Molecular Immunology Flashcards
What are potential “Threats from within” the immune systems combats?
Cancer, Necrosis, Prions
What are potential “Threats from the external environment” the immune systems combats?
Viruses, Bacteria, Fungi, Protozoa, Parasites, Prions
Very broadly, how does the immune system coordinate immune responses? (Lay mans terms)
Distinguishes between host tissues and foreign elements, and identifies danger signals produced during cell or tissue damage, to stimulate a specialised response.
What are the two branches of the immune systemn?
The innate immune system and the adaptive immune system.
Innate vs adaptive immune system:
Innate: Broad specificity, not affected by prior contact, immediate response, non-clonal activation, PRRS are inherited to recognise MAMPS, potential for damage to host, good at distinguishing self and non-self
Adaptive: highly specific, enhance by prior contact (e.g. in immunisation), slow response (days-weeks initially)
Both involve white blood cells + soluble factors, clonal activation, randomly generated antigen receptors, doesn’t reliably distinguish self and non-self, specifically targets affected cell or pathogen.
Components of the innate immune system:
- Barriers (physical and chemical)
- Preformed mediators -Soluble proteins (Complement, interferons, etc.)
-Local and systemic responses (inflammation, fever)
-Leukocytes( Myeloid : phagocytes + lymphocyte NK )
Components of the adaptive immune system:
- Response mediated by B and T lymphocytes -> both can develop into memory cells
- B cells secrete soluble antibodies - Humoral immunity
- T cell develop into cytotoxic T cells (kill infected cells) or helper T cells (secrete cytokines that act on other cells) - Cell mediated immunity
What are the two main lineages of leukocytes?
Myeloid (innate) and lymphocyte (adaptive + innate NK)
What does the adaptive immune system recognise to trigger a response
Antigens expressed by foreign body. (identified by specific antigen receptors)
What does the innate immune system recognise to trigger a response
Pathogen (microbes) Associated Molecular Patterns (MAMPS /PAMPS)
What are pathogen associated molecular patterns?
Conserved structures unique to microbes that are essential for their function, e.g. LPS or peptidoglycan.
Clonal Selection Hypothesis:
Thousand of B cells are produced at a given time, those with highly specific antigen receptors are selected for and cloned, differentiating into plasma or memory cells. Those that are unspecific or target host cells/ tissues are deleted early in development to prevent autoimmune disease.
How does the innate immune system activate the adaptive system?
Upon stimulation of the innate system, the activation of complement and cytokines, stimulates the activation of cell-mediated immunity, the developed T helper cells, then activating humoral B cells. These systems
Is the effects of the innate system on the adaptive one sided?
NO, the adaptive immune system can control and focus innate immunity, releasing cytokines to migrate components of the innate immune system to areas affected by the pathogen.
What is the site in which T cells mature?
The thymus
What is the site in which B cells mature?
The bone marrow.
What is meant by a cell maturing?
Cells acquire their specific receptors
What occurs at secondary lymphoid tissue (Adaptive immunity):
Mature lymphocytes are stimulated by the antigen and coordinate a response.
Broad outline of activation of adaptive immunity in the draining lymph node:
A macrophage or dendritic cells (innate) engulf a bacterium by phagocytosis, presenting antigens on their MHC’s, these migrate across a lymphatic vessel into a lymph node, the APCs (antigen presenting cells) bind to lymphocytes, maturing them (both T and B cells)
What is the major form of immunity in young children?
Innate immunity
What is the oldest form of immunity?
Innate immunity, shared by insects, plants, and mammals.
What are the 3 types of barriers in innate immunity?
Mechanical, Chemical, Microbiological
What is the major microbiological barrier to infections?
Components (normal microbiota)
What are the examples of different mechanical barriers to infections?
Skin: Air flow
Gut: Fluid Flow
Lungs; Cilia (move mucous)
Urogenital Tract: Flow of urine
Eyes/Nose/mouth: Flow of fluid (tears + nasal cilia)
-All have epithelial cells joined by tight junctions
What are the examples of different chemical barriers to infections?
Skin: Fatty acids, Enzymes
Gut: Low pH enzymes
Lungs: Pulmonary surfactant
Urogenital tract: Low pH urine
Eyes/nose/mouth: Enzymes
^Mucins
All contain antimicrobial peptides (e.g. defensins)
How do mechanical and chemical barriers to infection help prevent infection?
They make environments unfavourable for the growth of the pathogenic microbes, thereby reducing the capacity of a pathogen to reach viral load.
What symptom of cystic fibrosis causes greater risk of infection?
Malformation of mucous, leading to greater infection risk at mucosal surfaces
What are the features of keratinised skin?
Keratinised skin is a barrier to infection. Contains Keratinocytes which produce keratin (physical barrier) and sebum (containing fatty acids - chemical barrier)
Sweat glands produce sweat at a high salt concentration, deterring microbial growth. Skin sheds and new layers are produced, effectively repairing damage.
Home to many commensals that compete against opportunistic microbes.
What are the features of Mucous Membranes?
o Largest interface with the environment
o Mucus, cilia (resp tract), secreted enzymes (e.g. lysozymes)
o Low pH (gut, vagina), peristalsis (gut)
o Shedding of epithelia
o Commensals
What are the key examples of pre-formed mediators?
Lysozymes, Antimicrobial peptides, and Complement
Lysozymes: (As preformed mediators)
- present in many secretions -> break ester bonds between MurNAc and GlcNAc subunits in peptidoglycan -> destabilises Gram +ve bacteria
Antimicroibal peptides: (As preformed mediators)
Large variety (e.g. Defensins) -> produced by many epithelial cells and neutrophils, these peptides are cationic, disrupting lipid bilayers in bacteria, fungi, and enveloped viruses.
What are the 3 pathways of complement activation?
The Classical Pathway (Antibody binding), the mannose-binding lectin pathway (MBL), the alternative pathway
General features of Complement: (as a preformed mediator)
- Works in conjunction with antibodies to kill bacteria, made up of 20 soluble proteins, these components are normal inert until activation by antibody binding to antigen or pathogen presence.
What is the classical pathway also known as?
Antibody Binding Pathway
What is the order of complement protein activation in the classical pathway?
C1, 4, 2, 3, 5, 6, 7, 8, 9
What is the most abundant complement protein in the body?
C3
What are the products of the activation of C3 (by cleavage)?
C3b + C3a
What is C3b?
A complement protein that is a reactive thioester that can bind to adjacent proteins/carbohydrates on the surface of pathogens (e.g. LPS)
How and why is the action of C3b localized?
By its rapid inactivation in the fluid phase, its action is localised to more specifically target the pathogen.
What is the role of C3 convertase?
To cleave C3 into C3b and C3a
What is a lectin?
A molecule that binds tightly to carbohydrates on the surface of pathogens (in the case of mannose-binding lectin)
Which enzymes are activated unique to the MBL complement activation pathway?
MASP1 and MASP2
What is the sequence of activation in the MBL pathway?
MASP1 -> MASP2 -> C4, C2, 3, 5, 6, 7, 8, 9
(Note absent of C1)
What triggers the activation of the alternative pathway?
LPS (lipopolysaccharide) usually on the surface of bacteria
What is lipopolysaccharide (LPS)
a structural component of outer membrane of gram-negative bacteria, that provides integrity to the PG cell wall
What is the sequence of activation in the alternative complement activation pathway?
C3 -> Factor B, Factor D, Factor P (properdin stabilises the complex) (formation of C3 convertase) -> C3, 5, 6, 7, 8, 9
What is the C3 convertase in the alternative pathway?
C3bBb
What does the alternative complement activation pathway mainly rely on?
The C3 tickover mechanism, wherein C3 is spontaneously cleaved into fragments by proteases, usually the C3bs generated will deactivate, but if close to bacterial membrane it’ll bound to the LPS on the bacterial membrane. Alternatively C3b can be sourced from the other pathways,
What is the C3 convertase in the classical and MBL pathways?
C4bC2a
What is unique about the alternative complement activation pathway?
It can accept C3b from any pathway to amplify the innate response by complement.
What stabilises C3bBb (C3 convertase of alternative pathway)?
Factor P (properdin)
What does Factor D do to C3bB?
Factor D cleaves the bound Factor B into Factor Bb, to form C3bBb
What are the roles of C3a and C5a in complement activation?
Recruit phagocytes (act as chemoattractants) and induce localised inflammation (anaphylatoxins)
How do C3a and C5a act as chemoattractants:
Phagocytes have receptors that bind to C3a and C5a -> allowing for the detection of them, by moving up their concentration gradient until at the site of the pathogen.
How do C3a and C5a act as anaphylatoxins:
They act on local blood vessels to increase their permeability, increasing the volume of tissue fluid, and increasing blood flow.
