Important ones

Question 1

Describe TLRs

Answer 1

• Cause a change in gene expression i.e. allowing production of cytokines
• 10 TLRs in humans, each recognising distinct MAMPs
• examples:
  
  • TLR1 recognises lipopeptides
  • TLR3 recognises dsRNA
  • TLR4 recognises LPS etc.
• always membrane associated
  
  • Can be on the cell-surface or endosomal membranes!!
• The extracellular domain of TLR3 has a horseshoe shape formed by leucine-rich repeats (recognises MAMP (ligand)).
• The inner surface has β-sheet structure and forms the ligand binding domain (dsRNA in this case)
• Usually 2 subunits need to come together: dimerisation triggers signalling

Question 2

Describe what NOD-like receptors are and their function

Answer 2

Cytoplasmic receptors

NOD-like receptors (NLR)

• large group of cytoplasmic receptors that recognise bacterial components e.g. peptidoglycan, flagellin

function:

• trigger cells to make cytokines, particularly pro-inflammatory cytokines
• signal expression of pro-inflammatory cytokines
• trigger assembly of inflammasomes
  
  • inflammasomes are multisubunit complex that cleaves inactive cytokine precursors into active cytokines

Question 3

Describe the mechanism of action of TLRs, NLRs and inflammaosomes upon MAMP recognition

Answer 3

• Cell recognises MAMP through TLR → induces signalling → changes in gene expression → cytokines produced
• Many cytokines are initially made in an inactive form (important so they aren’t innapropriately produced) these are called pro-cytokines
  
  • they need to be cleaved to become activated cytokines
• Bacterium been taken up by phagocytosis → material from phagolyssome released (MAMPs like LPS) → recognised by NLRs (in cytoplasm), and also signal nucleus to start making different genes? → inflammasome produced
• Inflammasome protein complex needed to process pro-cytokines by cleaving them
• Cytokines produced fully
• NLRs important for bacterial infections
• Receptors in cytoplasm for dealing with viruses
  
  • RIG-I-like receptors detect viral RNA produced within host cells
  • induce different response → induce cells to start making inteferons (sub-type of cytokine)

Question 4

What are RIG-I-like receptors?

Answer 4

RIG-I-like receptors

• viral sensors that detect viral RNA produced within host cells
• signal expression of interferons

Question 5

Describe the 5 types of inflammatory mediators produced by sentinel cells, and give examples of each type

Answer 5

• Lipid mediators
  
  • e.g. prostaglandins
    
    • stimulate dilation of blood vessels and act on pain receptors
    • e.g. aspirin stops synthesis of prostaglandins (analgesic)
• Vasoactive amines
  
  • e.g. histamine, bradykinin
  • chemicals which call dilation of blood vessels
• Chemoattractants
  
  • e.g. fmet-leu-phe
  • help phagocytes move into the tissues
• Complement proteins
  
  • e.g. C5a
• Cytokines
  
  • e.g. TNF
  • production of by sentinel cells

Question 6

Name the 5 sub-families of cytokines and their functions

Answer 6

1. IL-1 family:
   
   1. most produced as inactive precursors that must be cleaved by inflammasomes
   2. important in inflammation
2. Haematopoietin superfamily:
   
   1. includes factors involved in leukocyte differentiation e.g. GM-CSF but also IL-2, IL-4, IL-6 (important in T cell responses).
   2. broadly, they stimulate the generation of new white blood cells from bone marrow
3. Interferons:
   
   1. involved in responses to viruses
4. TNF family (e.g. TNFα = tumour necrosis factor):
   
   1. many are transmembrane proteins that are shed, important in inflammation.
   2. very potent and toxic
5. Chemokines:
   
   1. involved in cell movement (e.g. IL-8 a.k.a CXCL8 → induce neutrophil movement out of blood stream into tissues)
   2. induce cell movement
   3. act like C5a
      
      • Cytokine receptors for the various subfamilies also tend to have similar structures and to signal in a similar way

Question 7

Describe the local and systemic effects of each of the 5 types of cytokines that may be produces by tissue-resident macrophages in the early stage of immune response

Answer 7

• IL-1-beta
  
  • Local effects:
  • Pro-inflammatory
  • Activates lymphocytes
  • Local tissue destruction increases access of effector cells
  • Systemic effects: Fever & production of IL-6
• IL-6
  
  • Local effects: activates lymphocytes(B & T cells)
  • increaes antibody production
  • System effects: fever, induces acute-phase protein production
• CXCL8
  
  • local: chemotactic factor recruits neutrophils, basophils and T cells to site of infection
• IL-12
  
  • local: activates NK cells, induces differentiation of CD4 T cells into TH1 cells
• TNF-alpha
  
