Revision immunity deck Flashcards
GALT =?
Gut associated lymphoid tissue
Thymus =?
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?
Beginning of original deck
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 = ?
Serum = ?
Plasma without clotting factors
Where is the immune system?
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
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?
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?
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
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?
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?
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
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
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 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?
go on original deck
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?
Get from original deck
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
What are the first responders to tissue detected microbes?
Neutrophils
What are the types of secreted molecules released by
In general deck
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,
The MAC is much more active towards Gram-negative than gram positive bacteria
Ion influx and efflux causes the cell to lyse
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:
More details on original flashcard 67
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
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
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
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?
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
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?
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
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
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?
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?
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?
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
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?
get from main deck
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?
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?
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?
Might be more important to learn this rather than all the subsets
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?
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?
Get from main deck
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?
Papain cleavage on antibody?
Fab 1
Fab 11
Intact Fc 111
pepsin cleavage on antibody?
pepsin cleavage on antibody?
F(ab)2
Fc fragments
Antibody structure - the Y shape?
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) Get Me An Edo Dad
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? And what are ITAMs
is flashcard 126 complex probably don’t need to know
and 127 and 128
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?
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?
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?
In main deck
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
Steps of this on flashcard 134
What are collections and ficolins?
In Main deck
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
More details of their mechanism on 135
Lectin pathway details?
On card 136
Alternative pathway?
on card 137
C3a, C5a functions?
C3a, C5a functions?
peptide mediators of inflammation, phagocyte recruitment
C3b function?
Binds to complement receptors on phagocytes
Opsonization of pathogens
Removal of immune complexes
Equation on card 139
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?
On 141
Allergy =?
Excessive immune responses to harmless antigens (allergens)
- Most allergic reactions are due to IgE-mediated responses
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?
Get from main deck
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)
If a question on allergy mechanisms comes up =
149 to 155
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
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
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
GP encodes?
In main deck
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
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
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?
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
Features of aids?
Acquired Immune Deficiency Syndrome (AIDS) is caused by
the Human Immunodeficiency Virus (HIV)
HIV infects and destroys CD4+ T cells
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 getting into lymph nodes and CD4 T cell and infection cycle and infection cycle and immunological consequences
191, 192, 192, 193, 194
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
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?
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)
mycosis = ?
Fungal infection of humans
Host recognition of fungal infection?
in the main deck:
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?
In the main deck:
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?
In main deck
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.
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!)
Against parasites what type of T cell is better at killing them?
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
Overall summary on parasites?
Overall summary on parasites?
Type 1 immunity for protoza
Type 2 immunity for Metazoan
Balanced immunity is essential to avoid host damage
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?
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?
In main deck
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?
In main deck
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
What’s reverse vaccinology?
Predict vaccines from the genome sequence
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
Sub unit vaccines are effective at stimulating protective antibodies which will?
Agglutination
Opsonization
Neutralization
Activation of complement
Inflammation
Antibody - dependent cell mediated cytotoxicity
s there immunological cross reactivity between the different polysaccharides on bacteria?
No they are unique
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?
nset 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?
In powerpoint
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
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
What is phage display?
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?
259
Describe the expression of scFv on the surface of phage?
260
Describe the expression of F(ab) fragments on filamentous 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?
In main deck
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 mice?
265
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
What does immunotherapy do?
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
New monoclonal antibodies for cancer
272-281
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 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
More details on 287
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
293
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 direct immunofluorescence?
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?
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
Rhizoctonia solania case study?
301 and 302
Aspergillus case study
304 to the end