How does C3b promote opsonisation?
Pathogens coated by C3b peptides are recognised by phagocytes with C3b receptors -> facilitating binding and phagocytosis.
Why is complement regulation needed?
To prevent damage to the host cells, such as the damage seen in macular degeneration (lack of factor H), Paroxysmal nocturnal hemoglobinuria (lack of CD59), autoimmune diseases (rheumatoid arthritis factor I deficient)
What is CD59?
A regulatory protein that protects host cells from MAC formation, by binding and sequestering C9
What infections is complement crucial in the treatment of?
Extracellular bacterial and fungal infections.
How does the complement system actively help activate the adaptive immune system?
Helps activate B and T cells via aiding opsonisation for phagocytes and dendritic cells.
What is membrane Attack Complex (MAC)?
A structure formed by complement proteins that forms within lipid bilayers of pathogens, causes an influx of fluid and cell lysis of the pathogen. Have a crucial role in treating Gram -ve bacteria, allowing for access to PG in their Inter membrane space.
What are the two general ways complement action is regulated?
Complement proteins rapid hydrolyse and inactivate in fluid phase, and are inactivated or inhibited by soluble and membrane bound proteins.
What is Factor H (complement activation):
Factor that competes with Factor B to inhibit the formation of C3bBb and later activation of C3 by the alternative pathway
What is Factor I (complement activation):
protease that cleaves C3,b preventing the rest of complement activation -> as it’s cleavage inhibits the cascade
From which stem cells do all leukocytes derive from?
Haematopoietic stem cells
Which leukocytes are examples of polymorphonuclear leukocytes:
Neutrophils, Eosinophils, Basophils
Basophils: Features
- few in blood
- Express receptors for C3a, C5a, and IgE
- granules stain with blue basic dye
Basophils: Functions
- Bind to C3a, C5a, and IgE to induce an inflammation response through the release of heparin and histamine to evoke inflammation of local tissue.
Basophils: Purpose
Defend against large extracellular parasites and can play a role in allergy (similar in purpose to MAST cells but different mechanistically)
Examples of inflammatory mediators:
Heparin and histamine
Eosinophil: Features
- granules stain with acidic red dye
- Very specialised
-Few in blood but more abundant beneath mucosal surfaces - Express receptors for C3b, and the Fc regions of IgG, IgA, and IgE
Eosinophil: Functions
-Release toxic proteins and free radicals via exocytosis of their granules -> contents attach to plasma membranes of pathogen and cause damage to their cell membranes -> lysis
Eosinophil: Purpose
Defend against multicellular parasites (e.g. tape worm) + role in allergenic reactions
Neutrophil: Features
-Granules not stained with acidic of basic dye.
-Most important/prominent out of the 3 granulocytes.
-Most numerous WBC in the blood (60-70% of the WBCs in the blood)
-Usually Short lived (living for less than 24 hours in the blood, undergoing apoptosis and dying) however life span can be extended upon infection
-Release in large numbers from the bone marrow
-Express receptors for C3b, IgG, and IgA (Their Fc regions)
- Fast moving and first leukocytes in immune response
- has an extracellular chromatin matrix impregnated with antimicrobial peptides e.g. defensins
Neutrophil: Functions
-Will enter tissues in response to chemoattractants
- Phagocytosis
- can also undergo “NETosis” - > extracellular chromatin net helps to trap/sequester pathogens, particularly bacteria.
Neutrophil: Purpose
Phagocytosis of foreign bodies
Macrophages:
Derived from monocytes in the blood during an infection, they act as ‘sentinel cells’ to detect infections and phagocytose pathogens to present antigen to develop adaptive immune response. They can survive months to years.
Why are macrophages mostly found in tissues?
seeded out into the tissues during foetal development and-so are resident to the tissues since before birth.
Dendritic cells:
Found in skin and lymphoid tissues, the phagocytose foreign material and display peptide fragments on their surface (on MHCII complexes) -> presenting these to naive T cells to stimulate the adaptive immune response.
Difference between macrophages and dendritic cells?
Dendritic cells have a specialised shape.
What protein on host cells is recognised phagocytes and prevents them from being targeted by phagocytosis?
CD47
Why don’t red blood cells express CD47?
CD47 protects cell from phagocytosis. However allowing phagocytosis ensures less efficient RBCs approaching cell death are disposed off.
Stages of phagocytosis:
Bacterium binds to the surface of phagocytic cell -> phagocyte pseudopods extend and engulf the foreign body -> invagination of phagocyte membrane traps this within a phagosome -> a lysosome fuses with the phagosome to form a phagolysosome -> Enzymes cleave macromolecules and generate reactive oxygen species to destroy the organism.
Which phagocyte bactericidal agents are examples of free radicals?
Toxic oxygen-derived products and nitrogen oxides.
What is the oxidative burst in phagocytosis?
A transient increase in oxygen consumption following phagocytosis due to activation of membrane-bound NADPH oxidase.
What is NADPH oxidase?
A multi-component enzyme on the membrane of the phagosome, catalyses the formation of superoxide ions, free radicals that bind to the pathogen and cause damage.
What are the different mechanisms by which phagocyte bactericidal agents may operate?
Acidification, Toxic oxygen-derived products, Toxic nitrogen oxides, antimicrobial peptides, enzymes, and competitors
What is an example of a toxic oxygen-derived product used by phagosomes?
Superoxide ions (catalysed by superoxide dismutase) -> hydrogen peroxide (catalysed by peroxidase enzymes to form radicals and bleach bacteria)
What is the peroxidase enzyme in neutrophils used to catalyse radical ion formation from hydrogen peroxide?
myeloperoxidase
Natural Killer (NK) cells: Functions
- Kill infected host cells
- Recognise changes in expression of MHCI proteins
- induce apoptosis
Natural Killer (NK) cells: Purpose
- Important in viral and intracellular bacterial infections, especially until adaptive immunity is triggered.
- Trigger apoptosis
- Active against some cancers
Natural Killer (NK) cells: Features
-Have receptors for IgG (allows for killing of antibody coated infected host cells)
NK cell induce apoptosis:
- NK cells produce perforin, which inserts into the membrane of the infected host cell (similar to the membrane attack complex of complement.
- The granules of the NK cell polarise to face the target cell, releasing their contents through the perforin pore so that the enzymes can enter the target cell -> activates the apoptosis pathway
How can NK cells kill bacteria?
Their hydrolytic granules (granzymes) can enter infected cells and directly kill intracellular bacteria.
What are Damage-associated molecular patterns (DAMPs) ?
Danger signals released during injury or cell damage.
What are Pattern Recognition Receptors (PRRs)?
Inherited germline Receptors the recognise MAMPS and DAMPs
What are the two types of PRRs?
Membrane receptors (e.g. Lectin receptors, or Tol-like receptors) and cytoplasmic receptors (e.g. NOD-like receptors)
Examples of DAMPs?
DNA, uric acid, fragments of extracellular matrix proteins (e.g. fibronectin)
Function of TOLL-like receptors (TLRs)?
TLRs are PRRs that signal the presence of microbial components or stimulate action against viral infection (TLR 3 + 9)
What responses may PRRs initiate?
Phagocytosis, chemotaxis, or signalling. The latter caused by changes to gene expression to trigger the secretion of pro-inflammatory cytokines and the assembly of the inflammasome
By what mechanism are Toll-like receptors activated?
Ligand binding to the receptor causes their dimerization, causing them to cross phosphorylate.
What is the inflammasome?
A multisubunit complex that cleaves inactive cytokine precursors -> the function of this complex is to upregulate the inflammatory pathway.
What is the purpose of inflammation?
To increase the permeability of blood vessels to allow phagocytes in the circulatory system to access microbes in tissues and fight infection.
What are the 4 characteristics of inflammation?
-Redness, swelling, heat, pain
What are the different types of inflammatory mediators?
Lipid mediators (prostaglandins)
Vasoactive amines (histamine)
Chemoattractants (fmet-leu-phe)
Complement proteins (C3a and C5a)
Cytokines (TNK, IL-1, IL-6)
What is the role of neutrophils in the inflammatory response?
Stick to and disrupt capillary walls to increase their permeability.
Summary of the inflammatory Response;
Bacteria infects tissue -> macrophage engulfs the pathogen and releases chemical mediators -> cytokines induce expression of selectins on the capillary endothelia to bind to neutrophils -> vasoactive factors induce integrin expression on neutrophils -> tight junctions loosen and prostaglandin synthesis induced -> chemoattractants then attract the neutrophils to migrate into the tissue.
What commonly causes chronic inflammation?
