Session 2 PDF - Immune System

Sensations: Receptors and signalling Dr. Chirag C. Sheth supporting literature Kuby Chapters Research Chapter 3 AP-1 NF-kB NFAT Pathways Innate Lymphoid Cells CXC44 and HIV TCR / TLR HIV-membrane Fusion Overview of TCR CXCR4-HIV...

Sensations: Receptors and signalling Dr. Chirag C. Sheth supporting literature Kuby Chapters Research Chapter 3 AP-1 NF-kB NFAT Pathways Innate Lymphoid Cells CXC44 and HIV TCR / TLR HIV-membrane Fusion Overview of TCR CXCR4-HIV TLR Minireview discuss… 01. What are the cytoplasmic signalling molceules, and what do they do?. 02. What are the principal functions of TNF? 03. What are the three main transcription factors participating in antigen-dependent T-cell activation, and how do they work? session 2 "Good communication is as stimulating as black coffee, and just as hard to sleep after." - Anne Morrow Lindbergh let’s bond... 01 non-covalency In immunology, cell-to-cell communication occurs via receptor- ligand interactions (non-covalent) affinity 02 Normally, the strength of the individual bonds tend to be weak avidity 03 Multiple bonds occur, resulting in a high cumulative bind strength valency 04 Increasing valency of the receptors is a mechanism to increase avidity ““We are only as strong as we are united, as weak as we are divided.” — J. K. Rowling ‘Harry Potter and the Goblet of Fire’  space Receptor expression can be targeted and oriented in response to external cues. time Receptor expression can change during the course of an infection process. when receptor meets ligand… When a ligand binds to a receptor, In turn, these it causes*: changes in the receptor result in*: Conformational change Enzyme activation Dimerization + clustering Intracellular remodelling Changes in membrane location Relocation of molecules Covalent modifications *any combination *any combination benefits of multimerization Cell-cell interactions rely on a binding affinity to maintain contact over long periods of time Extended contact facilitates signal transduction and exchange of cytokine signals adaptive immune receptors  Ig BCR Immune receptors bear The B-cell receptor binds immunoglobulin domains foreign antigens. It is known as an antibody when secreted. Quick Tm | Cs | Se TCR Immune receptors can be The T-cell receptor recognises reference transmembrane, cytosolic foreign or self-peptides or secreted presented on MHC molecules COO- CD4 | CD8 The carboxyl terminus is These are T-cell co-receptors lacking in secreted that help to define T-cell subset immunoglobulins functions. co-receptors are key Antigen-immune system receptor interactions are enhanced by co- receptor binding These are separate receptor-ligand interactions that may take place near the original interaction Often times, a single type of interaction may be insufficient to lead to an activation event A co-receptor interaction may provide a second signaling interaction to further signal the cell to proceed with activation immunoglobulin 2-D model space-fill model Two heavy chains (variable and constant regions) Two light chains (variable and constant regions) Held together by intra-/interchain disulfide covalent bonds  structural differences Secreted antibodies are grouped into five major isotypes (classes) IgA: alpha (α) heavy chain IgD: delta (δ) heavy chain IgE: epsilon (ε) heavy chain IgG: gamma (γ) heavy chain IgM: mu (μ) heavy chain Each class performs different functions during immune responses Light chain isotypes are Kappa (κ) Lambda (λ) immunoglobulin (BCR) Extend the “arms” of Antigen binding domain the antibody away Each Ab can bind two from the hinge region antigen molecules Glycosylation helps Contains proline, making it spread heavy-chain flexible domains apart Also cysteine, facilitating Also play a functional heavy-chain dimerization role through interchain disulfide bond formation Membrane-bound Ab has an extracellular hydrophilic 26 amino acid “spacer”, a 25 amino acid hydrophobic transmembrane segment and a very short three amino acid cytoplasmic