Partridge Flashcards
What is the role of the IS?
- provides protection or immunity against infectious disease
- distinguishes between self and non-self
- may also recognise danger signals caused by damage to cells and tissues
What external threats are there to the IS?
- viruses
- bacteria
- fungi
- protozoa
- parasites
- prions
Do most MOs cause diseases?
- no
- but infectious disease accounts for 1/3 of all deaths
- some commensal, some pathogenic under certain conditions
- but can be opportunistic and cause infection if have access to specific area don’t usually
Why is the IS a ‘double edged sword’?
- can cause disease if inapprop activated
- doesn’t usually react against normally self or innocuous env materials (tolerant of these), eg. food stuffs, but can break down causing allergy
What is active immunisation?
- vaccination
- give mod form or component of pathogen to activate immune response, to gen memory so will recognise pathogen
What is passive immunisation?
- administration of immune components from immunised source, eg. antisera, antibodies
What are the main differences between the innate and adaptive IS?
INNATE:
- limited specificity
- resistance not improved by repeat infection
- rapid response (hours)
ADAPTIVE:
- highly specific
- resistance improved by repeat infection
- slower response (days-weeks)
What leucocytes are part of the innate and adaptive IS?
- innate = phagocytes, NK cells
- adaptive = B and T lymphocytes
What soluble factors are part of the innate and adaptive IS?
- innate = lysosyme, complement, interferons etc.
- adaptive = antibody
Where are cells of the IS derived from?
- all hematopoietic cells derived from pluripotent stem cells
- give rise to 2 main lineages, 1 for myeloid cells and 1 for lymphoid cells
What cells are derived from the common myeloid progenitor?
- megakaryocytes (platelets) and erythrocytes
- granulocytes = eosinophil, neutrophil, basophil, mast cell
- monocyte –> macrophage
What cells are derived from the common lymphoid progenitor?
- T helper lymphocyte
- T cytotoxic lymphocyte
- B lymphocyte –> plasma cell
- NK cell
What are the types of professional phagocytes?
- neutrophils
- mononuclear phagocytes
- mast cells
- dendritic cells
What are the characteristics of neutrophils?
- main phagocytes in blood
- short lived and fast moving
- specialised lysosomes release enzs, H2O2 etc.
- DIAG*
What are the characteristics of mononuclear phagocytes, and what diff cells are they in diff organs?
- long lived (months-years)
- help initiate adaptive responses
- brain = microglial cells
- lungs = alveolar macrophages
- liver = Kupffer cells (all slightly diff characteristics)
- monocyte in blood and macrophage in tissues
- DIAG*
What professional phagocytes can act as sentinel cells?
- macrophages
- mast cells
- dendritic cells
What are the characteristics of mast cells?
- often underlie mucosal surfaces
- release inflammatory mediators (eg. histamine)
- important in responses to parasites and allergy –> express high affinity Fc receptors for IgE
- DIAG*
What are the characteristics of a dendritic cells?
- in skin, mucosa and lymphoid tissue
- specialised in presenting antigen to T cells
- related to monocytes/macrophages but v specialised function
- DIAG*
What are the characteristics of NK cells?
- type of lymphocyte
- kill infected cells “non-specifically”
- poss anti-tumour role –> can detect alt self cells from infection, or mutation causing cancer
- receptors recognise alt self
What are the characteristics of receptors on phagocytes and other myeloid cells?
- broadly specific for large categories of pathogen
- pattern recognition receptors (PRRs) recognise pathogen assoc molecular patterns (PAMPs)
What types of infection are diff soluble factors involved in?
- defensins = disrupt bacterial cells
- complement = in bacterial infections
- interferons = in viral infections
What is the inflammatory response?
- integrated response to local infection
What is the difference between B and T lymphocytes, and the process by which they differentiate to fight infection?
- DIAG*
- both dev from same stem cell precursor in bone marrow
- MATURE: B in bone marrow, T in thymus –> this is antigen indep differentiation, in central lymphoid tissue
- RECEPTOR: B is antibody, T is T cell receptor –> this is antigen dep differentiation, in peripheral lymphoid tissue
- only differentiate further if encounter antigen, in lymph nodes, spleen etc.