An unresolved infection (e.g. In TB, macrophage themselves are infected, making the innate immune system ineffective)
How many types of Toll-like receptor is there?
10 in humans
What do cytokines do? (generally)
Regulate immune responses by changing cell behaviour or gene expression.
What are the 4 classifications of cytokines?
- IL-1 family
- Interferons
- TNF family
- Chemokines
How are cytokines classified?
They are classified on the basis of their structural similarities, not by function.
What is the TNF family?
The tumour necrosis factor family is a family of cytokines. They are synthesised as transmembrane proteins that are released by proteolysis.
What cells are the primary producer of TNFs
macrophages
How do TNFs transduce signals?
TNFs tend to be trimers, that will cross-link 3 receptors to stimulate downstream signalling (of cell stimulation of inflammation or apoptosis)
Why are TNF concentrations highly regulated by the immune system?
They have the capacity to be very damaging in high doses.
What are the local effects of TNFs?
-Increased blood flow + capillary permeability (inflammation)
- Influx of platelets (clotting of capillaries reduces spread of infection)
- Increased drainage to lymph nodes -> to stimulate adaptive immunity.
What are the systemic effects of TNF at low concentrations (<1mg/ml)
can activate receptors all over body -> Can induce fever, acting on the hypothalamus, to inhibit the growth of some bacteria/viruses additionally providing the optimum temperature for B and T cell activation
Why are TNFs often targeted in the treatment of autoimmune disease?
The inappropriate production of TNFs is often responsible for tissue damage via chronic inflammation, causing rheumatoid arthritis, inflammatory bowel diseases, psoriasis, etc.
What are the systemic effects of TNF at high concentrations (>1mg/ml)
- Widespread increase in vascular permeability -> low blood pressure
- Disseminated clot formation -> heart attacks and organ damage
- Consumption (exhaustion) of clotting factors leads to internal bleeding and spread of infection
-Septic Shock with 80% lethality
When are concentrations of TNF extraordinarily high?
In the event of severe infection and sepsis
What are the types of interferons and their functions:
Type 1: IFN-a IFN-B -> induce expression of interferon-stimulated gene (ISGs)
Type 2: IFN-y (gamma) -> modulates immune response
What is endoribonuclease?
A hydrolytic enzyme that degrades viral RNA and RNA enzymes
What is eukaryotic initiation factor 2?
A factor stimulated by type 1 interferons that is phosphorylated to inhibit the translation of viral proteins and thus the assembly of viral enzymes.
What are the cell-local effects of type 1 interferons (IFN-a and IFN-B):
- Induce resistance to viral replication
- Increase MHC I expression
- Activate NK cells to kill virally infected cells
- Induce chemokines to recruit lymphocytes
What is the importance of type 1 interferons stimulating MHC I expression in virally infected host cells?
important in presenting viral antigen to cytotoxic T cells to more effectively target infected cells.
What cells make type 2 interferons?
neutrophils, NK cells, T cells
What cells make type 1 interferons?
All cells upon viral infection
What is the effect of type II interferons?
- Increase the expression of MHCI and MHCII to aid T-cell responses
- IFN-y secreted from helper T cells activates macrophages
What causes a cytokine storm?
An exaggerated immune response to an infection, caused by elevated levels of pro-inflammatory cytokines (e.g. TNF, IL1B, IL6)
What are used to treat cytokine storms?
Corticosteroids, or cytokine/ cytokine receptor inhibitors.
Describe briefly the interdependent relationship of the innate and adaptive immune responses:
Innate immune responses initiate and ‘steer’ adaptive responses, which in turn utilise and focus innate mechanisms to eliminate pathogens.
Why is innate immunity still important in higher organisms?
Innate immunity offers an immediate response, being important in inducing adaptive immunity
Why do defensins have little activity against eukaryotic cells?
Because bacteria have greater transmembrane potential that eukaryotes (more negatively charged inside)
What are the most important killing mechanisms in phagocytosis?
Reactive oxygen and nitrogen species (following oxidative burst)
Structure of antibodies:
Antibodies are Y-shaped immunoglobulins. The Fab arms are structures that bind to antigen, the Fc tail interact with immune system components and determine the response. These regions are joined by a flexible hinge region, and the structure made up of two light and two heavy chains.
What make up the Fab arms of antibodies?
Fv region and a constant region, with the Fv region being variable and binding to the antigen.
What does Fab stand for? (antibodies)
Fragment antigen binding
What does Fc stand for? (antibodies)
Fragment crystallisable
What are the 5 classes of immunoglobulins (antibodies)
IgG, IgM, IgA, IgD, IgE
What are the two types of light chains?
Kappa and Lambda -> these aren’t class restricted
What is the folding pattern of immunoglobulin domains known as?
The immunoglobulin fold - folding pattern is conserved however the loop regions can tolerate variability.
What are the globular domains of immunoglobulins stabilised by?
intra-chai disulphide bonds.
What is the structure of the Light Chain Fab region?
Made up of 2 domains, the C domain and the V domain. Both are beta pleated sheets joined by loops, with an internal disulphide bond.
What kind of interactions occur between antigens and antibodies?
Non-covalent. Instead electrostatic interactions, hydrogen bonds, Van der Waals, and hydrophobic interactions may occur.
How are antibody / antigen interactions strong despite being non-covalent?
Individual interactions are weak, however if many simultaneously form across the complementary residues, the combined attraction of these interactions can be very specific and of high affinity.
What regions of the antibody contact the antigen?
Complementary determining regions (CDRs)
What specific part of the antigen binds to the antibody (via CDRs):
The epitope
B cell receptors: (membrane immunoglobulins)
These are immunoglobulins associated with the B cell membrane, via 26 amino acids at its C-terminus. They recognise and bind to antigen. By themselves they normally cannot transduce a signal themselves, requiring interaction with additional membrane proteins (Iga and IgB the exceptions, having long tails which can associate with other immunoglobulins and transduce signals)
What feature of IgA and IgG tails’ allows them to not require association with additional membrane proteins to transduce a signal?
They contain ITAMs (immunoreceptor tyrosine activation motifs) allowing them to cross phosphorylate when cross-linked.
What is the structural basis of antibody diversity?
- Variations in sequence and length of CDRs are the main determinants of antibody diversity (in terms of binding)
- CDR3 (furthest C terminus domain) tends to be most variable in length a sequence
- Heavy chains generally contributes more to antigen binding and is more variable than the light chain,
What is the extent of the ‘immune repertoire’ -> possible variations of immunoglobulin receptors:
~ 10^14 antibody receptors
~10^18 T cell receptors.
What is the genetic basis of antibody diversity (brief)
The somatic recombination and mutation of a limited number of inherited gene segments which make up the V regions.
How many genes in the human genome encode antibody variations?
20-25,000 genes.
Where are the 3 sets of immunoglobulin genes located?
Heavy (H) chains- chromosome 14
Kappa (K) chains - chromosome 2
Lambda chains - chromosome 22
How was it proved that before B cell differentiation the variable region genes don’t lie next to constant region genes?
Tonnegawa:
- Used DNA from embryonic immature plasmid cells and from a mouse myeloma cell line producing antibody (mature).
- DNA from each was extracted and ran on a gel, the band were probed to identify variable or constant regions.
- V and C probes were in separate fragments in the embryonic but together on the same fragment in the matured.
What makes up the light chain loci
The light chains are encoded by 2 or more exons. The V kappa and C kappa, joined by a J segment
What makes up the heavy chain loci?
encoded by 3 segments of DNA. Heavy chain variable regions -> made of V exon, C exon, J segments, and D segment which gives rise to the increased diversity of the heavy chain variable region.
What will be the first class of antibody produced in an immune response?
IgM
What is the identity of the C exon of the heavy chain loci of antibodies (in the initial response to a pathogen)
IgM heavy chain / Cµ exon
During what process does the recombination of immunoglobulin chain coding region recombine to give rise to unique antibody chains?
B cell differentiation
What occurs during the DNA recombination of B cell differentiation to Kappa Chains.
a particular V segment aligns next to a particular J segment, during the folding of the DNA. In most cases the V gene is spliced to a J segment with the intervening DNA ( between the V gene and particular J gene) is excised, within somatic recombination.
Why is DNA recombination crucial for the expression of immunoglobulins?
The somatic recombination of the chain DNA, rearranges such that the V promoter is closer to the enhance, thus allowing transcription.
What occurs during the DNA recombination of B cell differentiation to Heavy Chains.
A particular D segment comes to lie next to a J segment, with the intervening DNA excised and lost. This is known as D-J joining. Following this V segment joins onto the adjacent D-J joining in V-D-J joining, which is how the DNA is formatted in a mature B cell.
