Molecular and Cellular Immunology 2017 Overview of ... · immu3902 – molecular and cellular immunology 2017 1 overview of immu3902 x l1–introduction to innate immunity1 x ntroduction

IMMU3902 – Molecular and Cellular Immunology 2017

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Overview of IMMU3902 x L1 – INTRODUCTION TO INNATE IMMUNITY 1 x L2 – INTRODUCTION TO INNATE IMMUNITY 2 x L3 – MHC STRUCTURE AND FUNCTION x L4 – MHC BIOSYNTHESIS PATHWAYS AND ANTIGEN PRESENTING CELLS x L5 – TCR AND ASSOCIATED MOLECULES x L6 – REGULATION OF T CELL ACTIVATION x L7 – CD4+ T HELPER AND INNATE LYMPHOID CELLS: LINEAGE COMMITMENT AND

EFFECTOR FUNCTIONS x L8 – MECHANISMS OF CELL MEDIATED IMMUNITY: CTL AND NK CELLS x L9 – IMMUNOLOGICAL MEMORY x L10 – DEVELOPMENT AND FUNCTIONS OF PHAGOCYTES x L11 – CYTOKINES x L12 – B LYMPHOCYTE ACTIVATION AND ANTIBODY PRODUCTION x L13 – EFFECTOR FUNCTIONS OF ANTIBODIES 1: COMPLEMENT AND FC RECEPTORS x L14 – EFFECTOR FUNCTIONS OF ANTIBODIES 2: COMPLEMENT PATHWAY x L15 – PROTECTIVE VS PATHOGENIC IMMUNITY x L16 – REGULATION OF IMMUNE RESPONSES


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LECTURE 1 (INTRODUCTION TO INNATE IMMUNITY 1) What is the importance of innate immunity? What are innate immune activators and how are they sensed? What signal transduction and effector molecule expression does innate immunity activate? Overview of PRRs

1. Soluble/secreted; complement system, collectin (MBL, SP-A, SP-D) 2. Membrane-associated; TLRs, C-type lectins (Dectin-1, Mincle) 3. Cytoplasmic; NOD-like receptors (NLRs), RIG-I-like helicases (RLHs)

Effector MoA x Cytokine production/signalling x Chemokine production x Effector molecule activation

Innate Adaptive

- Immediate response - Recognise conserved motifs - Germline encoded - Constitutively expressed/rapid synthesis - No memory

x Repeated exposure Æ no increase in response

- Does not change over course of an individual’s lifetime

- Delayed response - Recognise antigen-specific motifs - Require somatic recombination (VDJ) - Require induction - Immune memory

x Repeat exposure Æ ↑response - Immunological roadmap of immunogen encounters Æ different per person

Importance of innate immunity

x Immediate non-specific immunity (min Æ hrs) o Adaptive immunity (days Æ weeks)

x Intact innate immune system required to guide or direct adaptive immunity x Present in virtually all organisms

Cell types involved in innate immunity and sensing Macrophages: phagocytose foreign objects, destroy them and present to adaptive immune system Dendritic cells (DCs): phagocytose foreign objects, and present to adaptive immune system Neutrophils: rapidly infiltrate damaged tissues and kill them

x Short-lived but high turn-over rate Natural killer cells (NKCs): specifically target non-self cells for killing Innate lymphocyte cells (ILCs): newly discovered innate lymphocyte cells Location of innate immune sensors

1. Circulation and extracellular 2. Extracellular/cell surface

a. Bacteria/fungi 3. Cytosolic

a. Bacteria/virus b. Damage

4. Endosomal a. Bacteria/virus


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What does the innate immune system recognise? Pathogen-associated molecular patterns (PAMPs)

x Intrinsic motifs on/of pathogens x Evolutionarily highly conserved (immune system has adapted to recognise) x Not found in host cells Æ intrinsically non-self

Damage/danger-associated molecular patterns (DAMPs) x Present when homeostasis is disturbed x Can be extrinsic or intrinsic molecules (i.e. aggregated proteins, K+ efflux, cytosolic DNA)

