32. Specific Immune Response Flashcards
Describe the two main lineages in haematopoiesis.
- Common myeloid progenitor -> These give rise to all of the innate immune system
- Common lymphoid progenitor -> These give rise to all of the adaptive immune system
What are the two main types of pathogen that could provide an immunological challenge?
- Extracellular pathogens
- Bacteria, fungi and protozoan parasites
- Inhabit cornified epithelium, mucosal surfaces and body fluids
- Obligate intracellular parasites
- Primarily viruses and intracellular bacteria
- Distribution defined by the tropism of individual organisms and the expression of appropriate receptors by the target cells
What are the two main types of adaptive immunity and what pathogens do they target?
- Humoral immunity
- Involves the production of immunoglobulins
- Targets extracellular pathogens
- Cell-mediated immunity
- Involved cytotoxic T cells and natural killer (NK) cells
- Targets obligate intracellular pathogens
What are some important characterstics of the adaptive immune response?
- Kinetics of the response measured in days rather than minutes
- Exquisite specificity capable of exploiting the vulnerabilities of each microorganism
- Capacity for immunological memory facilitating more rapid and vigorous responses upon secondary exposure
- Capacity for immunological tolerance to limit deleterious responses to innocuous substances or self components
Draw a graph of the antibody titre in an immune response during a primary and secondary infection.
Compare the pathogen molecules that the innate and adaptive immune responses recognise. What are the implications of each?
Innate immune system:
- Recognises PAMPs
- These are carbohydrates, nucleic acids and proteins that differ greatly between eukaryotes and prokaryotes, so it is easy to distinguish the two
- The receptors (PRRs) are highly conserved between species and there are not many required
Adaptive immune system:
- Recognises antigens
- These are infinitely diverse and a complementary diversity of receptors is therefore required
- There is significant likelihood of cross-reactivity between prokaryotic proteins and their eukaryotic homologues
- A system of antigen recognition is required with exquisite specificity and precision, so as to minimize collateral damage to tissues
What does the adaptive immune system use to bind to antigens?
Immunglobulins
Draw and explain the structure of an immunoglobulin.
- Two heavy and two light chains
- Complementarity-determining regions (CDRs) are part of the variable region at the end of each arm
- Fab fragment -> Binds to the antigen
- Fc fragment -> Fixes complement
Which immunoglobulins are produced in the primary immune response?
- First IgM, which are less effective and do not penetrate as far into the tissue due to their pentameric formation
- Then after class switching there is IgG
What are the two forms of immunoglobulin (antibodies)?
- Cell-associated
- Secreted
Compare the functions of the cell-associated and secreted forms of immunoglobulins (antibodies).
- Cell associated -> Signalling receptor for detecting antigens
- Secreted -> Elimination of foreign molecules
What lymphocytes are immunoglobulins found on?
B lymphocytes
(T lymphocytes also have similar molecules though, called T-cell receptors)
Are B-lymphocyte immunoglobulins and TCRs (T-cell receptors) just found on cells or are they also found as secreted individual structures?
- Immunoglobulins are found on both B-lymphocytes and as secreted structures
- TCRs are found only on T-lymphocytes
What cells produce immunoglobulins and what cells are they derived from?
- Plasma cells
- These are derived from B lymphocytes
What types of molecules are immunoglobulins?
Glycoproteins
Are immunoglobulins soluble?
Yes
What secondary structures are found in antibodies?
Beta sheets
For each immunoglobulin class, state whether they are monomers, dimers, etc.
For each immunoglobulin (antibody) class, state the symbol, molecular size and concentration (mg/ml).
For each immunoglobulin (antibody) class, state whether it is/has:
- Involved in the classical pathway of complement activation
- Transferred via the placenta
- Low affinity binding to phagocytes
- High affinity binding to macrophages and activated neutrophils
- High affinity binding to basophils or mast cells
What are the different classes and subclasses of immunoglobulins?
- IgM
- IgG -> IgG1, IgG2, IgG3, IgG4
- IgD
- IgA
- IgE
Which immunoglobulin class is the first to be produced by B lymphocytes? What is its affinity and avidity?
- IgM
- It has:
- Low affinity
- High avidity (overall strength of binding between an antibody and an antigen)
What are the two main regions of immunoglobulins? Which is variable and which is constant?
- Fab (variable)
- Fc (constant)
How many binding sites for antigens does each class of antibody have? [IMPORTANT]
- IgG = 5
- IgM = 10
- IgD = 14
- IgA = 12
- IgE = 12
What does the Fab region of an immunoglobulin do?
It is the region that binds to the antigen.
What does the Fc region of an immunoglobulin do?
