Module 3.1: Basic Immunology Flashcards

Question

Desribe the importance of collectins in the gut

Answer 1

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

Answer 2

Mammalian C-type collagenous carbohydrate binding proteins C - as they require calcium for their action

Answer 3

``` surfactant protein A (SP-A) SP-D Mannan binding lectin (MBL) Collectin Liver 1 (CL-L1) Collectin Placenta 1 (CL-P1) ```

Answer 4

* Carboxy Terminal C-Type Lectin Domain * Alpha-helical coiled coil * Collagen-like triple helix * N-Terminal collagenous region

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

``` 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" ```

Answer 9

Sensing and Responding

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

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)

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

TLR9 KO - decreases response to Helicobacter + decreased inflammation

Answer 21

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

Answer 22

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?

Answer 23

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

Answer 24

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

Answer 25

TLR-inducible molecules NEGATIVELY regulate TLR signalling ``` IRAK-M SOCS1 MyD88s SIGIRR ST2 ```

Answer 26

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

Answer 27

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

Answer 28

NOD2 | RIG1

Answer 29

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

Answer 30

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

Answer 31

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

Answer 32

• 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: 1. Alternative (main) - Continuous tick-over production - Full activation upon fixation to foreign substances (bacteria) 2. Classical pathway - Activated by antigen-antibody complexes 3. 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

Answer 33

* 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

Answer 34

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)

Answer 35

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

Answer 36

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)

Answer 37

* 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

Answer 38

• 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”)

Answer 39

* 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)

Answer 40

• 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.

Answer 41

• 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

Answer 42

1. Extraction a. Isolation and purification of DNA and/or RNA 2. Amplification a. DNA – PCR b. RNA – RT-PCR (reverse transcription to convert to cDNA + PCR) 3. 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

Answer 43

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

Answer 44

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

Answer 45

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

Answer 46

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

Answer 47

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

Answer 48

* 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

Answer 49

* 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

Answer 50

• 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

Answer 51

* 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)

Answer 52

* 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)

Answer 53

* Diagnosis * Therapy selection * Therapy monitoring * Surveillance * Infection control * Predisposition

Answer 54

* Malignant lymphoma - clonal rearrangement analysis B and T cell Lymphoma * Thalassemia detection * HLA tissue typing for transplantation (these days using luminex)

Answer 55

• 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

Answer 56

• 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)

Answer 57

* Chromosomal translocation in various solid tumors and lymphomas * Breast cancer and herceptin treatment - her-2/neu testing * Glioma diagnosis and prognostication - chromosome 1p19q deletion

Answer 58

* The ability to scale up rapidly a molecular method makes it ideal for screening programs * Used for personalised treatment

Answer 59

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

Answer 60

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

Answer 61

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

Answer 62

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

Answer 63

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

Answer 64

* Ignorance * Anergy - lack of co-stimulation * Anergy – engagement of inhibitory receptors * APC control of T cell activation (Innate:adaptive interface) * Regulatory T cells

Answer 65

* 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

Answer 66

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

Answer 67

‘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.

Answer 68

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.

Answer 69

o anti-inflammatory cytokine production o Expression of co-inhibitory molecules eg CTLA4 o Direct cytotoxicity against T effector cells

Answer 70

* 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

Answer 71

- 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…

Answer 72

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

Answer 73

• 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])

Answer 74

* 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)

Answer 75

IFNa/b | TNFa

Answer 76

* 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

Answer 77

* Upregulation of MHC I * Immunoregulatory, antiviral pathways activated * Stimulates cell proliferation * Anti-apoptotic factors * Stimulates liver cells  produce C-reactive proteins

Answer 78

• 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

Answer 79

* 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

Answer 80

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

Answer 81

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.

Answer 82

* 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

Answer 83

* 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

Answer 84

* 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 ```

Answer 85

* 2’,5’-oligoadenylates (2-5A) * Protein kinase R (PKR) * Adenosine Deaminase (ADAR) * Mx Proteins (MxA)  binding proteins * Others (e..g inos)

Answer 86

* 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)

Answer 87

* 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

Answer 88

Catalyses the deamination of adenosine to inosine in viral dsRNA --> changes protein coding capacity of the RNA (site specific amino acid substitutions)

Answer 89

* 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

Answer 90

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)

Answer 91

• 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

Answer 92

* 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

Answer 93

* 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

Answer 94

cytotoxicity | Cytokine release

Answer 95

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

Answer 96

o IFN gamma supporting monocyte/macrophage responses o TNF alpha – proinflammatory, triggers apoptosis/cell death in target cells o Chemokines – recruit other immune cells

Answer 97

``` - 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 ```

Answer 98

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

Answer 99

- 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

Answer 100

* 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

Answer 101

* 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

Answer 102

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.

Answer 103

* 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

Module 3.1: Basic Immunology Flashcards

(128 cards)