1
Q
Specificity in immunity
Q: What does “specificity” mean in immunology?
A
A: The ability of the immune system to distinguish and respond to specific targets (e.g., pathogens) while ignoring others.
2
Q
Specificity in immunity
Q: What is the key idea behind specificity?
A
A: Discrimination — the immune system distinguishes self vs non-self.
3
Q
Specificity in immunity
Q: What does specificity ensure?
A
A: That immune responses are directed only at harmful pathogens, not the body’s own cells.
4
Q
Specificity in immunity
Historical Insight
Q: What did Thucydides observe about immunity?
A
A: People who recovered from disease were protected from reinfection, suggesting immune specificity and memory.
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Q
Specificity in immunity
Biological Definition
Q: How is specificity defined in biology?
A
A: The narrow range of substances an antibody or immune molecule can bind to or act against.
6
Q
Specificity in immunity
Biological Definition
Q: Why can specificity have different meanings?
A
A: It varies across disciplines, but in immunology it focuses on target recognition and discrimination.
7
Q
Specificity in immunity
Molecular Basis of Specificity
Q: What underlies specificity at the molecular level?
A
A: Molecular recognition — interactions between immune molecules and their targets.
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Q
Specificity in immunity
Molecular Basis of Specificity
Q: What determines molecular recognition?
A
A: Binding affinity between molecules (e.g., receptor and ligand).
9
Q
Specificity in immunity
Molecular Basis of Specificity
Q: What does this equation represent?
A
P + L ⇌ PL
A: Binding between a protein (P) and ligand (L) forming a complex (PL).
10
Q
Specificity in immunity
Molecular Basis of Specificity
Q: What is the equilibrium constant (Ka)?
A
Ka = [PL] / ([P][L])
It measures the strength of binding between molecules.
11
Q
Specificity in immunity
Molecular Basis of Specificity
Q: What is high-affinity binding?
A
A: Strong, stable interactions → high specificity
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Q
Specificity in immunity
Molecular Basis of Specificity
Q: What is low-affinity binding?
A
A: Weak interactions → lower specificity
13
Q
Specificity in immunity
Linking Concepts
Q: How are binding affinity and specificity related?
A
A: Higher affinity = more precise molecular recognition = greater specificity.
14
Q
Specificity in immunity
Linking Concepts
Q: What is the core principle of immune specificity?
A
A: Differential binding affinities enable the immune system to distinguish targets.
15
Q
Specificity in immunity
Q: Is the innate immune system specific?
A
A: Yes, but less specific than adaptive immunity — it recognizes general patterns rather than unique targets.
16
Q
Molecular Recognition vs Human Recognition
Q: What is an example of human recognition?
A
A: Recognising a familiar face at a distance, often triggering an emotional response.
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Q
Molecular Recognition vs Human Recognition
Q: How does human recognition differ from molecular recognition?
A
A: Human recognition is sensory and cognitive, while molecular recognition is physical binding between molecules.
18
Q
Molecular Recognition vs Human Recognition
Molecular Recognition Basics
Q: What is molecular recognition?
A
A: The formation of multiple non-covalent interactions between complementary binding partners.
19
Q
Molecular Recognition vs Human Recognition
Molecular Recognition Basics
Q: What types of forces are involved in molecular recognition?
A
A: Hydrogen bonds, ionic interactions, van der Waals forces, and hydrophobic interactions.
20
Q
Molecular Recognition vs Human Recognition
Molecular Recognition Basics
Q: What is a classic example of molecular recognition in biology?
A
A: DNA double helix formation via complementary base pairing mediated by hydrogen bonds.
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Q
Molecular Recognition vs Human Recognition
Molecular Recognition in Immunology
Q: What does “recognition” mean in immunology?
A
A: It is the binding event between immune receptors and their ligands.
22
Q
Molecular Recognition vs Human Recognition
Molecular Recognition in Immunology
Q: Why is binding essential for immune recognition?
A
A: Without binding, no recognition occurs, and no immune response is triggered.
23
Q
Molecular Recognition vs Human Recognition
Molecular Recognition in Immunology
Q: Is immune recognition intentional?
A
A: No — it occurs when molecules happen to bind with sufficient affinity (a probabilistic event).
24
Q
Molecular Recognition vs Human Recognition
T Cell Recognition (Using Diagram)
Q: What molecules are involved in T cell recognition?
