Immun 532. B (and T) cell Signaling:

Immun 532 B (and T) cell Signaling: Feb 2, 2017 David Rawlings, MD Director, Center for Immunity and Immunotherapies Chief, Division of Immunology Sea=le Children s Research Ins>tute Professor of Pediatrics and Immunology, UW School of Medicine drawling@u.washington.edu

Reading Assignments Basic Signaling Biochem. Dal Porto et al Molec Immun 2004 41:599 = minimal reading Scharenberg and Rawlings, Nature Rev Immun 2007 7:778 TIRF/Microsignalosome Studies Treanor et al. Biochemical Society Transactions, 2009 37:1014. Depoil et al. Early Events of B Cell Activation by Antigen Sci. Signal., 24 March 2009 Vol. 2, Issue 63 View the associated Slide show which makes all of this much more easy to understand: http://stke.sciencemag.org.offcampus.lib.washington.edu/cgi/content/abstract/sigtrans;2/63/pt1 Signal Initiation Models: Yang and Reth Nature 2010 467:466 Tolar et al, Immunological Reviews, 2009 232:34 Tonic Signaling Srinivavasan Cell 2009 139:573 Unique features of IgD vs. IgM and role for IgG tail Hobeika, Trends in Immunology; 2016, 310 320 Wienands, Immunology Letters, Volume 178, 2016, 27 30

Lecture Overview: Brief review of signal transduction concepts and major receptor systems utilized by lymphocytes Unique features of Antigen receptors (BCR or TCR) Review of key signaling cascades initiated by BCR engagement Newer Insights into BCR signaling

Major Goal of B-cell development: B-lymphocytes which: As a population express diverse Ig molecules on their surface Are able to respond when antigen is encountered Are appropriately tuned for the internal milieu - e.g. don t respond inappropriately

Signal integration at the organism level: Stem cell Antigenindependent lymphoid development mtor and Hematopoieitins - proliferative signaling to create new lymphocytes Antigendependent lymphoid development Ag Receptors - drive proliferation and survival of lymphocytes bearing appropriate antigen specific receptors TNFRSF Receptors - regulate differentiation and homeostasis of lymphocyte numbers Chemokine and Adhesion Receptors - Regulate localization in lymphoid and nonlymphoid tissues

Information type generally correlates with signaling mechanism TLR4/7/9 Pro-inflammatory - Homologous to Toll receptors involved in recognition of viruses and bacteria IL4, 7, 21,TSLP-R Regulate differentiation and growth of specific lineages CXCR4/5 Regulate chemotaxis and localization TGF-β R Anti-inflammatory - Complex biochemistry and functions BAFF-R/TACI/CD40 Regulate differentiation and survival of specific lineages Flt-3 Growth promoting - Better referred to as receptor tyrosine kinases (RTK s) - Primarily support hematopoiesis and lymphocyte development

Unique problem of infinitely variable an>gen receptors Surface receptors designed for specific ligands can achieve precise changes in shape or location to induce signaling BUT: How do you design a signal transduction system to recognize any shape of ligand? How do you control signaling when you can t control the abundance of the ligand? How design a system to recognize ligands with markedly different binding properties?

Antibodies made to immune cell surface proteins were able to induce cell activation Concept that receptor clustering was involved in immunoreceptor signaling

Antigen receptor subunit structures No kinase domain No GPCR type domains Tiny little tails How could these receptors generate a signal?

Antigen receptor tail clue Immuno-receptor tyrosine based activation motif (ITAM) Definitive experiment: transfer tails to a heterologous extracellular domain/tm span Clustering stimulus full gain of capacity to activate pathways that antigen receptors are able to activate

Clustering induced tyrosine phosphorylation initiates antigen receptor signals Tyrosine based motif lead to examination of role of tyrosine phosphorylation in immune receptor signaling Mutation of tyrosines established their absolute requirement for signaling via antigen receptors and designation as the immuno-receptor tyrosine based activation motif (ITAM) Src family tyrosine kinases identified as crucial to the initiation of ITAM phosphorylation (in most cases)

Structural basis of ITAM function: bind tandem SH2 domains Activated ITAMbearing receptor - bound to tandem SH2 kinase resembles a growth factor receptor ITAM phospho-tyr are precisely spaced to match tandem SH2 domains of a lymphocyte-specific tyrosine kinase e.g. Syk in B cells; ZAP-70 in T cells

