Cellular basis of immunity Flashcards
Does everything living have an immune system?
yes, most basic have only phagocytes, then first lymphocytes appear as more advanced then lymphocytes split into T and B cells as well as phagocytes
Has adaptive immunity become more efficient in vertebrates?
yes
GALT =?
Gut associated lymphoid tissue
Thymus =?
Specialised primary lymphoid organ where T cells mature
Spleen does?
synthesises immunoglobulins and is reservoir continuing half the body monocytes
Makes dendritic cells and macrophages for the innate immune system
What is AID?
Deaminase mediating class switch recombination and somatic hypermutation of antibody encoding genes
Essential for removal of auto reactive B cells
Order from GALT have developed from most simple to most complex
Immune system number of cells, types and number of connections?
10^12
Less than 10
Infinite
Brain number of cells, types and number of connections?
10^11
2
10^14
Where do all immune cells originate from?
Hematopoietic stem cells in the bone marrow
Goes into the lymphoid lineage, melodic lineage or erythroid lineage
Serum = ?
Plasma without clotting factors
Blood = ?
55% plasma = water, dissolved proteins, glucose, clotting factors, electrolytes, hormones, CO2, and oxygen
1% WBC
44% RBC
Where is the immune system?
In the lymphatics system and lymph organs
White blood cells circulate with blood
Some WBCs and quite a bit of plasma leave the capillaries in tissues
Circulation of plasma and WBC through interstitial space/tissues and back via lymphatics to lymph nodes allows surveillance of tissues for forge in molecules and pathogens
Around 70% of the immune system is associated with the gut
Describe neutrophils eosinophils and basophils?
Attack a variety of pathogens by phagocytosis or with antimicrobial or with anti-microbials and histamine released after degranulation
Describe plasma cells derived from B lymphocytes?
Secrete large amounts of antibody
Describe B and T lymphocytes?
Orchestrate the adaptive immune response and provide immunological memory
Describe monocytes and macrophages?
Phagocytes and regulate many aspects of innate and adaptive immunity
What does each pathogen have?
Antigens on its surface known as pathogen associated molecular patterns
Immune cells have invariant receptors on their surfaces known as pattern recognition receptors that recognise many of these PAMPS
Once a pathogen is recognised a cascade of events occurs activating an immune response to destroy it
Examples of PAMPs recognised by the innate system?
Cell wall components:
Peptidoglycan (bacteria)
Lipopolysaccharide (gram positive bacteria)
Galactomannans
Nucleic acid:
CpG motif DNA (bacteria)
ssRNA (viruses)
dsRNA (viruses)
Conserved surface proteins
Flagellin (bacteria)
Conserved stress proteins”
Heat shock proteins (bacteria)
Pattern recognition soluble receptors?
Natural antibodies Complement Pentraxins Collectins Ficolins
Pattern recognition cell receptors?
Toll-like-receptors NOD-like receptors RIG-like receptors C-type lectin like receptors Scavenger receptors N=formyl met-leu-phe receptors
Features of innate system?
Fast
Doesn’t require immunological memory
Activated by binding of PAMPS by PRRs on phagocytic cells
can discriminate self and non self
Receptors encoded in germline
Limited diversity
Typically recognise structures common to different microbes
Features of adaptive system?
Slower
Activated once the innate has been activated
Activated by a cascade of chemical signals from sites of infection , and physical interactions with phagocytic cells of the innate immune system known as dendritic cells that migrate from sites of infection into the lymphatic system to activate lymphocytes
Can discriminate self and non self
Receptors encoded by gene recombination - massive diversity
Typically recognise structures unique to different microbes
What decides the extent to how innate or adaptive system is used?
Severity, context and duration of disease, innate is mainly for something like a minor cut or splinter
What are cytokines and chemokines?
signalling molecules released at sites of infection by resident macrophages recruit neutrophils from the bloodstream leading to localised inflammation
Types of lymphocytes?
All key in adaptive immunity
B-lymphocyte/Bcell - express antibodies as B cell receptors
Helper T lymphocytes
Cytotoxic T lymphocyte
Natural killer T cell
T-lymphocytes express T cell receptors
What happens with B and T cells during an adaptive response?
Have receptors specific for particular pathogen molecules proliferate
Clonal selection and expansion forms the basis of protective immunity and immunological memory through which the body is protected against future attack
Homeostasis in which specific molecular interactions keeps the immune system in check?
Antigen receptors and antigens
Cytokines/chemokines and their receptors
Cell-cell interactions
we get infected or colonised in?
Cytoplasm Intracellular vesicles of cells Interstitial spaces Blood or lymph Epithelial cells
First barrier broken through during infection?
Skin
Cuts or burns
Main portals of entry is the mucosal epithelia of the gastrointestinal respiratory and urogenital tracts
Immunity requires?
The recognition and elimination/containment of infectious organisms
Most front line defences provided by the innate
Consists of molecules and cells that distinguish host cells from those of infectious agents by recognising conserved constituents of microorganisms
Activated within hours of contact and efficacy not significantly increased by previous exposure
Describe immune cell differentiation?
Cells of the innate and adaptive immune systems are derived from the pluripotent hematopoietic stem cell
The HSC divides to replace itself and produce a progenitor cell with the potential to give rise to all of the hematopoietic line ashes by generating further progenitor cells committed to progressively narrower ranges of differentiated fates
The erythroid lineage gives rise to?
erythrocytes and to megakaryocytic which shed fragments that form the platelets that initiate blood clotting
The myeloid lineage gives rise to?
Phagocytic and inflammatory cells of innate immunity
The lymphoid lineage gives rise to?
T and B cells of adaptive immunity and to natural killer (NK) cells which are specialised cytotoxic cells
Killer cells count as innate as don’t require activation
Hematopoietic stem cell lineages?
SCF = stem cell factor
TPO =Thrombopoietin (platelet production)
EPO = Erythropoietin (erythrocyte production)
What protects epithelial surfaces, especially the mucosa of the gastrointestinal, respiratory and urogenital tracts?
Basophils, mast cells and eosinophils
Mast cells have a sentinel role, whilst basophils and eosinophils are circulating cells recruited from the bloodstream
Whereas neutrophils destroy internalised micro organisms these cells typically provide immune defence against pathogens too large to be internalised
On activation they release the contents of their cytotoxic granules to the exterior creating an environment hostile to an invading organism or directly killing it
They also release histamines that mediate allergic and inflammatory responses such as coughing sneezing and vomiting
These expulsive responses may be mechanisms that are evolved to expel parasites
All 4 are known as the granulocytes
Who discovered phagocytosis?
Ilya Mechnikov (1898)
The phagocytic cells of the immune system comprise of?
neutrophils and macrophages which are effector cells, specialised to internalise and destroy micro organisms, and dendritic cells which internalise micro organisms for presentation to T lymphocytes of the adaptive immune system
Features of neutrophils?
Front line effector cells of innate immunity
After differentiation they circulate for a few hours before entering tissues to engulf microorganisms and destroy them via microbicidal products stored in vesicles
Features of macrophages?
Long lived cells that provide immune surveillance
They are derived from monocytes that circulate in the blood, differentiating as they leave the blood stream
Like neutrophils they ingest and destroy micro organisms
Features of dendritic cells?
Are involved in the induction of adaptive immunity
As immature cells they operate as phagocytes but rather than destroying the micro organisms they ingest their function is to display the ingested particles on their surface for recognition by T lymphocytes
Describe macrophage receptors?
Scavenger receptors - recognise particles released by dead or damaged tissues
Complement receptors and antibody receptors
Similar on dendritic cells
Activation of the innate immune system?
Directly through recognition of micro organisms or indirectly through recognition by cells of the adaptive immune system that then activate the innate immune defences
This is done as a lymphocyte of the adaptive immune system produces antibodies whose variable regions recognise a surface component of the bacterium A non variable region of the antibody is then recognised by a receptor of the phagocyte which in turn is activated engulf it. In this way, bacteria that have masked the conserved components can be recognised and destroyed by the innate immune system
Whats opsonin?
A general term for soluble components of the immune system that coat micro organisms and stimulate uptake by phagocytes
2 ways phagocytes are pivotal role in the immune system?
On activation by microbial surfaces they release cytokines and chemokines that amplify the response to infection
Cytokines and chemokines are signalling molecules. They increase the permeability of blood vessels and recruit additional cells and molecules of the immune system to sites of infection - the inflammatory response
Hence they activate the adaptive immune response
What does the chemokines IL8 (CXCL8) do?
From a macrophage
Recruits neutrophils, basophils and T cells to site of infection
Recruitment of neutrophils to inflammatory sites?
Fluid matrices contain antibacterial peptides and toxic proteins that are constitutively secreted and further induced in response to cell wall components such as lipopolysaccharide
B-defensins in lung and skin
a - defensives in paneth cells of the intestinal tract
Lysozyme in fluids bathing the cornea
Cathelicidins (another group of antimicrobial defensins) are produced by activated neutrophils and epithelial cells in addition to antimicrobial activity they trigger signaling through the formyl peptide receptor an innate immune recognition element thereby aiding recruitment of neutrophils to inflammatory sites
what are fMLPs?
powerful chemoattractants and activators of neutrophils
Derived from BH2 terminal extensions of newly synthesised polypeptides
fMLP directs the transport of secreted proteins the possess NH2 terminal signal peptides
Cleaved by a signal peptidase following polypeptide transport and released into the extracellular space
what are fMLPs?
powerful chemoattractants and activators of neutrophils
Derived from BH2 terminal extensions of newly synthesised polypeptides
fMLP directs the transport of secreted proteins the possess NH2 terminal signal peptides
Cleaved by a signal peptidase following polypeptide transport and released into the extracellular space
What is chemotaxis?
neutrophils are attracted to bacterial chemical products like the peptide fMLP
Describe the inflammatory response?
Memory T cells monitor keep the tissues of the body under surveillance
If they detect no antigen they return to circulation via the lymphatic system
If there is an antigen it is processed by antigen presenting cell, and presented as MHC peptide complexes
This activates memory T cells
Which release cytokines such as TNF-a which act on local blood vessels
TNF-a activates the vascular endothelium
Loosens gaps in them so more blood leaks out, swelling occurs and looks more red due to RBC
The activated endothelium also releases adhesion molecules that allows phagocytes out the blood vessel
Takes 1-3 days
The phagocytes uptake the antigen and also can present it
Memory and effector T cells also leave the vessel
Describe cytokine signalling?
Cytokine receptor consist of 2 chains
Each having an extracellular binding domain
An a intracellular binding domain which has JAKs kinases on
Without cytokines these 2 receptors are not associated
Cytokines stabilise the heterodimer bringing them together, including the 2 JAK kinases
The JAK kinases can then phosphorylate the cytoplasmic tails
STAT molecules then bind to the chains at the bottom of the receptors which get phosphorylated by the JAKs
The STAT molecules can now dimerise and migrate into the nucleus where they can directly activate gene transcription
Describe rolling adhesion?
Blood vessel endothelium at sites of infection expresses selectin molecules (P-selectin and E-selectin) the selectins are triggered by cytokines released by sites of inflammation
Leukocytes such as neutrophils express the ligand for these selections in the form os s-Lex
These creates a weak bond with the selectins and the neutrophils rolls along the blood vessel forming and breaking bonds
These neutrophils will find somewhere they can squeeze for the endothelial cells out of the blood vessel
Only happens in veins
Overview of the innate system?
Immediate response minutes to hours
Semi specific, recognition through invariant specifc receptors (PAMP)
Includes Granulocytes DC/Mono/Mac
NK cells
TLR/NLR/RLH
Cytokines Chemokines Antimicrobial molecules& complement
No memory
Ovreview of the adaptive immune response?
Functional 4-7 days after exposure
Specific through epitope specific receptors (TCR, lg)
Includes T cells/B cells DC
TCR/BCR
Antibodies Cytokines Chemokines
Memory
Does the innate system help healing?
yes
Most abundant white blood cell?
Neutrophils followed by lymphocytes
What are the first responders to tissue detected microbes?