How are unwanted regions removed from immunoglobulin chains post transcription?
mRNA splicing
General mechanism of lymphocyte recombination:
Involves sequences (recognition signal sequences RSSs)that are conserved that lie next to adjacent genes that need to be recombined together, the process is characterised by lymphocyte specific recombinases.
What are recognition signal sequences (RSSs)?
The RSSs comprise of a heptamer and a nonamer separated by 12 or 23 random nucleotides (acting as spacers). Found directly adjacent to the coding sequence of V,D, or J gene segments, guiding the rearrangement of them.
What is the 12-23 base pair rule?
a gene segment with a 12bp spacer can only join with a gene segment with a 23 bp spacer within the RAG1-RAG2 complex -> this ensures the correct arrangement of V-D-J joining / sequence (moreso important in heavy-chain genes)
What is V(D)J recombinase?
A complex of several enzymes (DNA cleavage and repair enzymes) required for somatic V-region gene recombination
What subunits of the VDJ recombinase are encoded by recombination activation genes?
RAG1-RAG2
What are RAG1 and RAG2?
Specialised endonucleases within VDJ recombinase, that recognise RSS sequences adjacent to the genes to be joined.
What conditions are caused by RAG deficiency?
severe combined immunodefficiency (scid) -> unable to make antibody receptors on lymphocytes
Mechanism of RAG1-RAG2 in somatic recombination of Kappa chain:
The RAG1-RAG2 complex recognises the RSSs adjacent to the gene segments to be joined, and aligns the DNA to joined. The complex cuts the DNA, excising the intervening DNA as a signal joint which is lost to the cell, the particular V segments that need to be joined together form a coding joint, which is successful coding region for the transcription of the kappa chain.
What is RAG1’s role in the RAG1-RAG2 complex?
RAG1 is an endonuclease, introducing 2 single stranded breaks into the sequence, this generates free 3’ hydroxyl groups that attack phosphodiester bond on the antiparallel strand of DNA) which react with the other strand of DNA to form covalently closed hair pin.
What is RAG2’s role in the RAG1-RAG2 complex?
RAG2 acts as a cofactor for the function of RAG1
What is the structure of RAG1-RAG2:
When not bound to the DNA, similar to pair of scissors, RAG2 handles and RAG1 blades. Two dimers of RAG1-RAG2 joined by a flexible domain.
What structure of the RAG1-RAG2 domain identifies RSS nonamer sequences?
A flexible nonamer binding domain within the flexible region of RAG1
What structure of the RAG1-RAG2 domain identifies RSS heptamer sequences?
Recognised between the RAG1 and RAG2 ‘head’ regions.
Why is RAG1-RAG2 evolutionarily significant?
The complex effectively acts as a transposase, they are thought to be evolutionary developed from a transposon and gave rise to adaptive immunity.
What occurs upon binding of RAG1-RAG2 to DNA?
A conformational change causes the complex to “close up”. Additionally once the RAG1 binds to a RSS separated by 12 random bp, this induces a tilt in the DNA towards 12 bp signal sequence, this action ensuring the 12-23 bp rule. (as the only piece of the DNA that can be accommodated by the rest of the enzyme is one with a 23bp sequence -> due to physical barrier).
What structure facilitates the bending of the DNA in somatic recombination (in RAG complexes)
High mobility group box 1 protein HMGB1
What are the 3 genetic mechanisms of antibody diversity?
-The multiple copies of V region gene segments
-Heavy and Light chain combination
-Imprecise recombination leads to junctional diversity (addition of nucleotides contributes to diversity of CDR3)
What is the role of terminal deoxynucleotide transferase in increase antibody diversity?
It’s active during recombination and can randomly add nucleotides to VDJ joins which increasing diversity.
Mechanism of junctional diversity:
Following RAG1-RAG2 action and the joining regions between the RSSs is excised, the covalently closed hair pins are then cleaved, and their ends joined together by normal DNA repair enzymes. Depending on the type of repair the hair pin may become a palindromic overhang or added nucleotides (N-regions as they’re non-templated encoded and not in the genome) -> the DNA repair machinery then fills in the overhang/ gaps -> this introduces a lot of variability due to machinery being imprecise and different nucleotides are lost and added.
What is an antigen dependent genetic mechanism of antibody diversity:
Somatic Hypermutation:
Point mutations introduced in rearranged V regions, theoretically these mutations are random, however are focused on the variable region, usually focused on the CDRs.
Where does antigen-independent recombination processes occur?
In the bone marrow
Where does antigen-dependent recombination processes occur?
Secondary lymphoid tissue.
What is the main function of somatic hypermutation?
To increase the diversity of antibodies produced by B lymphocytes in response to a specific pathogen, to affinity maturation.
What is the process of affinity maturation of B lymphocytes?
B lymphocytes undergo differentiation by somatic hypermutation to diversify their antibody structures. They’re then selected by their binding to antigen; therefore as the number of B cell increases or the amount of pathogen decreases, there’s increased competition amongst B cells, with only the ones with high affinity being selected for.
What enzyme is somatic hypermutation dependent on>
Activation-induced cytidine deaminase -> expressed by B cells responding in secondary lymphoid tissue.
What is class switching (anitbodies):
Process that occurs in secondary lymphoid tissue, wherein the C region changes but the V region stays the same. This means that the same antigen is recognised, however the change in heavy chain will vary and thus cause a different response, altering localisation and effector function.
What additional cells are required to aid the antigen dependent recombination methods?
cytokines from T helper cells
Brief description of class switching:
Once the heavy chain VDJ sequence has been established by somatic recombination and upon antigen binding in the secondary lymphoid tissue, the VDJ unit comes to lie next to a constant region gene by another recombination event across switch regions (adjacent to heavy chain genes).
What are the switch regions in class switching of antibodies?
The switch regions are very G-rich repeated DNA sequenced found close to C region genes
(Activation induced cytidine deaminase) AID enzyme:
Change cytidine into uracil allowing for point mutations during transcription.
What feature of switch regions facilitates AID action?
G-rich repetitive regions stall the process of transcription, allowing time for point mutation by AID.
Mechanism of AID:
AID deaminates cytidine to form uracil in the DNA (can also act on RNA during RNA processing) -> the uracil is identified by DNA repair proteins, triggering DNA repair pathways. In B cells these pathways are error-prone, leading to different mutational outcomes.
Why is AID essential for class switching?
AID action on switch region causes more intense point mutations, leading to single stranded nicks -> changing to double stranded nicks -> which are repaired leading to the excision of the intervening DNA.
How is it known that AID is essential for class switching?
Mutation to AID lead to Hyper-IgM syndrome type 2 -> - Characterised by elevated IgM levels due to lack of class switching antibodies.
Why is it important that AID is tightly regulated?
Because it causes mutations which when not regulated could cause cancers (particularly leukaemia)
What are the two types T cells?
T helper cells (CD4 +VE)
T cytotoxic cells (CD8 +VE)
Function of T helper cells (CD4 +ve)
augment and regulate immune responses by secreting cytokines
Function of T cytotoxic cells (CD8 +ve)
specifically kill infected host cells (a bit similar to NK cells in the innate response, however much more specific)
CDRs:
Hyper variable region in the DNA
Structure of T lymphocyte Receptors:
- Membrane protein made of two chains (heterodimer of alpha and beta chains). The α and β chains only have a small intracellular region -> insufficient for signalling -> in order to signal they must be associated with additional proteins.
- A disulphide bond holds the chains together, and the stalk segment exhibits flexibility (short space between transmembrane region and constant region) -> similar to a hinge region.
- The domains closest to the membrane are relatively constant in sequence (highly conserved), whereas domains further away are very varied in sequence.
- Each variable region (domains) contain 3 CDRs -> the CDR3 is the most variable in sequence and length (similar to antibodies)
- Associated with an adaptor protein that reaches intracellularly, with a ITAM domains.
What are the accessory proteins that associated with T-cell receptor complexes?
CD proteins e.g. CD3 or CD4 or CD8 etc.
Where does T cell receptor recombination occur?
Only in the thymus and antigen independent
MHC I
Expresses endogenous proteins ordinarily host peptides, but upon viral or intracellular bacterial infection will present pathogen endogenous proteins which will be identified by NK cells and T cytotoxic cells.
Terminal Deoxynucleotide transferase: action between B and T cells
Only active in B cells during the rearranging of heavy chain genes, in T cells these are active all the time, with action concentrated at CDR3 regions of the alpha and Beta subunits
What different types of infection does humoral and cell-mediated immunity target?
B cell immunity: extracellular pathogens - recognise free, unprocessed antigens
T cell immunity: intracellular pathogens - recognise endogenous processed antigen
Why must endogenous peptides be presented on MHC I complexes?