- Complement activation
- Binding to receptors on different cell types:
- Macrophages and neutrophils -> Triggers phagocytosis and activation
- Mast cells -> Triggers degranulation
- Epithelial cells -> This causes the immunoglobulin to be secreted into tears, saliva etc.
What are the different types of immunoglobulin based on their affinity for the receptor for the Fc region? What is the function of each?
- High affinity-Ig
- Often pre-bound to receptor (since the affinity is high)
- Waits for antigen to come along
- Low affinity-Ig
- Fc receptor not normally occupied
- The receptors are only occupied after antibody has become complexed to an antigen (useful in phagocytosis)
Which immunoglobulin classes are secreted into:
- Tears
- Saliva
- Colostrum
- Gut
- Across placenta
- Tears, Saliva, Colostrum, Gut -> IgA
- Across placenta -> IgG
What process underlies the generation of diversity of antigen-binding regions on immunoglobulins?
[IMPORTANT]
VDJ recombination (a.k.a. somatic gene rearrangement)
Describe the V, D and J regions on an immunoglobulin.
- They are variable regions on the Fab domain of the immunoglobulin that determine its specificity for antigens
- The heavy chain contains a V, D and J region
- The light chain contains a V and J region
They regions are known as variable (V), diversity (D) and joining (J).
How does VDJ recombination (somatic gene rearrangement) work in B cells?
- Immunglobulins break the rule that each polypeptide is encoded by one gene.
- They are made up of 2 copies of 2 different polypeptides, but 7 genes encode these.
- Within each V, D and J gene, there are several different versions of that region that exist (segments) -> One segment is randomly selected from each and they are recombined to give the final immunoglobulin structure
- For the light chain (lambda version!), there are 30 versions of the V region, 4 versions of the J region and 1 version of the constant region (there is no D region)
- For the heavy chain, there are 102-103 versions of the V region, 20-30 versions of the D region, 4 versions of the J region and 8 versions of the constant region
What is the mechanism of VDJ recombination in B cells?
- VDJ recombinase consists of the RAG1 and RAG2
- It selects a random segment from each VDJ gene and then makes a loop out of the DNA between the segments, so that the segments are brought close together
- It then catalyses a crossover event between the two, so that the two are now continuous in the DNA strand
- The excised DNA is lost from the genome, so that B cells (and T cells?) can be distinguished by having less DNA
Where does VDJ recombination occur?
[EXTRA]
In the bone marrow and thymus
What are some factors contributing to immunoglobulin diversity (not only VDJ recombination)?
- Multiple copies of V, D, J and C gene segments may be randomly recombined
- D region genes may be transcribed in multiple reading frames
- Imprecise joining may occur during rearrangement of genes and excision of the intervening DNA
- Nucleotides are randomly inserted or deleted from the regions flanking the sites where joining occurs
- Any H chain may pair with any possible L chain to generate up to 1016 antibodies with different specificity
What is meant by the clonotypic distribution of immunoglobulins and why is it important?
- The idea that any lineage of lymphocytes only expresses one distinct type of immunoglobulin
- This is the driving force behind clonal selection
What is clonal selection and clonal expansion?
- Clonal selection is the theory that specific immunoglobulins exist on lymphocytes and that an antigen causes the corresponding lymphocyte to be “selected”.
- After antigen presentation, selected lymphocytes undergo clonal expansion because they have the needed antigen receptor, multiplying rapidly.
What is affinity maturation and what drives it?
[IMPORTANT]
- It is the process by which activated B cells produce antibodies with gradually increasing affinity for antigen during the course of an immune response.
- With repeated exposures to the same antigen, a host will produce antibodies of successively greater affinities.
- It is driven by somatic hypermutation.
Describe somatic hypermutation and how it leads to affinity maturation.
- After activation, clonal expansion of B cells is associated with somatic hypermutation at hotspots within the immunoglobulin genes
- These hotspots correlate with the CDRs (complementarity-determining regions), which means that mutations there alter the affinity of the immunoglobulin for its antigen
- The mechanism:
- Activation induced deaminase (AID) deaminates cytosine to uracil at the HV hotspots
- Error prone DNA repair pathways create double strand breaks and introduce mutations
- The beneficial mutations are selected for, and therefore the B cells produced have progressively higher affinity for the antigen
In what types of cells does somatic hypermutation (leading to affinity maturation) occur?
B cells
What are the two types of T cell and what are the cell surface markers of each?
- Help T cells -> CD4+
- Cytotoxic T cells -> CD8+
How is immunoglobulin secretion controlled?
The conditions under which APCs present antigens to T cells controls the terminal differentiation of the T cell. These then control B cells.
What are the main types of antigen presenting cells (APCs)?
[IMPORTANT]
- Dendritic cells are professional APCs.
- Macrophages and B cells are also APCs.
What are dendritic cells?