Neutrophils
What are the types of secreted molecules released by
Lysozyme - released in fluids of cornea, saliva and gut (Paneth cells)
Breaks down peptidoglycan, a component of the bacterial cell wall
Phospholipase A2 - breaks down phospholipids in cell membrane of bacteria
Antimicrobial peptides - Defensins - (alpha-defensins in Paneth cells of the intestinal tract and beta-defensins in lung and skin) – disturb the cell membrane of bacteria and fungi
Cathelicidins (another group of antimicrobial defensins) are produced by activated neutrophils and epithelial cells
Histatins (oral cavity, active against fungi)
Lecticidins (creates a pore in microbial cell membranes)
Complement: exists in fluids and blood:
capable of direct killing of pathogens through membrane attack complex (MAC)
also potentiates phagocytosis and the recruitment of other inflammatory mediators
More details on complement?
Small proteins that are synthesised by the liver, tissue macrophages, blood monocytes and epithelial cells of the genitourinary system and gastrointestinal tract
They circulate the blood as inactive precursors
Can diffuse into tissues
Capable of direct killing of pathogens through membrane attack complex
Potentiaites phagocytosis
Potentiates the recruitment of other inflammatory mediators
What’s the complement cascade
Is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen’s cell membrane.
Its spontaneous and low level activation
If deposited onto pathogen = activation
If deposited into host cell = inhibited
What does C3 convertase do?
Cleaves C5 into:
C5a: potent anaphylotoxin
C5b: initiates Membrane attack complex
What is the membrane attack complex?
or terminal complement complex (TCC) is a structure typically formed on the surface of pathogen cell membranes as a result of the activation of the host’s complement system, and as such is one of the effector proteins of the immune system.
In order to permeabilize both the bacterial outer and inner membrane and thus kill a bacterium,
Where does phagocytosis occur?
Phagocytosis occurs mostly in neutrophils and in monocytes/macrophages/dendritic cells
What is phagosome killing?
The phagolysosome is matured through sequential fusion with endosomes and lysosomes and becomes a very specialised intracellular compartment
The bug is digested and degraded
Unpleasant environment for microbes
How does phagocytosis occur?
Microbes are recognised by phagocytic surface receptors:
- PAMPS-PRR (Toll-likereceptors, C-type lectins, scavenger receptors)
- Opsonization
Microbe coated with complement = binds CR3-CR4
Microbe coated with antibody = antibody binds FcRecepors
Recognition and subsequent signalling trigger phagocytosis
Describe how the phagolysosome is matured from a phagosome
The phagolysosome is matured through sequential maturation with lysosomes and becomes a very specialized intracellular compartment with:
Low pH (pH=4.5): mediated by a V-ATPase proton channel
Accumulation of reactive oxygen species (NADPH oxidase - subunit gp91 or NOX2, and others) oxidative stress
Accumulation of nitric species (iNOS) nitrosative stress
Antimicrobial enzymes : cathelicidins, defensins, acid hydrolases, which are secreted into lumen of phagosome.
Low nutrients: no sugars, no micronutrients (Fe, Cu, Zn, etc), which are pumped out of the phagosome or sequestered (lactoferrin).
Phagosome maturation to phagolysosome takes 30-60 min.
What’s nutritional immunity?
When for example the phagasome pumps out iron which the microbe needs to live for nutrition
How is the phagosome adapted in dendritic cells?
To perform antigen presentation, to degrade antigens and load them to bridge adaptive immunity
In neutrophils due to their specific granules?
The phagosome is basic (pH = 8 for about 30 min) to facilitate optimal activity of elastase and catches-in G
Extracellular killing?
Eosinophils (toxic granules):
They release mitochondrial DNA against bacteria
They also release granules are active against parasites, such as: major basic protein (MBP) eosinophil cationic protein (ECP) eosinophil peroxidase (EPX) eosinophil-derived neurotoxin (EDN)
Neutrophil extracellular traps (NETS) are also used catch microbes in sticky DNA fibrils
When killing a microbe is not possible what can happen?
Granuloma can contain microbes
Coordinated response of TH1 T-cells and macrophages which creates a layer of macrophages and outer layer of T cells which walls off the pathogen
Important in tuberculosis and in certain parasite infections
Means we can co live with the parasite
What organ is first to encounter blood after the gut?
The liver, gets rid of bad bacteria
This is done by liver macrophages called Kupffer cells using CRig functions as a pattern recognition receptor on Kupffer cells to directly bind gram positive bacteria via LTA, CRig-LTA is required for clearance of circulating bacteria
Example of regulation in the immune system?
Communication between all cells of the immune system
Phagocytosis occurs simultaneously with release of cytokines and chemokines
Complement deposition triggers phagocytosis and recruitment of other immune cells
Cytokines made by macrophages and dendritic cells trigger
recruitment of neutrophils and acute-phase response
Specific targeting and feedback mechanisms of the immune system?
Different PRR and cytokines signal integration adaptation of responses to each pathogen
Each microbe activates a set of PRRs different response based on ratio of signals allows a targeting of the response to pathogen and to tissue
Feedback mechanisms
Once other immune cells migrate into the tissue the cytokine and chemokine milieu modulates function of innate immune cells differential activation of macrophages and monocytes ( and perhaps other cells)
How is host damage regulated/prevented?
If its chronic can cause a degenerative or auto immune disease
This is prevented by:
Host cells have mechanisms to prevent activation of innate immune killing
Intracellular signalling cascades triggered by PAMPS and DAMPS direct the magnitude and nature of the immune response in a tissue specific manner
What’s the danger theory?
posited that the primary function of the immune system is to sense and respond to danger in the form of signals (ATP, adenosine) derived from damaged tissue, regardless of the source of damage. Danger-Associated Molecular Patterns (DAMPS)
What is pattern recognition theory?
posited that lymphocyte activation is controlled by evolutionarily ancient system of germline-encoded pattern-recognition receptors (PRR) that detected conserved pathogen-associated molecular patterns (PAMPS) - predicted that PRR-PAMP interactions would induce costimulatory molecules on APCs.
When the immune system is de regulated?
: CYTOKINE STORMS, IMMUNE-MEDIATED DAMAGE, ALERGY, FIBROSIS
Overview of immune memory?
The adaptive immune system consist of T and B lymphocytes
The T lymphocytes and immunoglobulins, and antibodies that B-lymphocytes produce can recognise an unlimited number of different targets
Become effective only after a delay of 2 to 4 days on first encounter
Lymphocytes and lymphocyte products specific for a given organism then persist as immune memory and are rapidly protective on re-exposure to the same infectious agent
This property of the adaptive immune system forms the basis of the protective effects of vaccination
What does vaccination do?
The ability of the body to remember an infectious agent to
which it has been exposed is the basis for natural and artificial
immunity against a disease
Vaccination initiates a primary immune response, generating
memory cells without making the person ill
Later, if the same or very similar pathogens invade the body,
specific memory cells already exist
They recognise the agent and produce massive number of
lymphocytes and immunoglobulins that overwhelm the invaders
Specific functions of each cell in adaptive immunity?
Adaptive immune responses are mediated by lymphocytes of the lymphoid lineage which fall into two major classes:
T lymphocytes - kill virus-infected and cancerous cells (CD8+ T cells/cytotoxic T lymphocytes or CTLs
or, activate other cells of the immune system (CD4+ helper T cells)
B lymphocytes - secrete immunoglobulins
Natural killer cells -capable of killing virus-infected or cancer cells immediately
without prior activation and without clonal expansion
They are generally regarded as part of the innate immune system
(because they don’t require prior activation)
Activation occurs via binding of activating receptors to surface molecules produced by cell damage e.g. cancer or that are encoded by infecting viruses
Definition of an antigen?
Any molecules or parts of molecules recognised by the variable antigen receptors of lymphocytes are known as antigens
Describe lymphoid lineage - cellular differentiation?
Mature lymphocytes that have not yet
encountered antigen are known as naïve
lymphocytes
Differentiation into effector cells is stimulated
by encounter with antigen and is preceded with
a vigorous proliferation that selectively expands
the numbers of those lymphocytes with receptors
specific for the inducing antigen
This is known as clonal selection of antigen-
specific lymphocytes
Describe the lymphatic system?
The critical interactions between
activated dendritic cells and
lymphocytes occurs in lymphoid tissues
Lymphocytes enter lymphoid tissues from the bloodstream Return to the bloodstream through the lymph if they are not activated Migration through lymphoid tissues, and cellular interactions, are guided and controlled by adhesive cell surface molecules whose expression is regulated by cytokines and chemokines
How do T lymphocytes recognise antigens?
T lymphocytes recognise antigens by means of T cell receptors generated during their differentiation - they interact with APCs (antigen- presenting cells - DC, macrophages and B cells) via complementary ligands
Each individual lymphocyte expresses
an antigen receptor of only one specificity,
but the total population of lymphocytes
in any given individual is collectively
capable of recognising virtually any antigen
What does CD stand for?
Clusters of differentiation
Features of the T cells CD4?
Marker of helper T cells
Is a co receptor for MHC call II molecules
These co-receptors play an important part in signal
tranduction on antigen binding by T cells
Features of CD8 T cells?
Marker of cytotoxic T cells
Co receptor for MHC call I molecules
These co-receptors play an important part in signal
tranduction on antigen binding by T cells
Features of dendritic cells?
Dendritic cells of the innate immune system play a crucial role in activating adaptive immunity
Begin life as phagocytes but when activated by conserved components of micro-organisms, or by inflammatory cytokines released by macrophages, become dedicated to displaying components of ingested micro-organisms for recognition by naïve T lymphocytes and subsequent differentiation of naïve T lymphocytes into effector T cells
Especially numerous in epithelia and at mucosal surfaces
Langerhans cells first recognised in the skin in the 19th century. They are a
subset of dendritic cells that reside in the keratinised epidermis for several months
Second population of skin-resident dendritic cells is found in the dermal layer - known as dermal dendritic cells or interstitial dendritic cells In mucosal epithelium of the gut, dendritic cells are concentrated at specialised sites of antigen collection that overlie lymphoid tissues Some have specialised surface properties that enable them to extend their long dendritic processes between the cells of the epithelium and into the lumen to sample antigens These cells are directly exposed to ingested antigens and to commensal bacteria as well as invading pathogens These cells have a specialised role in preventing inflammatory responses to harmless gut residents
M cells do what?
deliver antigen from the lumen of the gut to the underlying tissue where dendritic cells cluster
Describe how the dendritic cells activates the adaptive immune system?
A dendritic cell displays a component of a
micro-organism for recognition by a T lymphocyte
bearing a receptor for that component. This
stimulates it to proliferate and differentiate into
an effector cell. When the effector cell recognises
the same antigen displayed on the surface of a
B lymphocyte, the T cell activates the B lymphocyte,
which in turn proliferates and secretes antibodies
that recognise the micro-organism
Describe T cell targeting?
Unlike B cells whose surface immunoglobulin molecules are adapted to serve
as antigen receptors, T cells are unable to interact directly with
intact antigen and so must interact with other cells of the immune system
T cells are focused on their target cells by cell surface proteins known as
MHC molecules because they are encoded in the major histocompatibility
complex (MHC)
MHC molecules carry fragments of antigen, usually peptide fragments of
proteins, from internal compartments of the target cell to the cell surface
and display them for recognition by the T cell
All naïve T cells are activated by antigen fragments displayed on MHC
molecules on the surface of dendritic cells, and after differentiation into
effector cells they are triggered to kill or activate cells displaying the same
complex of MHC and antigen
Whats the Major histocompatibility complex?
Chromosomal region containing genes that encode cell surface proteins
that regulate interactions among cells of the immune system
During transplant the more similar they are the longer it will take for the body to reject the new organ
There is class I molecules (Activate CD8 T cell) and class II molecules (Activate CD4 T cell)
These monitor different internal compartments of cells and are recognised bu distinct classes of T cells
2 internal compartments of cells that need to be monitored for pathogens?
The cytoplasm
All viruses and some important bacterial pathogens replicate
in the cytoplasm
Vesicles of the endosomal/lysosomal pathway
Contain internalised antigens derived from extracellular pathogens
In the specialised case of macrophages, vesicles may harbour bacteria
and some fungi (e.g. Cryptococcus) adapted to survive phagocytosis
The cytoplasm is monitored by MHC class I molecules
Vesicular compartments are monitored by MHC class II molecules
Features of MHC class I molecules?