A
A: T cell receptor (TCR) binds to peptide–MHC complex on an antigen-presenting cell.
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Molecular Recognition vs Human Recognition
T Cell Recognition (Using Diagram)
Q: What does the diagram illustrate about recognition?
A: Recognition occurs at the interface between TCR and peptide–MHC.
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Molecular Recognition vs Human Recognition
T Cell Recognition (Using Diagram)
Q: What happens after successful recognition?
A: T cell activation → proliferation and differentiation.
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Molecular Recognition vs Human Recognition
T Cell Recognition (Using Diagram)
Q: Why is the correct peptide ligand important?
A: Only the correct peptide–MHC combination will bind effectively to the TCR and trigger a response.
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Molecular Recognition vs Human Recognition
Q: What determines whether recognition leads to activation?
A: The specificity and strength (affinity) of the binding interaction.
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Molecular Recognition vs Human Recognition
Q: What is the key takeaway about immune recognition?
A: Recognition = specific molecular binding event that triggers immune responses.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Pathogen-Associated Molecular Patterns (PAMPs)
Q: What are PAMPs?
A: Conserved molecular structures found on pathogens (e.g., bacteria, viruses).
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Pathogen-Associated Molecular Patterns (PAMPs)
Q: Why are PAMPs important?
A: They allow the immune system to recognise pathogens broadly based on shared features.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Pathogen-Associated Molecular Patterns (PAMPs)
Q: What is meant by “conserved patterns”?
A: Structures that are common across many microbes and do not change much.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Pathogen-Associated Molecular Patterns (PAMPs)
Q: Why are PAMPs good targets for the immune system?
A: They are essential for pathogen survival, so they cannot easily mutate.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Examples of PAMPs
Q: What is flagellin?
A: A protein component of bacterial flagella — recognised as a PAMP.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Examples of PAMPs
Q: What is peptidoglycan?
A: A structural component of bacterial cell walls, absent in mammalian cells.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Examples of PAMPs
Q: What is lipopolysaccharide (LPS)?
A: A molecule found in the outer membrane of Gram-negative bacteria.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Examples of PAMPs
Q: What do these examples show about PAMPs?
A: They are microbe-specific and not found in host cells.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Recognition of PAMPs
Q: What type of receptors detect PAMPs?
A: Pattern Recognition Receptors (PRRs), such as TLRs, NLRs, and RLRs.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Recognition of PAMPs
Q: Where are these receptors located?
TLRs: extracellular or endosomal
NLRs & RLRs: intracellular
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Receptors & Specificity
Q: How does the innate immune system achieve specificity?
A: Through germline-encoded receptors that recognise conserved PAMPs.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Receptors & Specificity
Q: Are innate immune receptors inherited?
A: Yes — they are passed on to offspring (germline encoded).
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Receptors & Specificity
Q: How does the adaptive immune system achieve specificity?
A: Through somatic recombination of gene segments, creating diverse receptors.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Receptors & Specificity
Q: What does “gene rearrangement” mean in adaptive immunity?
A: Receptor genes are reshuffled to generate new combinations.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Receptors & Specificity
Q: Which receptors are involved in adaptive immunity?
A: B-cell receptors (BCRs) and T-cell receptors (TCRs).
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Innate vs Adaptive Specificity
Q: What do innate receptors recognise?
A: Conserved patterns (PAMPs).
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Innate vs Adaptive Specificity
Q: What do adaptive receptors recognise?
A: Highly variable antigens.
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Innate vs Adaptive Specificity
Q: How does response time differ?
Innate: Immediate
Adaptive: Delayed
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PAMPs & Immune Specificity
Pathogen Recognition Overview
Q: What is the key difference in specificity between innate and adaptive immunity?
Innate: Fixed, inherited recognition of common patterns
Adaptive: Flexible, generated recognition of specific antigens
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Innate vs Adaptive Immunity
Receptors
Q: What receptors are used in the innate immune system?
A: Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-like receptors (RLRs).
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Innate vs Adaptive Immunity
Receptors
Q: What receptors are used in the adaptive immune system?
A: B-cell receptors (BCRs) and T-cell receptors (TCRs).
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Innate vs Adaptive Immunity
Localisation
Q: Where are TLRs located?