- Bone marrow derived leukocytes distributed throughout all interstitial and lymphoid tissues
- Serve as ‘Professional’ Antigen Presenting Cells (APC)
- Present components of foreign microorganisms to the lymphoid compartment
What are dendritic cells in the epidermis known as?
Langerhans cells
What are naive lymphocytes?
Lymphocytes which have not met a specific antigen and therefore did not have the opportunity to be activated by the antigen and subsequently be differentiated into memory and efficient cells. All lymphocytes that leave the central lymphoid organs are naïve.
Compare the format in which T cells and B cells recognise antigens.
[IMPORTANT]
T cells:
- Activated by peptide forms of the original antigen
- Peptide must be presented on an MHC complex
B cells:
- Activated by a wider range of antigens, including proteins, lipids, nucleic acids and polysaccharides
- They are able to bind to entire antigens (not just peptide segments) because their function involves release of antibodies that are complementary to the antigens on an undigested pathogen.
- Do not require an MHC to present the antigens but can instead bind to free antigens.
What is the MHC?
[IMPORTANT]
- The MHC is a large genetic locus spanning 3000kb
- It encodes the MHC class I and class II molecules
- It also contains many genes responsible for initiating and regulating the immune response including the complement genes
Describe the MHC locus in humans.
In humans it is called HLA (human leukocyte antigen), containing:
- MHC class I encoded by A, B and C genes
- MHC class II encoded by DP, DQ and DR genes
- Complement genes
What are MHC class I and II molecules? Why are they important?
- They are proteins on the surface of cells, used to present the peptide forms of foreign antigens to T cells
- They are also important because they are very highly polymorphic within the population, but each individual expresses only a limited subset of the many possible polymorphic forms -> This means that they are important in transplants, etc.
Give some clinical relevance of MHC molecules.
[EXTRA]
- The human leukocyte antigen (HLA) complex is the main MHC in humans, and the HLA genes have a high degree of polymorphism.
- This is relevant in transplants, where it is almost certain that an unrelated donor will trigger an immune response in the host (against the HLA of the donor).
- However, since the HLA genes are linked (have very close loci on chromosome 6), they tend to be inherited together, so that an individual can be viewed as having a maternal and paternal haplotype.
- Therefore, siblings have a ¼ chance of possessing the same haplotypes, while monozygotic twins have the same haplotypes, making them the best candidates for transplants.
Describe the structure of an MHC class I molecule.
- Composed of an alpha chain of 45kD folded into three distinct domains
- Non-covalently associated with beta-2 microglobulin, a secreted globular protein of 12kD
- The membrane distal portion is folded into a groove which may accommodate peptides of 7-9 amino acids in length
Describe the structure and function of the peptide-binding motif of MHC class I.
- The floor of the groove is composed of 8 strands of an antiparallel beta pleated sheet: the sides are composed of two alpha helices
- Peptide binding is very degenerate: a single class I molecules may bind up to 13 million different peptides
- However, all peptides capable of binding to a specific MHC molecule share a common ‘binding motif’
What is the significance of polymorphisms in MHC class I?
- Polymorphism in the MHC are primarily localised to the peptide binding groove
- MHC molecules differ in the number, orientation and distribution of hydrophobic pockets within the peptide binding groove
- These polymorphisms mean that each MHC molecule has a characteristic binding motif that determines what peptides it can present
Describe the structure of an MHC class II molecule.
- Composed of two integral membrane proteins: an alpha chain (33kD) and a beta chain (28kD)
- Each chain is folded into 2 extracellular domains
- alpha-1 and beta-1 domains fold to form a peptide binding groove similar to that of Class I
Compare the structures of MHC class I and II molecules.
- In MHC class I, the alpha helices are supplied by alpha chains, while in MHC class II, the alpha helices are supplied by an alpha and a beta chain
- MHC class I molecules are closed by disulfide bridges at each end, while MHC class II molecules are not -> The peptide binding groove is open at each end so that longer peptides can bind to MHC class II. It also means that the anchor residues are distributed throughout the peptide, not at the ends, like in MHC class I peptides, since these ends hang out of the MHC molecule
Can any peptide bind to any MHC molecule?
No, it depends on their structure. They must have the correct anchoring residues at the right positions.
Compare the peptides that are presented by MHC class I and MHC class II.
MHC class I peptides:
- 7-9 residues long
- Anchor residues are at the C and N termini
- Derived from intracellular pathogens
MHC class II peptides:
- Up to 30 residues long
- Anchor residues are throughout the molecule
- Derived from extracellular pathogens
Compare the cells that express MHC class I and MHC class II.
- MHC class I -> All nucleated cells
- MHC class II -> Antigen-presenting cells only
What are the two pathways for processing antigens and presenting on MHC molecules?