MHC class I molecules are expressed on all nucleated cells
(not RBC) including APCs and bind peptides generated by cytoplasmic
proteases from cytoplasmic proteins
Their critical role is to display antigens derived from pathogens that
replicate in the cytoplasm, and antigen displayed on MHC class I
molecules is recognised by cytotoxic T cells, which kill the infected
cells
This system ensures the destruction of cells in which cytoplasmic
pathogens are replicating and is believed to be essential for the
elimination of some of these pathogens
What’s a cytotoxic T lymphocyte?
A specialised white blood cell responsible for eliminating unwanted body cells (eg. cancer cells) for example killing a cell infected with influenza virus
Describe recognition of antigen and MHC class I molecules by cytotoxic T cells?
Peptide fragments of pathogens, such as viruses, that replicate in the cytoplasm compartments of cells are carried to the cell surface by MHC class I molecules and are recognised by cytotoxic T cells forming an immune-synapse, outer ring is an adhesion ring and the inner is a signalling zone and secretory zone
Upon activation, CD8 T cells produce:
- Perforins - proteins that form pores
in the membrane of target cells - Granzymes - proteases that induce
programmed cell death on entry into
target cells
Describe MHC class II molecules?
MHC class II molecules are normally expressed only on cells of the
immune system, and in particular on professional APCs (B cells,
macrophages and dendritic cells)
They bind to peptides generated from internalised antigen in the
endosomal compartments of cells
Antigen derived from internalised micro-organisms by lysosomal
proteases and displayed by MHC class II molecules on dendritic
cells is recognised by naïve T helper cells at the initiation of immune
responses
MHC class II processing?
The 2 chains of it form in the endoplasmic reticulum
An invariant chains binds to the peptide grove formed by the 2 chains
Preventing unwanted peptides in the ER from binding
The invariant chains takes it through the ER and Golgi apparatus via a vesicle
Proteases are released inside the vesicle cleaving the invariant chain in 2 places leaving the CLIP peptide bound to the MHC class II molecule
Engulfed proteins or pathogens that are degaded in the vesicle can’t mind on to MHC class II as it’s blocked by CLIP
HLADM allows the CLIP to be removed, its present in the vesicle and acts as a catalyst
The pathogen peptides can now bind and the vesicle is taken to the cell surface to present as as an antigen
Naive helper T cells are first activated when?
They recognise antigen bound by MHC class II molecules on the surface of dendritic cells
Recognition of antigen and MHC class II molecules on macrophages by helper T cells?
Helper T cells recognise fragments of a pathogen that has been internalised by macrophages and have undergone proteolytic digestion to generate fragments that are carried to the cell surface by MHC class II molecules.
The helper cell is then stimulated to
activate the macrophage to destroy
the internalised pathogen.
This is particularly important for defense
against bacteria that are able to grow
in the internal vesicles of macrophages
Helper T cell tells the macrophage via cytokines that what its doing is correct
Recognition of antigen and MHC class II molecules on B cells by helper T cells?
B cells internalise antigens bound to their surface immunoglobulin and deliver them to the endosomal compartments of the cell where they are digested into fragments. The antigen fragments then bind to MHC class II molecules which carry them to the cell surface where they are recognised by helper T cells, which then activate the B cell to proliferate and differentiate into an antibody- secreting plasma cell
What is CD4 or CD8?
Coreceptor from Tcell receptor binding to MHC
CD4+ T cell subsets?
There is TH1 and TH2 which release different cytokines which have different effects (picture in folder)
What do co-stimulator pairs do?
Enhance and sustain signal delivered by the MHC-antigen T cell receptor interaction (photo in folder)
Co-stimulator pair activation?
Professional antigen presenting cells present either MHC moelcules and co stimulator molecules such as CD80(B7.1)
When a complex is formed with the T cell signals are sent both ways, it tells the antigen presenting cell to produce more co stimulator pairs, and the T cell is induced to form pairs for the new co-stimulator pairs which can then form ligand making the connection stronger with the T cell
This then stimulates another co stimulator that is required when bound for full activation of the T cell
Summary of the immune response?
Pathogens manages to get through first layer of skin (through epidermis into dermis)
The bacteria establish an infection here
Phagocytic cells such as neutrophils and macrophages engulf the pathogen
Dendritic cells engulf as well and are stimulated by binding pathogens to leave the site of infection and migrate to a lmpyh vessel where the are drained into a lymph node
In the lymph node T cells are activated by the antigen presented on the dendritic cells and in turn activate B cells to release antibody
Effector T cells and antibody return to circulation
They return to the site of infection as inflammatory mediators have caused changes in the blood vessel endothelium
CD4 T cells activate macrophages to become more cytotoxic
antibody recruits complements of lysed bacteria directly and to opsonise them by inducing there uptake by phagocytes
In the case of a virus activated CD8 cells would kill any infected cells present
What does opsonise mean?
More susceptible to uptake by phagocytosis
What are antibody molecules?
Host proteins produced in response to the presence
of foreign molecules in the body
Synthesised primarily by plasma cells (lymphoid lineage)
and are components of the adaptive immune system
Circulate throughout the blood and lymph where
they bind to foreign antigens
Antibody-antigen complexes are removed from
circulation primarily through phagocytosis by
macrophages
Describe clonal selection of antigen specific lymphocytes?
The lymphocytes of any individual collectively express a large number of different receptors
Some cells bear receptors that recognise self-antigens. While still immature, they are eliminated
This process of clonal deletion is a critical mechanism for ensuring self-tolerance
Once mature, lymphocytes activated by recognition of foreign antigen differentiate into effector or memory cells through a process of clonal expansion
This is the mechanism for antigen-specific immune responses and immune memory
Receptor editing and clonal deletion of developing B cells?
The mechanism through which
self-tolerance is established differs between T cells and B cells
An immature B cell in the bone
marrow expresses membrane Ig antibody
together with Iga and Igb chains as the B cell receptor
If the BCR in is engaged by self antigens present in its local environment, maturation of the B cell is arrested and it stays in the bone marrow
Re-arrangement and recombination of genes encoding the BCR results in a new BCR possessing the original heavy chain but new light chain
This process is known as receptor editing
If the B cell is no longer auto-reactive, then it is exported to the periphery
If this BCR is still auto-reactive, then it can try again for a limited time before dying (clonal deletion)
Induction of B cell antibody response by T cells?
Germinal centres where B cells proliferate and undergo isotope switching hyper mutation
Germinal centres form from dendritic cells having antibody on their surface activating antigen specific CD4 T cells which proliferate into effector cells capable of activating antigen specific B cells
Once activated it proliferates to form a primary focus for antigen specific B cells
B cells from the primary focus migrate to nearby follicles whilst other B cells stay in the local area secreting antibody
B cells that enter to follicle proceed to proliferate rapidly under this time they also undergo somatic mutation to introduce new variation into the B cell receptor
The receptors are tested with their ability to bind to antigen those that fail to compete die
Antibody structure
Large family of glycoproteins
Share key structural and functional features
Functional - able to bind both to antigens and to
specialised cells or proteins of the immune system
Structural - composed of one or more copies of a
characteristic unit that form a Y shape
So any molecules or parts of molecules recognised by the variable antigen receptors of lymphocytes are known as antigens
Whats the epitope (antigenic determinant)?
The specific region of the antigen bound by the variable region of an immunoglobulin
What’s the paratope?
The antigen binding region of an antibody
What’s affinity?
Affinity is the measure of the strength of the binding of an
antigen by an antibody
Antibody binding to antigen is non-covalent and reversible
The affinity of an antibody (Ab) for an antigen (Ag) is related
to the ratio of the rates of the forward reaction for formation
of the complex to back reaction for decay of the complex
Like a reversible reaction in chemistry A level
Papain cleavage on antibody?
Fab 1
Fab 11
Intact Fc 111
pepsin cleavage on antibody?
F(ab)2
Fc fragments
Antibody structure - the Y shape?
Heavy chain is top part of arm of Y
Light chain is lower part of arm of Y
Each Y contains four polypeptides
Two identical heavy chains
(55kDa each)
Two identical light chains
(22kDa each)
The four polypeptide
chains are held together
by disulphide bridges
and non-covalent bonds
The two domains that carry the antigen binding sites are known as Fab fragments (fragment having the antigen binding site) (the 2 arms) (antigen binds at the end of the arm)
The third domain is known as the Fc fragment (the fragment that crystallizes) (the base)
Hinge region in the middle of the arms aids movement
The ability to bind antigen is determined by the N-terminal domains. These differ considerably between antibodies of differing specificities and are therefore termed ‘variable’ or ‘V’ domains - the end bits of each of the arms
Top part of the base of the Y is where complement binding happens
Bottom bit of the base of the Y is where binding to Fc receptors happens
What is hyper variability?
VH is top bit of the top arm of the Y, VL is the bottom bit of the top of the Y arm Sequence analysis of amino acids of VH and VL regions reveals small regions of hyper-variability within four conserved framework regions (FR1, FR2, FR3 and FR4)
In the 3-dimensional structure, the
hyper-variable regions form loops
that combine together to form the
principal antigen-binding surfaces
These surfaces are called the
complementarity determining regions
or CDRs (CDR1, CDR2 and CDR3)
Classes of immunoglobulin (different name for an antibody)
There are five main classes of antibody in serum
IgG, IgM, IgA, IgE and IgD
The five classes are divided on the basis of the
number of Y-like units and the type of heavy chain
polypeptide they contain (y, u, a, e and delta ) Greek symbols and associate same order as ones above
While there are five different types of heavy chains,
there are only two light chains (k or L) Greek again
One light chain always associates with one heavy
chain, so the number of light chains always equals
the number of heavy chains
There’s 2 heavy chains and 2 light chains per antibody
antibody in humans?
In humans, the IgG class has four sub-classes (G = gamma)
IgG1, IgG2, IgG3 and IgG4
These have y1, y2, y3 and y4 heavy chains
The IgA class has two sub-classes
IgA1 and IgA2
These have a1 and a2 chains respectively
What’s a J chain?
Holds more than 1 antibody together
What’s a secretory component?
In double antibodys
Movement of antibodies across mucosal tissues with secretory IgA?
IgA is the most prevalent immunoglobulin in mucosal
secretions
Binds to antigens and prevents the uptake of bacteria and
toxins
Epithelial cells in the intestine mediate the transfer of
dimeric serum IgA to the intestinal lumen via the polymeric
Ig receptor
IgA dimer with J chain attached binds
to the polymeric Ig receptor, a transmembrane
protein expressed at the basal surface of
epithelial cells of the gut, airways and various
secretory glands
Binding of IgA induces transcytosis of the polymeric Ig receptor Complex is delivered to the apical surface of the epithelial cell and into the lumen Proteases cleave the pIgR near the membrane, releasing the majority of the extracellular domain still bound to IgA dimer The ‘secretory component’ protects IgA from proteases present in mucus and anchors IgA at the desired location
After opsonisation with IgA, the receptor Fca/mR mediates
the uptake of organisms by macrophages and dendritic cells
A second receptor FcaRI mediates uptake by neutrophils
IgA deficiency is a common immunodeficiency in humans
IgM can compensate for IgA deficiency by also binding to pIgR
IgM opsonised organisms are phagocytosed via Fca/uR
Activating Fc receptors signal through ITAMs
What are ITAMS?
Antigen receptors, Fc receptors and some of the activating receptors of
natural killer cells signal via a common mechanism that is dependent on a conserved amino-acid sequence motif
This is known as the immunoreceptor tyrosine-based activation motif
The ITAM contains two precisely spaced tyrosines within a consensus sequence
When phosphorylated, the tyrosine residues provide a binding site for
one or two closely related intracellular tyrosine kinases
Syk in most immune cells ZAP-70 in T cells
These have tandem SH2 (Src Homology 2) domains spaced at exactly the right distance apart to dock onto the two phosphotyrosines, and that activate signaling events downstream of the receptor
Signal transduction by ITAM-containing immunorecetpors?