  • Local effects: Activates vascular endothelium and increases vascular permeability which leads to increased entry of IgG, complement, and cells to tissues and increased fluid drainage to lymph nodes
  • Systemic effects: Fever, mobilisation of metabolites & shock (can cause sepsis)

Question 8

What are interferons and what are the 2 types, how do they act

Answer 8

• Viral infection induces the production of interferons
• “Intruder alert cytokines”
• Interferons Interfere with viral replication
• Type I interferons: IFN-α ; IFN-β
  
  • IFN-α (several genes) & IFN-β (1 gene) act in a very similar way
  • many (almost any) cell types make type I interferons after viral infection.
  • Induce expression of interferon-stimulated genes (ISGs)
  • Some cell types (e.g. dendritic cells) are specialised for this – express high levels of endosomal TLRs e.g. TLR3 and TLR9.
• Type II interferon: IFN-γ
  
  • Main role is to modulate immune responses
  • different structure to IFN-alfa and beta

Question 9

Describe the mechanism of action of type 1 interferons and how they induce resistance to viral replication

Answer 9

• Virus infected host cell → IFN-α, IFN-β produced
• IF cell has TLRs → inteferon alfa and beta expressed and produced
  1. Induce resistance to viral replication in all cells.
  
  • not only the infected cell but secreted inferons can bind to surrounding cells and help them.
  • How they induced resistance to viral replication? →
    
    • Induce expression of endoribonuclease that degrades viral RNA and also, protein kinase phosphorylates eukaryotic initiation factor 2, inhibiting protein translation. (good because most proteins made by a virus infected cell are viral proteins)
      2. Increase MHC class I expression in all cells
  • MHCI is required for antigen presentation to cytotoxic T cells, so infected cells are more easily recognised and killed.
  • important in inducing adaptive immunity
    
    3. Activate NK cells to kill virally infected cells more efficiently
    4. Induce chemokines to recruit lymphocytes
       1. chemokines are chemoattractants

Question 10

Describe type 2 interferons, who makes them, their function

Answer 10

• Made by neutrophils, NK cells, T cells
  
  • unlike Type I inteferons, not all cells make it
• Primary role in adaptive immunity
  
  • particularly T-cell immunity
• Increases expression of MHCI and MHCII
  
  • helps induce formation of Cytotoxic T cells
  • MHC II expression increased → induce T helper cells
• IFN-γ made by T helper cells activates macrophages in responses to intracellular pathogens
  
  • activates macrophages to kill intracellular pathogens in the macrophage itsself

Question 11

Describe the B cell Receptor

Answer 11

• • NB! different to soluble antibodies as it is inserted into the lipid bilayer
  • Membrane immunoglobulins (antibodies) contain and extra ~ 26 hydrophobic amino acids at the C-terminus.
  • Most B-cells express IgM and/or IgD as B-cell receptors.
    
    • when developing
  • Recognise and bind to antigen, but cannot generate a signal itsself!
  • Membrane immunoglobulins are associated with two other proteins, IgALPHA and IgBETA.
  • Image:
    
    • Iga and Igb contain a single ITAM (Immunoreceptor Tyrosine Activation Motif) in their long cytoplasmic domains.
    • monomer of IgM
    • region that passes lipid bilayer, only a few amino acids ~3 pertrude past the lipid bilayer which is not enough to transduce a signal
    • through the Ig-alpha and Ig-beta which protrude further into the cell allows signalling

Question 12

How are immunoglobulins encoded in genes? what do genes consist of

Answer 12

• Heavy (H) chains - Chromosome 14
• Kappa chains - Chromosome 2
• Lambda chains - chromosome 22
• Each locus (Chromosome) has multiple VARIABLE region genes and one CONSTANT region genes.
• The variable regions (each) are encoded by 2 or more exons
• Light chain V regions are encoded by 2 segments of DNA:
  
  • Kappa chainsas example
  • single constant region gene, multiple downstream genes making variable region
  • most variable region encoded by Vkappa exon, some made by Jkappa exon
  • in intact kappa protein we will find most of sequence is made of constant region, variable region made mostly up of Vkappa gene(exon) and a little bit of J kappa gene(exon)
• Heavy chains are encoded by 3 segments of DNA
  
  • 3 exons/segments of DNA
  • V D and J exon. (D for diversity)
  • most made up by Constant region exon, rest made up of variable, mainly V exon

Question 13

Descsribe the process of somatic V(D)J recombination

Answer 13

• B cell differentiation triggered:
• Rearrangement of light and heavy chain genes occurs during B cell during differentiation from Lymphoid SC into B cell (before antigen contact) → permanent changes in the DNA
  
  • Using Kappa light chain as example:
    
    • DNA is permanently rearranged: a V gene is spliced to a J gene, and intervening DNA is excised.
    • V and J segments are spliced together and lie very close to the constant region
    • Each V gene has its own promoter → an enhancer element allows the gene to be transcribed: This occurs because now the V gene (including its promoter) are now close to the enhancer element after splicing (gene rearrangement occurred)
    • Intervening sequences are removed by RNA processing → mRNA produced for variable and constant regions → kappa light chain made

Question 14

Describe the mechanism of class switching

Answer 14

• Shown below at the top is the arrangement of all the heavy chain genes in the genome
  
  • Mature B cell VDJ + IgM (Constant region mu) closest → IgM
• Adjacent to the heavy chain genes is a gene sequence know as a switch sequence.
  