- Endogenous pathway
- Exogenous pathway
Describe the endogenous pathway for processing antigens and presenting them on MHC molecules.
- The process involves sampling of the intracellular space for foreign proteins
- Once found, the foreign proteins are tagged with ubiquitin for degradation by the same pathway as normal misfolded proteins (i.e. the proteasome, a multi-catalytic complex found in the cytoplasm of all nucleated cells)
- Upon infection and inflammation, the cell upregulates certain components of the proteasome, making it the immunoproteasome
- The immunoproteasome degrades the foreign proteins into peptides of 7-9 residue length (the right length for presentation by MHC class II)
- The peptides are moved into the endoplasmic reticulum by the TAP transporter
- Here they are attached to MHC class I molecules, which are then moved to the cell surface for presentation to CD8+ (Cytotoxic) T cells
Describe the exogenous pathway for processing antigens and presenting them on MHC molecules.
- The process involved APCs (e.g. dendritic cells) taking up foreign proteins from the extracellular space
- This can occur via phagocytosis or by endocytosis of just the antigens
- The endosomes carrying the foreign proteins fuse with lysosomes, leading to the digestion and breakdown of the proteins into peptides
- MHC class II molecules are translocated to the endosome, so that the peptides can be attached to them
- Then the MHC class II molecules are presented on the cell surface to CD4+ (Helper) T cells
Compare the endogenous and exogenous pathways in terms of the peptides presented, MHC class involved, T cells involved, and presenting cells involved.
Endogenous pathway:
- All nucleated somatic cells use this pathway
- MHC Class I involved
- Processes intracellular proteins
- Activates CD8+ (cytotoxic T cells)
Exogenous pathway:
- Only APCs use this pathway (since only they can take up cells/antigens)
- MHC Class II involved
- Processes extracellular proteins
- Activates CD4+ (helper T cells)
This makes sense because the endogenous pathway is triggered by infection of a cell (since the pathogenic proteins are found intracellularly), meaning that activating cytotoxic T cells makes sense, so that the cell can be killed. On the other hand, the exogenous pathway enables helper T cells to fight extracellular pathogens by stimulating B cells.
Can cytotoxic T cells be activated by cell presenting a peptide on its MHC class I?
- No, they can RECOGNISE the peptide on any cell, leading to its destruction.
- However, they can only be ACTIVATED by peptides presented by APCs (dendritic cells).
How can dendritic cells present viral peptides (for T cell activation) without being infected themselves?
[IMPORTANT]
- Most cells are able to present intracellular proteins as peptides on their surface following infection -> This is done via the endogenous pathway
- However, dendritic cells do not need to infected in order for this to happen
- They use a process called cross-presentation of antigens
- Usually, before a cell can present peptides on an MHC class I complex, a virus would have to infect a cell, insert its DNA and lead to expression of its proteins by the host cell, which can then be degraded and presented
- However, dendritic cells bypass this by endocytosing/phagocytosing the pathogen/virus, and then using a transporter to pump the proteins from the endosome into the cytosol, where it can enter the normal endogenous pathway of antigen presentation -> So it can present the peptide without necessraily being infected.
How do T cells bind to the MHC molecule carrying a peptide?
Using the T-cell receptor (TCR).
Describe the structure of a TCR.
- The TCR is composed of 2 chains (alpha and beta), each of 40-50kD.
- It is from the immunoglobulin supergene family, so it is similar in structure to the TCR.
How does signal transduction occur via the TCR?
The CD3 complex and zeta chains associated with the TLR are used to transduce the signal into the cell, activating it.
What determines whether a TCR binds to MHC class I or II?
- It depends on the presence of the co-receptors CD8 (on cytotoxic T cells) and CD4 (on helper T cells) stabilize the interaction between the TCR and either MHC class I or class II.
- CD4 recognises class II, while CD8 recognises class I.
Compare immunglobulins and TCRs.
What process underlies the generation of diversity of binding regions in TCRs?
VDJ recombination, similar to in B cells.
How does VDJ recombination (somatic gene rearrangement) work in T cells?
Note: It is basically the same as in B cells.
- TCRs break the rule that each polypeptide is encoded by one gene.
- They are made up of an alpha and beta chain, but 7 genes encode these.
- Within each V, D and J gene, there are several different versions of that region that exist (segments) -> One segment is randomly selected from each and they are recombined to give the final immunoglobulin structure
What are some factors contributing to TCR diversity (not only VDJ recombination)?
- Multiple copies of V, D, J and C gene segments may be randomly recombined
- D region genes may be transcribed in multiple reading frames
- Imprecise joining may occur during rearrangement of genes and excision of the intervening DNA
- Nucleotides may be randomly inserted or deleted from the regions flanking the sites where joining occurs
- Any alpha chain may pair with any possible beta chain to generate 109 – 1016 TCRs with different specificity
Where does lymphocyte maturation happen?