Because T cells cannot look inside the infected host cell to identify endogenous peptides.
What is the role of the major histocompatibility complex (MHC)?
Takes fragments of components within the cell to the cell surface to be recognised by T cells with the correct receptors.
What is a key feature of MHCs?
They’re very polymorphic, >10,000 alleles of HLA-B locus, alleles may differ by up to 20 substitutions
Inbred mouse experiment:
- Inbred mouse strains immunised with a virus, Infected cells from A were treated with T cells from both A and B strains -> only TcA could target the cell.
- Shows the MHC must be of self for T cells to recognise MHC associated antigens.
Which cells express MHC I?
All nucleated cells -> encoded by human gene loci HLA-A, HLA-B, HLA-C
Domains of MHCs: Functions
the domains closest to the membranes display the classic immunoglobulin folding pattern, however these are not the regions which bind peptides. The distal domains making up the peptide binding cleft have a very different structure and are the complex which bind peptide and are very polymorphic.
How specific are MHCs?
Very non specific, binding to a range of structurally related peptides.
Structure of MHC binding cleft:
Two alpha subunits with alpha helices joined by a beta sheet. In MHC I the binding cleft is smaller binding peptides 8-10 amino acids long, whereas MHC II can bind peptides of 13-18 amino acids long.
Role of interferons in presenting viral peptides on MHCI:
Interferons induce the expression of a component within the proteasome that facilitates the breaking down of viral peptides to lengths of 8-10 amino acids (so they fit in the MHC I binding cleft)
Transporter associated with antigen presentation (TAP):
Protein in the membrane of the endoplasmic reticulum that actively transfers peptides from the cytoplasm into the ER by ATP-hydrolysis.
Where in the cell do peptides associate with MHCs?
In the Endoplasmic Reticulum.
Process of antigen presentation by MHC II:
Antigen taken up by phagocytosis or endocytosis into an endocytic vesicle -> within the endosome the pH is acidic -> facilitating the unfolding of the proteins, and enzymes break the unfolded peptides down -> within the compartment association by peptide to MHCII occurs -> MHC II – peptide complex transported to the cell surface for recognition by helper T cell
Cross Presentation Function:
Allows some dendritic cells to take up exogenous peptide and associate with MHCI on the dendritic cell, to prime cytotoxic t cells to target cancer cells.
Cross Presentation Process:
Necrotic cell associated with a tumour cell -> taken up by a dendritic cell - proteins from the necrotic cell gain access to the cytoplasm and are loaded onto MHC I proteins -> a cytotoxic T cell will then recognise these complexes and be primed to identify tumours.
Difference between coding regions of MHCI and MHC I:
- There are 3 gene loci for the MHC I proteins -> each encoding for an alpha chain making up part of the MHC I molecule
- There are 3 genes loci for the MHC II proteins -> each encoding for both an Alpha and Beta chain.
How does variation in MHC structure occur across a population?
- These genes are very polymorphic, e.g. HLA-B has more than 10,000 alleles in the human population
- Co-dominant expression -> alleles inherited from each parent, both expressed on cell surface -> this widens the range of peptides you MHC proteins can bind.
What are the consequences of MHC polymorphism?
- Ensures wide recognition of foreign peptides (Side effect of graft rejection or transplants being identified as foreign)
- T cell responses that can be made are determined by an individual’s MHC type
This can be observed in very Inbred mice with some being unable to respond to certain types of antigens due to them not possessing the MHC alleles that bind to a particular set. - MHC polymorphism evolved in response to pathogens, therefore species that aren’t sociable possess less MHC polymorphism due to less selection pressure to fight contagious diseases.
What is within a TCR complex?
αβ subunits + CD3 + accessory protein that aid signalling
Why are co-receptors required in addition to TCR complexes?
To stabilise the interaction between the cells but also to facilitate signalling.
What are the co-receptors that aid TCR binding in T cells?
The coreceptor on T helper cells is CD4, and on cytotoxic T cells CD8.
How do the coreceptors aid TCR complex binding to antigen?
CD4 and CD8 are both associated with a tyrosine kinase (lck tyrosine kinase). When they interact with the MHC complex this localises the CD4/8 to the TCR and acts on the ITAM motifs of the accessory proteins in the TCR which facilitating signalling and eliciting a response.
By which process is the T cell repertoire developed?
Thymic selection
Why do T cells have to migrate to the thymus from the bone marrow (after differentiation from haemopoietic stem cells)
- T cells are originally produced in the bone marrow but have to migrate to the thymus in order to develop further and undergo rearrangement of the T cell receptor genes (αβ of γσ) to express TCRs on their surface.
Two stages of thymic selection:
-T cell receptors have to be able to bind to self MHC -> any cells without receptors which do will undergo apoptosis -> in a process of +ve selection
-Next -ve selection occurs, with TCRs that are able to recognise self MHC bound to self-peptide will be rejected and die by apoptosis
What gene allows the cells of thymus to express proteins/antigens from across the entire body?
AIRE gene (Auto immune regulator genes)
Why must Thymus cells express proteins/antigen from the entire body?
To ensure that host proteins that might be found in distant sites are still not selected for
IgM Structure:
- Instead of being a monomer it is a pentamer (5 antibody units) linked by disulphide bonds in the Fc part of the antibody, the pentamer also contains an additional peptide (J chain), which is needed to catalyse the formation of the pentamer in plasma cells.
- M.wt. 970,000 d
- IgM antibody units don’t have a hinge region, instead having an extra pair of constant region domains -> these domain are still slightly flexible -> and-so can “bend” to bind to antigen, although more restricted than IgG. (“Functional” hinge)
What is avidity?
the term used to describe the overall reaction between all the Fv arms and antigen
What is meant when an immunoglobulin has a high avidity?
It’s effective at “clumping” multiple antigens together -> this makes it an effective agglutinator of particulate antigen
Functions of IgM:
- Can activate complement very effectively
- Important in primary antibody responses.
Why do IgMs tend to have lower affinity?
- IgM is produced early in the immune response -> before the B cells have had the opportunity to undergo somatic hypermutation
What are the two forms of IgA?
Monomer IgA and Secretory IgA
Monomer IgA: Specific Role
not much is known
Monomer IgA: properties
Similar to IgG in serum
Secretory IgA properties:
- High valency -> able to bind to 4 antigens simultaneously
- Rapidly catabolised by environment despite protection by secretory component
- Secretory IgA cannot interact with Fc receptors on phagocytes.
- Present in milk -> important in maternal/inherited immunity
What suggests IgA has a role in maternal/inherited immunity:
Secretory IgA is present in milk
Secretory IgA structure:
- Dimeric with peptide J chain (facilitates formation of the polymer)
- Two monomers fused by their Fc regions.
- Wrapped around it is the secretory component (additional peptide -> thought to protect IgA in the environments of mucosal surfaces.)
Specialised transport mechanism of secretory IgA:
- In the submucosal lymphoid tissues, many plasma cells secrete secretory IgA -> binds to receptor on the internal membrane of the epithelial cells -> Upon binding the IgA is endocytosed -> travels across the cell -> to be released into the mucosal lumen. When its released, part of the binding region of the receptor remains attached, forming the secretory component.
What is the name of the receptor secretory IgA binds to stimulate its secretion?
Poly -Ig receptor
Poly -Ig receptor:
- Binds to polymeric IgA but also IgM (When IgA is deficient)
- Member of immunoglobulin gene superfamily (5 immunoglobulin like domains)
- Secretes Immunogobulins to the lumen
Why is the specialised transport mechanism of IgA useful for the treatment of bacteria?
If Bacteria penetrate the epithelial layer into the submucosal tissues, IgA can bind the bacteria and transport bacteria back into the lumen (unfavourable conditions).
What is the primary role/ function of IgA?
the role of IgA is to bind pathogens (with its high valency and agglutination) and stops them from sticking to the host’s cells.
Across which surfaces do most infections occur?
Mucosal surfaces.
IgD: Functions
produced particularly by B cells and plasma cells in the upper respiratory tract; interacts with receptors on basophils, inducing antimicrobial, inflammatory and B cell stimulatory factors.
IgD: Purpose
Unknown, however highly conserved
IgD: Structure
- Occurs as a monomer -> has a very long hinge region (differentiating it from IgG)
- M. wt 184,000
IgG: Structure
Monomer, bog standard antibody (Y shape)
- Carbohydrate associated with the paired CH2 domains
- Main antibody in tissues and blood
IgE: Structure
Monomeric lacking hinge region but still has residues providing a function hinge.
IgE: Functions
- Binds to high affinity to FcR on mast cells and basophils -> allergy response
IgE: Purpose
- Important in allergy + important in immune defence against large extracellular parasites e.g helminths.