A: Extracellularly and in endosomes.
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Innate vs Adaptive Immunity
Localisation
Q: Where are NLRs and RLRs located?
Intracellularly
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Innate vs Adaptive Immunity
Localisation
Q: Where are BCRs and TCRs located?
A: Extracellular (on the cell surface).
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Innate vs Adaptive Immunity
Ligands Recognised
Q: What do TLRs recognise?
A: Lipids, lipoproteins, LPS, DNA, dsRNA.
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Innate vs Adaptive Immunity
Ligands Recognised
Q: What do NLRs recognise?
A: Bacterial components such as flagellin.
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Innate vs Adaptive Immunity
Ligands Recognised
Q: What do RLRs recognise?
A: Viral dsRNA.
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Innate vs Adaptive Immunity
Ligands Recognised
Q: What do B-cell receptors recognise?
A: Carbohydrates, proteins, and other molecules.
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Innate vs Adaptive Immunity
Ligands Recognised
Q: What do T-cell receptors recognise?
A: Peptides (presented by MHC).
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Innate vs Adaptive Immunity
Nature of Recognition
Q: What type of patterns does the innate immune system recognise?
A: Conserved molecular patterns (PAMPs).
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Innate vs Adaptive Immunity
Nature of Recognition
Q: What type of antigens does the adaptive immune system recognise?
A: Highly variable antigens.
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Innate vs Adaptive Immunity
Genetic Basis
Q: What is the genetic basis of innate immune receptors?
A: Germline-encoded (inherited).
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Innate vs Adaptive Immunity
Genetic Basis
Q: What is the genetic basis of adaptive immune receptors?
A: Somatic recombination of gene segments.
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Innate vs Adaptive Immunity
Response Time
Q: How fast is the innate immune response?
A: Immediate.
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Innate vs Adaptive Immunity
Response Time
Q: How fast is the adaptive immune response?
Delayed
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Innate vs Adaptive Immunity
What is the key difference between innate and adaptive immunity?
Innate: Fixed, rapid, recognises conserved patterns
Adaptive: Flexible, slower, recognises specific antigens
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Direct Recognition (Innate Immunity)
Direct Recognition by Macrophages
Basics of Direct Recognition
Q: What is direct recognition in innate immunity?
A: The recognition of PAMPs by Pattern Recognition Receptors (PRRs) on immune cells.
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Direct Recognition (Innate Immunity)
Direct Recognition by Macrophages
Basics of Direct Recognition
Q: Which cells are key in direct recognition?
A: Macrophages.
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Direct Recognition (Innate Immunity)
Direct Recognition by Macrophages
Basics of Direct Recognition
Q: What do PRRs recognise?
A: Conserved microbial structures (PAMPs).
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Direct Recognition (Innate Immunity)
Direct Recognition by Macrophages
Macrophage Phagocytic Receptors
Q: What are the main macrophage phagocytic receptors?
Mannose receptor
Scavenger receptors
Dectin-1 (glucan receptor)
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Direct Recognition (Innate Immunity)
Direct Recognition by Macrophages
Macrophage Phagocytic Receptors
Q: Do all PRRs mediate phagocytosis?
A: No — not all PRRs are phagocytic.
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Direct Recognition (Innate Immunity)
Direct Recognition by Macrophages
Macrophage Phagocytic Receptors
Q: Are all phagocytic receptors PRRs?
A: No — there is overlap but not complete equivalence.
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Direct Recognition (Innate Immunity):
Dectin Receptors Overview
Dectin-1 Structure & Function
Q: What does Dectin-1 recognise?
A: β-glucans (fungal cell wall components).
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Direct Recognition (Innate Immunity):
Dectin Receptors Overview
Dectin-1 Structure & Function
Q: What is the structure of Dectin-1?
Single polypeptide chain
Extracellular carbohydrate recognition domain (CRD)
Intracellular ITAM motif
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Direct Recognition (Innate Immunity):
Dectin Receptors Overview
Dectin-1 Structure & Function
Q: What is the function of the ITAM motif?
A: Enables intracellular signalling via phosphorylation.
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Direct Recognition (Innate Immunity):
Dectin Receptors Overview
Dectin-2 Structure & Function
Q: What does Dectin-2 recognise?
A: Mannan (fungal carbohydrate).