A range of functionally independent lymphocytes is created during the process of maturation in the primary lymphoid organs (thymus and bone marrow).
Where does activation of naive lymphocytes occur?
Secondary lymphoid organs
What are the different signals required to activate a T cell?
- Signal 1
- Binding of the TCR to the MHC-peptide complex on dendritic cells
- Signal 2 (co-stimulatory molecules)
- Binding of CD28 receptor to B7 proteins (CD80 and CD86) on APCs
- Cytokines (sometimes called signal 3)
What are the different signals required to activate a B cell?
Signal 1:
- Binding of the BCR to an antigen
Signal 2 (co-stimulatory molecules):
- In T cell dependent activation:
- Binding of C28 receptor to CD40 ligand on T cells
- Signal 2 can also be elicited by binding of complement proteins to the CD21 receptor
- Cytokines (sometimes called signal 3)
- In T cell independent activation:
- No co-stimulation
Explain the concepts of T cell dependent and T cell independent B cell activation.
- Both types involve the same signal 1, elicited by binding antigens to the BCR.
- Then, T cell-dependent activation involves the B cell acting as an APC, which includes internalisation of a free or pathogen-bound antigen, degradation into a peptide form, and presentation on the cell-surface, bound to MHC Class II. This complex is bound to by helper T cells, which lead to signal 2.
- T cell-independent activation involves binding of antigens with repeating epitope units, such as polysaccharides in bacterial capsules, which can then bind to multiple BCRs, cross-linking them and activating the B cell. There is thus no need for signal 2.
The diagram shows T-cell dependent activation.
In signal 1 of B cell activation, how is signal transduction enabled?
Signal transduction from the receptor to inside B cells is enabled by the Igα/Igβ heterodimer associated with the BCR.
What is the function of co-stimulation of lymphocytes and how is ensure that it only happens at the right time?
- In both T cells and B cells, co-stimulation strengthens the intracellular signalling elicited by signal 1.
- Both signal 1 and 2 are required for fully activation, otherwise the lymphocyte is inactivated or undergoes apoptosis, which ensures that adaptive immune response is not triggered by self or by harmless molecules (Frauwirth, 2003).
- The main costimulatory proteins that act on T cells are the B7 proteins (CD80 and CD86), which are induced on APCs by the presence of microbes in the innate immune response, as well as positive feedback from the effector T cells themselves -> This means they are only activated during infection.
How do some viruses and tumours try to evade the adaptive immune system and how is this combatted?
- Some viruses and tumours avoid immune surveillance by down-regulating or inhibiting MHC class I expression and becoming invisible to CTL
- In order to avoid this scenario, natural killer (NK) cells are deployed which actively kill somatic cells devoid of MHC class I
What is the lineage of natural killer (NK) cells?
- NK cells derive from the common lymphoid progenitor and are closely allied to T and B cells
- However, they are considered components of the innate immune system, so they are frequently called ‘innate lymphoid cells’, which seems like somewhat of an oxymoron
Which immune system are natural killer (NK) cells part of?
They are part of the innate immune system, but they are activated by the adaptive immune system.
How do natural killer cells know whether to kill a cell?
- Natural killer cells lack either a TCR or surface Ig for antigen recognition
- Killing of target cells does not require prior priming
- The decision of whether to kill a target cell depends on the balance between activating and inhibitory receptor signaling
- Killing occurs only if the activating signal is dominant: dominant inhibitory signals pacify the cell
Give an example of an activating and inhibitory stimulus for natural killer cells.
Activating:
- NK cells express CD16, which is a receptor for the Fc region of immunoglobulins.
- Therefore, although NK cells are part of the innate immune response, they are stimulated by the adaptive immune response.
Inhibitory [EXTRA]:
- NK cells express KIRs (Killer Cell Immunoglobulin-like Receptors)
- They bind specifically to MHC class I, regardless of the peptide presented
- Stimulation of the KIRs inhibits the NK cell
- Failure to do so results in NK cell activation and killing of the target cell -> Thus this protects against certain virus infections or tumours that inhibit MHC class I expression
What are some functions of natural killer cells?
- Killing virally-infected/tumour cells, which involves the release of perforin and granzymes from cytotoxic granules at the synapse formed with the target cell
- Stimulating the action of macrophages by secreting pro-inflammatory cytokines
What do natural killer cells secrete to stimulate macrophages?
[IMPORTANT]
IFN-γ
What are the primary lymphoid organs and what is their function?
- Bone marrow and thymus
- It is where development of lymphocytes happens, as well as selection
What are the secondary lymphoid organs and what is their function?
