Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP /1/A

B-CELL RECEPTOR SIGNALING Tímea Berki and Ferenc Boldizsár Signal transduction Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP /1/A

TÁMOP /1/A B-cell development Haematopoietic development is a highly regulated multistep process in which pluripotent HSC differentiate through intermediate progenitors to mature cells in the blood These processes are regulated by transcription factors and signaling pathways The generationof lymphoid progenitors depends on: c-Kit, Flt3, and IL-7R

TÁMOP /1/A Early lymphopoiesis depends on: PU.1: crucial for myeloid and lymphoid progenitors IKAROS: controls development of lymphoid progenitors Bcl11a: zinc finger transcrition factor, in its absence development is blocked in CLP E2A: helix loop helix protein, B-cell fate determinant, turns on B-cell specific genes EBF: early B-cell factor, B-cell fate determinant, turns on B-cell specific genes Pax5: in its absence cells are blocked at pro-B stage, self renew, broad developmental potential. Pax5 represses non- B genes

TÁMOP /1/A The stage specific crucial events involved in B lymphoid commitment LMPP: lymphoid primed multipotent progenitor, CLP: common lymphoid progenitor, BCP: B lineage committed progenitor LMPP Lymphoid priming E2A/IKAROS/PU1 RAG1/2, TdT, IgH, IL-7Rα, Notch-1, Ebf1 BCP B-lineage commitment EBF1/PAX5 λ5, VpreB1, mb1, B29, OcaB, Pax5 CLP B-lineage priming E2A/EBF1 CD19, Tcf4, Aiolos, Irf4, Irf8, CD55

TÁMOP /1/A Genetic control of lineage commitment in early lymphopoiesis Low level of PU.1 → IL7Ra expression → lymphoid lineages (IL7R  +) PU.1 low Myeloid prog. HSC CLP Pro-T Pro-B Pre- NK Pre- DC Ikaros EA2EBFPax5 Id2 Notch PU.1 high IL-7R  + IL-7R  -

TÁMOP /1/A Early B-cell development in the bone marrow A schematic diagram of early B lymphopoiesis, showing the successive differentiation stages and the rearrangement status of IgH and IgL genes PU.1 Ikaros c-Kit Fit3 Bcl11a Il-7R E2A EBF Pax5 Foxp1 (D H ► J H ) D H ► J H V H ► D H J H Pre-BCR + BCR + V L ► J L Myeloid Erythroid TNK HSC CLP Pre- pro-B ELP Pro-B Large pre-B Small pre-B Imm. B

TÁMOP /1/A The Ig gene rearrangement DH–JH rearrangements are initiated in the earliest lymphocyte progenitors (ELPs) at a low level and are completed as the cell progresses to the CLP and pre- pro-B cell (also referred to as CLP2) stage. VH–DJH recombination takes place in pro-B cells, and successful rearrangement leads to expression of the Igm protein as part of the pre-BCR in large pre-B cells. Subsequent rearrangement of the IgL locus in small pre-B cells results in the expression of the BCR (consisting of m heavy and k or l light chains) on immature B cells (Imm. B). The approximate points of the developmental arrest in mice that have defective transcription factors PU.1, Ikaros, Bcl11a, E2A, EBF, Pax5 and Foxp1 (black) or signalling components involved in signalling through c-Kit, Flt3 or IL-7R (grey) are indicated above. E2A- and EBF-deficient progenitors in the bone marrow resemble pre-pro-B cells but lack all Ig rearrangements.

TÁMOP /1/A Expression pattern of transcription factors involved in B cell commitment and differentiationProgenitor CMLP, CLP Commitment to B lineage Bone marrow PreB → Imm.B Peripheralmaturation Germinal center Plasma cells PU.1 E2A EBF Ikaros Pax5 LEF-1 NF-kB Aiolos Bcl-6 Blimp-1 XBP-1

TÁMOP /1/A IT AM Immunreceptor Tyrosin-based Activation Motif Activating receptors IT Immunreceptor Tyrosin-based AMITAM Ativation Motif (ITAM): D/E-x2- YxxL/I-x6-9Yx2L/I Inhibiting receptors IT Immunreceptor Tyrosin-based IMITIM Inhibition Motif (ITIM): L/V/S-YxxL/V ACTIVATION ITAMITAM INHIBITION ITAMITIM