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Direct Recognition (Innate Immunity):
Dectin Receptors Overview
Dectin-2 Structure & Function
Q: How is Dectin-2 different from Dectin-1?
Does not contain its own ITAM
Signals via FcRγ chain containing ITAM
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Q: How does molecular shape affect recognition?
A: Small changes in structure → large changes in binding affinity.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Q: What determines specificity in these interactions?
A: The fit and affinity between receptor and ligand.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Q: What is the role of Ca²⁺ in lectin binding?
A: Required for carbohydrate binding in C-type lectin domains.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Q: What happens when a ligand binds to a receptor?
A: Can induce dimerisation or conformational changes, enabling signalling.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
CTLD / CRD Domain
Q: What is the CTLD (CRD)?
A: A conserved carbohydrate-binding domain found in lectin receptors.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
CTLD / CRD Domain
Q: Why is CTLD important?
A: It mediates recognition of carbohydrate structures on pathogens.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Evolution & Structure
Q: Are lectin receptors structurally conserved?
A: Yes — they show structural similarity despite evolutionary distance.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Evolution & Structure
Q: Are all residues conserved?
A: No — only key residues are conserved, while others vary.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Ligands for Dectin-2
Q: What types of ligands does Dectin-2 bind?
A: Mannan-containing carbohydrates from fungi.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Ligands for Dectin-2
Q: Why are these ligands diverse?
A: Different organisms have different carbohydrate structures and linkages.
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Direct Recognition (Innate Immunity):
Molecular Recognition & Specificity
Q: What is the key idea of direct recognition?
A: Innate immune cells use germline-encoded receptors to directly bind microbial patterns, leading to rapid responses.
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Macrophage Mannose Receptor (MR)
Structure of the Mannose Receptor
Q: What is the mannose receptor (MR)?
A: A macrophage receptor that recognises carbohydrate structures (e.g., mannose) on pathogens.
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Macrophage Mannose Receptor (MR)
Structure of the Mannose Receptor
Q: What is the structure of the mannose receptor?
A: A sequence of multiple C-type lectin domains (CTLDs).
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Macrophage Mannose Receptor (MR)
Structure of the Mannose Receptor
Q: Are the CTLDs in MR identical?
A: No — they are very similar but not identical, allowing variation in binding.
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Macrophage Mannose Receptor (MR)
Structure of the Mannose Receptor
Q: What happens to the mannose receptor at different pH levels?
A: It undergoes pH-dependent conformational changes.
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Macrophage Mannose Receptor (MR)
Structure of the Mannose Receptor
Q: How was this conformational change detected?
A: Using SAXS (Small-Angle X-ray Scattering).
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Macrophage Mannose Receptor (MR)
Structure of the Mannose Receptor
Q: What evidence supports conformational change?
Changes in:
Size
Shape
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Macrophage Mannose Receptor (MR)
Biological Function of Shape Change
Q: When do these conformational changes occur?
A: During cycling between the cell surface and endosome.
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Macrophage Mannose Receptor (MR)
Biological Function of Shape Change
Q: Why is pH important in this process?
A: The lower pH in endosomes triggers structural changes.
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Macrophage Mannose Receptor (MR)
Biological Function of Shape Change
Q: What is the functional consequence of this pH change?
A: It enables ligand release inside the cell.
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Macrophage Mannose Receptor (MR)
Recognition & Specificity
Q: Does the mannose receptor show specificity?
A: Yes — but less specific than adaptive immune receptors.
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Macrophage Mannose Receptor (MR)
Recognition & Specificity
Q: How does MR achieve specificity?
A: Through molecular recognition of carbohydrate structures.
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Macrophage Mannose Receptor (MR)
Recognition & Specificity
Q: Is MR recognition “all-or-nothing”?
A: No — it shows graded specificity, distinguishing between similar ligands.
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Macrophage Mannose Receptor (MR)
Recognition & Specificity
Q: What is meant by “molecular precognition”?
A: The ability of receptors to preferentially bind certain molecular patterns.
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Macrophage Mannose Receptor (MR)
Q: How does MR specificity compare to adaptive immunity?
MR: Broad, moderate specificity
Adaptive: Highly precise specificity
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Macrophage Mannose Receptor (MR)
Q: What is the key role of the mannose receptor?