The surface immunoglobulin (Ig)
that serves as the B cell receptor for antigen
brings the receptor into proximity with a Src-
family tyrosine kinase, which is held in the
plasma membrane by a lipid tail and phosphorylates
ITAM tyrosines in the receptor Iga and IgB chains
This creates a binding site for Syk, which binds to
the doubly phosphorylated ITAM sequence
Once bound, Syk becomes activated by tyrosine
phosphorylation and it then phosphorylates
other signaling proteins that ultimately bring about
changes in the behaviour of the cell e.g. antibody
production
Antibody-mediated cytotoxicity?
The two most widely recognised mechanisms of antibody-
mediated toxicity are:
- Antibody-dependent cell-mediated cytotoxicity (ADCC)
Antibody attracts cytotoxic cells by means of their Fc receptors
- Complement-mediated cytotoxicity (CMC)
Antibody binding results in the fixation of complement onto the target cell
Opsonisation and phagocytosis process?
It requires triggering of specific receptors on the phagocyte
Fc receptors that bind antibody coated bacteria are one of the receptors capable of triggering phagocytosis engulfing it and forming a phagosome
Lysosomes move in to degrade the content in the phagosome
What is complement?
The complement system, also known as complement, consists
of about 30 serum and membrane proteins that can mediate a
variety of immune reactions including:
Triggering inflammatory responses
Attraction of phagocytes to sites of inflammation
Degradation of membranes or virus envelopes
Stimulation of antibody production
Activation of complement?
The active components of complement are generated from
inactive precursors by a cascade of proteolytic reactions
These are triggered through 3 different pathways:
The LECTIN pathway (recognition of carbohydrate
moieties e.g. by collectins such as mannose-binding
lectin)
- The CLASSICAL pathway (antibody binding to antigen in
immune complexes)
The ALTERNATIVE pathway (directly at microbial cell
surfaces)
Activation of complement by any one of the pathways
initiates a cascade of cleavages in which each component
in the pathway is cleaved into two fragments:
The small fragment (a fragment)
The big fragment (b fragment)
The b fragment forms a subunit of the protease complex
mediating the next cleavage in the pathway
The reactions can be divided into early events, in which the
components are not the same for the same three pathways
and late events, in which they are identical
Early and late events of complement formation?
The function of the early events is to generate two functionally
equivalent forms of a protease, known as C3 convertase
C3 convertase then initiates the late events to produce the
effector components of complement
C3 convertase covalently attaches to the cell surface at
which the complement activation was initiated
Cleavage of complement component C3 leads to effector
activation
In this way, the effects of complement activation are confined
to the infectious organism that triggered
Pentraxins =?
Pentraxins = Pentameric serum
proteins that participate in innate
immunity and can also activate the
Classical pathway of complement
Antibody binds, C1r is activated by C1q C1r then cleaves and activates C1s
C1s cleaves C4 C4b attaches to cell surface C2 binds to C2 binds to C4b
C1s cleaves C2 bound to C4b producing C3 convert
C4bC2n and C3bBb conertases cleave C3 which provides C3a and C3b which are the effector actions, is an amplification loop for the alternative pathway as well
What are collections and ficolins?
The epithelium of the respiratory tract is lubricated by a
layer of phospholipids and proteins known as surfactants
Two of these, Surfactant Protein-A (SP-A) and Surfactant Protein-D (SP-D) are Collectins and function as opsonins, coating microorganisms (e.g. Pneumocystis in AIDs patients - Lecture 8) and stimulating uptake by phagocytes
The Collectin family also includes Mannose-Binding Lectin (MBL) - binds to mannose-containing carbohydrates on surface of viruses, bacteria and fungi Ficolins bind to N-acetylglucosamine which are polysaccharides outside of microorganisms
Each polypeptide chain of the collectin family of
proteins consists of an amino-terminal cysteine-rich region followed by a collagen-like region, an -helical neck region and a carboxy-terminal globular domain
(a C-type lectin known as the carbohydrate recognition domain [CRD])
The polypeptide chains trimerise Both Mannose Binding Lectin and Surfuctant Protein-A are hexameric structures. The multiple arrays of CRDs give high affinity binding to polysaccharide ligands in microbial cell walls Particle recognition is translated into immune action: 1. By phagocytes, which are stimulated to internalise bound microorganisms or, 2. By conformational changes in the collectin that activate pre-bound protease subunits and initiate the complement cascade
Lectin pathway?
MASP = Mannose binding lectin associated serine protease
MASp-2 cleaves C4
C4b attaches to cell surface
C2 binds to C4b
MASP-2 cleaves C2 bound to C4b
Forming C3 convertase
C4bC2n and C3bBb conertases cleave C3 which provides C3a and C3b which are the effector actions, amplifies alternative pathway
Alternative pathway?
C3b binds to microbial surface
B binds to C3b
D cleaves B bound to C3b forming C3 converts
C4bC2n and C3bBb conertases cleave C3 which provides C3a and C3b which are the effector actions
Amplifies alternative pathway
C3a, C5a functions?
peptide mediators of inflammation, phagocyte recruitment
C3b function?
Binds to complement receptors on phagocytes
Opsonization of pathogens
Removal of immune complexes
Can also results in terminal complement components C5b, C6, C7, C8, C9, which aid the membrane attack complex, lysis of certain pathogens and cells
C3b + C3 convertase → C5 convertase
C5 convertase + C5 → C5a + C5b
C5b + C6 + C7 + C8 + C9 ⇢ MAC
What is hypersensitivity?
Undesirable reactions produced by the ‘normal’ immune response
Can be an allergy or auto-immunity
allergy = reacting to something not normally in the body
Auto-immunity - reacting to stuff in our body
Different types of hypersensitivity reactions?
Type-I - IgE Causes degranulation of mast cells Allergy Asthma
Type II - IgG Can recruit complement and attack things such as RBC Autoimmune haemolytic anemia
Type III - IgG Binds to Fc receptor and recruits macrophages and neutrophils to the cell Rheumatoid arthritis
Type -IV Doesn't use antibodies Cellular effectors Multiple sclerosis
Old system they are all mixed up in fact
Allergy =?
• Excessive immune responses to harmless antigens (allergens)
- Most allergic reactions are due to IgE-mediated responses
• ~40% of adults show an allergic response to least one environmental allergen:
Atopy: individuals that have a predisposition to become IgE-sensitized to allergens
No major common characteristics between allergens has been identified!
• Airborne allergens – small, soluble proteins (some with protease activity)
Types of allergies?
Anaphylaxis Acute urticaria Allergic rhinitis Asthma Food allergy
What’s asthma?
Airway hypersensitivity
Lung epithelium inflammation, smooth muscle contraction
Cytokine environment orchestrated by what is crucial?
Which CD4+ Th subset it is
In allergy think its highly a Th2 mediated response can be termed Type 2 immunity
Th2 response leads to?
IgE production that is specific to the allergen after CD4+ Th2 has stimulated a B cell
Why can dendritic cells sample luminal antigens through the epithelial cells?
Can express tight junction proteins allowing interaction with epithelium
Epithelial cells can secrete factors that trigger DC activation:
Instructive cytokines
DAMPS - damage associated molecular pattern
Effector mechanisms in IgE mediated allergy?
Mast cells: abundant in mucosal tissues (lung, gut)
and connective tissues (e.g. surrounding blood vessels)
Th2 tells B cell via Type 2 cytokines (IL - 4, IL - 5, IL - 13)to class switch IgE antibody, which forms plasma cells which take the antibody to the Fc receptors on mast cells that causes degranulation when stimulated by the allergen
Most IgE is cell bound (on FcεR) not in body fluids (unlike other antibodies)
Mast cell granules?
Histamines • Proteases • Enzymes • Cytokines • Prostaglandins • Leukotrienes
In intestines causes fluid secretion and peristalsis that can case GI contents expulsion through diarrhoea or vomiting
Can decrease airway size increasing mucus
Increase blood flow and permeability - causing anaphylactic shock
How are eosinophils different to mast cells?
Circulating rather than in tissue
Originate from bone marrow progenitors and are continually circulating
In health (% of circulating leukocytes):
Eosinophils ~ 2-6%
Basophils ~ 0.4% (very rare!)
Recruited to tissues via chemokines to sites of inflammation…
Activated by type-2 cytokines (especially IL-5), complement and antibodies
Release enzymes, toxic proteins, cytokines chemokines and leukotrienes
Characteristic feature of allergic disease (yet role is unclear)
Time frame from these responses?
PEFR = peak expiratory flow rate = lung function
30 minutes huge dip in function due to mast cells but returns to normal in another 30 minutes
In late phase will dip not as steep around 8 hours
Allergy can lead to airway tissue remodelling which is mediated by which events happening?
Too much mucus made:
Goblet cell hyperplasia
Airway smooth muscle:
Hyperplasia and hypertrophy
ECM deposition:
deposition of collagen
Treating symptoms?
Antihistamines
Target H1 receptor
Adrenaline
Anaphylactic reactions, e.g. reforms endothelial tight junctions and relaxes smooth muscle.
Inhaled bronchodilators
Act on β-adrenergic receptors to relax constricted muscle
Immunosuppressive drugs (general anti-inflammatories) – corticosteroids Suppress immune inflammation (can be given via inhalation)
Developing therapie?
Antibody therapies:
Omalizumab (anti-IgE)
Reduces asthma symptoms;
• Allows for a reduction in inhaled corticosteroid (ICS) dose
Mepolizumab & Reslizumab (anti-IL-5 antibodies)
• Some evidence of efficacy, but only with patients where eosinophils are dominant (prednisone-dependent eosinophilic asthma).
Duplimab (anti-IL-4Rα – receptor for IL-4 and IL-13)
• Effective at controlling asthma with reduction in corticosteroid dose in mild to severe asthma patients that have high eosinophil blood count.
Low dose allergen therapies (allergen de-sensitization):
• Injection of low doses of allergen (that escalate).
• Mechanism unclear, though likely to involve Tregs and skewing toward IgG.
Hygiene hypothesis?
Autoimmune disorders high in Western culture
Much cleaner
Could be due to microbiota
The innate system recognises molecules that are?
Common to the pathogen is this lecture it is bacteria
Pathogen associated Molecular patterns (PAMPS)
The innate system is pre coded to recognise a limited repertoire of molecules
This enables it to respond quickly, but to provide a low level response
It kills by complement and interactions with phagocytes
Overview of the 3 pathways of complement activation again?
In all pathways the first step is the recognition of microbial components:
Alternative pathway:
Recognised molecule - Spontaneous activation but various cell surface molecules play a role
Classical pathway:
Recognised molecule - Bound IgM or IgG, so relies on antibodies and is far more effective in individuals who have been previously exposed
Lectin pathway:
Recognised molecule - Carbohydrates such as mannose or N-acetyl glucosamine
Early events of complement pathways results in?
C3 converts
C3 converts covalently attaches to the cell surface at which the complement activation was initiated
Turns into C3a (Inflammation - binding to receptors on mast cells, causing them to release histamine) and C3b (opsonization and phagocytosis)
In this way, the effects of complement activation are confined to the infectious organism
After recognition - late events of complement pathways?
C3B forms part of the C5b convertase helping to later form the membrane attack complex which causes lysis of the microbe
C5b also produces C5a which causes inflammation
Describe the membrane attack complex?
The Gram-positive cell wall tends to be thicker and more robust than the Gram negative cell wall
The MAC is much more active towards Gram-negative than gram positive bacteria
Ion influx and efflux causes the cell to lyse
How do we know complement is needed to kill bacteria?
Can see that deficiency in being able to produce certain types of complement results in not being Protected against certain types of bacteria
Indirect recognition of bacteria by phagocytes?
bacteria coated with antibodies are recognised by antibody receptors (Fc receptors)
Bactiera coated with C3b are recognised by C3b receptors
Direct recognition of pathogen associated molecular patterns?
PAMPS are recognised Pattern recognition receptors such as Toll-like receptor
Structure of a toll like receptor?
Extracellular domain - ligand recognition
Intracellular domain - signalling
(dimerisation is essential for signalling) so TL1/2 would be a heterodimer and TL5/5 would be homodimer
Consequences of TLR recognition by phagocytes?