Antigen Receptor Signaling: an overview antigen B cell receptor (pre-bcr or BCR) and signal transducers: Igα/Igβ Signal Initiation Receptor transducer Co-receptor or Modifier Response Modifiers: Positive: CD19/21; CD45, others Negative: CD22; FcγRIIB, PIR-B, others Signal integration Signalosome Downstream effectors ERK NF-AT JNK, p38 NF-κB Signal integration Lipid Raft-Associated Signalosome : Tyrosine Kinases: Src, Syk, Btk Lipid Kinases: PI3K Phosphatases: Signal propagation CD45 Lipases: PLCγ2 Linkers/Adaptors: BLNK, CARMA1 Serine kinases: PKCβ, PKD

Detailed Map of B Cell Receptor (BCR) Signaling Events Signal propagation Signal Initiation 500+ new py sites in our Mass Spec Studies 2000+ new ps/t sites in our Mass Spec Studies Signal Integration

ITAM signaling mechanism provides for multiple regulatory veneers In contrast to growth factor signaling, where strength is set by number of activated receptors, ITAM signaling mechanism allows additional levels of regulation Example: extracellular control of signaling via CD45/CSK/ SRC family kinase regulatory network

Coordinate regulation of Src family kinase activity by CD45, Csk and PTPN22- allows precise control of Signal Initiation PTPN22 (Lyp, PEP)

Sayak Mukherjee et al. Monovalent and Multivalent Ligation of the B Cell Receptor Exhibit Differential Dependence upon Syk and Src Family Kinases; ci. Science Signal., 1 January 2013

Signal Propagation: Early Events Activation of phospholipase Cγ Phospholipase C degrades phosphoinositide-4,5- bisphosphate (PIP2) to two different 2 nd messengers DAG: membrane delimited 2 nd messenger with function analogous to PIP3 IP3: freely diffusible 2 nd messenger

BCR-driven PLCγ Signaling Step 1- adapter assembly Scharenberg and Rawlings, Nature Rev Immun 2007

BCR-driven PLCγ Signaling Step 2- PI3K activation Scharenberg and Rawlings, Nature Rev Immun 2007

BCR-driven PLCγ Signaling Step 3- Btk-mediated PLC activation Scharenberg and Rawlings, Nature Rev Immun 2007

Immunoreceptor early event: elevation of cytosolic Ca 2+ Extracellular space [Ca 2+ ]= 1mM PM Ca/ATPase CRAC Ca 2+ + leak - - - - - - - - - - - current + + + DAG IP3 + + + + + + + Stim1 [Ca2+] ER release sustained Ca2+ entry Time + + - -? Other mechanisms PKC/PKD HDAC intracellular stores ER Ca/ATPase (e.g. SERCA) IP3 receptor cytosol [Ca 2+ ]= 100 nm

Store-operated calcium entry Scharenberg and Rawlings, Nature Rev Immun 2007

Ca 2+ as a 2 nd messenger Set up for rapid action Mechanisms Calmodulin: modulates function of many proteins Calcineurin: calcium regulated phosphatase Calpain: calcium regulated protease Ca 2+ signaling in-vivo: STOP N. Bhakta and R.S. Lewis

Downstream signal propagation to the nucleus via PLCγ: Three parallel pathways 1) IP3-calcium-NFAT-calcineurin 2) DAG/PKC/Carma/NFKb 3) DAG/PKC/PKD/HDACs Distinct functions for each pathway Key target for inhibitory receptors Increased cytosolic Ca 2+ Calcineurin activation Regulation of NFATdependent target genes Phosphorylation of CARMA1 Phosphorylation of IKBα Regulation of NFkBdependent target genes Phosphorylation of PKD Phosphorylation of HDAC Epigenetic regulation of multiple genes Clinically important target pathways for immune disorders

Nuclear Factor of Activated T-cells Regulation via phosphorylation - dependent nuclear export Calcineurin (Cn) regulates dephosphorylation in the cytosol to allow Ca2+dependent regulation of target genes One key NFAT target is the IL-2 gene