TÁMOP /1/A Acute antigen signaling LYN SYK BCR P P P P Ig  Igβ P P P P PLC  2 BTK BLNK GRB2 VAV SOS RAS ERK DAG PKC IP3 Ca 2+ Transcription factors Proliferation, activation and antibody secretion Proliferation, activation and antibody secretion Antigen PlP 3 PlP 2 PI3K

TÁMOP /1/A SYK P P BCR ITAM PLC  2 PI3K Ig  Ig β PIP 3 PIP 2 AKT1 RAS RAF DAG MEK ERK PKC β TRAF3 NIK IP 3 + Ca 2+ NFAT NF-  B1 Cytoplasm Nucleus Canonica l NF-  B1 pathway Non- canonical NF-  B1 pathway Non- canonical NF-  B1 pathway REL P100 Anti-apoptotic proteins Such as BCL-X L and MCL1 Anti-apoptotic proteins Such as BCL-X L and MCL1 LYN Plasma membrane TRAF3 TRAF2 BAFFR CD19 NF-  B2 Co-stimularory pathways of BcR signaling

TÁMOP /1/A LYN SYK BCR P P P P Ig  Ig β P P P P PLC  2 BTK BLNK GRB2 VAV SOS RAS ERK DAG PKC IP3 Ca 2+ Transcription factors Proliferation, activation and antibody secretion Proliferation, activation and antibody secretion SHIP1 DOK Migration Survival CXCR4BAFFR ? Pl (3,4)P 2 Pl(3,4,5)P 3 Pl(4,5)P 2 PI3K Long term BcR stimulation

TÁMOP /1/A The positive (CR2) and negative (FcgRIIb) B cell regulation model

TÁMOP /1/A Role for lipid rafts in B- cell activation BCR CD45 Ig  / β CD22 ITIMITAM BCR P P P P P P P P LYN CD45 Ig  / β CD22 SYK Signal transduction Internalization Antigen targeting Receptordownregulation Lipid raft Antigenbinding

TÁMOP /1/A Lipid rafts The plasma membrane is composed primarily of sphingolipids, (glycerol)phospholipids and cholesterol. Sphingolipids differ from most phospholipids in that they have long, largely saturated acyl chains that allow them to pack tightly in a bilayer, forming a gel phase in which there is very little lateral movement or diffusion. The gel phase of the sphingolipids is altered by the association of cholesterol, which condenses the packing of the sphingolipids by occupying the spaces between the acyl chains. So, cholesterol-containing sphingolipid microdomains exist in a liquid-ordered phase that is significantly more fluid than the gel phase.

TÁMOP /1/A BCR Ig  Ig  Antigen RapL Riam Rap Dok-1 ezrin Bam32 clathrin Cbl Bam32 BLNK GRB2 LAB Dok-3 Shc GRB2 TSC2 lBlB lBlB Lyn c-RAF MEK1/2 Erk1/2 PKC TAK1 IKK CaMK Akt GSK-3 mTOR P70 S6K P70 S6K Akt Btk P13K p110 P13K p110 SOS Ras GRP Ras GRP Ras GAP Ras GAP CaM Nck BLNK PRK2 VAV Rac/ cdc42 Rac/ cdc42 Rho Rac Ras RhoA SHIP1 SHIP2 PTEN Gab BCAP p85 Rheb IP 3 DAG Pl(4,5)P 2 DAG PLC  2 CARMA1 Bcl 10 MALT1 lBlB lBlB NF-  B NFAT NF-  B FoXO MEKKs PKC PIR-B SHIP2 Cytoskeletal rearrangments and integrin activation Cytoskeletal rearrangments and integrin activation Proteasomal degradation Proteasomal degradation Protein synthesis Protein synthesis Glucose uptake Glucose uptake Glycolysis ATP generation ATP generation Lipid raft aggregation Lipid raft aggregation BCR Internalization BCR Internalization CD22 CD19 CD40 FcgRIIB1 Ca 2+ ER IP 3 R Intracellular Ca 2+ store Intracellular Ca 2+ store STIM1 Transcription Growth arrest, apoptosis Growth arrest, apoptosis Ca 2+ Calcineurin Bam32 MKK3/4/6 p38 MKK4/7 JNK1/2 JNK NFAT Ets-1 Oct-2 Bfl-1 ATF-2 Egr-1 Bcl-6 Elk-1 Bcl-xL CREB Jun CD19 Transcription Cytoplasm Nucleus CD19 CRAC channel PIP 3 Syk Lyn CD45 PIP 3 Fc  RIIB1 SHIP1 Overview of BcR signaling