tail Secreted Ab is formed by alternative RNA splicing mechanisms that remove/replace these regions T-cell receptor Contains immunoglobulin domains (similar to BCR) Two subunits, α and β, each have a Constant region and Variable region Variable regions have three CDRs forming peptide specific binding site Constant regions each contain transmembrane regions Two TCR types, αβ (more common) and γδ, have diverse antigen binding characteristics  TCR signal transduction via CD3 complex. Receptor-associated molecules CD4 (avidity) and CD28 (required for naive cell activation). BCR signal transduction via Iga and Igb. Receptor-associated molecules CD19, CD21 and CD81 (transmit and relay signals to cell interior innate immune receptors pattern recognition receptors (PRRs) PRRs recognize Pathogen Associated Molecular Patterns (PAMPs) Receptors for PAMPs may uniformly recognize large numbers of pathogens that share the same PAMPs Receptors for PAMPs are not clonally distributed but are expressed equally on the same cell types Receptors for PAMPs may be integral membrane proteins or intracellular proteins PAMPs represent motifs of recurring patterns on bacteria, yeast, and parasites  human PRRs Full name Cellular location(s) Ligands Cellular functions TLR Toll-like receptor Plasmamembrane, Microbial carbohydrates, Production of antimicrobials, endosomes, Lysosomes lipoproteins, fungal mannans, antivirals, and cytokines; bacterial flagellin, viral RNA, inflammation self-components of damaged tissues, etc. CLR C-type lectin receptor Plasma membrane Carbohydrate components of Phagocytosis, production of fungi, mycobacteria, viruses, antimicrobials and cytokines; parasites, and some allergens inflammation RLR Retinoic acid inducible Cytosol Viral RNA Production of interferons and gene-I (RIG-I)-like receptor cytokines NLR Nucleotide oligomerization Cytosol Fragments of intracellular or Production of antimicrobials domain (NOD)-like receptor extracellular bacteria cell wall and cytokines; inflammation peptidoglycans ALR Absent-in-melanoma Cytosol and nucleus Viral and bacterial DNAs Production of interferons and (AIM)-like receptor cytokines now for the signalling molecules Basic information… Cytokine signals are usually generated by the binding of a ligand to a complementary cell-bound receptor Cytokine-receptor binding is noncovalent, although it may be of generally high affinity Cytokine-signaling end results often induce a change in the transcriptional program of the target cell A cytokine signal is any event that instructs a cell to change its metabolic or proliferative state How they work Cytokines are proteins that mediate the effector functions of the immune system They can act in several different ways… Endocrine action ‒ released into the bloodstream to effect distant cells Paracrine action ‒ released to effect nearby cells Autocrine action ‒ released, but then bind to receptors on the cell that produced them  pleiotropic cytokine action… 01 activity induces different biological effect dependent on target cell redundant 02 activity mediates similar effects on target cell synergy 03 effect combines two cytokine activities to be greater than additive effect antagonistic 04 effect inhibits one cytokine’s effect by another’s action cascade 05 effect of one cytokine on one target cell to produce additional cytokine(s) practical examples  the six cytokine families Family name Representative members of family Comments Interleukin-1 family IL-1α, IL-1β, IL-1Ra, IL-18, IL-33 IL-1 was the first noninterferon cytokine to be identified. Members of this family include important inflammatory mediators. Class 1 (hematopoietin) IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-13, Members of this large family of small cytokine molecules cytokine family IL-15, IL-21, IL-23, GM-CSF, G-CSF, exhibit striking sequence and functional diversity. growth hormone, prolactin, erythropoietin/hematopoietin Class 2 (interferon) IFN-α, IFN-β, IFN-γ, IL-10, IL-19, IL-20, IL- While the IFNs have