Module 3.1: Basic Immunology Flashcards
Define T-Cells
Lymphocytes with key importance to the immune system.
They are at the core of ADAPTIVE immunity, the system that shapes the body’s immune response to specific pathogens
Mature T-cells are derived from the THYMUS gland and participate in a variety of cell-mediated immune reactions
Subsets of T-cells
CD4+ —> T-helper cells
- They lead the attack against infections
- Four fundamental subsets
- – Th0
- – Th1
- – Th2
- – Th17
CD8+ –> cytotoxic T-lymphocytes (CTLs)
Subsets of T-helper cells
Th0 - undifferentiated
Th1
- responsible for cell-mediated mechanisms
- eliminate INTRACELLULAR pathogens
Th2
- role in regulating Ab production
- eliminate EXTRACELLULAR pathogens
Th17
- key regulator of inflammation
Describe the polarisation of Th cells
Th0 can differentiate into Th1, Th2 or Th17 depending on the cytokine environment that it is in
Signature cytokines will be produced as a prime response of the different T-helper cells
In IL-12/STAT4 –> Th1 –> secrete IL-2/IFNg/TNF (transcription factor Tbet)
In IL-4/STAT6 –> Th2 –> secrete IL-4/IL-5/IL-10 (transcription factor GATA-3)
In TGFb/IL6/+-DC/IL-23/IL-1b –> Th17 –> secrete IL-17/IL-21/IL-22 (transcription factor RORg/RORa)
Describe the role of T-cells in immune responses
ANTIGEN recognition on the cell surface by MHCI or MHCII
The antigen specific TCR on the T-cell surface interacts with the appropriate peptide-MHC complex
T-Cells play a critical role in the regulation of immune responses and are responsible for mediating many of the effector mechanisms of the immune system
Describe the steps required to take before assessing T-cell function
Isolation - Measure - analyse and interpret
Describe how T-cells can be isolated
For in vitro assays
- Human or animal blood
- Animal spleen
Sorted by
- magnetic bead separation
- flow cytometry sorting
Describe the principles of Ficoll Paque Isolation of peripheral blood mononuclear cells in T-cell extraction
Completed via density gradient centrifugation using Paque/Ficoll-Paque Plus
- Layer blood on Ficoll-Paque and centrifuge
- Due to the density gradient between whole blood and Ficoll-Paque, you end up with different layers based on the density of each component
Allows for purer isolation and enables freezing to be used at a different time
Describe the principles of Conjugated Magnetic Beads in T-cell extraction
Give two examples in commercial use
EXAMPLES: Dynabeads, MACS system
DYNABEADS
- add beads to sample
- they bind to desired target
- respond to magnetic field allowing bound material to be rapidly separated
- – These beads are quite big –> don’t want them in culture
Soooo
MACS microbeads
- smaller, biodegradable beads (won’t be in culture)
- easier separation
Positive selection vs negative selection
Positive: target cells isolated directly
- best purity
- best recovery
- fast
Negative: all the unwanted cells are collected as the fraction
- may be considered for avoiding activation of cells
Describe cell sorting via Flow Cytometry
Flow sorting = fluorescence microscopy whereby single cells in liquid suspension can be identified and physically separated from each other according to unique charateristics
e.g. for CD4+ cells: stain the cells and sort them
Start with a mixed population
Stain cells with colour conjugated Ab againt CD4+
Incubate
Wash and sort cells
Data = DOT plot or HISTOGRAM
Describe methods of measuring T-cell function
3H-thymidine incorporation assays
CFDA-SE (CFSE) labeling
Limiting dilution analysis
Measurment of cytokine production
- immunoassays or bioassays
- ELISPOT
- intracellular cytokine staining
- chromium release
- ELISPOT for CD8+ T cells activity
- Tetramer analysis for direct visualisation of CD8+ T cells
- RNA extraction and real time PCR
Describe the principles of 3H-thymidine incorporation assays in T-cell function measurments
measures the strength of response of T-helper cells by measuring the proliferation of T-cells
Occurs following stimulation of cells using different stimuli
Hence T cells are counted
- Positive control: incubated in the presence of a known mitogen e.g. ConA - used to ensure that cells are not responding due to a specific defect they have in proliferation if they are blind to specific antigen hence the need for a positive control
- Negative control: incubated in the abcence of any stimuli
- incubated in the presence of specific antigen being tested
When cells proliferate, they will incorporate the radioactive thymidine into the DNA of the dividing cells
Incubated foe 3-7 days and harvested
Radioactivity is measured
Describe the principles of CFDA-SE in T-cell function measurments
CFDA is not radioactive
passively diffuses into cells
its acetate groups are cleaved by intracellular esterases
highly fluorescent amine-reactive carboxyfluorescent succinimidyl ester interacts with intracellular amines to form fluroescent conjugates
The label is inherited by daughter cells
HOWEVER, cell division causes halving of the fluorescnece - limited to 8-10 divisions
Describe the principles of ELISA in T-cell function measurments
Enzyme linked immunosorbent assays
specific Abs are coated on a plate
specimens are incubated on the plate to measure cytokines directly
another layer of Ab conjugated to an enzyme is added
Followed by the addition of a substrate to the enzyme
ELISA uses a standard curve –> cytokine conc can be extrapolated
Describe the principles of ELISPOT in T-cell function measurments
similar to ELISA
looks at the response of SINGLE CELLS that are cultured with other cells
Data will be quantified differently through DETECTION OF SPOTS on nitrocellulose lined microtitre wells
Allows for qualitative and quantitative analysis
Detects cytokine release at single cell level
each spot is the footprint of a single cell that reacted to the antigen
Can be quantified by eye or automated readers
Amount of cytokine released is proportional to spot size and intensity
ELISA vs ELISPOT
ELISPOT 200x more sensitive
however the scoring of the spots involve manual enumeration in ELISPOTT
Describe the principles of MSD (meso scale discovery) in T-cell function measurments
Multiplexing platorm of more than one cytokine –> uses electrochemiluminescence (ECL) detection
ultra-low detection limit and minimal background
Up to 5 logs of linear dynamic range
minimal sample - as low as 10-25 microlitres
easy handling and protocol
Describe the principles of intracellular staining in T-cell function measurments
Flow cytometry used
T cells are stimulated in vivo
the transport cytokines through the golgi is blocked in order to prevent the secretion of cytokines
T cells are then fixed and permeabilised to allow cytokine specific Abs to enter the cell