Activation of phagocytes which leads to:
Cytokine and chemokine production
Ingestion and killing
TLR signalling and inflammatory responses?
Recognition of bacteria molecule by TLR
Dimerisation of the TLR
Signalling and inflammatory responses
The adaptive system does what to bacteria?
Recognises antigens that are unique to an individual pathogen
It takes time to develop and mature but can provide a very powerful protective response
Normally recognises proteins or polysaccharides
MHC presentational pathways?
Protein within the cytosol:
Processed by the proteasome generates peptides
Go to endoplasmic reticulum where become processed with MHC class 1 to form a complex which is recognises by CD8+ T cells
Activated CD8 T cels kill host cells infected with bacteria by releasing cytotoxins such as perforin, granzymes and granulysin
Protein taken up into the phagosome: Endocytosis of extracellular protein in it's vesicle it forms a complex with with MHC class 11 from the ER, this complex is presented on the cell surface and recognised by CD4+ T cells
CD4+ T cells releases different cytokines that result in inflammation, macrophage activation, antibody response
Bacterial diseases where CD8+ T cells play a role in protection because bacteria grow within host cells?
Tuberculosis
Salmonellosis
listeriosis
meliondosis
What’s agglutination?
Clumps the bacteria so cleared better by phagocytes
Its triggered by antibodies
What’s complement binding?
Antibodies bound to bacteria promote complement binding and complement activation by the classical pathway
Complement can now effect them
What’s opsonisation?
Antibodies can make it easier for phagocytes to uptake bacteria
Can antibodies neutralise toxins?
yes
Overview of viruses?
Responsible for the commonest recurring infections of humans
Although usually transient, they have massive socio-economic consequences
Viruses that have ‘jumped’ species barriers (zoonotic) generally cause substantially greater mortality than endemic viruses that have adapted over greater evolutionary time to their hosts
Human Immunodeficiency virus from chimpanzees in Africa
Ebolaviruses from bats (via bushmeat e.g. duikers) in Africa
Severe Acute Respiratory Syndrome (SARS) SARS-CoV-1 coronavirus from horseshoe bats (via civets?) in China
Middle East Respiratory Syndrome (MERS) MERS-CoV coronavirus from camels in Middle East
Coronavirus disease 2019 (COVID-19) SARS-CoV-2 coronavirus from bats (via pangolins?) in China
Epidemics are?
Epidemic: classification of a disease that appears as new cases in a given human population, during a given period, at
a rate that substantially exceeds what is ‘expected’, based on recent evidence
An epidemic may be restricted to one locale (an outbreak), more general (an epidemic) or even global (pandemic)
Pandemic: an epidemic that spreads through human populations across a large region e.g. a continent, or even worldwide
Viral diversity?
Exist as obligate intracellular pathogens - dependent on host proteins
for replication
May contain as few as 3 to 4 to up to several hundred genes expressed
from single- or double-stranded RNA or DNA genomes
Genomes are enclosed within capsids (coat proteins)
Cytopathic (lytic) viruses e.g. poliovirus and influenza virus, lyse host
cells by inducing apoptosis or autophagy
Some cytopathic viruses e.g. herpes viruses can infect cells without
producing infectious virions until immunity wanes or until re-activated
by various physiological signals - known as latent viruses
Non-cytopathic viruses e.g. hepatitis B replicate without destruction of
cells
GP encodes?
GP encodes Ebola virus glycoprotein (GP). Two forms:
- Trimeric trans-membrane complex targets virus to
cells via receptor binding - allows virus to introduce
its contents into monocytes/macrophages leading to
inflammatory cytokine dysregulation - Soluble dimeric form (sGP) secreted after infection –
prevents neutrophil activation
What’s a cytokine storm from ebola?
Immune cells get caught in endless loops releasing extreme levels of cytokines - proteins within cells which cause inflammation and attracting yet more immune cells
What’s septic shock from ebola?
Infected cells detach from blood vessels causing massive haemorrhage. Loss of blood leads to kidney and liver failure
Virus breaking and entering?
Like bacteria, viruses must overcome epithelial barriers
of skin and mucosa to establish infection
Intestinal viruses e.g. poliovirus enter via antigen-sampling
M cells
Respiratory viruses e.g. influenza virus and rhinoviruses
establish infection in the epithelial cells of the airways
Some viruses e.g. Zika, Dengue and West Nile viruses require
insect vectors for transmission
Bloodborne viruses e.g. HIV and hepatitis B can invade
through mucosa or epithelia following physical trauma
Tissue specific receptors and viral tropism?
Viruses exploit specific molecules on cells as receptors for invasion
While some viruses bind widely expressed surface molecules, most viruses invade using receptors with restricted tissue expression patterns e.g. HIV
They are said to display TISSUE TROPISM
SARS-CoV-2 (COVID-19) accesses host cells via Spike Glycoprotein S binding to angiotensin-converting enzyme 2 (ACE2) on type II alveolar cells in lungs
Virus immunity?
Major innate immunity is mediated via type 1 interferons*, complement and natural killer (NK)
cells
Major adaptive immunity is mediated via antibody
and cytotoxic T lymphocytes (CTLs)
Interferons are Group of proteins that induce host cell enzymes that affect transcription and translation of viral genes
Adaptive immune response to acute virus infection?
Appearance of virus is followed
rapidly by a massive expansion of
virus-specific CD8 T cells
CD4 T cell expansion and contraction
also occurs but at a lower magnitude
Neutralising antibodies are produced
relatively early after acute infection
with cytolytic viruses
What do neutralising antibodies do?
Prevent viruses leaving and entering cells
Mechanisms of protective antibody responses to viruses?
The C1q/r/s complex couples
antibody binding to the
classical pathway of complement
Its structure is closely analogous to MBL and H-ficolin complexes with MASPs (lectin pathway)
Features of aids?
Acquired Immune Deficiency Syndrome (AIDS) is caused by
the Human Immunodeficiency Virus (HIV)
HIV infects and destroys CD4+ T cells
GRID (later called AIDs) first recognised clinically in US in
1980s in intravenous drug users and gay men with no
apparent cause for impaired immunity but who showed
symptoms of Pneumocystis carinii pneumonia (PCP)
~50,000 people infected/yr (USA) and 39+ million deaths
globally since start of pandemic in 1980s
2/3 of infected people live in Sub-Saharan Africa, 1/5th in Asia
Vast majority of human AIDS infections caused by HIV-1
Whats PCP
yeast like fungus pneumocystis jirovecii
Is opportunistic so found in healthy people lungs as well
Seen in people undergoing chemotherapy
History of HIV?
HIV-1 originated in southern Cameroon from a simian immunodeficiency virus (SIVcpz), a retrovirus that infects non-human primates e.g. Pan troglodytes (chimpanzee) in West-Central Africa
SIVcpz introduced into humans through bushmeat activities with
subsequent mutation into HIV – requires one or more high-risk
transmission channels for human-to-human spread
Channels absent in Africa prior to 20th Century but growth of large Colonial African cities led to societal changes such as prostitution (with increased frequency of STDs (genital ulcer diseases e.g. syphilis)) that allowed sexually transmitted infection
Earliest documented case of HIV in a human in Congo in 1959
In US as early as 1966
Important HIV glycoproteins?
gp 120 nicking glycoprotein
gp 41 transmembrane glycoprotein
HIV epithelial entry?
HIV-1 can cross mucosal surfaces through M cells - prevalent in tonsils and rectal epithelia
It can also gain access via epithelia damaged by ulcerative infections, or by trauma or injection (i.v. drug use or blood transfusion)
Vaginal epithelia lack M cells - HIV gains access via interdigitating processes on
Langerhans cells
Epithelial cells in the small intestine express
CCR5, a chemokine receptor
HIV-1 binds to CCR5 (once tethered by
galactosylceramide) via the viral envelope
glycoprotein gp120 and the virus is transcytosed
HIV transport to lymph nodes?
Once across mucosal epithelial barriers,
HIV-1 encounters sub-epithelial dendritic
cells
C-type lectin receptors (CLRs) on dendritic cells bind high-mannose N-linked
oligosaccharides on viral envelope
glycoprotein gp120
Interaction with CLRs initiates
dendritic cell migration to regional lymph
nodes
Once in lymph nodes, intact virus, internalised with CLRs, is re-exposed at the
surface and displayed to T cells
Regardless of the route of infection, the
predominant site of virus replication early
in disease is in the small intestine - this reflects the large number of activated T cells
that express CCR5 in that organ
HIV getting into the CD4 T cell?
Entry of HIV into cells mediated by viral envelope glycoprotein gp120
Binds to CD4 and
chemokine receptors
(CCR5 or CXCR4) on
host surfaces
Binding with receptor
leads to a conformational
change in gp120 that exposes the transmembrane component of the envelope protein gp41
gp41 mediates fusion of
the virus envelope with the host cell membrane
HIV infection cycle?
HIV infects cells via 2 cell surface molecules
CD4 is the primary receptor for the virus, whilst the chemokine receptors CCR5 or CXCR4 act as co receptors for the viral infection of macrophages and T cells
HIV binds initially to CD4 via the envelope like protein gp120, this interaction allows the uncoating of the virus and the entry of the nucleocapsid containing the viral genome
The viral reverse transcriptase copies the RNA genome of HIV into double stranded DNA which integrates into the chromosomal DNA of the host
In this state HIV is latent, it becomes transcripted when the host cell is activated this results in the accumulation of viral proteins as well as genome length transcripts of the virus
Viral proteins assemble at the cell membrane and bud off to form a new viral particle maturation occurs once its left the cell
Immunological consequences of HIV infection?
Dendritic cells translocate HIV from mucosa within 30 minutes
of infection
A wave of viral proliferation in lymph nodes peaks 4-7 days after
infection
Viremia peaks at 14 days and all lymphoid tissues are infected
by 3 weeks
HIV is quiescent in resting CD4 T cells, but on T cell activation,
virus production is also activated and the host cell dies
> 99% of virus is produced by newly infected CD4 T cells
~107-108 CD4 T cells are producing virus at any given time
Loss of helper activities (mainly through cytokine stimulation)
means that maintenance of cytotoxic CD8 T cells is lost as
is the antibody response of B cells
Infection typically results in flu-like illness from days to weeks
after exposure - associated with a drop of CD4 T cells in the blood
The CD4-dependent antiviral cytotoxic CD8 T cell response develops
and is responsible for a prolonged period of stable viremia
This is known as the VIRAL SET POINT
Patients are normally asymptomatic until the CD4 T cell counts decline
further - infection of developing lymphocytes in the bone marrow and
thymus results in failure to replace lost T cells
Opportunistic infections begin when CD4 T cell counts reach levels
<200/l
Death then typically ensues in about 2 years
Common one is crypto coccus meningitis kills 600,000 lots of them with aids each year
Overview of influenza virus?
There are 3 classes of influenza
viruses - A, B and C
Influenza A and B are major
causes of human disease
Only influenza A is responsible
for pandemic outbreaks
Influenza A viruses are avian
viruses that adapt to humans
The virus is spread by aerosol droplets and establishes cytolytic infections in the epithelial cells of the upper and lower respiratory tract
Features of influenza A?
Influenza A is a spherical or rod-
shaped enveloped virus covered
with 2 spike-like glycoproteins:
Trimeric hemagglutinin (HA)
Tetrameric neuraminidase (NA)
HA mediates binding to cell
surfaces and internalisation
NA cleaves sialic acid and
promotes viral release from
cells
Features of influenza genotypes?
Influenza genotypes are diverse in birds (migratory waterfowl) and express a
variety of HA (H1 to H15) and NA (N1 to N9) subtypes
Avian viruses prefer receptors, primarily on intestinal cells, that have the -2,3
sialic acid linkage to galactose
Human viruses prefer the -2,6 linkage expressed on respiratory epithelia
Pigs express both types of linkage on respiratory epithelial cells
Influenza viruses enter the human population in regions where humans, pigs
and waterfowl, as wells as domestic chickens, are in close proximity
Pigs can be infected with both human and avian influenza, and exchange
of segments between viral genomes in the pig can give rise to variants expressing novel surface proteins together with human-adapted virulence determinants
Immune response to infleunza virus infection?