CsA and FK506: Target NFAT for immunosuppressive effects Chemically distinct and different targets, but similar gain-offunction mechanism Identical end target and very similar clinical effects

Downstream signal propagation to the nucleus via PLCγ: Three parallel pathways 1) IP3-calcium-NFAT-calcineurin 2) DAG/PKC/Carma/NFKb 3) DAG/PKC/PKD/HDACs Increased cytosolic Ca 2+ Calcineurin activation Regulation of NFATdependent target genes Phosphorylation of CARMA1 Phosphorylation of IKBα Regulation of NFkBdependent target genes Phosphorylation of PKD Phosphorylation of HDAC Epigenetic regulation of multiple genes

CARMA1 is critical adapter that assembles the BCR-driven NFKB (and JNK) signaling cascade Rawlings, Sommer, Moreno-Garcia, Nat. Rev Immun 2006

Nuclear Factor kappa Beta Canonical NF-κB pathway: IκB NFκB PKC Carma-1 (adaptor) IκB Kinase P IκB P + NFκB ΙκΒ (degradation) Transcription factor implicated in regulating lymphocyte survival Phosphorylationregulated nuclear import via Inhibitor of NFκΒ (IκΒ) Gene Transcription e.g.bcl-x L

P Summary: signal propagation Ras PLCγ DAG Ca 2+ Distinct pathways lead to nuclear translocation of distinct transcription factors MAPK P NFAT IκB NFκB NFκΒ P P IκB ΙκΒ + (degradation) Each transcription factor will have cell type specific access to target genes IKK NFκB A P P A A NFκB B B B

Signal integration Example 1: Inhibition of PLCγ activation by FcγRIIb Extracellular space [Ca 2+ ]= 1mM PM Ca/ATPase ER Ca/ATPase (e.g. SERCA) intracellular stores CRAC Ca 2+ + + + + + + + + + + + - - - - - - - - - - - DAG PKC/PKD HDAC IP3 receptor [Ca2+] Dimunition of PLCγ activation by SHIPmediated PIP3 hydrolysis IP3 ER cytosol release Time [Ca 2+ ]= 100 nm? Other + + - - mechanisms

Signal integration: ITIM inhibitory signaling: SHIP and SHP Concept of ITAM lead to exploration or role of tyrosine motifs found in receptors previously implicated in down-regulating immune responses Numerous immunoreceptor tyrosinebased inhibitory motifs (ITIMs) identified in tails of various receptors Identifcation of proteins bound by ITIMs: SH2-containing inositide phosphatase (SHIP) - degrades PIP3 SH2 containing tyrosine phosphatase (SHP1 and SHP2)

Signal integration: Example 2: Signal amplification by CD19 B-cell CD19/CD21/ CD81 coreceptor complex enhances B- cell antigen receptor signaling when co-clustered with surface IgM Orders of magnitude more sensitivity to antigen to which complement has been fixed

Signal integration Example 3: Nuclear signal integration via PLCγ Three parallel pathways 1) IP3-calcium-NFAT-calcineurin 2) DAG/PKC/Carma/NFKb 3) DAG/PKC/PKD/HDACs Increased cytosolic Ca 2+ Calcineurin activation Regulation of NFATdependent target genes Phosphorylation of CARMA1 Phosphorylation of IKBα Regulation of NFkBdependent target genes Phosphorylation of PKD Phosphorylation of HDAC Epigenetic regulation of multiple genes

Epigenetic modulation of transcription factors by HDACs AgR Signaling Complex Syk BLNK Bam-32 Ag R Lyn Btk PLC-γ2 Another family of protein kinases, designated the protein kinase D (PKD) family, is dependent on DAG and PKC for activation their major direct target has been shown to be class II histone deacetylases (epigenetic regulatory protein) PKD regulates nuclear import of HDACs via direct HDAC phosphorylation (either masks nuclear import signal or activates nuclear export signal) Ca ++ DAG PKCβ PKD P HDAC P PKD HDAC HDAC pathway: IKK NF-κB Class II histone deacetylases Gene Transcription

HDACs regulate accessibility of chromatin to transcription factors Wound DNA nucleosome Histone acetylation is thought to control chromatin accessibility through a form of histone code PKD-dependent HDAC export would tend to lead to enhanced chromatin accessibility and transcriptional activation Recruitment of HDACs to chromatin is a highly regulated process