A: To bind, internalise, and release pathogen-derived ligands efficiently via pH-dependent mechanisms.
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TLR Signalling & Specific PRRs
Overview of TLR Signalling
Q: What does TLR5 recognise?
A: Flagellin (bacterial flagella protein).
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TLR Signalling & Specific PRRs
Overview of TLR Signalling
Q: What is the NAIP2-NLRC4 inflammasome?
A: A cytosolic PRR complex that detects bacterial components (e.g., flagellin) and activates inflammation.
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TLR Signalling & Specific PRRs
Origin of Toll Receptors
Q: Where does the name “Toll” come from?
A: A gene in Drosophila involved in development, later found to have immune functions.
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TLR Signalling & Specific PRRs
Location & Structure of TLRs
Q: Where are TLRs located?
Cell surface
Endosomal compartments (for internalised pathogens)
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TLR Signalling & Specific PRRs
Location & Structure of TLRs
Q: What is the basic structure of TLRs?
Membrane-bound monomers
Extracellular domain (ECD)
Intracellular TIR domain (signalling)
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TLR Signalling & Specific PRRs
Activation Mechanism
Q: What happens when a TLR binds its ligand?
A: It dimerises, bringing intracellular domains together.
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TLR Signalling & Specific PRRs
Activation Mechanism
Q: What is the role of the TIR domain?
A: Initiates intracellular signalling cascades.
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TLR Signalling & Specific PRRs
Activation Mechanism
Q: What is special about TLR7, 8, and 9?
A: They are endosomal and can form constitutive dimers.
111
TLR Signalling & Specific PRRs
General TLR Signalling Steps
1. Detection of PAMP
2. Ligand binding
3. Receptor dimerisation
4. Intracellular signalling cascade
5. Activation of transcription factors
6. TFs enter nucleus
7. Gene transcription (e.g., cytokines)
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TLR Signalling & Specific PRRs
Transcriptional Outcomes
Q: What happens after transcription factors are activated?
They induce expression of genes involved in:
Cytokine production
Inflammation
Cell proliferation & differentiation
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TLR Signalling & Specific PRRs
TLR Ligand Recognition
Q: What do TLRs bind?
A: Various PAMPs (e.g., LPS, flagellin, nucleic acids).
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TLR Signalling & Specific PRRs
TLR Ligand Recognition
Q: What is the shape of TLR extracellular domains?
A: Horseshoe-shaped.
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TLR Signalling & Specific PRRs
TLR Ligand Recognition
Q: Do all TLRs assemble the same way?
A: No — assembly varies depending on the ligand.
116
TLR Signalling & Specific PRRs
TLR4 & LPS Recognition
Q: What is required for TLR4 to bind LPS?
TLR4
MD-2 adaptor protein
LPS
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TLR Signalling & Specific PRRs
TLR4 & LPS Recognition
Q: Why is MD-2 important?
A: It enables proper binding of LPS to TLR4.
118
TLR Signalling & Specific PRRs
TLR5 & Flagellin
Q: What does TLR5 bind specifically?
A: Flagellin (FliC protein).
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TLR Signalling & Specific PRRs
TLR5 & Flagellin
Q: What is unique about flagellin structure?
Appears rigid
Actually flexible, making crystallisation difficult
120
TLR Signalling & Specific PRRs
Structural Features
Q: Are TLRs structurally similar?
A: Yes — they share sequence and structural similarities.
121
TLR Signalling & Specific PRRs
Structural Features
Q: Do all TLRs function identically?
A: No — they have similar architecture but different ligand specificities and behaviours.
122
TLR Signalling & Specific PRRs
Q: What is the key principle of TLR function?
A: Ligand binding → dimerisation → signalling → gene activation → immune response
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TLR5 Experimental Challenges
Expression Challenges
Q: Why is expressing functional TLR5 (ECD) difficult?
A: It is hard to produce a stable, soluble form that behaves properly outside cells.
124
TLR5 Experimental Challenges
Expression Challenges
Q: Which species’ TLR5 ECD expression was successful?
A: Zebrafish (others like human, mouse, frog, trout failed).
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TLR5 Experimental Challenges
Expression Challenges
Q: What does this suggest about TLR5?
A: It is conserved across species, but expression properties differ.
126
TLR5 Experimental Challenges
Engineering Solutions
Q: How was TLR5 modified to improve expression?