Antibody mediated immunity:
Can bind to HA and block viral attachment
Can also bind to NA protein and prevent release of new virions
Cell mediated immunity: CD8 T cells recognise the infected cell due to its MHC class 1 the T cell then causes cell lysis
What’s antigenic shift and antigenic drift?
Epidemics occur when point mutations (red dots) accumulate in surface HA (ovals) or NA (diamonds)
This leads to ANTIGENIC DRIFT so
that, in many previously immune individuals, HA and NA are no longer recognised by neutralizing antibodies produced in response to earlier infections
Pandemics occur when avian influenza viruses and human influenza viruses infect pigs and re-assortment of the two genomes leads to the expression of avian HA or NA in a virus otherwise adapted to infect humans (ANTIGENIC
SHIFT)
WHat’s hypercytokinemia?
A ‘cytokine storm’ (hypercytokinemia) is the systemic expression of a
healthy and vigorous immune system
Caused by release of >150 inflammatory mediators (pro-inflammatory cytokines,
anti-inflammatory cytokines, free radicals, coagulation factors etc.)
Precise mechanisms for cytokine storms are not known, but may be
caused by an exaggerated response when the immune system encounters
a new and highly pathogenic invader
They have the potential to do significant damage to body tissues and organs
e.g. if in the lungs, large concentrations of macrophages accumulate restricting
the airways
Cytokine storms occur in response to a number of infectious and non-infectious agents
e.g. avian influenza, adult respiratory distress syndrome (ARDS), sepsis and Ebola
Overview of fungi?
Because they are eukaryotes, and share many similarities with mammals, antifungal drug design is difficult (cross reactivity and thus toxicity to the host).
Unicellular (yeasts_ and multicellular (filamentous) organisms
Ubiquitous components of terrestrial environments where they contribute to nutrient recycling
Endemic mycoses = living in geographic areas with distinct natural conditions
Many fungi dispersed by air borne spores that can reach terminal airspaces in the lung
Although many are transmitted from the environment, fungi that cause disease can also be normal commensal microbial flora = Candida albicans
Malassezia furfur and other species of this genus lives on skin = deregulated immune response associated with dandruff and may play a role in skin exacerbating skin immune pathologies
mycosis = ?
Fungal infection of humans
Top killers in immunosuppressed patients?
Developing countries are less affected by HIV-AIDS due to access to antiretroviral regimens
HOWEVER MORTALITY RATES IN SUSCEPTIBLE POPULATIONS ARE VERY HIGH
3% of patients develop infection
dependent on transplant type, Candida most prevalent
30-40% mortality rate in transplant recipients in US
Candida albicans virulence?
In response to microenvironmental signals of the host (changes in pH, cell density, exposure to serum and iron deprivation), the fungus switches from the yeast form to filamentous form.
The ability to switch morphologies has been linked to pathogenicity.
Both forms are likely to contribute to virulence:
Yeast form more easily disseminated through bloodstream
Filamentous form facilitates invasion and evasion of phagocytosis
Mutants unable to make the dimorphic switch are commonly non-pathogenic in animal models.
Host recognition of fungal infection?
Innate recognition of fungi is mediated by interactions with conserved cell wall constituents (glucans, mannans, galactans).
PRR Dectin-1 (gene is CLEC7A) is highly expressed on the surface of dendritic cells and in lesser amounts on macrophages
It has a C-type lectin carbohydrate recognition domain that mediates
the recognition of beta 1,3- and 1,6-linked glucans on the surface of intact cells and zymosan (zymosan=cell wall fragments consisting of beta-glucans, mannans, mannoproteins and chitin)
So Dectin-1 is essential for immune system
Engagement of Dectin-1 results in tyrosine phosporylation of its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) Syk (Il-10) and others (TNFalpha is Syk-independent)
Dectin-1 acts synergistically with TLRs to induce production of inflammatory cytokines and the production of reactive oxygen species (ROS)
What are toll like receptors?
Dendritic cells and other immune cells must be able to distinguish between apoptotic particles generated by normal tissue turnover and particles that are indicative of infection
The molecules mostly responsible for making this pivotal distinction are those of the family of Toll-like receptors (TLRs)
Stimulation of immune cells through their TLRs leads to synthesis and secretion of proinflammatory cytokines, thereby initiating the inflammatory response that recruits both soluble immune components and immune cells from the blood
TLR stimulation of dendritic cells also induces the initiation of an adaptive immune response
Acts synergistically with TLRs
Features of Mannose-binding lectin (MBL) and complement?
Candida expresses surface adhesins and mannoproteins that activate the alternative and lectin pathways of complement
MBL is a collectin, and is one of a number of serum proteins collectively known as opsonins
MASPS (Mannose-binding lectin-Associated Serine Proteases) couple collectins to the complement pathway
Before ligand binding, MASPs lack protease activity.
Binding of multiple carbohydrate recognition domains of MBL to the microbial cell surface leads to conformational changes in the molecule, which promotes proteolytic activation of the MASPs, which the initiate the complement cascade
Defence against Candida albicans?
Activation of complement + Interactions of cell wall components, such as mannoproteins and beta- glucan, with phagocyte receptors such as TLR2 and dectin-1 stimulate
release of cytokines including IL-1, IL-6 and TNF.
phagocytosis by neutrophils and macrophages.
Phagocytosis triggers the production of appropriate innate fungal killing mechanisms
Production of cytokines by activated neutrophils, together with direct interaction of neutrophils with dendritic cells* within inflammatory sites, promotes the maturation of dendritic cells, = adaptive immunity is activated.
Chromoblastomycosis and Fonsecaea pedrosoi?
non-fatal disease of skin and subcutaneous tissues high socio-economic burden
frequently occurs in tropical and sub-tropical regions of America, Asia and Africa
infection occurs following transcutaneous trauma (mostly farmers)
chronic disease that can take decades to develop (polymorphic skin lesion)
difficult to treat (antifungals, surgical debridement, with limited success)
Cooperation between receptors on immune cells can result in?
Synergy and singla intergration
Resulting in enhanced cytokine production protective (curative anti-microbial immunity
Why is it difficult to kill cryptococcal meningitis?
Cryptococcus is the exception in human fungal pathogens:
requires a Th1 protective response and killing seems to be more dependent on macrophages than neutrophils
independent of Dectin-1, since capsule hides glucan epitopes
no hyphae are produced in vivo. However giant cells are produced Titan cells.
Sugar capsule = invisibility cloak, harder for the body to make antibodies against sugar epitopes
2 different types of eukaryotic parasites?
Proliferating unicellular = Protoza Malaria Trypanosomes Leishmania Toxoplasma
Non-Proliferating multicellular helminths = Metazoa Pinworm Ascaris Hookworm Ascaris Schistosomiasis Filariasis
Complicating factors of Parasites?
Numerous development stages Numerous hosts Vertebrate and Invertebrate Many environments Inside and outside host
Can then lead to long term chronic infections:
Immune evasion
Immune modulation
Immune pathology
= Absence of sterilising immunity
Features of malaria?
Spread mainly by female Anopheles mosquitoes
Obligate intracellular parasite
All of the symptoms of severe malaria occur during the erythrocytic stage of infection
The liver stage is clinically silent
Different immune responses are required against liver and blood stages of infection
Stronger immune responses develop to the blood stage of infection
Precise mechanism of how malaria infection causes sudden and life-threatening complications is unclear
Cerebral malaria is when it gets into the brain 15-20% case fatality rate
Features of Leishmania?
Primary host is the sandfly
Numerous different species of Leishmania parasites, each of which can give rise to a different type of disease
Obligate intracellular parasite that primarily invades macrophages
Can result in Mucocutaneous Cutaneous
Features of Trypanosomes?
Extracellular parasite
• Causes “sleeping sickness”
What does some parasites being intracellular and some being extracellular mean?
Too different for adaptive immune system to work on different ones as well
But there are still similar themes to protective immunity
Innate cell killing of parasites?
Neutrophil mediating killing:
Neutrophil extracellular traps…
(Trojan horse)
Macrophage:
PRR activation and take up parasites (and infected neutrophils) via phagocytosis and enter phagosome
Killing:
Reactive oxygen species (ROS), triggered by phagocytosis
Production of nitric oxide…
Induced by IFNγ (Need help from Th1 CD4+ T cells!)
How do parasites live inside macrophages?
Subversion and evasion
Sand fly salivary proteins protect against neutrophil mediated death
Against parasites what type of T cell is better at killing them?
Th1 cell rather than a Th2 cell
The Th1 produces IFN-y cytokines that activate M1 macrophages which produce iNOS which produces nitric oxide which kills the parasites
Regulatory T cells can also suppress immunity and macrophage function
The effector functions of the pathogen specific CD4+ T cells determines the outcome of disease
Immune response to plasmodium parasites?
Skin and liver stage - clinically silent and no hint of acquired immunity.
Symptoms appear during blood stage
Rupture of iRBCs and the release of merozoites = clinical symptoms of malaria
Variant surface antigens (VSA) – binding to endothelium and rossetting of RBC..
Immune responses:
Th1 CD4+ T cells, NK cell secrete pro-inflammatory cytokine
macrophages production of NO
B cell producing antibody (blocks invasion)
But VSA antigen shift – immune evasion…
Trypanosome evasion via antigenic variation?
Trypanosome population can express up to 100 VSGs at any time.
And this can be shed (avoiding complement)
Sophisticated immune evasion strategy…
3 types of metazoan parasites?
CESTODE - Tapeworms, Taenia, Echinochoccus
NEMATODE - Round worms, Filaria, Ascaris, Hookworm,Trichinella
TREMATODE - Flat worms, Schistosomiasis
Gastrointestinal, vs. tissue dwelling, vs. both!
TH2 T cells needed to kill worms instead of TH1, explain?
Key events causing response:
Parasite damage
- Tuft cells in intestine
- Epithelial cell response
- Parasite antigen stimulation on APCs
Impact of type-2 cytokine:
Stimulates mast cell degranulation (increase gut motility, fluid secretion)
Increase number of goblet cells –mucus hypersecretion (trap parasites).
Release of toxic proteins, directly harm parasites.
Increased epithelial cell turnover and intestine peristalsis.
All lead to parasite expulsion
Type 2 causes collagen synthesis and fibrosis due to arginase converting argine
Granuloma responses?
Caused by macrophages
Can parasite products be used as treatment?
yes - they can suppress the immune system in useful ways
Overall summary on parasites?
Type 1 immunity for protoza
Type 2 immunity for Metazoan
Balanced immunity is essential to avoid host damage
Is there resistance against vaccines?
Low levels of resistance to vaccines are a consequence of the timing of the treatment (prevent rather than treat infection) and the multiplicity of target sites
Main types of vaccine?
Live vaccines
Living, but cannot cause disease
Killed vaccines
Killed by heating, or exposure to chemicals
Sub-unit vaccines
Fragments of the microorganism e.g. proteins (or polysaccharides)
Almost always antibody mediated
In some cases an immune response against a single protein antigen elicits protection
Nucleic acid vaccines (DNA or mRNA)
Direct protein expression after injection
Vectored vaccines
DNA encoding the sub-unit is cloned into another “safe” microorganism
What is formaldehyde detoxification?
Removes toxicity of a protein
The immunogenicity of the protein is preserved
Problems:
Must balance cross linking with retention of antigenic structure
Batch to batch variation
Reversion
Requires pure toxin at the starting point:
Needed to grow pathogen
Need to purify and handle the toxin
What are genetic toxoids?
Modern approach to subunit vaccines against toxins
Modify the gene so that it encodes a non toxic (but immunogenic) protein
Describe e toxin?
causes enterotoxaemia in livestock
Produced in intestine crosses gut wall, targets kidneys and brain causing death
Can mutate the toxin now 1000 fold less toxic
Is a vaccine
Diphtheria toxin CRM197 genetic toxoid?
Mutation in the catalytic A-subunit blocks activity
Glycine to glutamic acid substitution at residue 52
Advantage of genetic toxoids?