1. Soluble PRRs Complement system

1. Classical Æ complement C1q complex recognises antibody-bound microbe 2. Lectin Æ recognition of carbohydrates on microbial proteins/lipids by MBL (Mannose-

binding lectin) a. MBL is part of the collectin family of proteins

3. Alternative Æ direct binding of complement protein C3b to microbe

2. Membrane-associated PRRs Toll-like receptors (TLRs)

x Transmembrane proteins x 10 TLRs in humans and 13 in mice

TLR4 Æ recognise LPS (G-ve bacteria) x Lipid A = immunogenic PAMP component of LPS x Cytoplasmic adaptor protein MyD88 determine:

o Anti-viral (MyD88-independent) Æ IFN-α, IFN-β (both type I interferons) OR o Pro-inflammatory response (MyD88-dependent) Æ TNF-α, IL-1, IL-6, IL-12

Location: Blood plasma Outcome:

x Opsonisation x Cell recruitment Æ

stimulation of inflammatory responses

x Direct lysis of microbe


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TLR signalling is critical in host control of endogenous retroviruses (ERVs)

x ERVs account for ~8% of the human genome x Prevention of de novo ERV integration into oncogenic genes

PAMPs from different organisms may not be detected by TLRs C-type lectins (CLEC)

x Binds mannose/mannan (bacteria, virus, fungi) & β-glucans (fungi) o Mannan = plural of mannose

x C-type Æ Ca2+-dependent carbohydrate binding x Soluble C-type lectins (secreted PRRs)

o Collectins (MBL, surfactant protein-A/D) x CLEC mainly expressed on cell surface of APCs due to their phagocytic potential

(macrophages, DCs) Dendritic-cell-associated C-type lection-1 = Dectin-1

x Expressed on DCs, monocytes, macrophages and neutrophils x Highly expressed in lungs and intestine x Ligand = β-glucan (fungi wall component) Æ anti-fungal

Effects of Dectin-1 x Induce pro-inflammatory cytokines (IL-6, IL-12, TNF-α)

o IL-12 Æ inhibitory effect on Th17 formation x Produce anti-inflammatory IL-10 x Instruct T cells to secrete IL-17 and become Th-17 cells via IL-23 x Induce ROS production in neutrophils


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Macrophage-inducible C-type lectin (Mincle) & Macrophage C-type lectin (MCL) x Expressed on macrophages, DCs, neutrophils and subsets of B-cells x Expression induced by stimulation with TLR-ligand and cytokines (IFNγ, TNF-α) x Activated by:

o Mycobacterial cord factor (also known as TDM), a glycolipid o Damaged cells release SAP130 (act as a Mincle ligand)

x Require FcRγ/ITAM (Immunoreceptor tyrosine-based activation motif) for signal transduction (since Mincle lacks cytoplasmic tail containing ITAM)

o Activates NF-kB Æ induce TNF-α, IL-1 and IL-6 for Th1/Th17 development


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LECTURE 2 (INTRODUCTION TO INNATE IMMUNITY 2) RNA/DNA sensing and induction of type I interferons (antiviral) Inflammasomes – components, activators/ligands, two signals Innate immune effector mechanisms (phagocytosis, antimicrobial peptides, cytokines – TNFα) Major immune cells Adjuvants and the role of innate immunity in instructing adaptive immunity 3. Cytoplasmic PRRs NOD-like receptors (NLRs)

x NOD = Nucleotide-binding oligomerisation domain x Intracellular sensor of PAMPs/DAMPs

o Muramyl dipeptide (MDP) of both G+ve and G-ve bacteria x RIP2 = central adaptor that activates NF-kB, which subsequently translocate into the

nucleus to induce expression of inflammatory genes RIG-I like helicases (RLH)

x Sense cytoplasmic viral RNA (since ss/dsRNA NOT normally found in the cytoplasm) RLHs lead to induction of type-I interferons (IFN-α/IFN-β) via IRF3/7 for anti-viral effects

x RIG-I (Retinoic acid-inducible gene I) Æ detects short ss/dsRNA (1kbp) x MDA-5 (Melanoma Differentiation-Associated protein 5) Æ detects long dsRNA (3-4kbp)