- Lymph nodes, spleen, Peyer’s patches and mucosal tissues
- They are where initiation of the immune response first occurs by activation of lymphocytes
- Secondary tissues sample antigens from different body compartments
Explain the concept of mature/immature and naive/activated lymphocytes.
- Immature lymphcytes differentiate into mature lymphocytes in the primary lymphoid organs
- Naive lymphocytes are those which have not yet encountered their antigen and are therefore not yet activated
What body compartments do each of the secondary lymphoid organs sample?
- Spleen is highly vascularised, so it samples the blood
- Peyer’s patches sample the gut
- Lymph nodes drain tissue fluid from interstitial tissues
Describe the structure and function of the spleen.
It is made of two parts:
- Red pulp -> Contains a store of erythrocytes in case of haemorrhage, No immunological function
- White pulp -> Wraps around arterioles (periarterial lymphatic sheath) and contains:
- Naive T cells freely arranged
- Primary follicles containing naive B cells
Describe the structure and function of lymph nodes.
- Afferent lymphatics bring tissue fluid in
- Tissue fluid accumulates in the marginal sinus (just inside the capsule), then percolates into the medulla, from which it exits via the efferent lymphatic
- Cords in the medulla are made up of plasma cells that can secrete IgM antibodies (they are the source of the IgM that is produced early in the primary immune response)
- The IgM exits at the efferent lymphatic and enters the blood at the subclavian vein
- The paracortex contains naive T cells
- The primary follicles contain naive B cells
Describe how lymphocytes enter lymph nodes.
- High endothelial venules (HEV) are found in the paracortex of the lymph nodes
- HEV constitutively express the adhesion molecules GlyCAM-1, CD34 and ICAM-1
- Binding of L-selectin on naïve T cells to GlyCAM-1 and CD34 induces rolling along the luminal surface
- Activation of LFA-1 results in tight binding to ICAM-1 and diapedesis
- This means that lymphocytes can move in and out of the lymph node via this route
Describe what happens to dendritic cells in the immune response.
- Dendritic cells are a type of antigen-presenting cell (APC) that are resident under epithelia.
- They have pattern recognition receptors (PRRs) that bind pathogens, leading to dendritic cell maturation.
- This leads to upregulation of co-stimulatory molecules, cytokine secretion and migration (via the lymphatic system) to the T cell zone of secondary lymphoid organs.
What is the function of cytotoxic T cells? How do they do this?
[IMPORTANT]
- Activated cytotoxic T cells bind (using their TCR) to peptide-MHC class I complexes on infected/tumour cells
- This triggers them to kill the cell by:
- Binding to CD95 receptors using Fas ligand, which induces caspase activity
- Degranulation of perforin and granzyme -> Perforin produces pores on the surface, via which granzymes are released into the cell, triggering apoptosis
What are the different subsets of helper T cells?
[IMPORTANT]
- Th1
- Th2
- Tfh
- Th17
- Treg
How are Tfh cells created and what is their function?
Creation:
- Created when a naïve CD+ T cell recognises an antigen presented by a dendritic cell that secretes IL-6
- This stimulates the up-regulation of Bcl-6 and commits to the Tfh cell lineage
Function:
- Expression of CXCR5 means that Tfh cells can migrate from the paracortex to the primary follicles -> Here, Tfh cells activate antigen-specific B cells through interaction of CD40L with the co-stimulatory molecule CD40
- Secretion of IL-21 sustains B cell proliferation and formation of germinal centres
Where in the lymph nodes can B cells be activated and what difference does the location make?
B cells enter the lymph node from the HBV, then they start travelling towards the primary follicles:
- If they encounter an antigen before they reach the follicle -> They become short-term plasma cells, which migrate and form the medullary cords (that secrete IgM antibodies in the early immune response)
- If they encounter an antigen in the follicle -> They go through clonal expansion, forming the germinal centre (which is a region of rapid B cell expansion)
Germinal centres are the site of…
- Somatic hypermutation and affinity maturation
- Immunoglobulin class switching
- Development of memory B cells
What is class switching, where does it happen and what is the mechanism?
- It is when an activated B cell changes its antibody production from IgM to usually IgG
- It happens in the germinal centres
- Different immunglobulins have different constant regions
- The gene segments for each of these is flanked by switch (S) regions)
- AID is used to excise segments out and then there is end joining, so that the VDJ region and constant region are juxtaposed
What are the 3 subsets of activated B cells?
- Short-lived plasma cells
- Long-lived plasma cells
- Memory B cells
Compare short-lived plasma cells, long-lived plasma cells and memory B cells in terms of:
- Location
- Length of memory
- Isotypes
- When they are generated
- Affinity of antibody
- Diversity of repertoire
Where within bone marrow are haematopoietic stem cells found?