Produce in harmless bacterium
High yields
Molecular structure more similar to active toxin
Reproducible properties
No reversion
Sub unit vaccine against plague?
F1 antigen - capsule and V-antigen part of the type 111 system are the protective components
Then produce F1 and V antigens using genetic engineering
Together produces solid protection
covid vaccine?
The spike S1 protein induces protective immune responses
Some antibodies against S1 protein epitopes are protective others make the disease worse
What’s reverse vaccinology?
Predict vaccines from the genome sequence
Neisseria meningitidis causes globally 1.2 million
invasive disease cases and 135,000 deaths per year
• Serogroup B strains are the main cause of disease in Europe, New Zealand, Australia, Argentina, Canada, Japan
• Traditional vaccinology had failed to identify a vaccine
pSORT predicts cellular location so can help us find proteins that will be on the surface will give lots of proteins then you have to test them
Meningitis B vaccine?
H binding protein (fHbp), fused with NA2091 protein, binds human factor H, a negative regulator of the alternative pathway of complement activation.
NadA, major adhesion protein involved in colonization, invasion, and induction of pro-inflammatory cytokines.
NHBA, heparin-binding protein that increases resistance against the bactericidal activity of human serum
PorA 1.4 Porin, A major outer membrane protein and the target for bactericidal antibodies
Sub unit vaccines are effective at stimulating protective antibodies which will?
Agglutination
Opsonization
Neutralization
Activation of complement
Inflammation
Antibody - dependent cell mediated cytotoxicity
Is there immunological cross reactivity between the different polysaccharides on bacteria?
No they are unique
Examples of polysaccharide vaccines?
Streptococcus pneumoniae - pneumonia
• Haemophilus influenzae - pneumonia
• Neisseria meningitidis - meningitis
• Salmonella typhi – typhoid fever
Features of Streptococcus pneumoniae?
Major cause worldwide of community acquired pneumonia, bacterial meningitis, bacteraemia and otitis media
Causes disease in young and elderly
Antibody against the capsular polysaccharide is protective
Antibody causes:
Opsonising Agglutinating Complement fixing
3 problems with S.pneumoniae vaccines?
Polysaccharide diversity so doesn’t work on slightly different strains
- At risk populations respond poorly to vaccines
- Poor long-term memory
How do they get around polysaccharide diversity?
Pick the serotypes most likely to cause disease and isolate these polysaccharides
Created a 23-valent vaccine
Are polysaccharides T cell independent antigens?
Yes
No involvement of antigen presenting cells, CD4+ or CD8+ T cells
• Polysaccharides interact directly with B-cells
• Clonal proliferation of B-cells
• Production of IgM antibody
• Poor antibody responses in children & in the elderly
• no memory
Can fix this by chemically coupling it to a protein which means it can now be processed
Mechanisms of T-cell activation by glycoconjugate (adding the protein) vaccines?
Glycoconjugate is internalized into an endosome of an antigen presenting cell (e.g. B cell).
• processedintoglycanP-peptides.The polysaccharide is broken down into ~10kDa glycans
• MHCII presentation of glycanP-peptide by MHCII to the αβ receptor of CD4+ T cells (αβTCR).
•The T-cells involved are called T cells carb
•Activation of the T cell results cytokines which mature the B cell to become a memory B cell (especially IL2 and IL4)
Protein used to bind to the carbohydrate?
diphtheria toxin CRM197 genetic toxoid
• glycine to glutamic acid substitution at residue 52 abolishes toxicity
Diseases where CD+ T cells play a role in protection?
Diseases where the microbe grows within host cells
tuberculosis
• salmonellosis • melioidosis
• viral infections yellow fever
influenza Measles SARS CoV2
How are CD8 + responses induced?
induced by live attenuated microbes that can invade host cells
Usually very effective – excellent mimetics of the natural microbe
Risk is that they will still cause disease in some individuals e.g. immunocompromised
Activates
Another approach:
Injected DNA or mRNA is taken up by host cells
The protective sub-unit is produced by host cells and seen as foreign by the immune system
Protein antigen is processed via the MHC I (and MHC II) pathways.
Combine the advantages of live and sub-unit vaccines
Work well in animals not really humans
Vector vaccine?
Inset gene encoding the protective subunit into a “harmless” microbe
• A wide range of carrier microbes have been suggested e.g.
– Disabled Salmonella – Lactococcus
– Vaccinia virus
Polyclonal vs monoclonal antibodies?
Because most antigens carry many different epitopes,
animals injected with a single antigen will produce a
complex mixture of antibodies, each made by a
different clone of B cells
This antibody response is said to be polyclonal and
will not discriminate between different antigens
To discriminate between different antigens,
antibodies need to be identified that bind to epitopes
specific to individual antigens
These are known as monoclonal antibodies
Do cancer cells or malignant cells have unique epitopes?
yes
What is hybridoma technology?
Allows the identification and culture of cells
secreting identical antibodies with pre-defined
specificity
A single clone of cells secreting a single
antibody is made by fusing a B cell (Ig+ splenocyte
with finite lifespan) with a myeloma cell (cancerous
Ig- B cell with infinite lifespan)
The resulting hydrid cells (HYBRIDOMAS) each
make a specific monoclonal antibody
Time course of a typical immune response when injected with antigen?
Primary injection:
Production of IgM
Multivalent
Low affinity
Secondary injection:
Production of IgG
Bivalent
Low to medium affinity
Tertiary injection:
Production of IgG
Bivalent
Medium to high affinity
Multiple boosts:
IgG
Bivalent
High affinity
Make sure to look at photo library
ok
Mechanism of hybrid cell selection in HAT medium?
HGPRT = Hypoxanthine:guanine phosphoribosyl transferase = blocks RNA and DNA transcription only can go through salvage pathway
Spleen cell = HGPRT+ but is not able to grow in long-term
Myeloma cell =HGPRT-, therefore not able to use the salvage pathway for RNA synthesis because the normal pathways are blocked
Hybridoma cell = has HGPRT from a spleen cell and immortality from a myeloma cell, therefore is able to grow in the HAT medium (providing there is an exogenous supply of Hypoxanthine and Thymidine)
What is the PTA-ELISA?
Plate-Trapped-Antigen-Enzyme-Linked
Immunosorbent Assay
Find out antibody is present when substrate added for target antigen goes from purple to yellow
What is phage display?
An issue with hybridoma technology is immuno-
dominance
Alternative technologies to generate mAbs include
phage display
A technique in which antibody fragments
are expressed as a fusion with a coat protein of a
bacteriophage
Fused proteins are displayed on the surface of the
virion, while the DNA encoding the fusion resides
within the virion
Making antibody combinatorial libraries?
B cell source (from immunised animal)
extract the mRNA
reverse transcribe to get cDNA
PCR to amplify each of the V-gene families
Assesmble the VH and VL genes ar random (creates novel antibody repertoire with new pairings and specificities not present in donor)
Clone as scFv or F(ab) onto pIII or pVIII
Describe the expression of scFv on the surface of phage?
scFv = VL and VH (V heavy and V light) domains of an immunoglobulin linked by a peptide spacer
Fusion proteins between scFv and pIII minor coat protein allows expression of scFv on surface of phage
Five copies of PIII expressed
Up to five scFv can be expressed on phage
Describe the expression of F(ab) fragments on filamentous phage?
F(ab) fragments of antibodies can
be expressed on the surface of
phage as fusion proteins with pIII
Multiple copies expressed along the
length of the phage particle
Uses of monoclonal antibodies?
Diagnostic pathology (cytology and histology)
In vitro diagnostic assays based on e.g. Enzyme-Linked Immunosorbent Assay (ELISA), Lateral-Flow Assay
(LFA) for pathogens and biomarkers of disease
(Lectures 15 & 16)
Affinity purification and characterisation of antigens
Limited use of mouse mAbs in vivo until relatively recently
The need to humanise monoclonal antibodies?
Immunogenicity - most mAbs used in vivo are of mouse
origin and are therefore XENOGENEIC resulting in
Human Anti-Mouse Antibody (HAMA) reactions
- Origin - Mouse origin diminishes their ability to elicit effector mechanisms such as COMPLEMENT-MEDIATED LYSIS or ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY (ADCC)
How does humanisation occur?
Use of xenoantibodies in human patients had
proved to be severely limiting owing to their
being seen as foreign proteins by the patient’s immune system
Strategies adopted to increase the human content of
antibodies which have proven clinical applications include:
Production of human hybridomas from human B lymphocytes
Replacement of the constant regions of mouse mAbs with those of human antibodies yielding CHIMERIC antibodies
Replacement of complementarity-determining regions (CDRs) of a human antibody with those of a mouse antibody via CDR GRAFTING to make a HUMANISED antibody
Production of FULLY HUMAN mAbs in transgenic mice
Human immune system (HIS) mice?
CRISPR-Cas9 technology used to knockout Prkdc and IL2rg loci
Prkdc and IL2rg are part of the SCID (Severe Combined Immuno Deficiency) family of genes affecting maturation and formation of T cells, B cells, NK cells and other leukocytes
Knockout renders mice immunodeficient
Transplantation and engraftment of human HSC leads to mice with HIS
Evidence for immunity and cancer surveillance?
Immunocompromised individuals more susceptible to cancer than general population
Some cytokines e.g. IL-2 are immuno-protective against cancer (IL-2 treatment is FDA approved for some cancers)
The tumour micro-environment is rich in Tumour Infiltrating
T-lymphocytes (TILs)
The 3 phases of cancer immune surveillance?
Elimination:
By B cells, antibody, T cells, Treg cells, NK caells
This is the innate and adaptive immune response active against tumour
Can result in tumour cells death
Equilibrium:
Tumour is contained by the immune system but not completely destroyed
Escape:
Tumour antigen editing via genomic instability causes immune evasion
Tumour microenvironment becomes immunosuppressive
The impediments to anti tumour immunity?
Immunocompromised individuals more susceptible to cancer than general population
Some cytokines e.g. IL-2 are immuno-protective against cancer (IL-2 treatment is FDA approved for some cancers)
The tumour micro-environment is rich in immune suppressive signals such as:
+ High levels of suppressive cytokines
+ Infiltration of Tregs (inhibit dendritic cells and therefore T cell activation)
+ Low MHC expression on tumour cells
+ High expression of immune checkpoint proteins on T cells
+ High expression of immune checkpoint protein inhibitors on tumour cells
What does immunotherapy do?
Specific:
Trains the immune system to recognise and target only cancer cells
Powerful:
Attacks the cancer systemically through out the body
Universal:
A treatment approach that can be applied to nearly all cancers
Memory:
Capacity for memory means durability of protection
Types of immunotherapy?
Passive vaccination (Therapeutic Monoclonal Antibodies)
Active vaccination e.g. HPV vaccine (Gardasil, Cervarix) and cervical cancer
Immune system modulators e.g. IL-2 (activates CD8 T cells)
Immune Checkpoint Modulation
Immune Cell Therapy
How do therapeutic monoclonal antibodies target cancer?
Carry cytotoxic agents to tumour
Induce Complement-Mediated Lysis or ADCC of tumour cells e.g. Rituximab
Block activity of tumour-specific proteins e.g. Herceptin
Steps:
Naked MAb
Immunoconjugates
Multistep targeting
What is non Hodgkins lymphoma?
Non-Hodgkin’s Lymphoma (NHL) is a malignant growth of
B cells that results in tumors in lymph nodes and
throughout the body
No cure for most patients, and only 60% of patients treated with
radiation and chemotherapy are still alive after 5 years
What is Rituxan?
Rituximab is the first monoclonal antibody treatment approved for
cancer
Rituxan is a chimeric human-mouse mAb targeted against CD20,
an antigen present on the surface of neoplastic B cells
It consists of a human IgG1k antibody, with mouse variable
regions isolated from a murine anti-CD20 mAb, IDEC-2B8
In vitro studies show the mAb is able to lyse CD20+ cells using
human complement or ADCC
Features of breast cancer?
Breast cancer is a common cancer and 1 in 9 women will develop it
during their lifetime. It also occurs in men.