What is the general role of soluble Immunoglobulins?
- Act as labels for pathogens to trigger their elimination / destruction, by specifically binding to their antigens
Avidity vs affinity
- Affinity is the interaction of a single FAB arm with a single antigen
- Avidity is the strength of interaction between a whole antibody and its antigen. (cumulative)
Which soluble immunoglobulins block binding of pathogens to host cells + neutralise toxins:
IgG, and IgA
Examples of pathogens that produce toxins?
tetanus, diphtheria, cholera.
What is the function of Fc tails of soluble immunoglobulins?
To Invoke or effect the destruction of labelled pathogen
Which soluble immunoglobulins can activate the complement by the classical pathway?
IgM and IgG
What is C1 in the classical complement activation pathway
a complex made of C1q, associated with C1r and C1s -> the C1q is component that binds to antibody, C1s and C1r are serine proteases which cleave pieces of other complement components
How many interactions must occur with C1q and antibody Fc regions for activation to occur?
2
Why is IgM a more efficient activator of complement than IgG?
IgG is a monomer, and-so 2 must bind to the C1q to initiate a response, whereas pentameric IgM and-so have multiple Fc regions available for interaction.
Which soluble immunoglobulins bind to receptors on leukocytes surfaces?
IgG, IgA, IgD (basophils), and IgE
What feature of IgM prevents it from activating complement when in mobile phase?
IgM undergoing a conformational change upon antigen binding -> adopting a “staple” form from a “planar” form-> this form being the conformation that can activate complement .
How many subclasses of IgG are there?
4: IgG1, IgG2, IgG3, IgG4
Which C1 component activates C4 and C2?
C1s
Order of IgG subclass ranked by their ability to activate complement:
Subclasses of longer hinge regions better activators of complement. IgG 3 > IgG1 > IgG2 > IgG4 (no activation)
Order of IgG subclass ranked by their ability to bind to FcRs:
(IgG1 = IgG3 > IgG4, IgG2 doesn’t interact with Fc receptors)
What are the roles of Fc receptors on phagocytes:
- Increases uptake of immune complexes (soluble antigen and antibody)
- Opsonisation -> aids phagocytosis and destruction of antibody-coated pathogens.
How are pathogens that are too big to phagocytose dealt with?
Eosinophils release the contents of their granules onto the bacterial surface in a process of “frustrated phagocytosis”
What are the roles of Fc receptors on mast cells and basophils:
- Mediate allergy/defence against large parasites
- Mast cells secrete inflammatory mediators and cytokines.
What are the roles Fc receptors on Natural Killer (NK) cells (IgG1 and IgG3)
- NK cell upon recognition via Fc receptor crosslinking to antibodies proteins -> produce perforin channels that pierce the infected host’s cell membrane, releasing hydrolytic granules into the infected cell. -> triggering apoptosis.
What is the process of NK cell meditated apoptosis initiated by binding to FCRs called?
Antibody-dependent cell-mediated cytotoxicity (ADCC
Which immunoglobulins recognise viral proteins associated with viral envelopes on infected host cells?
IgG
(Summary) Allergic response:
- IgE antibodies produced by plasma cells upon exposure to foreign body, these will then bind to Fc receptors on the mast cells under the skin and mucosal surfaces.
- Upon re exposure to the foreign body, the antibodies bound the Fc receptors, cross link and cluster together -> this clustering of Fc receptors triggers the fast (5-10 minutes) exocytosis of its granules (containing histamine etc.)
- Immediate hypersensitivity -> over reaction of immune response.
Thymus independent antigens 7(e.g. bacterial polysaccharides):
- Lots of repeating epitopes (also known as the antigenic determinant, part of antigen that is recognised by immune system) or shapes that antibodies on the B cell surface can recognise -> the cross linking induced by this causes the clustering of receptors (known as capping) -> this is enough to generate signal to B cell for response by production of IgM antibodies.
o Very few memory cells are generated
Thymus dependent antigens (e.g. proteins):
- Require T cells for differentiation of B cells into plasma cells.
o Long-live memory B cells may also be generated.
o Responses can involve somatic hypermutation for increase affinity and class switching for increased variability of effects.
Why don’t T cell receptor genes undergo somatic mutations?
- Risk (could generate ‘self’ recognising receptors)
- May lose the ability to recognise host-MHC I
- Don’t need high affinity, as they need to unspecific binding to multiple possible antigens
What occurs in the absence of co-stimulatory signals to naive T cells?
They will become inactive (unresponsive) or tolerise (apoptosis)
Why is T cell regulation by co-stimulatory factors important?
This mechanism ensure that T-cells don’t develop “self-targeting” i.e autoimmune disease.
What is the best characterised co-stimulatory signal molecule for T cells?
B7, which binds to CD28 (receptors) on the T cell surface.
What is the mechanism by which co-stimulatory molecules determine the differentiation of T cells?
B7 on the membrane of the APC binds to CD28 on the surface of the T cell, upon this binding the APC is stimulated to express and secrete cytokines (determine subset in addition to IL-2 (determine CD4+ve or CD8+ve).
Domain Nomenclature of soluble immunoglobulins
-Domain nomenclature:
- By convention the variable regions are Vl and Vh.
The constant region of the light chain is CL
The constant region of the heavy chain going back form the N terminus goes, CH1, CH2, CH3
Differing levels of IgG and IgM in a primary and secondary response:
initially IgM will be produced and a few days later IgG will be produced at similar levels -> upon secondary exposure -> IgM is produced (more quickly) but at the same level -> what differentiates this response however is the presence of high levels of IgG (long lived) -> depending on the nature of the pathogen IgA or IgE may also be produced.
IgG: Functions
- interact through their Fc regions with Fc receptors present on phagocytes and NK cells (and other cells)
- Crosses placenta (binds FcRn on trophoblast) during the last trimester of pregnancy -> allows the maternal antibody to enter the fetus
What is the receptor that binds to IgG in the last trimester of pregnancy?
FcRn – neonatal receptor
What is the function of neonatal IgG?
protects the new-born from infection -> babies born prematurely without their full complement of maternal antibody are more susceptible to infection
What is the benefit of having subsets of T helper cells?
Allows for a more flexible response.
T helper cell 1: (Th1)
(Activated by IL-12) Activates macrophages, causes inflammation, induce production of IgG and CD8+Ve cells
T helper cell 2: (Th2)
(Activated by IL-4) Class switch to IgE, activate mast cells
T helper cell 17: (Th17)
(Activated by IL-6 + TGF-B) Activate epithelial cells to recruit neurtophils + proinflammatory
T helper cell follicular: (TfH)
(Activated by IL-6) Induce B cell differentiation, class switching and affinity maturation
T regulatory cells: (Treg)
(Activated by TGF-B) Inactivate T helper cells, downregulating immune response once infection has been dealt with
What are the two types of T regulatory cells:
Natural and Induced
Natural T Regulatory Cells:
(Develop in thymus) upon recognition of self-MHC and self-peptide, produce IL-10 and TGFB to switch off T cells + dendritic cells (upon contact)
Induced T Regulatory Cells:
(Develop in peripheral lymphoid tissue) - recognise MHC and non-self peptide - produce IL-10 adn TGFB -> switch of immune response via secrete suppressor antigens
What are the two ways in which cytotoxic T cells may induce apoptosis?
- Uptake of proteases (granzymes) into target cell via perforin channels
- Fas ligand induces clustering of fas on target cell
What is the function of Fas receptors?
Upon binding to fas ligand they stimulate the caspase cascade inducing apoptosis of the cell.
Fas activation pathway (apoptosis further reading)
In Fas TNFs, the trimerization of the receptors allows for the binding of FADD, an adaptor protein that acts as a docking surface for pro caspase. Upon binding, the pro caspase is proteolytically activated by self-cleavage, forming an active enzyme complex which activates further downstream effectors and triggering apoptosis (Schulze-Osthoff, et al., 2001)
What are immune checkpoints?
Examples of proteins and ligands that down regulate the immune response to ensure it’s not constantly on to prevent unnecessary tissue damage.
Immune Checkpoint: CTLA-4
- induced on activated T cells, binding B7 on APCs with a higher avidity than CD28 -> down regulating T cells by preventing T cell survival
Immune Checkpoint: ITIM motifs
Found in the cytoplasmic region of proteins involved in immune responses, their phosphorylation leading to inhibitory signals to the cells.
Immune Checkpoint: PD-1
On activated B and T lymphocytes, interacting with PD ligands to initiate programmed cell death.
How do cancer cells subvert immune responses?
They take advantage of immune checkpoints, stimulating these checkpoints to downregulate the immune system.