A: By creating a chimeric protein with hagfish VLR replacing part of the structure.
127
TLR5 Experimental Challenges
Engineering Solutions
Q: Which region was replaced in the chimera?
A: The LRR-CT (C-terminal region of leucine-rich repeats).
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TLR5 Experimental Challenges
Engineering Solutions
Q: Why use hagfish VLR?
A: It is structurally stable and experimentally tractable.
129
TLR5 Experimental Challenges
Engineering Solutions
Q: What system was used for protein expression?
A: Baculovirus / insect cell expression system.
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TLR5 Experimental Challenges
Protein Truncation
Q: How was TLR5 further modified?
A: Truncated to 14 LRR modules (TLR5-N14).
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TLR5 Experimental Challenges
Protein Truncation
Q: Why truncate proteins?
A: To improve stability and solubility for experiments.
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TLR5 Experimental Challenges
Protein Truncation
Q: What modification was made to flagellin (FliC)?
A: The D0 domain was removed (ΔD0 truncation).
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TLR5 Experimental Challenges
Protein Truncation
Experimental Difficulties
Q: What are common problems when expressing proteins?
Protein truncation
Instability
Poor behaviour in solution
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TLR5 Experimental Challenges
Protein Truncation
Experimental Difficulties
Q: Why must proteins “behave well” in experiments?
A: They need to be stable and functional in vitro for structural and binding studies.
135
TLR5 Experimental Challenges
Testing Functionality
Q: How was functionality of TLR5-N14 tested?
A: Using binding assays and signalling assays.
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TLR5 Experimental Challenges
Testing Functionality
Q: What did native gel and size exclusion chromatography show?
A: The ligand CBLB502 binds truncated TLR5-N14.
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TLR5 Experimental Challenges
Testing Functionality
Q: What is CBLB502?
A: A TLR5 agonist (flagellin-derived ligand).
138
TLR5 Experimental Challenges
Signalling Assay
Q: What assay was used to test signalling?
A: Luciferase reporter assay.
139
TLR5 Experimental Challenges
Signalling Assay
Q: What did the luciferase assay show?
A: Both soluble TLR5 and TLR5-N14 can bind and compete for ligand, indicating functionality.
140
TLR5 Experimental Challenges
Q: Why are chimeric and truncated proteins used in immunology research?
A: To create stable, functional versions of difficult proteins for experimental study.
141
TLR5 Experimental Challenges
Q: What is the key conclusion from these experiments?
A: The engineered TLR5-N14 retains ligand binding and functional activity, despite modifications.
142
TLR Signalling & Flagellin Recognition
TLR Signalling Assay (CBLB / Luciferase)
Q: What is CBLB used for in TLR experiments?
A: A known agonist used to stimulate TLR signalling.
143
TLR Signalling & Flagellin Recognition
TLR Signalling Assay (CBLB / Luciferase)
Q: What happens when a TLR is stimulated by an agonist?
A: It triggers a signalling cascade.
144
TLR Signalling & Flagellin Recognition
TLR Signalling Assay (CBLB / Luciferase)
Q: How is TLR activation measured experimentally?
A: Using a luciferase reporter assay.
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TLR Signalling & Flagellin Recognition
TLR Signalling Assay (CBLB / Luciferase)
Q: How does the luciferase assay work?
A gene under an NF-κB promoter is inserted
TLR signalling activates NF-κB
NF-κB drives luciferase expression → light output
146
TLR Signalling & Flagellin Recognition
TLR Signalling Assay (CBLB / Luciferase)
Q: What does luciferase activity indicate?
A: That the receptor is functionally active and signalling.
147
TLR Signalling & Flagellin Recognition
TLR Structure & Sequence
Q: What is the main structural feature of TLR extracellular domains (ECD)?
A: Leucine-rich repeats (LRRs).
148
TLR Signalling & Flagellin Recognition
TLR Structure & Sequence
Q: What is the LRR sequence motif?
A: LxxLxLxxN (leucine-rich repeat).
149
TLR Signalling & Flagellin Recognition
TLR Structure & Sequence
Q: Are TLR sequences highly conserved?
A: No — low overall sequence conservation, but key motifs are preserved.
150
TLR Signalling & Flagellin Recognition
TLR Structure & Sequence
Q: What structural feature is conserved across TLRs?
