren-von Minden 2012). Activated B-cell-like diffuse large (ABC-DLBCL) mutations components, active NFκB increased (Davis (BL) aggressive derived germinal center transformed translocation c-Myc oncogene into locus (Dalla-Favera 1982). Knock-down experiments application specific inhibitors (Schmitz 2012, recent study model suggests (Varano 2017). used method code components assay showed subunit Igβ. mIg-independent fashion Results depends To role supporting gene-editing tool generate mutants lacking all (BCR-null) four L chains, Igβ, (Fig 1A). then compared BCR-null wild-type (WT) 1B). Surprisingly, expanded culture similar kinetics WT indicating they were compromised vitro next competition 1C) labeled GFP mixed them 1:1 ratio either unstained (GFP−) mutant FACScan analysis verified did (neither nor IgD-BCR) 1D). Interestingly, assay, gradually disappear within 8 thereby less fit 1E). Figure 1. Fitness schematic diagram showing route generation single- multi-BCR KO method. Cell Ramos-null using CytoTell™ UltraGreen. Null = HC/LC/Igα/Igβ tetra KO. depicting assay. retrovirally transduced pMIG empty vector (EV) create GFP-labeled (gWT). gWT about day 0, relative amount GFP− measured flow cytometry time points. levels IgD-BCR determined cytometry. Growth between serves control. represent mean standard error minimum three independent experiments. against Data One clone genotype. Download figure PowerPoint asked whether loss both caused reduced this, generated single well double none 1F). intra- extracellular Western blot (Appendix Fig S1). When cultured separately, S2). lost whereas competed 1G). exclude influenced variants during selection prolonged clones, repeated set clones 14 obtained same S3). Together, associated total rather expression. analyzed apparent function mIg BCR. HL HLα HLβ triple 2A) corresponding S1D E). anti-Igβ antibodies still albeit amounts comparison 2B). In 2C). finding prompted us test could stimulate exposed WT, HL-KO, HLα-KO, HLβ-KO monoclonal antibody 2D–G). shows expressing stimulated antibody, delayed manner respond treatment served negative control experiment 2G). As control, anti-Igα responded S4). 2. determines A. map generating multi- KOs B. C. D–G. Calcium stimulation antibodies. HL-KO HC/LC KO; HC/LC, Igβ-dependent carries (Y196 Y207) interaction Syk. signaling, vectors encoding ITAM-mutated 3A). co-cultures containing non-transduced Igβ-producing enriched over EV transfected effect 3B). growth-promoting abolished, when mutated phenylalanine 3C). Thus, mechanism. conclusion confirmed Igβ-α chimera, portion transmembrane region Igα. chimeric provided even higher advantage, truncation (Igβtl) increase 3. through contributes reconstituted B, percentage GFP-positive points form shown. express marker. D. constructs representative E. αβ F, G. proportion H. 3D). chimera displayed tail-less stimulus. correlated responsiveness toward stimulation. above 3E–H). formation fitness, induced release Taken together, cellular constant tyrosines. employs pathway exposure cognate antigen, kinases Syk, support dissociation mediate uses classical pre-treated DMSO, inhibitor PP2, R406 strongly lesser extent 4A). result demonstrates involved Igβ-mediated Indeed oxidant pervanadate, latter pSyk EV1). 4. though canonical indicated inhibitors. HLSLP65 tamoxifen-inducible SLP65 4-OHT. Cells without production controls. GFP-expressing Click here expand figure. EV1. Flow phosphoSykNormalized intracellular phosphoSyk pervanadate treated deviation P-values calculated paired t-test. SLP65/BLNK, (Fu 1998; Wienands 1998). S5). plasmids SLP65ERT2, (Trageser 2009). tamoxifen, SLP65ERT2-transduced flux, SLP65ERT2 no change level 4B). indicates agreement conclusion, disadvantage co-cultured 4C). improves employing Lyn, Igβ-induced All association tetraspanner stable (Maecker Levy, move closer receptors cooperate amplification (Fujimoto 2000; Klasener bulk transfection HLCD19 HLCD81 population S6) had maintained 5A B). After CD81, CD19-negative disappeared 12 5C). unable mount 5D). 5. A, rendered method, CD19- CD81-negative KO, F. Percentage HLCD19KO vector. HL-KO. batch sorted. fitness-promoting gain-of-function while CD19-transduced 5E). Compared EV, (GFP+)-transduced culture, Ig