Both interacts with mitochondrial bound adaptor MAVS (mitochondrial anti-viral signaling protein)

NLRs contain 3 domains: » C-terminal LRR (Leucine-rich repeat) » Central NACHT domain (NOD + NTPase) » N-terminal is variable

- CARD (Caspase recruitment domain) - PYD (Pyrin domain) -


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Cyclic GMP-AMP Synthase (cGAS) x Cytoplasmic DNA sensor

Sense microbial and endogenous dsDNA Æ catalyse synthesis of cGAMP (2nd messenger) ↓

cGAMP bind and activate ER-associated protein STING ↓

Trigger type-I interferons (IFN-α/IFN-β) production [Anti-viral]

TREX1 (cytosolic exonuclease)

x Degrade dsDNA in acidic state (exonuclease) x Deficiency in TREX1 Æ heightened inflammatory response

Inflammasomes

x Sense pathogen (PAMPs) and cellular (DAMPs) damage x Multi-protein molecular machines


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Comprised of: 1. Sensor protein (cytosolic PRRs)

a. NLRP3 (MDP, anthrax toxin, crystals) b. NLRC4 (Flagellin) c. AIM2 (dsDNA) Æ non-NLR

2. Adaptor protein (Link sensor to effector) a. ASC (Apoptosis-associated speck-like protein containing a CARD)

3. Effector protein a. Caspase-1 Æ Activate pyroptosis Æ IL-1β and IL-18 release

i. Cleaves pro-IL-1β and pro-IL-18 into their active, pro-inflammatory, mature form (IL-1β and IL-18)

Pyroptosis = highly inflammatory form of programed cell death x Immune cells recognise foreign danger signals within themselves, release pro-

inflammatory cytokines, swell, burst and die. x In contrast to apoptosis, pyroptosis require caspase-1

Inflammasome signalling pathway (2 signals) - 1st signal = PAMP binding to TLR to initiate signalling cascade leading to the activation +

translocation of NF-kB Æ release of proinflammatory cytokines + pro-IL-1β/pro-IL-18 - 2nd signal = Recognition of intracellular PAMP by NLR Æ assembly of the Inflammasome

(mediated by ASC) Æ recruitment + activation of caspase-1 Æ cleavage of pro-ILs into active form


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Innate immunity effector MoA x Trigger inflammation x Eliminating pathogens x Tissue repair/wound healing x Directing adaptive immunity

TNF-α

x Key role in inflammation and defence x Mainly produced by macrophages x Usu. undetected in healthy individuals Æ elevated in inflammatory & infectious conditions

TNF-α MoA x Induces local inflammation by promoting cell migration

o Act directly on endothelial cells to up-regulate adhesion molecules and increase permeability to allow extravasation

x Activates macrophages to trap intracellular bacterial and parasitic pathogens x Brain Æ fever x Liver Æ release of acute phase proteins (opsonins, complement factors, MBL etc.) x Bone marrow Æ ↑Leukocyte production x Pathological systemic effects

o Low BP Æ Shock o ↑coagulation o Muscle wasting

TNF-α blockade as therapeutic strategy to treat various inflammatory diseases x Side effects relate to inhibiting important inflammatory pathway

o Reactivation of latent TB o ↑ risk of opportunistic infections

Antimicrobial peptides

x Evolutionarily ancient group of anti-microbial compounds (found in virtually all organisms)

o I.e. defensins, cathelicdin, histatins x Constitutively produced and inducible via TLRs, TNF, IL-1 x Produced by many cell types:

o Neutrophils o Macrophages o Paneth cells o Epithelial cells o Mast cells

x Release/activated upon pathogen sensing

x Able to enter microbes Æ form pores Æ disrupt lipid bi-layer Æ lysis of microbe