Directly conjugated anti cytokine Abs are then used for staining
Large no of T cells can be analysed in a short time
More than one cytokine can be assessed at a time
Describe the use of Chromium release assays and ELISPOT assays in CD8+ cell analysis
Chromium release assays:
- radiolabel the target cells with sodium chromate
- incubate with test effector cells for 4-16h
- the amount of Chromium released in the supernatant is then quantified to provide a measure of target cell lysis
ELISPOT
- where the antigen is a peptide known to be an epitope recognised by CD8+ CTL
- look at CD8+ cells producing IFNg and/or TNFa
Describe the use of limiting dilution anaysis in T cell analysis
not favourable
LDA are used for quantitative estimates of the number/frequency of T cells that are specific for a particular antigen
Positive results indicate the presence of antigen specific precursor cells in PBMCs population at the start
Long incubation period - 2-3 weeks and risk to over/underestimate cell numbers
Summarise the characteristics of H. pylori
G-ve
spiral
microaerophilic
multiflagellated GI pathogen
colonises gastric mucosa
Colonises 50% of the world population, only 15% symptomatic
90% prevalence in developing countries, 30% in developed
Causes:
- gastritis
- gastric and duodenal ulcers
- MALT lymphoma
- atrophy
- non-ulcer dyspepsia
- adenocarcinoma
Summarise the virulence factors of H. pylori
Motility
Urease
- enzyme that hydrolises urea into ammonia and CO2 - provides protective cloud allowing it to move from acidic enviornments to epithelial cells
Adhesins
- Helicobacter outer membrane proteins (HOP) –> allows it to stick to epithelial surfaces
Pathogenicity islands (PAI)
- include the cytotoxin-associated gene A (CagA)
- immunodominant protein into target cells
- 90% CagA+ from ulcer patients - contributes to virulence and damage
Vacuolating cytotoxins (VacA) - cytoplasmic toxic vacuoles inside target cells
Lipopolysaccarides
- site of antigenic variation
- Formed by lipid A, core oligosaccharide and O-chain (most external part seen by host)
- O side chain expresses LEWIS ANTIGENS - extended chains with fucosylated carbohydrate structure
- phase variation is the occurence of reversible high frequency on and off switching of cell surface receptors
Define collectins
components of innate immune system (PAMP receptors)
innate immunity is an antigen-non-specific defence mechanism used by a host after exposure to antigen
Initial response by the body to eliminate microbes and prevent infection
Desribe the importance of collectins in the gut
the gut has many challenges
- food contains microoganisms that could thrive in rich growth medium of digested nutrients
Abundant potential sites of infections in the long tube
oral mucosa, intestinal mucosa comprised of only a single layer of epithelial cells
Describe the type of collectin family found in mammals
Mammalian C-type collagenous carbohydrate binding proteins
C - as they require calcium for their action
Which chromosome is the family of proteins implicated in innate immunity encoded on?
10
List the proteins implicated in innate immunity on Chr10
surfactant protein A (SP-A) SP-D Mannan binding lectin (MBL) Collectin Liver 1 (CL-L1) Collectin Placenta 1 (CL-P1)
Describe the 4 domains of collectins
- Carboxy Terminal C-Type Lectin Domain
- Alpha-helical coiled coil
- Collagen-like triple helix
- N-Terminal collagenous region
Describe the role of SP-D in immunity
Recognises and binds SELECTIVELY to the surface of viruses, bacteria and fungi and directly to the LPS on G-ve bacteria
Results in aggregation of microorganisms with ENHANCED PHAGOCYTOSIS by neutrophils and macrophages
First discovered in alveolar space, secreted by alveolar type II cells and non-ciliated bronchial epithelial cells
Describe the role of SP-D in H. pylori infections
SP-D is expressed and upregulated in the presence of H. pylori infection
binds and agglutinates and inhibits motility in a calcium dependent, lectin specific manner (Murray et al 2002)
Not all H. pylori agglutinate in the presence of SP-D - escape variants present (Khamri et al 2005)
Higher proportion of SP-D binding organisms in the mucus - suggesting that SP-D binding organisms are trapped in the mucus where they can be cleared through physical elimination (Khamri et al 2005)
H. pylori evade SP-D by varying the LPS antigenic structure
Describe the role of dendritic cells in innate and adaptive immune response
link between the two
Uptake of CFDA-SE labelled H.pylori by transferrin-Alexa fluor labelled DCs
After 15m of incubation in the absence and after 15m incubation of SP-D and calcium
Uptake by DC was significantly enhanced in the presence of SP-D
Concluded that:
In the absence of SP-D, mice are more suceptible to low dose of H. pylori
Neutrophil responses are diminished in the absence of SP-D due to the absence of chemotactic function of SP-D
In the absence of SP-D, T cell responses are diminished probably due to impaired Ag uptake by DCs
Summarise the differences between innate and adaptive immune responses
INNATE: immediate low specificity same every time hard wired reflex
ADAPTIVE: delayed high specificity (recognises specific aa sequences) increases with repeat exposure sophisticated control (e.g. T-reg) "voluntary complex movement"
What are the two divisions of the innate immune system?
Sensing and Responding
Describe innate immune sensing
Senses
- self vs non-self
- danger signals - you can present foreign antigens without a danger signal hence not responding e.g. chronic HepB infection
mainly related to detecting PAMP receptors
- LPS, flagellin, RNA
Describe the different types of PAMP receptors and what they recognise
Collectins (mannose binding lectin, SP-A, SP-D)
- bacterial/fungal carbohydrates
C-type lectins - recognise carbohydrates
- fungal cell wall (b-glucan)
Pentraxins
- phosphocholines
TLRs
- microbial genomes
- bacterial/fungal cell walls
- flagellin
NOD-like receptors (intracellular)
- Viral RNA
- bacterial cell wall
RIG-1 receptors (intracellular)
- viral RNA
AIM2-like receptors (intracellular)
- cytosolic DNA
Define Dendritic cells’ role in immunity
immature DCs are not localised and sample antigenic proteins in tissues
interaction with foreign Ag leads to MATURATION –> localisation to LNs
present the antigen to CD4+ –> proliferation
What is the danger model of DCs
Matzinger 2002
DCs want to find a non-self Ag or a danger signal
The immune system is more concerned with damage than with foreignness –> called into action by alarm signals from injured tissues rather than the recognition of non-self
HYPOTHESIS OF DAMPs - Danger associated molecular pattern receptors
How does the danger model work?
