Lymphocyte Receptor Signaling PDF

Lymphocyte Receptor Signaling Literature: Chapter 7, Janeway’s Immunobiology ETH Zurich Lecture on “Pharmaceutical Immunology I” Prof. Dr. Cornelia Halin Winter 535-0830-00L HS 2024  Revision from Cha...

Lymphocyte Receptor Signaling Literature: Chapter 7, Janeway’s Immunobiology ETH Zurich Lecture on “Pharmaceutical Immunology I” Prof. Dr. Cornelia Halin Winter 535-0830-00L HS 2024  Revision from Chapter 1 Many steps in the induction of an adaptive immune response require cell-cell interactions and signaling Examples: T cell activation by antigen-presenting dendritic cells: B cell activation by T helper cells: Þ In this Chapter we will revise general concepts of signaling and learn about the signaling events required for lymphocyte activation 2 Content 1. General Principles of signal transduction and propagation 2. Antigen receptor signaling and lymphocyte activation 3. Co-stimulatory and inhibitory receptors 3 Preview on exercise of Chapter 7 (next week): Essentials of T cell activation? 1) Self-Study “1. General principles of signal transduction and propagation” (p. 5-12) 2) Follow lecture on “2. Antigen receptor signaling and lymphocyte activation” 4  1. General Signaling – self-study 1. General Principles of signal transduction and propagation Transmembrane receptors convert extracellular signals into intracellular events. Signaling via immune receptors typically involves: Kinases, which phosphorylate proteins e.g. receptor tyrosine kinases or intracellular Ser/Thr kinases Phosphatase, which remove phosphate groups from Ser/Thr Phosphorylation (de)activates generates sites on several proteins proteins to which (e.g. enzymes, other signaling transcription proteins can bind factors) Dephosphorylation either blocks or activates a protein 5  1. General Signaling – self-study Enzyme-associated receptors of the immune system can use intrinsic or associated protein kinases Examples: Receptor tyrosine kinases: e.g. vascular endothelial growth factor receptors (VEGFRs) Receptors with non-covalently associated kinases: T cell receptor, B cell receptor, costimulatory molecules (CD28, CD40)  1. General Signaling – self-study Signaling proteins interact with each other and with lipid signaling molecules vial modular protein domains => present in adaptor proteins or signaling enzymes 7  1. General Signaling – self-study Assembly of signaling complexes is mediated by scaffold and adaptor proteins Scaffold proteins: large proteins with numerous phosphorylation sites bring many different signaling proteins together may confer membrane localization Adaptor proteins: brings two different proteins together Example: adaptor protein Grb2 containing SH2 and SH3 domains 8 Small G proteins important signaling molecules downstream of tyrosine kinase-associated receptors Family comprises more than 100 proteins Switched from inactive to active states by GEFs and the binding of GTP Ras: important in lymphocyte signaling, often mutated in cancers (oncogene) Other examples: Rac, Rho, Cdc42: important for cell migration (control changes in actin cytoskeleton) GEF: Guanine-nucleotide exchange factor  1. General Signaling – self-study Signaling proteins can be recruited to the cell membrane in 3 main ways Extracellular signals are sensed by receptors at the cell membrane. This is the site where signaling needs to start. Membrane-localisation of signal transducers occurs via: scaffolds and adaptor proteins bind to phosphorylated sites small G proteins that have lipid modifications => naturally localise to membrane protein domains recognizing lipid motifs (e.g. PH recognizing PIP3) 10  1. General Signaling – self-study Signaling must be turned off as well as turned on TWO important post-transcriptional control mechanisms: phosphorylation / dephosphorylation ubiquitinylation to induce targeted degradation (proteasome or lysosome) 11  1. General Signaling – self-study Signaling pathways