- They are tethered to osteoblasts in the osteolastic niche
- The osteoblast niche is found on the lining of the medullary cavity in bones
What is the importance of the osteoblastic niche?
- The osteoblastic niche maintains quiescence among HSC to prevent their exhaustion
- The endosteal surface offers physical protection from trauma and toxins:
- Bone absorbs environmental radiation, preventing DNA damage
- Significant distance from a blood supply ensures a low O2 tension, reducing exposure to oxidative stress
Describe how central tolerance (i.e. negative selection) happens in B cells.
- Stromal cells in the bone marrow express an Fc receptor that binds IgM immunoglobulins
- These immunoglobulins have a low specificity and are bound to various different self-antigens that they are essentially presenting to the B cells
- The B cell BCR attempts to bind to the self-antigens
- If one of the B cells appears to have produced an immunoglobulin with self-reactivity, they are given a chance to rearrange their binding region (light chain editing) -> This is not the case in T cells
- If they are still self-reactive, they are deleted by clonal deletion -> Caspases execute the apoptosis
- If not, then they can leave the bone marrow
Describe the structure and cells of the thymus.
- There are lobules separated by septa, each with cortical and medullary regions
- There are
- Haematopoietic stromal cells (macrophages and dendritic cells)
- Non-haematopoietic stromal cells (epithelial cells)
Describe the process of thymocyte (T cell progenitor) differentiation.
[EXTRA, but worth understanding]
- T cells progenitors in the outer thymus begin as double-negative T cells that express neither CD4 or CD8
- During this time, VDJ recombination occurs
- First the beta chain is rearranged (using VDJ recombinase).
- While the beta chain is being rearranged, it is complexed with an invariant pT-alpha chain. Ligation of the pre-TCR terminates beta rearrangement and alpha rearrangement can occur. This occurs when the the pre-TCR undergoes auto-dimerisation (it is its own ligand), signalling that it is somewhat functional.
- Then the alpha chain is rearranged (since the beta chain locus is inaccessible and the alpha chain locus is accessible to the VDJ recombinase).
- This induces the T cell to then become double positive, expressing both CD4 and CD8
- The T cell proliferates ready for positive selection
- Positive selection leads to only either CD4 or only CD8 being expressed
What are the three types of cell that are important in T cell development and central tolerance?
- Cortical thymic epithelial cells (cTECs) -> Positive selection
- Medullary thymic epithelial cells (mTECs) + Dendritic cells -> Negative selection
This means that cells travelling from the outer cortex to inner medulla first undergo postive selection and then negative selection.
How does positive selection of T cells occur?
- Cortical thymic epithelial cells (cTECs) have a stellate morphology and form a 3-dimensional network with no basement membrane
- This means there is interaction between developing T cells and the epithelial cells
- The cTECs constitutively express both MHC class I and class II
- If the TCR binds more to the MHC class I than class II, then the CD8 receptor is stabilised and the CD4 receptor is lost
- If the TCR binds more to the MHC class II than class I, then the CD4 receptor is stabilised and the CD8 receptor is lost
- If there is insufficient affinity, then the cell dies by apoptosis
How are the AIRE-derived self-antigens in mTEC cells also presented by dendritic cells in the thymus?
Exosomes containing the protein components are sent from mTEC cells to dendritic cells using exosomes.
Both positive and negative selection occur by recognition of an MHC complex with a peptide bound. How comes they have different outcomes?
- The result of the positive and negative selection process is dependent on the avidity of the TCR-MHC complex binding, known as the ‘affinity model’ (Starr, 2003).
- Only T cells with a moderate avidity for the antigen undergo maturation, while those with little/no avidity undergo apoptotic ‘death by neglect’ (during positive selection) and those with very high avidity undergo deletion (during negative selection).
- How the TCR differentiates between a low and high avidity interaction is not known for certain, but it has been suggested that high-avidity interactions may involve a zipper mechanism that joins the CD8 and α-chain of the TCR, leading to a signalling cascade (Palmer, 2009).
- Caspases cause the final apoptosis of the cell.
Give some experimental evidence relating to B cell peripheral tolerance.
[EXTRA]
- Transgenic mice expressing hen egg lysozyme are bred with transgenic mice expressing anti-lysozyme IgM and having B-cell receptors capable of recognising lysozyme (i.e. one parent expresses a protein and the other expresses B cells and IgM against it)
- The resulting mice produce lysozyme, but despite having lysozyme-recognising B-cells, they do not produce anti-lysozyme antibody after immunization with lysozyme
- This demonstrates that the simultaneous presence of lysozyme and lysozyme-reactive B-cells leads to B-cell anergy
- B-cells from the hybrid strain transferred into irradiated wild-type mice were indeed able to make anti-lysozyme antibodies
- This proves that anergy was related to continued stimulation by lysozyme, but could be reversed when B-cells were in a host without the tolerising effects of lysozyme
Draw the continuum of immune privilege of different tissues.