DCs stimulate CD4+ and co-stimulate it with CD40 only in the presence of a danger response –> appropriate Th1 response
If it doesnt encounter a danger response –> regulatory response instead –> Tr1 for immunoregulation activated
Describe the structure of TLRs
have an extracellular region with contains Leucine rich repeats (LRRs) + cytoplasmic tail with Toll/interleukin-1 receptor (TIR) domain
Different TLRs recognise different surface and intracellular components of microorganisms
Summarise the types of TLRs
TLR2 + TLRX
- lipoproteins
- peptidoglycan
- zymosan
- present in Gm4ve, yeast, fungal infections
TLR3
- double stranded RNA
TLR4
- LPS
- present in G-ve bacteria
TLR5
- flagellin
TLR7
- single stranded (viral) DNA
TLR9
- hypomethylated CpG DNA
- hypomethylation indicates bacterial DNA (not host)
Describe the distribution of TLRs
most expressed on cell surface
Some only useful inside endosomes - related to bacterial/viral invasions thar are engulfed in phagocytic cells
- 3,7,9 implicated
Describe the roles of TLR5
involved in FLAGELLIN DETECTION
non-flagellated e.coli is not detected by TLR5
If induction of listeria flagellin to e.coli – TLR5 expression increases
Salmonella –> normally has flagella and expresses TLR5
– KO flagella causes dampening of TLR5 expression
TLR5 muttion - Hayashi et al 2001
- causes non-response
- associated with recurrent UTIs
Describe the roles of TLR2
Detects a range of bacterial components
Arg677Trp mutation predisposes pt towards lepromatous (rather than tuberculoid) leprosy
tuberculoid leprosy: immune response can contain infection
lepromatous leprosy: inadequate T-cell response due to inadequate innate response
Describe the roles of TLR9
TLR9 KO - decreases response to Helicobacter + decreased inflammation
Describe the roles of TLR4
Does not induce signal by itself and REQUIRES CO-FACTORS
- Bacterial LPS interacts with liposaccharide binding protein (LBP)
LBP interacts with CD14 on cell membrane
this causes interaction with TLR4 with adaptor molecule MD2
SO: multiple host proteins are needed to respond to LPS - not single ligand
Describe the roles of TLR3
involved in recognition of double stranded RNA
HCV blocks TLR3 signalling via NS3/4 protease production thereby preventing body from inducing IFN responses via IFR3 and leading to PERSISTENT VIRAL INFECTION
2003 - FIND THE REFERENCE?????
Imran et al 2012?
Describe the interactions between TLRs and the TIR domain
triggers the activation of the innate immune system + development of acquired immunity
Cytoplasmic tails of TLR show similarities to IL-1 receptor
TLR signalling pathways originate from the TIR domain (cytosolic component) as a result of its recruitment of TIR-domain-containing adaptors
Crucial proline residue in all TLR TIR domains, except TLR3
All TLRs likely have a MyD88 pathway (Except TLR3)
MyD88 - common adaptor to TLRs - myeloid differentiation primary-response protein 88
MyD88 KO mice have no response to LPS - ESSENTIAL TO INFLAMMATORY SIGNAL (to activate NFkB)
MyD88s –> splice variant of MyD88 - downregulates the inflammatory response
Describe the role of TIRAP, TRIF, TRAM
MyD88 independent pathway
- TIRAP -
TIR domain-containing adaptor protein
Common mutation: S180L
Wild-type TIRAP –> strong NFkB signal
- TRIF -
TIR domain containing adaptor protein inducing intrferon (TLR-3)
TRIF binds to IRF-3 to produce IFN
- TRAM -
TRIF related adaptor molecule
What regulates the TLR-signalling pathways?
TLR-inducible molecules NEGATIVELY regulate TLR signalling
IRAK-M SOCS1 MyD88s SIGIRR ST2
What genes are regulated by NFkB?
Activation of NFkB by binding of TLRs (except TLR3) leads to the activation of:
Inflammatory cytokines - TNF, IL-1, IL-6, IL-12, IFNb
Chemokines - IL8
Adhesion molecules - ICAM-1, VCAM-1
Immune effector mlecules - FasL, iNOS
Pro-survival molecules - Bcl-XL, A1, cIAP1,2
Summarise the role of TLRs in immunity
TLRs have an extracellular region containing LRRs and a cytoplasmic tail which has a Toll/IL1 receptor (TIR) domain
Different TLRs recognise different surface and intracellular components of microorganisms
The interaction between TLR and a microbial component triggers the activation of the innate immune response as well as the development of acquired immunity
TLR-signalling pathways originate from the TIR domain
as a result of its recruitment of TIR-domain-containing adaptors such as MyD88, TIRAP, TRIF and TRAM
Signalling through each TLR requires MyD88 for the production of inflammatory cytokines
HOWEVER a MyD88-independent pathway exists for signalling through TLR3 or 4 and leads to the production of IFN1
The TLR ignalling pathways are negatively regulated by TLR-inducible molecules such as IRAK-M, SOCS1, MyD88s, SIGIRR, ST2
Examples of cytoplasmic PAMPr
NOD2
RIG1
Describe the function of NOD2
Bacteria cell walls contain peptidoglycan which can be broken down to MDP
MDP interacts with NOD2 through the LRR region which can lead to two pathways
1-
binding results in oligomerisation of NOD2 and recruitment of RIP2 through homotypic CARD-CARD interactions
RIP2 activates IKK complex through IKKy/NEMO, resulting in phosphorylation, ubiquitination and degradation of IkB and release of associated NFkB members
2-
NOD2 activation can also result in the activation of MAPkinase pathways resulting in induction of AP-1 transcription factors
Peptidoglycan can also activate the TLR pathway (TLR2/6)
Signals via MyD88 to stimulate MAP kinases: p38, JNK, ERK causing crptidins and defensins
Describe the function of RIG-I
RIG-1 signalling is in the same pathways as TLR - NS3/4 pathways also interferes with this system
HCV proteins NS3/4A eliminates antiviral signalling by cleavage of MAVS and TRIF
Production of dsDNA by HCV during infection should result in synthesis of IFNb which in turn leads to production of antiviral proteins known as ISGs
PAMP receptors RIG1 and TLR3 signal through kinases IKKe and TBK1 to activate IRF3 and cause the phosphorylation and degradation of IkB which allows for the nuclear translocation of NFkB.
HCV overcomes this response and establishes chronic infection through cleavage of MAVS and TRIF
Give examples to the innate immune effectors
PHYSICAL BARRIERS
- skin
- oral and GI mucosa - acid and pepsin
- mucus membranes - mucociliary clearance
CHEMICAL BARRIERS
- complement system
- defensins
- proteases, DNAses, RNAse
CELLULAR BARRIERS
- macrophages
- NK cells
Describe the complement system in immunity
• Key role in innate and antibody-mediated immunity
• Made up of a complex series of proteins and glycoproteins
- Majority are found in solution in the blood
- Some are membrane bound
- These compounds are mainly produced by the liver, some by monocytes and macrophages
- Triggered by enzyme cascade system in blood
- Once triggered, produces a very rapid and highly amplified response
- Activation occurs through three distinct pathways:
- Alternative (main)
- Continuous tick-over production
- Full activation upon fixation to foreign substances (bacteria) - Classical pathway
- Activated by antigen-antibody complexes - Lectin pathway
- Antibody-independent activation of Classical pathway
Classical and Alternative pathways converge at C3, the third component of complement, leading to the final common pathway
The common pathway leads to the formation of the Membrane Attack Complex (MAC), which causes cell lysis and death
What are the roles of the complement pathway?