- RESPONSE: B secrete antibody, T kill infected host cells and make cytokines
- IMMUNITY: B is humoral, T is cell-mediated
- INFECTIONS: B is ec bacterial and 2° viral, T is viral, intracellular bacterial and intracellular parasitic
- infection can result in prod of long-lived, specific memory B and T cells
What are the immunoglobulin (antibody) classes and their roles?
- IgG = main class in serum and tissues, important in 2° responses
- IgM = important in 1° responses
- IgA = in serum and secretions, protects mucosal surfaces
- IgD = don’t know much about role
- IgE = present at v low levels, involved in allergy and protection against large parasites
What are 2 subpops in cell mediated (T cell) immunity?
- T helper cells (CD4 +ve)
- T cytotoxic cells (CD8 +ve)
What is the role of T helper cells?
- help B cells make antibody
- activate macrophages and NK cells
- help dev of cytotoxic T cells
What is the role of T cytotoxic cells?
- recognise and kill infected host cells
- act a bit like NK cells, but much more specific (only kill those infected by particular type of infection)
How do cytokines differ from hormones?
- most act locally
- but can have systemic actions
What are cytokines and what is their role?
- small (5-20kDa) secreted glycoproteins involved in communication between cells of immune response
- immunomodulators –> usually prod and act locally, bind to specific cytokine receptors on target cells
- secreted in response to immune activation
- pleiotropic = diff effects on diff cell types
- stimulatory and inhibitory
- synergy or antagonism
- redundancy
What are some of the main groups of cytokines, and how are they prod?
- interleukins (eg. IL-1, IL-38, IL-39) –> usually made by T cells
- interferons (IFNs) –> some prod by any cell in response to infection, eg. IFNα, IFNβ and some prod by immune cells for cell activation, eg. IFNγ
- chemokines –> cell movement or chemotaxis, eg. IL-8
How is lymphoid tissue organised?
- 1° = lymphocytes reach maturity
- 2° = mature lymphocytes stimulated by antigen
What are the characteristics of innate immunity?
- oldest form –> elements shared by plants, insects and mammals, changes on evolutionary timescale
- always available –> prior exposure not req and little/no memory
- major form of immunity in young children –> approx 6 month period between loss of maternal antibodies and formation of own
How does the innate immune response aid the adaptive?
- innate crucial role in initiating and directing adaptive, and dictating type of response adaptive mounts
- adaptive take 4-6 days to dev, so innate critical in controlling infections before this (takes time for B and T to recognise antigen and divide)
What is the consequence of the delay in adaptive immune response dev?
- failure to dev immunity for organisms that mutate quickly (antigenically unstable), eg. parasites, influenza, HIV
What are the phases in initial response to infection?
pathogen
preventative barrier –> no infection
- FAIL-
infection
pre-formed broadly specific effectors –> pathogen removed
- FAIL -
innate cells recruited = early induced innate response (4-96 hrs)
recognition, activation, inflammation –> pathogen removed
- FAIL -
antigen - lymphoid tissue
recognition by naive B and T cells = adaptive immune response >96 hrs
clonal selection - effector cells –> pathogen removed
What are physical innate immunity barriers in the skin, GI tract, UG tract and resp tract?
- in all epithelial cells joined by tight junctions
- in resp tract cilia to move mucus and in others flow of air or fluid
What are chemical innate immunity barriers in the skin, GI tract, UG tract and resp tract?
- in skin FAs
- in GI tract low pH and enzs (pepsin)
- in UG and resp tracts lysozyme
What are microbiological innate immunity barriers in the skin, GI tract, UG tract and resp tract?
- in all antibacterial peptides (defensins) and commensals (microbiota)
How do secreted chemicals, antimicrobials and commensals help protect from infection?
- make unfavourable env for pathogens
How does keratinised skin help protect from infection?
- generally impermeable (unless damaged by burn/cut/insect bite etc.)
- keratinocytes prod keratin, ebum (contains FAs and defensins
- shedding
- commensals
How do mucous membranes help protect from infection?
- largest interface w/ env, semi-permeable (eg. gut, needs to take up nutrients)
- mucus, cilia (resp tract) and secreted enzs (eg. lysozyme in tears and saliva
- low pH (gut, vagina), peristalsis (gut)
- shedding of epithelia
- commensals
What are the diff types of pre-formed effectors, and their characteristics?