Summarise all the outcomes of selection in the thymus.
- If the avidity of a T cell to MHC complexes presenting self-antigens is too low, then it undergoes death by neglect
- If the avidity of a T cell to MHC complexes presenting self-antigens is too high, then it undergoes clonal deletion
- If the avidity of a T cell to MHC complexes presenting self-antigens on the upper end of the range in between these, then it becomes a regulatory T cell (Treg)
Summarise the cytokines produced by each subset of T cells.
[IMPORTANT]
Give a list of diseases that have been linked to autoimmunity.
Give some examples of HLA alleles and how they affect the relative risk of developing various autoimmune conditions.
[EXTRA]
HLA-B27 is the only allele that encodes MHC class I which explains why it has such a huge effect on the risk of developing ankylosing spondylitis.
Describe how molecular mimicry can lead to autoimmunity.
- An infectious microorganism with similar epitopes to a self-antigen may infect the human
- Peptides from this microorganism are presented with many immunogenic and inflammatory stimuli, so they lead to activation of T cells
- Once activated, these T cells no longer need co-activation so they can mount a response against self-antigens
Describe the different ways in which the antibodies in myasthenia gravis cause damage.
- Activation of complement, which leads to simplify the end-plate
- Internalisation and degradation of the AChR
- Steric hindrance of ACh binding
Describe an experimental model for studying multiple sclerosis.
[EXTRA]
What is some evidence for epitope spreading in autoimmune diseases?
Many autoimmune conditions feature periods of relapse and remission. The remissions correspond to a new epitope being attacked each time.
Describe how epitope spreading occur.
- Infection leads to an immune response against the bacterial epitopes
- This leads to T cell activation and macrophage activation
- The macrophages lead to tissue damage that releases self-proteins
- These self-proteins may be taken up by APC and presented to T cells, leading to an autoimmune response against the self-proteins
- This is a positive feedback loop, where each round of self-protein release leads to triggering of an autoimmune response against the protein and therefore epitope spreading, so that there is more damage and more self-protein release
Explain in detail how a flow cytometer works.
- Cells are labelled using antibodies and then fed one by one into the path of a laser
- Forward scatter detector -> The larger the cell, the more forward scatter there will be
- Side scatter detector -> The more granulated, the higher the scatter
- Fluorescent detectors -> Detect fluorescence from antibodies that are used to label the cells
Label this graph produced by flow cytometry.
How can monoclonal antibodies be produced for therapeutic purposes?
Hybridoma -> This is a fusion of a B cell and a plasma tumour cell, leading to the mass production of monoclonal antibodies.
What is antibody-dependent cellular cytotoxicity?
A mechanism of cell-mediated immune defense where an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies.
Describe the evolution of engineering of monoclonal antibodies.
- First, mouse antibodies were used
- Then, chimeric antibodies were used, which were a mix of mouse and human antibodies, becoming increasingly human
- Finally, pure human antibodies were achieved
The advantage of using more human antibodies is that they tend to be less immunogenic than mouse antibodies.
Summarise the different potential targets in rheumatoid arthritis.
Describe how monoclonal antibodies (and other treatments) can be used to treat rheumatoid arthritis.
[IMPORTANT]
- Monoclonal antibodies are used to target the cytokines that are used to recruit immune cells (i.e. the ‘master’ cytokines)
- These include:
- Anti-TNF antibodies (e.g. infliximab, etanercept, adalimumab) -> Combined with methotrexate
- IL-6 receptor antibodies (e.g. Tocilizumab)
- Can also target immune cells directly:
- B cells (e.g. Rituximab targets CD20+ B cells)
- T cells (e.g. Abatacept blocks T cell co-stimulation)
What are some problems with anti-TNF antibody use in treating rheumatoid arthritis?
What are the main pitfills of anti-rheumatoid arthritis therapy?
Describe how monoclonal antibodies can be used to reduce the risk of cardiovascular disease. Give some experimental evidene for this.
[EXTRA]
Anti-PCSK9 antibodies:
- PCSK9 is a protein secreted from cells
- It binds to LDL receptors on the cell surface, leading to their internalisation
- Anti-PCSK9 antibodies stop this from happening and thus increase the number of LDL receptors on the cell surface
- In turn, this leads to increased uptake of LDL into the cell, so that plasma levels are reduced and there is a decreased risk of CVD events
(Robinson, 2014):
- Found that alirocumab in addition to maximal statin therapy reduced plasma LDL by 62% compared to maximal statin use alone
- The fall in LDL was rapid