- Opsonisation of microorganism for phagocytosis
- Direct killing via MAC
- Promotion of inflammation
- Chemotaxis of neutrophils and leukocytes
- Processing of immune complexes (dysregulation of this can lead to conditions such as SLE)
- Augments induction of specific antibody
Describe the recruitment of monocytes and macrophages
Inflammation leads to chemokine release, which recruits monocytes via concentration gradient
recruitment also involves using adhesion molecules that attache these and allow transmigration into the cell
Monocytes are localised and enter tissue to become macrophages
Different signals result in different types of macrophages (M1 + M2)
M1
- Pro-inflammatory
- Phagocytose bacteria/viruses/dead cells
M2
- Involved in the resolution of inflammation
- Deactivated, alternatively activated, activated (more complex)
- Produce anti-inflammatory cytokines + TGFb + efferocytosis (apoptotic/necrotic cells are removed by phagocytic cells)
Describe how microbes are killed by monocytes and macrophages
The microorganism is enclosed in a phagolysosome within the phagocyte
Many hostile factors within the lysosome
- Low pH
- Hydrolyases, proteases, defensins
- ROS (most important chronic granulomatous disease = enzyme producing ROS Is defective)
Some microorganisms can escape/survive
- TB can survive acidity with the waxy coat
- TB also inactivates IFN-y signalling, which is required for ROS production
When the microorganisms are broken down, their antigens are loaded onto MHC II, to be presented to Th molecules
Describe how monocytes and macrophages achieve antigen uptake
Phagocytosis: Involves the ingestion of particulate material including whole pathogenic microorganisms. The plasma membrane expands around the particulate material to form large vesicles called phagosomes (10-20times larger than endosome) [endocytosis is macromolecule ingestion]
Phagocytosis begins with engagement of the bacteria with cell surface receptors
These trigger changes in the cytoskeleton thereby triggering phagocytosis
End result: pathogen broken down in the endosomal compartment so that it is expressed on cell surface receptors MHC Class I and II so that it can stimulate T-cell responses (link between innate and adaptive immunity)
Describe the role of defensins in immunity
- Short (25aa) peptides, containing multiple disulphide bonds
- Very toxic to bacterial cell walls
“punch holes in bacterial cell walls”
• Produced by Paneth cells in the crypts
Describe the applications of Molecular Diagnostic Methods in Pathology
• Infectious disease
o Virus, Bacteria, Parasites, Fungi
• HLA typing (transplantation)
• Cancer (genomics)
• Genetic diseases (e.g. cystic fibrosis)
• Chromosomal abnormalities (Trisomy 21)
• Immune deficiencies diagnosis
o (e.g Severe combined immunodeficiency – “bubble baby disease”)
What is the Key to Success of Molecular Methods
- For a new diagnostic technology to become a mainstream tool it has to be faster, cheaper and better than existing alternatives
- Concept coined as the iron triangle (Daniel Goldin – NASA)
Advantages to Molecular Diagnostic Methods
• Fast results
o Results in 2h some POC systems in 15-20 min
• More specific and sensitive than traditional tests
o Viral culture / Minimal Residual Disease Monitoring
• Can screen multiple targets / pathogen at the same time
o (Multiplex e.g. Respiratory viruses)
• Tests can be developed quickly in response to a need
o H1N1 swine flu outbreak
• Cost effective
o Can be integrated to full automation minimising costs
Automation has superceeded previous techniques so that a lot of molecular diagnostics is carried out in massive automated machines (even in a single cartridge). Future direction: moving this to point of care/bedside, away from a lab for the quickest results.
What are the variants in molecular testing
• Qualitative - Detection presence or absence of DNA or RNA
• Quantitative - Measurement levels of DNA/RNA in a sample
• Genomic analysis – Mutation screening / HLA-typing transplantation/pathogen analysis
• Detection of gross chromosomal abnormalities - (Trisomy 21)
o Array-based comparative genomic hybridization (CGH)
o Chromosomal translocation – i.e. no need to amplify just looking for a mass abnormality
Describe the technology used in quantitative and the qualitative technology used in molecular testing
- Extraction
a. Isolation and purification of DNA and/or RNA - Amplification
a. DNA – PCR
b. RNA – RT-PCR (reverse transcription to convert to cDNA + PCR) - Detection
a. Real-time PCR (amplification by fluorescent dyes light emission)
b. Detection by gel PCR product separation (Multiplex)
c. Microarrays/Next Generation sequencing (fluorescent dye)
d. Luminex (laser light emission/detection – Multiplex)
e. Electrospray Ionization Mass Spectrometry
f. Electrochemical – pH change – nanotechnology
Describe the process of EXTRACTION in molecular testing
High quality is a key element to a reliable molecular test
Steps involve mainly lysing the cells and solubilise the DNA/RNA and removing contaminating molecules [silica resin is used to separate DNA/RNA from other molecules, and centrifugation to remove the purified DNA (by changing pH)]
Available isolation kits from a number of companies (eg:Roche, Qiagen, Ambion) – so it can be done manually or automated
- Current automated platforms use liquid handling with packed silica resin or magnetic bead technology
- – Reduces staff time: can take as little as 5-10mins to 2 hours
- – Introduces traceability
- – Minimises potential contamination
Describe the process of AMPLIFICATION in molecular testing
In order to amplify a specific genomic region, you need to design forward and reverse primers by starting and finishing thee primers where differences in bases lie between different types of viruses
E.g. if designing a sequence for Neisseria menigitidis, you will pick primers where sequences differ from Neisseria gonorrhoea
If using real-time PCR you need to use a method of detection hence the use of a fluorescent DNA probe that can be detected by the machine as amplification carries on (instead of forward and reverse primers)
Amplification process:
PCR detects by making replicates of the target region to the point that it is visible to the naked eye or instrumentation
Describe the basic PCR protocol
A tube that contains reagents to produce copies of DNA:
Buffer –> create optimal conditions for activity of Taq DNA polymerase
Mg++ –> acts as a cofactor and is a catalyzer in PCR. That means, higher concentrations of MgCl2 increases higher productivity of Taq polymerase.