amplify the initial signal one activated kinase (e.g. Raf) can activate many further kinases enzymes activated by signaling generate many products (e.g PLC-g => DAG and IP3) release of preformed second messenger Ca2+ can activate many further molecules (e.g. calmodulin) 12  2. Antigen receptor signaling 2. Antigen receptor signaling and lymphocyte activation T cell receptor (TCR) complex The TCR complex is made up of the variable antigen- recognition proteins (TCR) and invariant signaling proteins (CD3 e, d, g, e chains and the 2 z chains) ITAMs Immunoreceptor Tyrosine-based Activation Motif cytoplasmic recognition motifs containing two tyrosine residues binding of cognate peptide/MHC to the TCR induces phosphorylation of the ITAM motifs the T cell receptor complex contains 10 ITAMs  2. Antigen receptor signaling B-cell receptor (BCR) complex The BCR complex is made up of a cell-surface immunoglobulin with one each of the invariant signaling proteins Iga and Igb The immunoglobulin recognizes the antigen but cannot by itself generate the signal. Signaling is started via the phosphorylation of the ITAM motifs of the accessory chains Iga and Igb BUT: Signaling via the B cell receptor complex is mainly important during B cell development (see Chapter 8). Regular activation of B cells occurs by the help of T cells and requires CD40L/CD40 signaling and cytokine signaling (see Chapter 10). 14  2. Antigen receptor signaling ITAMs recruit signaling proteins that have tandem SH2 domains Kinases recruited to phosphorylated ITAMs: T-cell receptor: ZAP-70 (Fig. 7.9) B-cell receptor: Syk (Fig. 7.26) 15  2. Antigen receptor signaling Engagement of the co-receptor CD4 or CD8 with the T cell receptor enhances phosphorylation of ITAMs Þ Kinase Lck bound to cytosolic part of CD4 Þ Once CD4 associates with the TCR complex, Lck phosphorylates ITAMs Þ Role of CD4 and CD8 is two-fold: Stabilization of TCR-MHC complex Phosphorylation of ITAMs 16  2. Antigen receptor signaling Also other receptors that pair with ITAM-containing chains deliver activating signals: Example: FcgRIII on NK cells NK cells recognize antibodies bound to target cells (e.g. a virally infected cell or cancer cell) Antibody-Fc binding to FcgRIII induces antibody- dependent cell cytotoxicity (ADCC) Þ Mechanism of action of several anti-cancer antibodies (see Chapter 3 & 10) Þ e.g. Rituximab (anti-CD20)  2. Antigen receptor signaling ZAP-70 phosphorylates the scaffold proteins LAT and SLP-76, initiating four downstream signaling modules: activation of transcription factors increased metabolic activity cytoskeletal rearrangement enhanced integrin activation and adhesiveness How do these processes relate to T cell activation? DC T cell  2. Antigen receptor signaling ZAP-70 phosphorylates the scaffold proteins LAT and SLP-76, initiating 4 downstream signaling modules: activation of transcription factors increased metabolic activity cytoskeletal rearrangement enhanced integrin activation and adhesiveness How do these processes relate to T cell activation? Immune T cell T cell Effector synapse DC proliferation differentiation T cell formation T cell 19  2. Antigen receptor signaling ZAP-70 phosphorylates the scaffold proteins LAT and SLP-76, initiating 4 downstream signaling modules: activation of transcription factors increased metabolic activity cytoskeletal rearrangement enhanced integrin activation and adhesiveness How do these processes relate to T cell activation? DC T cell  2. Antigen receptor signaling Recruitment and activation of phospholipase C-g (PLC-g) Initiated by phosphorylation of the scaffold proteins LAT and SLP-76 PLC-g is activated by phosphorylation PLC-g activation ultimately results in activation of transcription factors: NFAT, NFkB, AP1 NFAT, NFkB, AP1 are jointly required for expression of interleukin-2 21 Phospholipase C-g (PLC-g) cleaves inositol phospho- lipids to generate two important signaling molecules Diacylglicerol (DAG): Remains in the membrane and recruits PKC-q and RAS-GRP (=> => activation of AP-1 and NFkB) Inositol-triphosphate (IP3): Opens calcium channels that induce Ca2+-release from the endoplasmatic reticulum (ER) (=> => activation of NFAT)  2. Antigen receptor signaling Ca2+-influx in activated T cells leads to activation of the trans- cription factor NFAT Ca2+ binds to and activates calmodulin Ca2+-complexed calmodulin binds and activates calcineurin (phosphatase) calcineurin dephosporylates NFAT => translocates to the nucleus Interleukin-2 (IL-2) 23 NFAT : nuclear factor of activated T cells  2. Antigen receptor signaling Activated T cells secrete and respond to IL-2 NFAT is essential for IL-2 transcription in activated T cells Blocking IL-2 transcription or blocking autocrine IL- 2 signaling inhibits T cell activation Therapeutic immunosuppression: - Cyclosporin - Basiliximab (antibody directed against IL-2Ra) 24 Cyclosporin A Trade name: Sandimmun, Neoral cyclic peptide of eleven amino acids natural substance from fungus (discovered 1971 at Sandoz) strong immunosuppressant used for treatment/prevention of transplant rejection forms a complex with an immunophilin, what inhibits Ca2+-induced activation of the phosphatase calcineurin as a consequence, NFAT is not activated and the autocrine production of interleukin-2 is reduced => suppressed T cell activation  2. Antigen receptor signaling ZAP-70 phosphorylates the scaffold proteins LAT and SLP/76, initiating four downstream signaling modules: activation of transcription factors increased metabolic activity cytoskeletal rearrangement enhanced integrin activation and adhesiveness  2. Antigen receptor signaling TCR signaling activates the serine/threonine kinase Akt Akt promotes cell survival by inhibiting cell death and promotes metabolic activity via mTOR = mammalian target of rapamycin 27  mTOR inhibitors Drug: Rapamycin (sirolimus) First isolated in 1972 from bacteria (streptomyces hygroscopicus) from Easter Island (Rapa Nui) Hence the name: mammalian target of rapamycin (mTOR) inhibitor Rapamycin binds the FK-binding protein (FKBP). The rapamycin-FKBP complex inhibits cell growth and proliferation by selectively blocking activation of the kinase mTOR by Raptor Approved for treatment of transplant rejection 28 Raptor: Regulatory Associated Protein of mTOR  2. Antigen receptor signaling ZAP-70 phosphorylates the scaffold proteins LAT and SLP/76, initiating four downstream signaling modules: activation of transcription factors increased metabolic activity cytoskeletal rearrangement enhanced integrin activation and adhesiveness  2. Antigen receptor signaling Interaction of T cells with antigen-presenting cells Antigen recognition and co-stimulation-induced signaling lead to the formation of an “immune synapse” large zone of cell-cell contact stabilized by adhesion molecules peptide-MHC/TCR complexes and co- stimulatory molecules are drawn into the synapse cell-cell interactions may last for > 60 min https://www.sigmaaldrich.com/technical-documents/articles/biology/cancer- immunotherapy/response-modeling.html Outcome of long-lasting T cell-APC interactions: T cell will start to proliferate and differentiate into an effector cell Immune synapse T cell T cell effector cells 30 formation proliferation differentiation  2. Antigen receptor signaling Composition of the immune synapse cSMAC: inner ring, comprising peptide-MHC/ TCR complexes and costimulatory molecules pSMAC: outer ring, comprising adhesion molecules for stabilisation (integrin LFA-1 on T cells and ICAM-1 on DC) enable effective signaling Purpose of targeted release of cytokines immune synapse (important of T cell differentiation) Essentials of T cell activation? 