P HDAC Signal integration in the nucleus: MAPK P P P Ras NFAT PLCγ Ca 2+ DAG HDAC PKD HDAC P HDAC IκB NFκB NFκΒ P P IκB ΙκΒ + (degradation) Distinct pathways lead to nuclear translocation of distinct transcription factors Each transcription factor will have cell type specific access to target genes A B HDAC C IKK NFκB NFκB Target gene access further regulated by histone acetylation B B C

Developmental Regulation: Example-Transitional B cells are programmed at the nuclear level not to transcribe BCR dependent survival genes T1 NFAT NFκB AP-1 Pol II A1, Bcl-x L, c-myc S. Andrews J Immunology, 2009

Developmental Regulation: Example-Transitional B cells are programmed at the nuclear level not to transcribe BCR dependent survival genes RNA Polymerase II recruitment to gene promoters: Anti-IgM (min) S. Andrews J Immunology, 2009

BCR Signaling: overview Review of major surface receptor systems utilized by lymphocytes Unique features of B cell Antigen receptors (BCR) Review of key signaling cascades initiated by BCR engagement New Insights into BCR signaling

B cells must respond to membrane-bound antigen Antigen may be mono- rather than poly-valent. Previous biochemical studies (such as F(ab) 2 cross-linking) fail to mimic these conditions.

Imaging of B cells using planar lipid bilayers containing GPI-linked integrins +/- antigen

High-resolution tracking using TIRF-Microscopy: monitoring single molecule dynamics in lymphocyte activation Total internal reflection- light strikes the interface between two optical media of different refractive indices. Light incident at an angle greater than the critical angle undergoes total reflection. Beyond the angle of total reflection, an electromagnetic field from incoming/reflected light extends into the z direction. This evanescent wave, decreases exponentially, and extends only a few 100nm Molecules at cell membrane within the evanescent field are excited to emit fluorescence and allowing single molecule imaging.

How do membrane-bound antigens trigger signal initiation?? Antigen leads to assembly of BCR microclusters and, ultimately, an immune synapse Model: Antigen binding leads sequentially to microcluster assembly, followed by B cell spreading, contraction, and mature synapse formation See multiple published studies from F. Batista and colleagues

BCRs diffuse randomly in resting cells

Antigen exposure triggers localized BCR signaling

Signaling promotes microcluster formation and signalosome assembly

Microcluster assembly drives membrane spreading, contraction, and formation of mature B cell immune synapse

Antigen and BCR Aggregate in Microclusters Free diffusion of BCR in non-stimulated conditions. Antigen and BCR rapidly form Microclusters Microclusters initially spread Microclusters then gather and fuse to form a csmac Depoil et al Nature Immunology 2008 9:63-72

Figure 1 B-cells spread and contract after BCR recognition of antigen presented on the surface of a cell Biochemical Society Transactions www.biochemsoctrans.org Biochem. Soc. Trans. (2009) 37, 1014-1018

PLEASE SEE SCIENCE SIGNALING WEBSITE FOR EXCELLENT OVERVIEW: Depoil et al. Early Events of B Cell Activation by Antigen Sci. Signal., 24 March 2009 Vol. 2, Issue 63 View the associated Slide show which makes all of this much more easy to understand: http://stke.sciencemag.org.offcampus.lib.washington.edu/cgi/content/ abstract/sigtrans;2/63/pt1

How does antigen trigger signal initiation?? Opposing Models Model 1- BCR Monomer Model- Susan Pierce and colleagues Methods- TIRF and FRET BCRs primarily exist as randomly moving monomers Monovalent antigen leads to assembly of immobile signaling-active BCR oligomers Initial oligomer assembly is independent of signaling activity!! Clustering is driven by a membrane proximal domain of BCR (Cµ4) Model: Antigen binding induces a conformational change in the Fc portion of the BCR revealing an interface that promotes clustering

Model 2- BCR Oligomer Model- Michel Reth and colleagues; Methods- BiFC (bifluorescence complementation) Nature 2010 467:465 Surface BCRs are primarily comprised of closed auto-inhibited oligomers (requires transmembrane region of mhc and linker region of Igα) Antigen independent (low level) equilibrium shift to/from active monomers permits constitutive/survival signaling Antigen encounter alters the equilibrium, shifting to more active monomer. Polyvalent Ag is required to maintain this apart activity Model: Antigen binding leads to release inhibitory conformation, thereby triggering signaling.