Nucleotides (A, C, T, G)
Taq DNA polymerase enzyme
Primers located at two defined positions
A thermocycler (35-40 cycles)
Heats the tube >95oC for extracted DNA to open the double strand to two single strands (Denature)
Cools tube to a temperature optimum for “complementary” Primer DNA single strand to bind to one of each open extracted DNA strands (Anneal)
Raises the temperature marginally to optimum for the Polymerase enzyme to make copies of DNA starting from the each primer (Extension)
Repeats the same process 35 to 40 times
• Key points:
o Detection of the unique region is defined by designed short
o single strands of “complementary” DNA Primers
o Primers are designed to produce products of a specific size
o (nucleotide base pairs (bp) (typically: 80bp -500bp -2000bp)
o PCR only works with DNA. RNA has to be converted to DNA by the Reverse Transcriptase Enzyme before PCR
Describe the process of DETECTION in molecular testing:
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis – End Point Detection
• Primers binding on specific locations on each DNA strand and define the size of PCR product
• DNA has Negative Charge
• Small DNA PCR products move faster in a separation matrix than larger products
• Dyes such as Ethydium bromide or SybrGreen bind to double strand DNA and fluoresce when exposed to UV light
• Example: detection of chromosomal translocation (T: 11, 14) in a patient with suspected Mantle Cell Lymphoma
Real Time PCR
• Useful when time-constrained
• Detection of amplification as it is occurring by PCR machine (in real life report not made until the process is finished)
• Machine: TaqMan Probe
o Addition of primers with labelled flourescent dyes on the 5’ end and a quencher at 3’ end
o At the 5’ end fluorescence emits a wavelength to quencher at 3’ end quencher absorbs this and emits a second wavelength detector records second wavelength emitted by quencher
• Fluorescence increases in the reaction vessel and accumulates at the end of each PCR cycle.
• Fluorescence increases exponentially due to logarithmic increase in PCR product
• CT= Cycle Threshold - PCR cycle where you observe the first detectable level of fluorescence above background (baseline) – Lower CT equates to higher the DNA concentration input
• Quantitation requires a reference standard to read level against
Describe the process of DETECTION in molecular testing:
Real Time PCR
Real Time PCR
• Useful when time-constrained
• Detection of amplification as it is occurring by PCR machine (in real life report not made until the process is finished)
• Machine: TaqMan Probe
o Addition of primers with labelled flourescent dyes on the 5’ end and a quencher at 3’ end
o At the 5’ end fluorescence emits a wavelength to quencher at 3’ end quencher absorbs this and emits a second wavelength detector records second wavelength emitted by quencher
• Fluorescence increases in the reaction vessel and accumulates at the end of each PCR cycle.
• Fluorescence increases exponentially due to logarithmic increase in PCR product
• CT= Cycle Threshold - PCR cycle where you observe the first detectable level of fluorescence above background (baseline) – Lower CT equates to higher the DNA concentration input
• Quantitation requires a reference standard to read level against
Describe the process of DETECTION in molecular testing:
Digital PCR
- Digital PCR is a real-time PCR amplification of single template molecules
- Nucleic acid molecules are partitioned to hundreds to thousand reaction wells dilution
- Some wells receive 0,1,2, 3 or more molecules.
- Following PCR, positive and negative wells are counted and Poisson distribution is used to identify how likely it is that a positive well contains one or more templates given the fluorescence data.
- The key to accurate Poisson analysis lies in optimizing the ratio of the number of positive
- events to the total number of independent events
- Unlike real time PCR - No need to rely on references or standards to quantify
- Enables linear detection of small-fold changes
Describe the process of Sanger sequence analysis
- Termination sequencing reaction template DNA (double stranded)
- Primer targeting one strand (specific to your target)
- DNA polymerase (heat stable, e.g., Taq), Mg2+, Suitable buffer
- a small amount ofdeoxytriphosphates ddATP / ddCTP/ ddGTP/ddTTP [stops further nucleotide addition each fluoresce differently]
End Result: generation of a range of primer lengths that can be separated so that each base can be read out to determine the sequence of the template
Sequencing Products separated on Size by Acrylamide Gel Seperation
• Capillary sequencer modification one sequencing reaction containing dideoxy nucleotides ddATP, ddTTP, ddCTP ddGTP
• Each terminator with a different label previously sequences read by hand but now substituted by fluorescent dyes that can be translated by software (see right)
• This is useful to determine defects in single nucleotide sequences
o E.g. complement defect in a patient this can be sequenced and compared to normal and you can see SNP difference in the 80th position
• Useful for determining sequence for 1-6 genes
Describe the process of next generation sequence analysis
• Next generation sequencing principle à take whole DNA and chop it into different pieces using a microchip that separates each reaction
• Each position on the microchip will complete seperate individual reactions (can do around 30000 reactions for one particular position), amplifying through primers
• Hence you are looking at multiple sequences, so must use bioinformatics to assemble the information together
o Examples: identify a new species that causes disease, identify something in the DNA that is causing a specific disease
• Process takes weeks and months, however if you have a target NGS this allows for a quicker processing (used in cancer e.g. chronic leukaemia panel)
• Useful for determining whole genome sequencing i.e. several thousands of genes
Describe the procedure of DNA/CGH Microarrays
- Array comparative genomic hybridisation (CGH)
- Analyse whether sections of DNA are either missing or present in extra copies
- The main benefit of array CGH is that it is able to detect small genetic changes and can provide accurate information on the size and the possible effects of the genetic changes found.
• Process:
o Exploits the ability of a DNA molecule (strand) to bind/ hybridise to another DNA
o molecule
o DNA from the patient is “digested” to small fragments
o These fragments are labelled with a coloured fluorescent dye
o Reference DNA, from a person / pool of people without genetic abnormalities is labelled with a different coloured fluorescent dye.
o Reference and patient samples are mixed together and applied to the chip and hybridisation takes place
o The chip is then scanned in a microarray machine scanner which measures the amount of red and green fluorescence on each probe.