  • this allows recombination of the VDJ gene with another class constant region gene e.g. IgA heavy constant region.
  • Looks similar to somatic recombination but enzymes and process is different
• DNA inbetween is lost (irreversible). This means that the B cell cannot switch back to e.g. IgM
• switch regions aren’t conserved but tend to be rich in G which AID likes to bind to.
• How is the enzyme acting → next

• Class switching by DNA recombination between switch regions:
  
  • intervening DNA lost
  • irreversible
• NB. Different mechanism to V-D-J joining. Initiated by AID acting at switch regions (G –rich tandem repeated DNA sequences found close to C region genes).

Question 15

Describe the mechanism of action of AID

Answer 15

• Mechanism of action of activation-induced cytidine deaminase (AID)
  
  • AID deaminates cytidine to form uracil (C→U)
  • AID is expressed in activated B lymphocytes, only active on ssDNA
  • Activity triggers DNA repair pathways
  • Repair pathways in B cells are error-prone, leading to different (2) mutational outcomes:
    
    • Mismatch repair, base excision repair resulting in : Somatic hypermutation
    • If it occurs in the G-rich switch sequence you get more Uracil added (AID likes to bind to these regions) ultimately causing double stranded breaks resulting in Class switching
  • AID mutation causes immunodeficiency: Hyper-IgM Syndrome Type 2

Question 16

Describe the concept of MHC restriction

Answer 16

• T lymphocytes can only recognise antigen in the context of self-MHC molecules
  
  • this is known as MHC restriction
• Experiments with inbred mouse strains and virally infected cells (had the same MHC proteins on their surfaces)
• Mouse Strain A and mouse strain B immunised with Virus, T cell from the mice were isolated and cultured in vitro with cells infected with the same virus
  
  • if you took T cells from mouse strain A and mixed with cells from mouse A → would kill mouse A infected cells
  • if you took T cells from mouse strain B and mixed with cells from Mouse B → T cells can’t kill infected cells from mouse A
  • SO T cells will only recognise antigen thats being presented to them by ?? (31 min)
• why did this happen? → 2 ideas
  
  • 2 receptors on T cells – one (TCR) for antigen, one for MHC?
    
    • 1 thats recognised antigen, and one recognising MHC
  • 1 receptor on T cells (TCR) – recognises antigen + MHC?
    
    • only 1 receptor on t cell that recognises antigen and MHC

x-ray crystallography proved answer
- Unknown peptide antigen bound as part of the structure
- At the tip of the molecule in a groove, there was a peptide → proved that T cells recognise MHC and foreign peptide.
- MHC, they bind foreign peptide and transport it to the cell surface

Question 17

Describe how MHC interacting with co-receptors leads to T cell signalling?

Answer 17

• Engagement of the CD4/CD8 co-receptors with the TCR complex enhances phosphorylation of the ITAMs, promoting T cell signalling
  
  • how does it enhance signalling: CD4 as example
  • CD4 with MHC protein enahnces signalling because Cterminus of CD4 protein has a protein kinase associated with it called lck tyrosine kinase, which can phosphorylate the iTAM motifs present on CD3 complex associated with TCR.
  • Engagement of the CD4/CD8 co-receptors with the TCR complex enhances phosphorylation of the ITAMs, promoting T cell activation
  • CD4/8 associate with lck tyrosine kinase.

Question 18

In what 2 forms can IgA be found in? and why/where + describe IgA structure

Answer 18

• In blood: monomer
  
  • do not know what it does in monomer
• In secretions: dimer
  
  • tears, saliva, mucose secretions that coat the mucosal surfaces
  • this is where most infections occur so its important
  • can be found as other polymers
  • dimer needs a J chain to form properly
  • has a secretory component (wrapped around the Fc regions of the dimer)
    
    • helps protect IgA against proteolysis, so e.g. in the gut its important for protection against degradation
    • Secretory component can also bind bacteria itsself in a fairly non-specific way
    • secretory component is a member of the immunoglublin super gene family
    • made up of 5 immunoglobulin domains that interact with Fc region of the dimer

Question 19

Describe how a Naive T cell becomes an effector T cell

Answer 19

• Naive T cell will go into lymphoid tissue, if it meets antigen displayed on appropriate MHC molecule + signal from the co-receptor CD4 or CD8
• NB! T cells get chosen for cytotoxic or helper cell in the thymus during thymic selection.