What is the 1988 hygiene hypothesis?
children brought up on farms or from large families were less prone to develop allergy -> repeated childhood infections may be protective against allergy.
- Insufficient exposure to certain types of infection skew the TH1/TH2 balance towards TH2.
DISPUTED as there is a negative correlation between helminth (parasitic) infections and allergic disease.
What is the counter regulation hypothesis? (better than hygiene hypothesis)
o Childhood infections protect against allergy by promoting IL-10 (Treg increase, TH1 and TH2 decrease)
o Infection with microbes or larger parasites plays a critical role in driving immunoregulation e.g. promotes formation of Treg IL-10
o Human immune system and “old friends” co-evolved
o May also explain rise in autoimmune disease (If its TH1/TH17 driven)
What are γdelta T cells?
T cells with receptors made of gamma delta subunits -> found in the mucosal epithelium -> recognise broader range of antigens -> don’t require presentation by MHC -> can make cytokines -> role in various infections and cancers
Advantages of passive immunity treatments?
-Quickly neutralise toxins and venoms
-Faster than conventional immune response
-Limit/prevent highly virulent pathogens
-If no vaccine then it may be the only form of treatment
Disadvantages of passive immunity treatments?
-Does not activate immunological memory
-No long term protection
-Could react with anti-sera (if cross-species) -> cause allergic response?
What are the different type of passive immunity treatments?
Antitoxins - treats toxins e.g. botulins
used prophylactically -> reduce chance of infection after exposure -> used for hepatisis, measles and rabies
Antivenins - to treat vemons e.g. snake bites
Convalescent plasma
Hyperimmune globulin
Monoclonal Antibodies
What are the two categories of active immune treatments?
Natural (in response to infection) + Artificial (In response to vaccine)
What are the principles of active immune treatments:
Manipulating immune response to generate protective response -> this is done by mimicking natural infections to generate an immune response.
What is passive immunity?
Transfer of preformed antibodies into circulation for immediate treatment/ protection from disease.
Natural passive immunity:
Occurs naturally by transfer of maternal antibodies across the placenta.
When is artificial passive immune treatments used?
-When the immune system is defective
-When there is no time for active immunisation
-There is acute danger of infection
Advantages of active immunity treatments?
- Upon secondary infection of the real pathogen, a rapid response is induced as the infection if recognised by memory cells.
- Because immunisation saves a pathogen to the immunological memory, protection to the disease is long term.
Disadvantages of active immunity treatments?
What is a vaccine?
A type of active immunity treatment underpinned by the principles of immunisation. A vaccine is a biological substance that can safely stimulate an immune response to recognise and defend against specific pathogens to prevent future infections and disease.
What are the 3 primary types of vaccine?
Attenuated (pathogen with reduced virulence), Killed (non-living + the pathogen unable to replicate), Subunit (non-living molecular components of the pathogen)
What is adjuvant?
Adjuvant is an ingredient added to vaccines to enhance immune responses by inducing inflammation.
Examples of adjuvant:
Aluminium salts, lipids
What types of vaccine are adjuvants most appropriate for?
non-living vaccines (subunits and dead), with attenuated vaccines usually able to elicit an appropriate response by themselves.
What are the desired features of Vaccines:
Safe
Protective
Long term + sustained protection
Effective inducer of antibody and activator of protective T cells
Practical considerations
Attenuated live vaccines: How effective?
- Most effective as it’s a real infection at the appropriate body site and-so induces an appropriate immune response
What are the methods that can be used to attenuate a pathogen for use in immunisation?
- Repeated selection for colonies of the pathogen adapted to temperatures not found in the body, to ensure the pathogen’s functions aren’t working at their operational maximum -> inactive riboswitching
- Genetic manipulation
Killed Vaccine: Features
- Pathogen inactivated by chemicals or heat treatment
- Unable to replicate
- Some side effects possible
Subunit Vaccines: Types
Recombinant Protein (e.g. Hepatitis B antigen in yeast - produced by cell line)
Subcellular Fractions (e.g. polysaccharide capsules)
Toxoids (inactive toxins)
Conjugate Vaccines ( Component linked to carrier protein)
DNA/RNA Vaccines
Subunit Vaccines: Features
- Safer form of vaccine with fewer side effects
- Require adjuvants and multiple injections
- Immunity may be short lived
Recombinant Vector Vaccine:
Use genetically engineered viral vector to deliver genetic material encoding antigen from target pathogen in host cells.
RNA/DNA vaccines:
- Use DNA or RNA to transiently express pathogen antigen in host cells -> e.g. Pfizer of Moderna SARS-Cov-2 vaccines.
- Different to recombinant vector vaccine as the DNA/RNA is encased in a lipid coat
History of Vaccination:
-Practice first seen in Türkiye
- Used in treatment of small pox
- Inoculation through scratch
Edward Jenner -> developed principles of vaccination + found infection with cowpox provided protection from small pox without significant risk of disease.
What is the First step of vaccine development?
Antigen discovery and characterisation
What occurs during the Antigen discovery and characterisation step of vaccine development?
- Potential antigens identified -> using genomics, proteomics, and bioinformatics to target and identify proteins that can serve as potential targets of the immune system
- Once identified these antigens are characterised using analytical techniques (e.g. PCR, ELISA, Western Blot) to confirm identity, purity, and immunogenic properties.
What are the two parts of designing a vaccine once a target antigen has been identified?
- Choice of vaccine platform (live attenuated, inactivated, subunit etc.)
- In silico modelling strategies (identify epitopes + help economise stages of development)
What techniques can be used to change antigen structure when developing a vaccine?
- Amino acids substitution can be used to enhance the immunogenicity of an antigen, by stabilising it’s pre-fusion conformation and structural rigidity, so it’s more easily recognised.
What occurs during the preclinical testing of a vaccine?
- the safety, immunogenicity, and potential efficacy of a vaccine is assessed -. to gauge response in humans
- provides data on the vaccine’s safety and ability to elicit a response
Methods of preclinical testing:
In vitro studies, animal models, toxicology studies
What are the 3 phases of clinical trials?
Phase I - assess safety and dosage (small group of healthy volunteers)
Phase II - optimise dosage and safety (larger sample size of people with target characteristics similar to target group)
Phase III - confirm the vaccine efficacy and monitor rare side effects (Thousands of participants)
Regulatory approval and licensure of vaccine process:
Clinical data submitted to regulatory body for a regulatory review (ensure it meets safety, efficacy, and quality standards). If it meets the requirements its granted license for public use and monitored in the general population (post-license surveillance).
What are the steps of large scale vaccine manufacturing?
- Antigen production, formulation, purification, filing and packaging, quality control and assurance.
What are the different components of a Vaccine?
Antigen, Adjuvant, preservatives (e.g. 2-phenoxyethanol), stabilisers, surfactants, residuals (yeast or antibiotics), diluent
Batch testing:
Each batch of the vaccine is tested for quality and consistency before it is released for distribution. This includes tests for sterility, potency, and absence of contaminants.
Good Manufacturing Practices:
All vaccine manufacturing processes must adhere to GMP standards, which ensure that products are consistently produced and controlled according to quality standards. This includes maintaining aseptic conditions, proper documentation, and regular inspections.
What are the 3 methods of quality control and assurance:
Good manufacturing practices, Batch Testing, Post-market surveillance
What are the methods used to store and distribute vaccines?
Cold Chain Management and Distribution Networks
What is meant by vaccine equity?
A global effort to ensure the fair distribution of vaccines worldwide, to compensate differences in wealth and available infrastructure.
Cold Chain Management
Most Vaccines require refrigerated storage at temperatures between 2’C and 8’C. Somme vaccines, like mRNA vaccines, require ultra-cold storage at temperatures as low as -70’C. specialised equipment and protocols are used to maintain these temperatures during transportation and storage.
Distribution networks:
Vaccines are distributed through a network of a refrigerated trucks, warehouses, and portable iceboxes to ensure they remain within the required temperature range until they reach the point of administration
mRNA vaccines: Function
- Use mRNA to instruct cells to produce pathogens that trigger an immune response (once expressed by MHCI)
What are the challenges in vaccine development?
- Emerging infectious diseases are difficult to predict
- adaptation and the development of new infection strains
- Require rapid research
- Misinformation leads to a lack of public trust
- Cost associated with vaccines leads to unequal distribution
mRNA vaccines: mechanism
mRNA vaccines deliver genetic instructions to cells to produce viral proteins (e.g. spike proteins) -> these are recognised by the immune system and stimulates an immune response
mRNA vaccines: Advantages
Developed and produced more quickly than traditional vaccines and are highly adaptable.