32 Summary of antigen receptor signaling and T cell activation Antigen receptors associate with chains carrying ITAM (immunoreceptor tyrosine-based activation motifs) Activation of antigen receptors results in ITAM phosphorylation, leading to the recruitment of further kinases (Zap-70 and Syk), phosphorylation of scaffold proteins and activation of PI3-kinase and of PLC-g. PI3-kinase activation results in the deposition of PIP3 in the cell membrane, to which the kinase Akt can dock, leading to its activation and enhancement of survival and metabolic activity via mTOR. Recruitment and activation of PLC-g results in generation of IP3 and DAG, important second messengers, resulting in activation of transcription factors Ultimate outcome: activation of various transcription factors: NFAT, NFkB, AP-1 altered metabolic activity and survival adhesiveness, cytoskeletal rearrangements (stabilization of the immune synapse) 34  3. Co-stimulatory / co-inhibitory signals 3. Co-stimulatory and co-inhibitory receptors modulators of antigen receptor signaling in T and B lymphocytes 35  3. Co-stimulatory / co-inhibitory signals T-cell co-stimulatory protein CD28 transduces signals that enhance antigen receptor signaling pathways T cells: phosphorylation of CD28 enhances signaling => activation of PI3K, PLCg (DAG, IP3) => => activation of NFAT, NFkB, AP1 36  3. Co-stimulatory / co-inhibitory signals Simplified scheme depicting multiple signaling pathways that converge on the IL-2 promoter IL-2: autocrine T cell survival factor! Autocrine IL-2 signaling is therapeutically targeted by Cyclosporin A and Basiliximab (antibody directed against IL-2Ra). Both products are approved for treatment of graft rejection.  3. Co-stimulatory / co-inhibitory receptors CTLA-4 T cell-expressed inhibitory receptor for B7 molecules Induced on activated T cells Downregulates T cell activation Competes with CD28 for binding of B7 molecules (expressed by antigen presenting cells (APCs) Has higher affinity and avidity for B7 than CD28 and consequently outcompetes CD28-B7 binding, thereby inhibiting costimulatory signaling via CD28 Genetic knockout or mutations in CTLA-4 are associated with autoimmunity  3. Co-stimulatory / co-inhibitory signals Several CTLA-4-blocking antibodies (e.g. Ipilimumab) have recently been approved for anti-melanoma cancer therapy: Þ support the induction of anti-tumor T cell responses Þ known as “checkpoint inhibitors” What could be side effects of anti- CTLA treatment? Other co-inhibitory receptor on T cells that is targeted by antibody is PD-1 39 Inhibitory receptors downregulate the immune response by recruiting protein or lipid phosphatases ITAM: Immunoreceptor tyrosine-based activation motif Þ Recruit activating kinases ITIM: Immunoreceptor tyrosine-based inhibition motif Þ Recruit inhibitory phosphatases SHP: SH2-containing phosphatase; SHIP: SH2-containing inositol phosphatase  Slide from Chapter 3 NK cells express activating and inhibitory receptors to distinguish between healthy and infected cells Activating receptors recognizes cell-surface proteins induced in target cells by stress or damage => signaling activates killing Inhibitory receptors recognize surface molecules that are constitutively expressed at high levels, such as MHC class I molecules Þ signaling inhibits killing Important: The host cell’s MHCI expression is often reduced by the virus!! The balance between activating and inhibitory receptors determines whether an NK cell kills a target cell 40 Take-home messages Chapter 7 Common features of many signaling pathways: - activation of Ser/Thr kinases and tyrosine kinases - generation of second messengers (Ca2+, PiP3, DAG) - recruitment of receptors at plasma membrane, formation of signaling complexes Signaling typically leads to the activation of transcription factors (eg. NFAT, NFkB, AP-1) Signaling results in events like proliferation, activation, differentiation, changes in cell shape/migration Activating and inhibiting receptors / receptor complexes exist, which frequently contain ITAMs (immunoreceptor tyrosine-based activation motifs) or ITIMs (immunoreceptor tyrosine-based inhibitory motifs) 44