The reasons for breast cancer are not well understood. Only 5-10% of
cases are thought to be inherited
Two breast cancer genes have been identified: BRCA1 and BRCA2
The most frequent mutations in BRCA1 - common in a single group, the
Ashkenazi Jews
Women with mutations in the BRCA2 tumor suppressor gene have a
greatly elevated risk of developing breast and ovarian cancers
Breast cancer cells express an antigen (protein) on
their surface called HER2
Breast cancer cells divide and grow when the
protein Human Epidermal Growth Factor attaches
itself to HER2
What is Herceptin?
Herceptin is a humanised monoclonal antibody
It blocks attachment of human epidermal growth
factor to HER2
Herceptin is mainly used to treat women with
advanced breast cancer and is given in
combination with chemotherapy drugs such
as Taxol
After surgery, Herceptin treatment in combination
with chemotherapy halved the rate of recurrence
Ahzheimers disease treatment called sola?
Solanezumab (Sola) neuroprotector
mAb binds to amyloid-b peptides
Binds to central epitope KLVFFAD
with picomolar affinity
Epitope is nucleation site for Ab
polymerization
Oligomers of Ab toxic to neurons
Tocilizumab for rheumatoid arthritis?
Tocilizumab
Humanised anti - human IL - 6R receptor antibody
Blocks binding of IL - 6
Mechanism of Immune Checkpoint Protein Inhibition?
Co-stimulation via CD28 ligation transducer T cell activating signals
CTLA-4 ligation on activated T cells down regulates T cell responses
Blocking CTLA-4 ligation enhances T cell responses
Immune Checkpoint Proteins (e.g. CTLA-4) keep immune responses in check by preventing overly intense responses that might damage normal cells
Blocking the activity of immune checkpoint proteins releases the “brakes” on the immune system, increasing its ability to destroy cancer cells
The first such drug to receive FDA approval, ipilimumab (Yervoy®), for the treatment of advanced melanoma, blocks the activity of a checkpoint protein CTLA-4 which is expressed on the surface of activated Cytotoxic T-Cell. This increases T-Cell activation.
What is Nivolumab approved for?
approved for patients with melanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colon cancer, liver cancer
What is pembrolizumab approved for?
approved for patients with melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma and stomach cancer
What is theralizumab - TGN1412?
Investigated immune modulatory drug for rheumatoid arthriris
Binds to CD28 co-stimulator pair on T cells
Potent agonist - stimulates anti inflammatory cytokine production
However caused severe inflammatory reactions due to cytokine release syndrome
What is adoptive immune cell therapy: Normal TILs?
Idea behind this approach is that the TILs have already shown the ability to target tumor cells, but there may not be enough of them within the tumour to eradicate it or overcome the immune suppressive signals that are being released there
Introducing massive amounts of activated TILs can help to overcome these barriers and shrink or destroy tumors
In some experimental cases these treatment responses
have lasted for years
TILs are collected from samples of the tumor
TILs that show the greatest recognition of the patient’s tumor in lab are selected
Cells are then activated with cytokines and infused into the patient’s bloodstream
What are transgenic TILs?
T cells are engineered to have a specific transgenic TCR which we know has high affinity to a certain tumour antigen
What are CAR-T cells?
Another form of ACT that is being actively studied is Chimeric Antigen Receptor Modified T Cells (CAR-T cells)
A patient’s isolated T cells are collected from the blood and genetically modified to express a protein known as CAR
CARs are modified forms of a T cell receptor, which is expressed on the surface of T cells. These receptors allow the modified T cells to attach to specific proteins on the surface of cancer cells. Once bound to the cancer cells, the modified T cells become activated and attack the cancer cells
The modified cells are grown in the laboratory to produce large populations of the cells
CAR-T cells are then infused into the patient.
Features T cell Receptors in treating a tumour cell?
Sensitive signal amplification derived by evolution
Low avidity
Targets intracellular proteome
Requires MHC class 1 expression and HLA matching on tumor cell
Possible misfiring with endogenous TCR
Features of CAR targeting tumour cells?
Signal amplification derived by synthetic biology
Avidity controllable
Targets only surface structures
HLA independent antigen recognition
No misfiring with endogenous TCR
What is antibody guided molecular imaging?
Radio-labeled antibodies used in nuclear medicine to locate cancer cells (or infections) by molecular imaging using:
Computed Tomography (CT)
Magnetic Resonance Imaging (MRI)
Positron Emission Tomography (PET) - PET/CT or PET/MRI
Antibody Capromab reacts with prostate membrane specific
antigen (PMSA) on prostate cells
Can be conjugated to 111I with chelator pendetide and the radio-labeled antibody (trade name Prostascint) used to locate cancerous prostate cells. Others include antibody J591.
Summary?
Monoclonal antibodies produced using hybridoma or phage display technologies are highly specific and can be used in immunotherapy and molecular imaging of cancers
Tumour associated antigens can be recognized by the immune system
Tumours adapt to evade immune clearance by mechanisms that either prevent recognition or that directly interfere with immune mediators
There at least 5 different modalities to circumvent this immune evasion
These modalities are collectively known as cancer immunotherapy
The most promising tools are immune checkpoint inhibitors and CAR-T T cells
What is Ouchterlony immunodiffusion?
OID is a qualitative technique for detecting antibody-antigen
interactions in which antigens and antibodies are placed in a
gel of agar and allowed to diffuse towards one another
A positive reaction is indicated by the presence of PRECIPITIN lines due to a Antibody-antigen equivalence = immune complex formation
Antigen excess or antibody results in no precipitin
Precipitin that forms when both antigen sources react with the antibody as they both have shared properties?
An arc
Precipitin Line that forms when each antigen doesn’t share a common determinant of the antibody but both have a determinant of it?
A cross sword
Precipitin line that forms when antigens share one common determinants but one of them has both required for the antibody
Arc on the side which doesn’t have an extra shared one
Spur forms on side that does, so cross bridge shape there
What is Western blotting?
A technique in which specific antibodies are used to identify
particular antigens in mixtures of proteins that have been
resolved and transferred to a membrane
The proteins are separated by an appropriate technique,
usually SDS-PAGE (POLYACRYLAMIDE GEL ELECTROPHORESIS)
Transfer is carried out by electroblotting to a sheet of
nitrocellulose or PVDF, which is then incubated with labeled
antibodies specific for the antigen of interest
Radiolabeled or enzyme-linked antibodies can be used
Unbound antibodies are washed away, and the position of the
labeled antibodies is revealed using an appropriate technique
Chemical substrates (Chemiluminescent or Colourimetric) Radiography
Features of an SDS page?
The speed of migration in an electrical field depends on the dimension form and charge of molecules
For deaggregation and denaturation of the proteins SDS, B-mercaptoephanol or DTT (reducing agents) and heat is used
SDS provides negative charge to the proteins
Right to left = separated by charge by isoelectric focusing (IEF)
Up to down = separated by size by SDS electrophoresis
What is immunofluorescence?
A qualitative technique in which antibodies tagged with
fluorescent reagents (Fluorescein IsoThioCyanate or
Texas Red) are used to detect molecules of interest.
FITC and TR fluoresce under UV at different absorbance
and emission spectra
IF is one of the most widely used techniques in immunology,
cell biology and clinical medicine
Used to localise antibodies or antigens on cell surfaces or
inside cells and to identify pathogenic organisms in tissues
or exudates
IF can be DIRECT or INDIRECT
What is direct immunofluorescence?
Treatment on cell with labelled antibody
Unbound antibody washed away and UV light applied
Fluorescence observed where antigen is located
What’s indirect immunofluorescence?
Treatment with unlabelled primary antibody
Unbound antibody washed away
Treatment with labelled secondary conjugate
Unbound conjugate antibody washed away UV light applied
Fluourescnce observed where antigen is located
What’s immune-electron gold microscopy?
A qualitative technique in which antibodies tagged
with gold particles are used to detect molecules of
interest
EM-gold allows localisation of antigen-antibody
interactions at the nm scale (cf. IF at the m)
A powerful method of visualising intracellular antigens
and organelles
What is Enzyme-linked immunosorbent assay?
ELISA is a quantitative technique for detecting antigens or
antibodies using enzyme-substrate reactions
The enzymes are usually coupled to antibodies either
DIRECTLY (to the antibody specific for the antigen) or to anti-immunoglobulins (INDIRECTLY)
The two main ELISA formats are:
- Plate-Trapped-Antigen (PTA)-ELISA
- Sandwich-ELISA
Quantification in ELISA is achieved using standard calibration curves
4 types of plate ELISA?
Direct
Indirect
Sandwich ELISA
CompetitiveELISA
Rhizoctonia solania case study?
Ubiquitous soil-borne plant pathogen with worldwide distribution and wide host range
Difficult to control using chemicals
Soil dwelling
Widespread resistance to fungicides
Soil fumigants are banned (Class II ozone depletors)
Most effective way to prevent disease is to ‘evade’ the pathogen altogether - test soils for infestation prior to sowing with susceptible crops
R. Solon-specific mAbs = Monoclonal antibodies raised against a surface glycoprotein antigen
mAb EH2 - protein epitope
mAb EE1 - carbohydrate epitope
Antigen is secreted by live cells only
Proces:
Stimulate active growth using baits
- Detect R. solani using specific mAbs
- Recover positive isolates for further testing
Sandwich-ELISA format for R.solani?
Coat plates with mAb EH2 16h at 4 degrees
Bait incubation 2h and 23 degrees
add mAb EE1, 1h at 23 degrees
Anti-IgM (u-chain specific) peroxidase conjugate (1h at 23 degrees
Substrate formed 30min at 23 degrees
What’s Batrachochytrium dendrobatidis
amphibian killer
Spores swim towards spogs
Burrow into them
Mature into sporangium
Spores released
Kills the frog within 120 days
Kills by colonising keratinised skin layer
Hyperplasia + Hyperkeratosis = disrupted electrolyte balance
Leads to cardiac arrest and death
Features of Aspergillus disease?
Caused by Aspergillus fumigatus
Most important opportunistic mould pathogen of immuno-compromised humans esp. haematological malignancy and bone marrow transplant patients
Can result in -
Invasive Pulmonary Aspergillosis >300,000 cases/year worldwide with 30-95% mortality
Chronic Pulmonary Aspergillosis ~3 million cases worldwide in patients with underlying lung diseases including asthma
Allergic Bronchopulmonary Aspergillosis ~4 million cases worldwide in patients with asthma and cystic fibrosis
Covid-19-Associated Pulmonary Aspergillosis (CAPA) / IAPA?
Diagnosis for Aspergillus infection?
Diagnosis notoriously difficult - patient history, culture from invasive biopsy, and biomarkers in invasive BALf or in blood
Hospital treatments ‘fever-driven’ or by non-specific chest CT - patients can receive inappropriate treatment with costly and toxic antifungal drugs
Imperative that diagnosis is made without delay - prognosis worsens rapidly over time with high mortality - exacerbated by poor diagnosis
Drug resistance in clinical strains of Aspergillus is increasing
Mouse mAb JF5 for detecting Aspergillus?
IgG3 immunoglobulin
Recognises extracellular glycoprotein antigen
Antigen is secreted during active growth of hyphae
and is not produced by dead or dormant spores
JF5 used to develop a rapid, user-friendly, diagnostic test (lateral-flow assay) for detection of IPA using BALf and serum
What is Bronchoalveolar lavage?
Lung fluid
Commercialisation process of a monoclonal antibody lateral flow company?
Institute to industry
Patent to product
Bench to Bedside
What’s non invasive antibody guided molecular imaging with immunoPET/MRI?
Can use it show up Aspergillus infection in a scan
It acts as a tracer
Depletion of neutrophil granulocytes in C57BL/6 mice via injection (i.p.) of the anti Gr-1 antibody RB6-8C5
Intratracheal (i.t.) infection of neutropenic C57BL/6 mice with A. fumigatus conidia
In vivo & ex vivo bio-distributions of [64Cu]-labeled antibody tracers measured in lungs via how much radioactivity there is
Next step of developing the technique -
Humanisation of JF5 by CDR grafting
Proved that the target epitope (b1,5-galactofuranose
) of the antigen is not present in humans