Viral Vector Vaccine: Mechanism
Virus vector is engineered to be harmless and unable to replicate -> carries genetic code for antigen assembly which is then inserted into host cells. They are then assembled and expressed, inducing an immune response.
Vaccine Components: Stabilisers
Protect the antigen from degradation
Vaccine Components: Nanoparticles
- Stabilise antigen and protects from degradation
- allows for specific targeting of tissues / delivery
- enhance immune response
What are the 3 types of nanomaterial?
Lipid, polymeric, inorganic
Personalised Vaccines: What are they?
Vaccines tailored to the individual characteristics of a patient’s disease (notably for cancers)
Personalised Vaccines: Mechanism
Designed based on specific mutations of antigens present in pathogen (or tumour) -> elicit strong response against the unique antigens
Personalised Vaccines: Types
Autologous nucleic acid - whole tumour lysates
Autologous antigen peptides - autologous tumour cells + adjuvants
Autologous subunits
Factors favouring the global eradication of diseases (e.g. polio)
- No animal vectors (only infects humans)
- No long term carrier state (easily identifiable)
- Few unrecognised clinical cases (distinct symptoms)
- Few serotypes (single vaccine adequate)
- Stable, cheap, effective vaccine
- Cost effective eradication program.
Global Importance of vaccines:
Preventing disease outbreak, protect vulnerable populations (via herd immunity), global health security.
Examples of pathogens lacking effective vaccines:
Malaria - (used to be)
HIV/AIDS - global pandemic - no effective vaccine currently - therapeutic treatments have been developed and preventative medications
Covid-19 - vaccines developed however -> multiple serotypes + animal vectors prevent eradication. - 704 million infections -> 7 million deaths
What are the desired properties of therapeutic antibodies?
Specific (high affinity)
Stable structure
Multi-valent -> for improved binding and cross linking (to induce signal transduction)
Useful effector properties
Immuno-electro microscopy:
Method used to see where the antibodies bind to antigen or receptor by electron microscopy (uses gold particle labels)
Immunoprecipitation:
Method used to separate a protein of interest from cell lysate (use sepharose label)
What is a linear epitope:
When the binding sequence in the antibody binds to adjacent amino acids in the protein antigen.
What is a discontinuous epitope:
When the binding sequence in the antibody binds to amino acids in 3D proximity but are not adjacent in the antigens residue sequence.
What are the future area of growth for immunotherapy?
Development of soluble TCRS e.g. Kimmtrak
Tumour infiltrating lymphocyte therapy
Modulation of innate immune system
In vivo gene editing of antibody genes (via CRISPSR Cas9)
use of gamma delta T cells in cancer treatment
Which antibodies can be administered to treat inflammation and related autoimmune disease:
TNF a, IL1, IL6, C5 -> block cytokine binding to effector cells and downregulate the inflammatory pathway
Passive Immunisation for Covid: Advantages
- Provides immediate protection for up to 4 weeks
-suitable for immunocompromised patients - relives severe symptoms
Passive Immunisation for Covid: Disadvantages
- No long lasting protection
- Expensive
- Loses effects against variants
- May promote evolution of the pathogen
-Limited efficacy and supply
Passive Immunisation for Covid: Process
Convalescent sera from patients who already recovered from the infection is processed and administered (example of polyclonal antibodies).
Future Antibody prospective uses:
- Development of antibodies that target conserved epitopes
- Computational redesign of existing antibodies to broaden specificity
- Cocktails of antibodies to effect multiple targets
- Monoclonal antibodies to treat excessive inflammation
- Prevent damage to lungs caused by cytokines (chronic inflammation)
Immune checkpoint inhibition by antibodies:
Bind to CTLA-4, PD-1 (immune checkpoint) and inhibit their action, preventing/ limiting the downregulation of the immune response. This allows for the targeting of cancers that’d usually downregulate the immune response by exploiting these checkpoint systems.
Antibodies as ‘magic bullets’ in tumour cell treatment: Examples
anti-CD52 antibodies: Found on all WBCs -> recognise leukocytes and activate complement and ADCC -> used in treatment of leukaemia and lymphoma.
anti-CD20 antibodies: Found on B cells -> activator of complement and ADCC -> used in treatment of leukaemia and lymphoma.
anti-Her2 antibodies: An RTK expressed on some breast cancer cell -> binding to Her2 inhibits tumour growth.
Generating fully human antibodies from animals:
Transgenic mice in which their antibody genes have been replaced by human antibody genes (Xenomice). These mice can be immunised to generate human antibodies by conventional monoclonal techniques.
What is the function of residuals as vaccine components?
Residuals help prevent contamination of the vaccine (e.g. antibiotics or yeast)
What are antibody chimeras?
Antibodies with mice V regions spliced to be associated with human C region genes
What are the problems with using animal antibodies in the treatment of humans?
The antibodies differ in structure and will be targeted and cause serum sickness
Disadvantages of antibody engineering?
Time consuming
May lose affinity / specificity of antibodies
Monoclonal Antibodies: Uses
- ## Recognise and bind to cell surface molecules to identify Cell types (e.g. subpopulations of T cells) + cell surface complexes of varying function (e.g. CD63 - member of tetraspanin family)
Advantages and Disadvantages of Monoclonal Antibody use:
Advantages
- highly specific
-Can be standardised
Disadvantages
-Expensive
-Conformation sensitive
What is the immunogenicity of an antigen?
The ability of an antigen to induce an immune response
What can be used to overcome the tendency for small molecule antigens (<1000Da) to be washed out of the body?
Carrier proteins
What is antisera?
- Product of several B cell colonies
- contains mixture of antibodies specific to different “epitopes” on the antigen.
What are the advantages of using antisera?
- Cheap and robust (likely that some of the antibodies will bind to target protein even when partially unfolded/denatured)
What are the disadvantages of using antisera?
- Because specific to multiple epitopes, need pure antigen to immunise, can be difficult to standardise.
Why aren’t human chimera antibodies fully humanised?
They still contain foreign V region and-so can still provoke an immune response
What is a disadvantage of humanised antibodies?
the antibody may lose some of its binding capacity and are very time consuming to approach.
Antibody gene libraries:
- Antibody V genes are cloned from naïve Immune B cells using a suitable vector and used to make a “library”. The antibodies can then be expressed in bacteria or on the surface of the bacteriophage, antibodies with the desired antigen are selected from the library, using phage display tehniques.
process of phage display (in the generation of human antibody):
Isolate mRNA from antibody producing cells -> reverse transcribe the mRNA, amplifying the Fab or Fv cDNA by PCR -> clone or express the cDNA in bacteria/phage (phage display) -> antibody library -> screen library, coating surface with antigen, the phage with specific antibodies will then bind to the dish coated with antigen of interest -> can then select phages -> this can be repeated in process of “panning”.
What is the synthetic human antibody gene library?
large gene library -> 109 members -> if then the library is screened there will be some antibodies produced which are seen to bind to the target antigen-> selected antibodies then further mutated and screened to further improve specificity and/or affinity
How are cell lines for monoclonal antibody secretion developed?
Mouse is immunised with an antigen -> their lymphocytes are harvested -> fused with myeloma cell line (mutated to not secrete antibody or produce purines) -> unfused myeloma cells remain unable to produce purines and die in HAT medium -> hybridomas that secrete the specific antibody selected and clones.
Advantages and Disadvantages of Polyclonal Antibody use:
+ves
- relatively cheap
-Robust (may recognise partially denatrued or unfolded antigen)
-ves
-Specific to multiple epitopes
- Need pure antigen to immunise
- difficult to standardise
How are polyclonal antibodies produced?
Antisera taken from immunised animal from blood sample -> containing mixture of antibodies. Instead of targeting a single epitope, they target multiple
Which functional groups increase the immunogenicity of antigen?
aromatic residues and charged group facilitate binding to antibodies.
Chimeric antigen receptor T cells (CAR-T cells):
T cells taken from patient with cancer engineered in vitro. Cells are infected with genetic material for chimeric receptor -> grown in lab and stimulated using CD3 and CD28 to activate and divide the T cells. These T cells are injected back into the patient, overcoming the need for MHC recognition.
Functions of Antibody Engineering:
Improve the half-life (FcRn interaction), improve or remove effector functions.
Why kind of type of antibody is better for tumour penetration?
Antibody fragments, e.g. single chain Fvs (scFv) - in which paired V regions are linked by a peptide chain.
What are Bi-specifci antibodies?
Antibodies with dual specificity. Can link tumour cells to effector cell proteins (e.g. tumour antigen with CD3 on T cells)
Car T cell significance:
since 2017 6 CAR-T cell therapies approved by FDA, all treating blood cancers.
What is the chimeric receptor in Car-T cells?
A receptor engineered to target cancer-associated antigens.