How does the actin cytoskeleton regulate initial steps in B cell activation? Immunological Reviews Volume 237, Issue 1, pages 191-204, 19 AUG 2010 DOI: 10.1111/j.1600-065X.2010.00943.x http://onlinelibrary.wiley.com/doi/10.1111/j.1600-065x.2010.00943.x/full#f1

Dual-view TIRF movie BCR diffusion is not entirely random!

Visualizing a role for the actin cytoskeleton in the regulation of B cell activation

Visualizing a role for the actin cytoskeleton in the regulation of B cell activation Immunological Reviews Volume 237, Issue 1, pages 191-204, 19 AUG 2010 DOI: 10.1111/j.1600-065X.2010.00943.x http://onlinelibrary.wiley.com/doi/10.1111/j.1600-065x.2010.00943.x/full#f4

ARs interrogate APC surfaces Current AR signaling model: Upon antigen encounter ARs become immobilized forming oligomers/ (or activated monomers) microclusters. Clustering is signalling independent and driven by intrinsic structural features of the BCR Lipid rafts condense around microclusters leading to a conformational change in the ITAMcontaining intracellulur chains. This conformational change and proximity to raft-resident src kinases leads to recruitment of key initial signaling effectors (e.g. src/syk family kinases). Initial signaling events trigger transient actin depolarization and ezrin dephosphorylation PLC/Vav-dependent signals then trigger membrane spreading and additional microcluster formation Microclusters progressively enlarge by trapping additional ARs Large clusters are actively transported to the central synapse with integrin molecules in periphery. CD45/CD148 are excluded from csmac due to their strutural features (thereby limiting phosphatase activity).

The cytoskeleton (picket-fence) may help to explain constitutive signaling

Key Question: What constitutive signals mediate BCRdependent survival of mature B cells??

Key Question: What constitutive signals mediate BCR dependent survival of mature B cells?? Ablation of BCR on mature B cells (CD21+) results in rapid cell death (Lam et al, Cell, 1997) Cretargeting Cremediated of ROSA locus allows IgH concurrent deletion expression creates of candidate BCRmature survival B signaling cells gene Srinivasan et al, PI3 Kinase Signals BCR-Dependent Mature B Cell Survival, Cell 2009 139:573

Key Questions in BCR Signaling How does BCR structure impact signaling? Does this change with Ig isotype and cell stage? What makes memory B cell more responsive to BCR engagement

Differential IgM and IgD Expression during B cell development Elias Hobeika et al: Control of B Cell Responsiveness by Isotype and Structural Elements of the Antigen Receptor; Trends in Immunology; 2016, 310 320

Unique structure of IgD and the flexible orientation of its antigen-binding Fab arms Elias Hobeika et al: Control of B Cell Responsiveness by Isotype and Structural Elements of the Antigen Receptor; Trends in Immunology; 2016, 310 320

Protein island organization of IgM and IgD and their interaction with antigens Elias Hobeika et al: Control of B Cell Responsiveness by Isotype and Structural Elements of the Antigen Receptor; Trends in Immunology; 2016, 310 320

IgG Tail facilitates increased BCR signaling in memory B cells IgG-BCR-intrinsic signal amplification of switched memory B cells. Compared to naïve or IgM-memory B cells, GC-independent as well as GC-dependent IgG-memory B cells have a lower activation threshold because the canonical ITAM-based signaling Wienands, Control of memory B cell responses by extrinsic and intrinsic mechanisms; Immunology Letters, Volume 178, 2016, 27 30

Key Open Questions in BCR Signaling How does BCR structure initiate signaling? Does this change with Ig isotype and cell stage? What signals specifically operate within the synapse? How are distinct signaling cascades assembled and segregated? What are timing and spatial requirements for co-receptor signals? How do developmental-specific changes impact BCR signaling? Proximal events? Integration of nuclear signaling? How do these processes differ in anergic cells? How do heritable change in BCR signaling effectors modulate protective responses and/or predipose to autoimmunity?

Thanks for your attention.