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Phagocytosis and microbial killing by reactive oxygen/nitrogen species Microbes bind to phagocyte receptors

↓ Phagocytosis (microbe ingested in phagosome)

↓ Fusion of phagosome with lysosome Æ phagolysosome

↓

Activation of iNOS (Inducible nitric oxide synthase) by TNF-α/IFN-J

Major innate immune cells Macrophages

x Phagocytose and digest foreign molecules, microbes and cellular debris x Different organs Æ different resident population

o Kupffer cells (liver) o Alveolar macrophages o Microglia (CNS)

x Monocytes recruited to sites of infection/inflammation Æ differentiate into macrophages x M1 macrophage: activated by LPS/IFN-γ Æ microbicidal x M2 macrophage Æ involved in homeostasis and wound healing

Neutrophils

x Classified as a granulocytes (Neutrophils, basophils, eosinophils) o Harbour cytoplasmic granules containing enzymes and antimicrobial peptides

x Major leukocyte in blood (50-70%) x Produced in the bone marrow (1011/day) x IL-17 regulate neutrophil production by inducing G-CSF (granulocyte CSF)


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Dendritic cells (DCs) x Professional APCs x Many different types

o Langerhans cells (LCs) of the skin/mucosa o Splenic DCs o Plasmacytoid DCs

x Constantly sample environment for foreign molecules/antigens to process x Required for initiation of adaptive immune response x Activation signals affect type of T-cell response

o IL-12 (from DCs) Æ Th1 (IL-12 also prohibit Th17 development) o IL-4 (from basophils & mast cells) Æ Th2

Immature DC (Surveillance cells) Mature DC (APCs)

- Peripheral locations (skin, lung, mucosa) - Specialised for Ag uptake (potent phagocytes) - Poor at Ag presentation (low MHC and costimulatory molecules) - Activation leads to “maturation” + migration

- Lose ability to capture Ag - Upregulate MHC and costimulatory molecules - Produce cytokines, i.e. IL-12 - Migrate to draining lymph nodes to activate T cells

Plasmacytoid DCs (pDCs)

x Doesn’t present Ag to T cells (unlike other DC subsets) but instead express large # of IFN-α in response to TLR signalling

o Via TLR7 (ssRNA) and TLR9 (unmethylated CpG DNA)

x MyD88 activates IRF7 directly Æ inducing IFN-α directly (as opposed to MyD88-dependent pro-inflammatory pathway Æ NF-kB)

Adjuvants

x Compounds added to vaccines to elicit/enhance the immune response (vaccine antigens have limited ability to instigate immune response)

x Inducing inflammation @ site of vaccination aids the recruitment of immune cells + presentation of vaccine antigens to adaptive immune cells

x Activate innate sensors (PRRs) Æ create an antigen depot Freund’s adjuvant

x Most commonly used experimental adjuvant x Incomplete (IFA) = water in oil emulsion only

o Elicits Th2 + Ab response x Complete (CFA) = water in oil + killed M. tuberculosis

o Elicits Th1/Th17 response Æ highly inflammatory o Mycobacterial TDM (cord factor) induced Th17 response via Mincle/CARD9/NF-kB o Prohibited from human use


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Alum Æ activates NLRP3 inflammasome x Hydrated potassium aluminium sulfate [KAl(SO4)2·12H2O] x Most common adjuvant licensed for human use x Induces strong humoral immunity and Th2 dependent immunity

Crystals (alum, silica, asbestos) disrupt the phagolysosome Æ NLRP3 activation

Importance of adjuvants in vaccine research x Adjuvants determine the type of immune response elicited (Th1, Th2, Th17) x Critically important to select the right adjuvant for the disease in question x Harder to find new adjuvants due to stringent requirements to show MoA before licensing

Documents

Molecular and Cellular Immunology 2017 Overview of ... · immu3902 – molecular and cellular immunology 2017 1 overview of immu3902 x l1–introduction to innate immunity1 x ntroduction