o The scanner calculates the ratio of the red to green fluorescent dyes
o Correct amount of DNA (YELLOW) -Too much DNA (RED = duplication) - Too little DNA (GREEN = deletion)
Describe the procedure of Flourescent in situ Hybridisation (FISH)
- Is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity
- Uses fluorescent probes to targets a specific nucleic acid sequence within a histology sample / chromosomes
- Useful in detecting translocation in cancer i.e. if you paint chromosomes in different colours, you can see where big translocations have taken place as two different colours will be present on a single chromosome (cancer)
Describe the applications of Molecular Diagnostics in Pathology
- Diagnosis
- Therapy selection
- Therapy monitoring
- Surveillance
- Infection control
- Predisposition
Application of PCR product size separation
- Malignant lymphoma - clonal rearrangement analysis B and T cell Lymphoma
- Thalassemia detection
- HLA tissue typing for transplantation (these days using luminex)
Application of real time PCR
• Virology viral detection and viral loads
o HIV, HCV, HBV, CMV, VZV, HPV, EBV, FluA, FluB, etc
• Microbiology
o Screening tests Gonorrhoea/Chlamydia (GC/CT)
o Mycobacterium Tuberculosis (antibiotic resistance)
o Invasive fungal disease post bone marrow transplantation
o Genito-Urinary Medicine - Sexual Health associated pathogens – rapid diagnostics
• Oncology (diagnosis, staging, prognosis, monitoring) (+/- ) DIGITAL PCR
o Colorectal cancer-KRAS & NRAS -Activating mutation-Resistance to anti-EGFR treatment
o Non-small cell lung cancer –EGFR- Activating mutation- Response to anti-EGFR TKI treatment
o Non-small cell lung cancer –ALK- Translocations -Response to crizotinib treatment
o Leukaemia BCR-ABL Fusion Gene level detection
Application of PCR + Sequencing or NGS
• HIV treatment resistance mutation analysis
o Allows practitioner to change the regime in HIV therapy if patient is not responding sequence baseline virus typing to identify mutations that can be targeted by specific regimes
• HCV genotype identification
o Some drugs work better under certain mutations
- Identify by sequencing bacteria / fungi from aseptic fluids
- HLA–> High resolution tissue typing to enable bone marrow transplantation (need very good match if you do BM transplant so you need sequence a few target regions)
- Risk factors / predisposition (e.g. Coeliac Disease HLA-DQ2.5, DQ8 or DQ2.2 – used particularly in paediatrric patients)
- Mutation screening in cancer cells and gene defects in primary immunodeficiency (e.g complement genes)
Application of FISH
- Chromosomal translocation in various solid tumors and lymphomas
- Breast cancer and herceptin treatment - her-2/neu testing
- Glioma diagnosis and prognostication - chromosome 1p19q deletion
Other benefits of molecular diagnostics
- The ability to scale up rapidly a molecular method makes it ideal for screening programs
- Used for personalised treatment
Summarise innate vs adaptive immunity
INNATE
- primitive
- cellular contents: monocytes, macrophages DCs, NK, mast cells
- humoral contents: complement, cytokines, acute phase reactants, antimicrobial peptides
- receptors: PRR receptors e.g. TLRs, phagocytic receptors
- rapid
- limited diversity
- low specificity
- some memory
- no self-discrimination
ADAPTOVE
- advanced
- cellular: T, B cells, APCs, NKT cells
- humoral: immunoglobulins
- receptors: TCR, BCR
- slow
- diverse
- specific
- long lived memory
- self-discriminatory
Describe the structure of the TCR
95% composed of a and b chains
(5% y and d chains)
diversity arises from genetic recomination of the DNA encoded segments in individual T cells by recombination using RAG1 and RAG2 recombinases
Describe T cell development
T cells derive from haematopoetic stem cells from BM
migrate and colonise thymus
undergo beta selection, positive selection and negative selection
- DN development and Beta selection
first, they lack both CD4 and CD8 = DN
progress through development characterised by CD44 and CD25 expression
During DN2/DN3 RAG dependent recombination, B-chain production occurs
Signal for proliferation, survival and upregulation of CD4 and CD8 = DP
Cells that don’t suceed die by apoptosis
- Positive selection
DP move deeper into cortex of thymus
interact with cTEC presenting self-antigen
2% receive survive signal
98% die by apoptosis
ones that recognise MHCII downregulate CD8 –> become SP CD4 cells
ones that recognise MHCI downregulate CD4 –> become SP CD4 cells
migrate into medulla of thymus
- negative selection
process of autoregulation
DCs in medulla cross present antigen to CD4+ thymocytes on MHCII
Ones that react with too high an affinity are eliminated by apoptosis or commited to become Tregs
Cells that survive exit as naive t cells
B cell central tolerance
heavy chain rearrangement by RAG1 and 2
Formation of a pre-BCR with heavy chain pairing with a surrogate light chain
Antigen independent triggering of pre-BCR promotes RAG downregulation and proliferation
Differentiation to small pre-B-cell with downregulation of SLC and reexpansion of RAG - light chain rearrangement
Autoreactive BCR eliminated by apoptosis OR receptor editing of light chain
Compare B cell and T cell central tolerance
SITE:
Thymus vs BM
RAG-mediated recombination
- T: sequential RAG mediated recombination of B chain locus then a chain locus
- B: sequential RAG mediated recombination of heavy chain and then light chain
Formation of pre-receptor
- T: pre-TCR formed by b chain plus pre-Ta
- B: pre-BCR formed by heavy chain plus surrogate light chain
Positive selection
- T: MHC recognition and binding
- B: tonic BCR signalling
Negative selection
- T: death by neglect, no editing
- B: receptor editing alter antigen specificity by changing light chain structure OR death by neglect
Regulatory cell formation:
- T: Treg produced during -ve selection
- B: none
Methods of peripheral tolerance
- Ignorance
- Anergy - lack of co-stimulation
- Anergy – engagement of inhibitory receptors
- APC control of T cell activation (Innate:adaptive interface)
- Regulatory T cells
process of ignorance
- Naïve T cells circulate blood to secondary lymphoid organs to lymph then back to blood
- Limited access to many TRA
- Immunologically privileged sites e.g testes, brain, placenta, where T-cells can’t cross to
Anergy in the absence of appropriate co-stimulation
Two Signal Model
o In the interaction between an APC and naïve T cell, two signals are required in order to activate the T cell:
Signal 1: TCR receptor ligation with MHC-peptide complex
Signal 2: Appropriate co-stimulation via CD80/CD86 interacting with CD28
If both signals are present T cell activation and clonal expansion will occur
In the absence of signal 2 T cell anergy (functional inactivation) occurs
NB Kupffer cells and LSEC – induce tolerance to gut derived and dietary antigen
Anergy due to enagement of inhibitory molecules
‘Signal 2’ can also be actively blocked by an inhibitory receptor called CTLA4
Competitive inhibition of CD80/86 for binding to CD28
Sends inhibitory signal which induces T cell activation
CTLA4 is itself induced by T cell activation – provides a natural negative feedback loop (brake/checkpoint)
Multiple other inhibitory receptors exists, which limit T cell activation or effector functions.
Anergy in absence of appropriate co-stimulation & engagement of inhibitory receptors
APC, such as DCs, will alter their immunogenicity depending on the local milieu
In the presence of innate immune stimulation in a pro-inflammatory environment (presence of pathogens, necrotic cells – TLR stimulation) DCs will upregulate co-stimulatory factors and down regulate co-inhibitory receptors.