important roles in antiviral cytokine family 22, IL-24 responses, all are important modulators of immune responses. Tumor necrosis factor TNF-α, TNF-β, CD40L, Fas (CD95), BAFF, Members of this family may be either soluble or family APRIL, LT-β membrane-bound; they are involved in immune system development, effector functions, and homeostasis. Interleukin-17 family IL-17 (IL-17A), IL-17B, IL-17C, IL-17D, IL- This is the most recently discovered family; members 17F function to promote neutrophil accumulation and activation and are proinflammatory. Chemokines IL-8, CCL19, CCL21, RANTES, CCL2 All serve chemoattractant function. (MCP-1), CCL3 (MIP-1α)  IL-1 family Cytokines of the IL-1 family promote inflammation IL-1 stimulated by viral, parasitic, or bacterial antigens IL-1 Secreted very early in immune responses by macrophages and dendritic cells Acts locally on capillary permeability and to pull leukocytes to infected tissues Acts systemically to signal the liver to produce acute phase proteins Can help to activate adaptive immune responses  class 1 family Class I cytokines are diverse in action and cell target Structurally class I cytokines are a single protein Class 1 family Most of class I cytokines are made up of multiple subunits Cytokines recognized by Common cytokine receptors bearing that common receptor subunit subunit γc IL-2, IL-4, IL-7, IL-9, IL-15, IL-12 βc IL-3, IL-5, GM-CSF gp130 IL-6, IL-11, LIF, OSM, CNTF, IL- 27  IFN family Type I interferons IFN-α and IFN-β are 18–20 kDa dimers with antiviral effects Secreted by activated macrophages and dendritic cells IFN Induce synthesis of ribonucleases and inhibit protein synthesis Type II interferon (IFN-γ) Dimer produced by activated T/NK cells Potent modulator of adaptive immunity Type III interferon family (IFN-λ) Secreted by plasmacytoid dendritic cells Upregulate genes controlling viral replication and host cell proliferation  TNF family Tumor Necrosis Factor (TNF) regulates development, effector function, and homeostasis of cells of the skeletal, neuronal, and immune system TNF TNF cytokines may be soluble or membrane bound TNF-α is proinflammatory and produced by activated macrophages and other cell types TNF-β (lymphotoxin-α) is produced by activated lymphocytes, delivering signals to leukocytes and endothelial cells.  Lymphotoxin-β mem. bound TNF 01 induces lymphocyte differentiation BAFF and APRIL 02 required for B-cell development and homeostasis TNF 03 CD40 Ligand (CD40L) involved in transmitting the T-cell differentiation signal to B cell Fas Ligand (FasL) 04 programmed cell death - induces cell death (apoptosis) upon ligand binding  IL-17 family IL-17 family cytokines are proinflammatory molecules expressed on a variety of cells Receptors found on neutrophils, keratinocytes, IL-17 and other nonlymphoid cells Tend to work at the interface of innate and adaptive immunity Generally exist as homodimers Monomers range from 17.3–22.8 kDa All are transmembrane proteins  chemokines Direct leukocyte migration They are small (7.5–12.5 kDa) proteins Ch Possess highly conserved disulfide bonds that dictate both structure and category (six categories) Share two, four, or six conserved cysteine residues chemokine receptors Are G-protein-coupled receptors (seven-pass transmembrane proteins) Transduce signals via interactions with a polymeric GTP/GDP- binding G protein Many receptors can bind to more than one chemokine Several chemokines are able to bind to more than one receptor Signaling through chemokine receptors helps cells move to different body areas  remember… A cellular signal is any event that instructs a cell to change its metabolic or proliferative state Signals are usually generated by the binding of a ligand to a complementary cell-bound receptor A cell can become more or less susceptible to actions of a ligand by increasing or decreasing expression of the receptor for that ligand  remember… Cell signaling often induces a change in the transcriptional program of the target cell Sometimes multiple signals