• Naive T cell:
  
  • Recognition of MHC + peptide + co-receptor (CD4/8) → SIGNAL 1
    
    • this allows the T cell to recognise antigen and become activated, however this is not the whole story
    • to activate a naive T cell in the first place, more is required, signal 2
  • Recognition of CO-STIMULATORY molecule(s) → SIGNAL 2
    
    • T cells exposed to Signal 1 in the absence Signal 2 become unresponsive or “tolerised”
    • co-stimulatory molecules are additional proteins found on the T cell and antigen-presenting cell that also have to engage in order for the T cell to become activated.
    • many of these, example is CD28 (on T cell) interacting with B7 (on antigen presenting cell)
    • → this is known as signal 2. without signal 2, the T cell becomes unresponsive or tolerised
  • Cytokines convert activated T cells into different subsets → (SIGNAL 3)

Question 20

Describe TH1 cells

Answer 20

TH1 cells - IL-12 gamma interferon (Signal 3)
- typically most abundant in blood.

• TH1 cells are generated in response to IL-12 and gamma-interferon
  
  • i.e. thats the TH1 signal 3
• TH1 cells
  
  • Produce IL-2, gamma-interferon and TNF which →
    
    • Activate macrophages → inflammation (Classic CELL-MEDIATED immunity)
      
      • particularly gamma-inteferon is good at activating macrophages and inducing inflammation
      • activated macrophages become much better at killing organisms they ingested, e.g. they may be able to kill tubercle bacilli where before they weren’t able to
    • Important in intracellular infections
    • Induce B cells to make IgG1 and IgG3 (opsonizing) antibodies
      
      • produces a pro-inflammatory response
    • Important for the development of cytotoxic T cells
• overall TH1 cells are important in intracellular infections, but also important for extracellular infections (through stimulating IgG1 and IgG3 production)

Question 21

Describe TH2 cells

Answer 21

• TH2 cells are generated in response to IL-4
• TH2 cells produce IL-4, IL-5, and IL-13
  
  • activate eosinophils and mast cells
    
    • allergy- and parasitic-associated cells
  • IL-4 and IL-13 induce B cells to make IgE (promotes mast cell degranulation)
• important in helminth infections (large EC parasites) and allergy

Question 22

Describe TH17 cells

Answer 22

• tend to be found in lymphoid tissue near mucosal surfaces
• TH17 cells generate in response to TGF-beta and IL-6
• TH17 T cells produce IL-17 and IL-22.
  
  • Activates epithelial cells (which line mucosal surfaces) and fibroblasts.
    
    • Proinflammatory, especially at mucosal surfaces.
  • Recruit neutrophils to sites of infection
    
    • good at getting neutrophils to move out of the blood and onto the mucus surface where the infection is
• Important in fungal and extracellular bacterial infections, role in AUTOIMMUNE disease?

Question 23

Describe T-FH cells (Follicular helper T cells)

Answer 23

• Found specifically in lymphoid follicles
  
  • Lymph node anatomy:
    
    • Lymph node is divided into areas predominantly T cells (blue), and follicles around the outside, where B cells differentiate into plasma cells
      
      • when B cells undergoes clonal selection and clonal expansion, it forms these lymphoid follicles
      • TFH reside in these follicles, normally T and B cells separate in lymph nodes, but here they mingle
• TFH cells generate in response to IL-6
• Help naïve B cells differentiate into plasma cells and memory cells.
  
  • thymus-dependent antigens
• Promote somatic hypermutation + class switching
  
  • through producing cytokines that switch on the expression of AID (activation induce cytesinedeaminase) which promotes somatic hypermutation and class switching
• Contact dependent (requires interaction between co-stimulatory molecules CD154 and CD40), but also produce IL-21.
  
  • for this to occur, B and TFH cells have to interact with each other (in the lymph follicles), B cell presents antigen to T cell, co-stimulatory molecules CD154 (T cell) and CD40 (B cell) → activation of somatic hypermutation + class switching
• also produce IL-21

Question 24

Describe T Regulatory cells

Answer 24

• down regulates the immune response
• generated in response to TGF-beta (Signal 3)
• 2 types:
  
  • Natural Treg
    
    • Develop in the Thymus, recognise MHC + self-peptide
      
      • against what you would expect, recognising MHC + self-peptide, they should normally be selected against, but these don’t
    • Produce cytokines IL-10, TGF-beta which downregulate T cell responses
    • dependent on T cells interacting with cells directly → contact-dependent
  • Induced Treg
    
    • Developed in secondary lymphoid tissues, particularly mucosal lymphoid tissue
    • Recognise MHC + non-self peptide
    • produce cytokines that downregulate immune responses
    • act on T cells e.g. TH1 TH2 etc.
    • Downregulate inflammatory responses etc.