How to Treg limit T cell activation
o anti-inflammatory cytokine production
o Expression of co-inhibitory molecules eg CTLA4
o Direct cytotoxicity against T effector cells
Peripheral Tolerance Summary
- Multiple peripheral mechanisms exist to limit T cell activation and prevent autoimmune mediated tissue destruction
- T cell anergy (functional deactivation) can be induced during antigen presentation in the absence of appropriate co-stimulation or presence of inhibitory molecules
- Important role for innate:adaptive interaction, with APCs sensing inflammatory milieu and altering threshold of T cell activation
- Negative feedback mechanisms through immune checkpoints (eg CTLA4) limit excess T cell activation
- Regulatory T cells (both central and peripheral) have important roles in maintaining tolerance through their control of T cell activation
Consequences of breakdown in tolerance
- Impaired thymic negative selection…
• APECED – Autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy OR autoimmune polyglandular syndrome 1 (APS1
• AIRE (Autoimmune Regulator) gene mutation
• mTEC unable to generate tissue restricted antigens to developing T cells
• Peripheral escape of autoreactive T cells - Impaired regulatory T cell function
• IPEX Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome
• Rare X linked recessive condition
• Mutation in FoxP3 gene which is essential (master transcription factor) for Treg development
• Patients develop multiple autoimmune complications including enteropathy, type 1 diabetes, dermatitis and autoimmune anaemias - Impaired peripheral negative regulation
• Spontaneous autosomal dominant CTLA4 mutations described – Schubert et al Nat Medicine 2014
• Multiple autoimmune phenomena including enteropathy, thyroiditis, arthritis
• Immune checkpoint blockade for cancer therapy associated with Immune mediated tissue injury…
Induction and Function of Cytokines
A stimulus (e.g. viral RNA) induces receptor (e.g. TLR) on a cell triggering a response intracellular signalling pathways are activated and cytokine gene is activated, transcribed, translated and cytokines synthesised + released
Receptors for stimulus can be membrane bound at the cell surface, membrane bound in intracellular vesicles or soluble in cytoplasm
Cytokine then binds to target cell by a specific receptor (e.g. Type 1 interferon) and intracellular pathways are activated (e.g. JAK/STAT pathway) target genes synthesised (e.g. PKR) and tagrget genes will provide effects (e.g. intracellular inhibition of virus replication)
Cytokine effects:
o Autocrine: self stimulation
o Paracrine: stimulation of local cells
o Endocrine: distant cells
Nomenclature of cytokines
• Interferons
o Type I (IFN,a,b,o,t)
o Type II (IFNy)
o Type III (IFN-lambda1,2,3)
- Interleukins (IL-1, IL-2… IL-36) related in structure
- Tumour necrosis factors (TNFa, TNFb, FASL [FAS ligand], TRAIL [TND-related apoptosis-inducing ligand])
Classificaiton of cytokines
- Initial and innate cytokines act straight away
- Adaptive cytokines second layer of defence
- Chemokines attract cells of immune system when infection occurs
- Haematopoietic growth factors stimulate cells of blood system (crosses barrier of hormones and cytokines)
Initial and Innate Cytokines
IFNa/b
TNFa
Roles of IFNa/b
- There is one IFNb and multiple IFNa genes
- Made by most cells in response to viral infection
- Stimulates specific expression of interferon specific genes (ISG) (100s of ISGs)
• Other activities
o Activates NK cell cytotoxic activity
o Enhances MHC I antigen presentation
o Facilitation of T-cell IFNy responses
Roles of TNFa
- Upregulation of MHC I
- Immunoregulatory, antiviral pathways activated
- Stimulates cell proliferation
- Anti-apoptotic factors
- Stimulates liver cells produce C-reactive proteins
What are adaptive cytokines
• Produced mainly or exclusively by T cells
IL2, IFNy
IL2 • T-cells express IL2R • Autocrine growth factor • T-cell proliferation • Induces IFNy expression
IFNy
• Enhanced antigen processing and MHC presentation
• Switching of immunoglobulin classes
• Stimulates nitric oxide synthase (iNOS) required for T-cell killing of infected cells
What are chemokines
- Chemotactic cytokines of 8-12kd in size (smaller than other cytokines)
- Four families: (named according to number of intramolecular cysteine bonds by letter C)
C: one disulphide bridge
CC: two disulphide bridges
CXC: 2 disulphide bridges separated by any amino acid
CX3C: 2 disulphide bridges separated by any three amino acids
• Chemokines are a homing beacon for cell migration – concentration increases with the direction of chemotaxis
What is CCL3
macrophage inflammatory protein
- Induces the synthesis and release of other pro-inflammatory cytokines IL1, IL6 and TNFa from fibroblasts and macrophages
- Migration of protective NK cells into CMV infected liver
- Tissue inflammation – influenza, HSV, Coxsackie infections
Summarise when each cytokine is secreted
in all cases IFN-a/b are stimulated as initial and innate cytokine and IFN-y are simulated in adaptive response. These 3 are central to many virus infections.
Describe the role of TLRs in infection
- Currently 13 which recognise different PAMPs (microbial components e.g. dsRNA)
- Role in viral recognition on endosomal membrane
- Forms a major part in innate immune responses
This may result in interferon production
Explain how HCV is able to block TLR/RIG-I activation
- NS5A blocks Myd88 when TLR7/8 is activated, reducing its activity
- NS3/4A cleaves both TRIF and MAVS
- This therefore shuts off IFNa/b production and is one of the ways it can persist
- Virus has multiple layers of defences to host responses outcome of infection will depend on which way the balance of these two will tip
Describe IFN function
- IFNa binding its receptor causes activation of Janus kinase pathways or JAK/STAT pathway
- JAK is present in the intracellular part of the IFN receptor, and binding causes its binding to the STAT molecules in the cytoplasm (phosphorylation)
- STAT activation causes dimerisation, which migrates the nucleus –> stimulation of ISG expression expression of IFN effectors (antiviral, antiproliferative, immunoregulatory responses)
- Different IFNs stimulate different JAK molecules (Tyk2, part of the Jak family) AND different STAT molecules –> different effects
Cytokine Jak STAT IFNy Jak1/2 1 IFNa/b Tyk2, Jak1 1, 2, 3 IL2 Jak1, Jak3 3, 5 IL6 Tyk2, Jak1/2 1, 3 IL12 Tyk2, Jak2 3, 4
Summarise the different mechanisms of IFN action
- 2’,5’-oligoadenylates (2-5A)
- Protein kinase R (PKR)
- Adenosine Deaminase (ADAR)
- Mx Proteins (MxA) binding proteins
- Others (e..g inos)
Describe how the 2-5A pathway works in IFN action
- IFN1 stimulated
- Enzymes stimulated by dsRNA binding
- Binding synthesises 2-5 adenosine from ATP
- 2-5 adenosine binds and activates RNAaseL (ribonuclease L) –> dsRNA degradation –> this will reduce viral replication (if this does not occur, the cell will apoptose)
Describe how the PKR pathway works in IFN action
- Activated when it binds dsRNA
- PKR is normally inactive in uninfected cells
- Two PKR molecules bind to the segment of dsRNA, and phosphorylate each other (autophosphorylation) activation
- Activated PKR causes inactivation of eIF-2a inhibition of translation
• Other viral inhibitors:
o Adenovirus: VA RNA
o Influenza: P58IPK
Describe how the ADAR pathway works in IFN action
Catalyses the deamination of adenosine to inosine in viral dsRNA –> changes protein coding capacity of the RNA (site specific amino acid substitutions)
Describe how the Mx protein pathway works in IFN action
- MxA and MxB GTPases are induced by type 1 IFNs are involved in endocytosis and vesicular transport
- Mx protein monomers oligomerise, trapping viral components and inhibiting transcription, transport and assembly
- So although the viral particles are created, they cannot be exported out of the cell
Describe the role of SOCS in immunity
Suppressors of Cytokine Signalling (SOCS)
• Induced by cytokines and negatively regulate their activity, which viruses can exploit to reduce antiviral responses (HIV1, HCV, HBV, HSV1, RSV)
Describe the process of SOCS degradation of JAK/STAT proteins
• SOCS protein contains:
o Kinase inhibitory region (KIR)
o SH2 region which binds phosphotyrosine
o SOCS Box which binds ubiquitination complex
- SH2 region binds the phosphotyrosine on JAK + the SOCS Box binds a ubiquitination complex –> JAK degraded by proteasome
- Also by binding to JAK, reduces its binding to STAT –> competitive inhibition
- So: both competitive inhibition of binding + degradation of JAK –> decreased JAK/STAT response
Describe how SOCS can be used as an antiviral therapy
- HCV core protein induces SOCS3 increases virus replication
- It can lead to glucose intolerance, as SOCS mediates ubiquitination of the insulin receptor
- Genotype 1 patients have highest SOCS3 level and are most resistant to IFNa therapy
- Suppression of SOCS, using SOCS siRNAs which mimic the peptide of phosphorylated JAKs activation loop are being trialled
What are NK cells?