A: The spacing of leucine residues.
151
TLR Signalling & Flagellin Recognition
TLR Structure & Sequence
Q: What shape do LRRs form?
A: A horseshoe-shaped structure.
152
TLR Signalling & Flagellin Recognition
TLR Structure & Sequence
Q: Why is this horseshoe shape important?
A: It creates a concave binding surface for ligands.
153
TLR Signalling & Flagellin Recognition
Flagellin (FliC) Structure & Recognition
Q: What are the domains of flagellin?
A: D0, D1, D2, D3.
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TLR Signalling & Flagellin Recognition
Flagellin (FliC) Structure & Recognition
Q: What is flagellin (FliC)?
A: The main protein component of bacterial flagella.
154
TLR Signalling & Flagellin Recognition
Flagellin (FliC) Structure & Recognition
Q: Which domain is most important for TLR5 recognition?
A: The D1 domain.
155
TLR Signalling & Flagellin Recognition
Flagellin (FliC) Structure & Recognition
Q: Why is the D1 domain important?
Makes most contact with TLR5
Is highly conserved across species
156
TLR Signalling & Flagellin Recognition
Flagellin (FliC) Structure & Recognition
Q: What about the D3 domain?
A: It is flexible and variable, not consistently structured.
157
TLR Signalling & Flagellin Recognition
TLR5–Flagellin Interaction
Q: How does TLR5 bind flagellin?
A: Through dense interactions at specific binding interfaces.
158
TLR Signalling & Flagellin Recognition
TLR5–Flagellin Interaction
Q: What happens to TLR5 upon ligand binding?
A: It dimerises, enabling signalling.
159
TLR Signalling & Flagellin Recognition
TLR5–Flagellin Interaction
Q: How were important binding regions identified?
A: By mutating contact regions and testing function.
160
TLR Signalling & Flagellin Recognition
TLR5–Flagellin Interaction
Q: What happens when key binding interfaces are mutated?
A: Signalling is reduced or lost, showing functional importance.
161
TLR Signalling & Flagellin Recognition
TLR5–Flagellin Interaction
Q: What does conservation of binding surfaces suggest?
A: These regions are critical for function and evolutionarily preserved.
162
TLR Signalling & Flagellin Recognition
Q: What is the overall mechanism of TLR5 activation?
Flagellin binding → TLR5 dimerisation → signalling → NF-κB activation
163
TLR Signalling & Flagellin Recognition
Q: Why can TLR5 detect many bacterial species?
A: It recognises the conserved D1 domain of flagellin.
164
TLR Signalling & Flagellin Recognition
Q: What do binding and luciferase assays confirm?
A: That structural binding interfaces are functionally relevant.
165
TLR Signalling & Flagellin Recognition
Q: What is the key principle linking structure and function in TLRs?
A: Conserved structural motifs (LRRs) enable recognition of conserved pathogen features, leading to immune activation.
Y3 Immunology
flashcards
Decks in class (25)
# Cards
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week 1 - Evolution of innate immunity41
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week 2 - non cellular immunity37
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week 2 - phagocytes69
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week 3 - Phagocytosis and phagosome maturation70
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week 3 - phagosome maturation notes from Freeman and Grinstein 201415
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week 3 - adaptive immunity94
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week 4 - adaptive immunity II108
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week 4 - adaptive immunity III138
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week 5 - Pattern recognition receptors166
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week 5 - pattern recognition receptors - NAIP2-NRCL4 inflammasome47
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week 5 - PRR - RIG-I PAPER NOTES (Nature 2011)26
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week 6 - b cell receptors128
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week 6 - t cell receptors - part 1113
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week 6 - t cell receptors - part 273
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week 6 - t cell receptors - part 350
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week 8 - allergy and autoimmunity155
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week 8 – Immune receptor signalling128
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week 8 - therapeutic antibodies - Why antibodies as therapeutic agents?36
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week 8 - therapeutic antibodies - How are (therapeutic) antibodies made?21
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week 8 - examples of therapeutic antibodies33
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week 8 - therapeutic antibodies - effector mechanisms113
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week 8 - therapeutic antibodies - new possibilities through genetic engineering30
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week 9 - cancer immunotherapy180
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week 10 - T cell function in solid cancers120
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week 10 - Imaging T cell function86