through multiple receptors are required to effect particular outcomes Integration of all signals received by a cell occurs at the molecular level inside the recipient cell putting it all together  step 02 tyrosine phosphorylation of ITAMs. CD3 (T cells) and Igα/β (B cells). Phosphorylated tyrosines dock adaptor proteins. step 01 antigen binds ligand. Receptor dimerization / multimerization. Clustering in lipid rafts. step 03 Adapter proteins help to gather members of signaling pathways Signal-inducing phosphatidyl bisphosphate (PIP2) breakdown by phospholipase C gamma (PLCγ) causes an increase in cytoplasmic calcium ion concentration Calcium ions bind Calmodulin (CaM) and activate it. Calmodulin dephosphorylates NFAT in the cytosol, activating it. NFAT migrates to the nucleus and activates immune-specific genes. step 04 Ras is a G protein Activated when GTP exchanged for GDP Guanine-nucleotide Exchange Factors (GEFs) activate Ras by inducing this exchange GTPase activating proteins (GAPs) inhibit Ras by stimulating its ability to break down GTP into GDP Ras (once active) participates in downstream signaling events step 05 The Ras/MAP kinase cascade activates transcription through AP-1 by these nuclear events Phosphorylated ERK (pERK) phosphorylates and activates Elk-1 transcription factor leading to production of the Fos protein Fos is phosphorylated by ERK and binds with phosphorylated Jun protein This pJun/pFos combination is known as transcription factor AP-1 AP-1 can facilitate transcription of the IL-2 gene step 06 Phosphokinase C (PKC) activates Nuclear factor kappa B (NF-κB) NFκB inactive in the cytoplasm when bound to inhibitor (IκB) PKC phosphorylates IκB releasing it from NFκB Diacylglycerol activates PKC, activating IKK complexes IκB phosphorylation and ubiquitination targets it for destruction NF-κB translocates to the nucleus to enhance transcription Immune responses and cell signaling Antigen signaling includes ○ Bringing dendritic cells into the required locations ○ Macrophages and neutrophils upregulate phagolysosome activity and cytokine production ○ Dendritic cells exhibit antigen peptides on MHC class I and MHC class II ○ Cytoplasmic proteasomes process antigen to peptides ○ Dendritic cells induced to secrete cytokine lift off… 01 leukocytes Increased migration into infected tissues. chemokine and cytokine prod. M∅ and neutrophils 02 pathogen destruction. activation of adaptive immunity. cytokine production. dendritic cells 03 enhanced antigen presentation. chemokine and cytokine production. motility B and T cells 04 activation. cell division and differentiation. cytokine production. NFAT and NFkB detail AP-1 detail Component B-cells T-cells Receptor B-cell receptor – similar to antibody T-cell receptor – heterodimer, either ab helpful summary structure. CH structure modified to bind to cell membrane (more common, binds MHC) or gd (mucosal localiztion, binds MHC and Short cytoplasmic tail other ligands). Accessory proteins Iga and Igb with ITAM (Immuno-receptor CD3 complex (3 dimers – de, ge and (signal transduction function) tyrosine activation motifs) [either zz or zh]. Intra-cytoplasmic tails of CD· complex contain ITAMs Ligand Antigens (sometimes bound to MHC class I and II complement components eg C3d) Co-receptors CD19, CD21 (C3d binding) and CD81 CD4 (MHC class II) and CD8 (MHC class I) - bind non-polymorphic regions of the MHC molecule CD28: naïve T-cells need CD28 co- stimulation – CD4 and CD8 alone are not enough. Key tyrosine kinases Syk and Lyn (membrane associated) Lck (membrane associated) and ZAP-70 Key adaptor proteins Phospholipase C; BLNK; LAT, SLP76 and GADS, PLC Key effector molecules NFAT, NF-kB, AP1 NFAT, NF-kB, AP1 session 3 – prepare… 01. Name and describe three components of the innate immune system. 02. What is the principal role of phagocytes (neutrophils and macrophages) in natural immunity, and what are their main properties? 03. If pus contains mostly neutrophils, does it mean that macrophages do not injure host tissues?