- Mononuclear lymphocytes
- Innate immune system
- Immediate immune response to pathogens
- No need for selection or clonal expansion
- Activation depends on a fine balance between signals from activating and inhibitory surface receptors
- Presentation of self peptides on MHC class I provides POTENT inhibition of NK cells via KIR (Killer cell Immunoglobulin-like receptors)
- Note that almost all cells in the body present MHC class I
- Loss of inhibition via KIR plus positive stimulation via activating receptors results in activated NK cells
- = the MISSING SELF hypothesis
- Virally infected or transformed malignant cells express abnormal peptides on MHC class I resulting in loss of NK inhibition via KIR
• NK cells activated against cells identified as ‘non-self’ i.e. virally infected, transformed cells or cells from allogeneic transplants
NK cell functional outputs
cytotoxicity
Cytokine release
cytotoxicity by NK cells
o release of lytic granules forming perforations in the target cell wall
Lytic granules contain perforin and granzyme
o measured experimentally directly (chromium release), indirectly (CD107a expression on NK cell surface, target cell death)
o Antibody mediated cytotoxicity
Antibodies produced by plasma cells (activated B cells) coat infected cells expressing antibody specific antigen
CD16 receptors on NK cells bind the Fc portion of the antibody and induce NK cell mediated cytotoxicity and lysis of the target cell
Cytokine release by NK cells
o IFN gamma supporting monocyte/macrophage responses
o TNF alpha – proinflammatory, triggers apoptosis/cell death in target cells
o Chemokines – recruit other immune cells
NK cells in Liver Immunology
- NK cells make up 50% of the normal lymphocyte infiltrate o Liver is inherently tolerogenic to prevent abnormal activation Inhibitory Cytokines: • IL18 • IL10 • TGFβ1 Inhibitory ligands: • PD-L1 • B7H6
Differences between hepatic and peripheral NK cells
o Stegmann et al
o CXCR6 defines liver NK cells
o Relative anti-inflammatory function in liver NK cells compared to peripheral NK cells
o Reduced CD107a post activation on CXCR6+ Liver NK cells compared to CXCR6- NK cells (peripheral NK cells)
o Reduced Granzyme B and Perforin secretion by CXCR6+ Liver NK cells – key molecules within lytic granules released by NK cells when killing target cells
o Increased TRAIL expression in liver NK cells – alternative method used by liver NK cells to induce target cell death by inducing apoptosis
Controlled apoptosis less inflammatory response
Cirrhosis mediated immune dysfunction
- Patients with cirrhosis are susceptible to infection
- Patients with cirrhosis are susceptible to hepatocellular carcinoma
- Some changes might be aetiology dependent:
o Chronic alcohol intake in mice impaired NK cell function
o Reduced NK cell cytotoxicity in chronic HCV infection despite increase activating receptor NKG2D expression
Why are NK cells important in Cancer?
- NK cells make up 50% of the normal lymphocyte infiltrate – cytotoxic tumour immunosurveillance
- NK cells within the tumour are hypofunctional and low in number – correlates with poorer HCC survival
- Reduced number of NK cells in advanced HCC tumour tissue
The ‘Missing Self’ Hypothesis
- Tumours express abnormal peptides on MHC-1 and downregulate MHC-1 expression
- Allows tumours to escape T cell immunosurveillance
- NK cells are potently activated when they interact with cells lacking the inhibitory MHC-1 expression
• BUT- Tumour infiltrating NK cells are not activated despite this – Why?
The Tumour Microenvironment
- Immunosurveillance
o Cytotoxic immune cells
- Immunoediting
o interplay between malignant and non-malignant cells resulting in modification of non-malignant cells
o Dysfunctional NK cells within HCC tumour – high levels of surface CD107a but low levels of perforin
NK cells in hepatocellular carcinoma – how are they modulated?
Kumar and Khakoo, 2018
- NK cells can produce stress ligands (MICA), which should activate them, but they also secrete
- Activated NK cells which are further away from the tumour
- By the time they get to the cell, they are exhausted
- The expansion of MDSCs (myeloid derived stem cells) they have an inhibitory effect on NK cells
o Express TGFb-1 directly inhibits NKG2d and inhibits cytotoxic granular release
o Interaxts with NKP40 inhibitory effect on interaction with MDSCs
- Formation of nest with increased expression of CD48/2B4 which interacts with tumour associated monocytes, resulting in a potent activation, resulting in exhaustion before being able to exert effects.
Can we use what we know about NK cells for immunotherapy?
- Pre-activating and expanding NK cells ex-vivo
- Manipulate the balance of activating and inhibitory signals – blocking inhibitory receptors and over expressing activating receptors
- Allogenic NK transplants – recognising ‘non-self’cells and being activated by the over expression of stress ligands on tumour cells
- Up-regulating TRAIL expression
- Specific antibodies against tumour antigens – CD16 mediated NK killing
- Adapting NK cell lines (eg NK-92) to express chimeric antigen receptors (CARs) that target tumour cells for a more specific response