- lysozyme –> breaks bond in peptidoglycan, more active against Gram +ve bacteria
- antimicrobial peptides –> eg. defensins, cathelicidins, histatins, all made as inactive precursors
- defensins –> large group peptides 25-30 AAs long, 3 subfamilies w/ activity against diff types of pathogens, amphipathic and disrupt lipid bilayer
What is complement, and how was it discovered?
- > 30 soluble proteins found in blood and other bodily fluids
- discovered as heat sensitive substance that could “complement” immune sera (antibodies) in killing bacteria (inactive at high temps)
How is complement activated?
- components normally inert, but activated by presence of pathogens or antibody bound to pathogen
- DIAG*
- central event is cleavage of C3, by C3 convertase
What is the order of activation of complement in the classical pathway?
- C1, 4, 2, 3, 5, 6, 7, 8, 9
How did complement originally evolve?
- as part of innate immune response
What is the role of complement?
- provides protection early in infection in absence of antibodies through other “older” activation pathways
Where are complement proteins made, and which is most abundant?
- mainly made in liver
- C3
What are many activated complement components?
- Ser proteases, act on 1 another to gen larger and smaller fragment
- C3 –> C3b + C3a
- b is larger and a is smaller
What is the result of C3 cleavage?
- exposes reactive thioester in C3b, which can bind covalently to adj proteins/carbs, eg. on pathogens surface (rapidly inactivated in fluid phase
What are the 3 pathways of complement activation?
- classical pathway
- mannose-binding lectin (MBL) pathway
- alt pathway
- DIAG
What is the C3 convertase in each pathway for complement activation?
- for classical and MBL = C4bC2a (from cleavage of C4 and C2 by C1r,s and MASP2, respectively)
for alt = C3bBb
What is the role of C3b, and how it gen, in alt pathway?
- most abundant component
- some gen spontaneously in body fluids by “tickover” mechanism –> but usually hydrolysed and inactivated quickly
- if C3b gen binds to pathogen surface, factor B binds, which is cleaved by factor D to C3 convertase
- C3bBb convertase stabilised by factor P (properdin)
- C3b gen by classical or MBL pathway can also bind factor B
- in alt pathway can get amplification –> feeds into other pathways once activated
What are the later stages of complement activation?
- C3 convertase + C3b –> C5 convertase –> C5 –> C5b, C6, C7, C8, C9 (membrane attack complex - MAC)
- C5a disperses and C5b triggers rest of cascade
What is the role of the membrane attack complex (MAC)?
- can insert into cell membrane of Gram -ve bacteria and prod pores that allow entry of membrane damaging molecules, eg. lysozyme, and makes bacteria susceptible to osmotic lysis
What are the 3 major biological activities of complement, and what components do they involve?
- opsonisation –> C3b
- activation of IS –> C5a (or C3a but not as active)
- lysis of foreign cells –> MAC, esp C9
What is opsonisation?
- binding of complement proteins or antibodies (=OPSONINS) to surface of pathogen, so phagocytes can recognise and bind
How does complement cause activation of IS?
- chemoattractants and anaphylatoxins
- stimulate mast cells to induce inflammation at local level
What occurs when allergy activates IS?
- allergy causes widespread activation of mast cells throughout body
- lots of histamine release
- causing pressure on heart and lungs
How does complement cause lysis of foreign cells?
- through MAC (C5b - C9)
- C9 forms polymer to form pores in eg. bacterial cells
- esp important for Gram -ve (as harder for complement to penetrate peptidoglycan in Gram +ve)
Why does complement need to be reg?
- “double edged sword”
- to prevent damage to host
How is the complement reg?
- components rapidly hydrolysed in fluid phase
- soluble and membrane bound reg proteins, eg.
- -> factor H competes w/ factor B for C3b binding
- -> C1 inhibitor inactivates C1
- -> carboxypeptidase N inactivates C3a and C5a
- -> CD59 on host cells bind C9, preventing MAC formation
What can deficiencies in inhibitors of complement system cause?
- increase risk of some diseases
- age related macular degen (factor H)
- paroxysmal nocturnal hemoglobinuria (CD59) –> body attacks own RBCs
What is the importance of the complement?
- esp important in ec bacterial and fungal infection, but may be active against some viruses, eg. pox
- interacts w/ adaptive IS –> classical pathway, aids clearance of immune complexes, role in activating B and T cells
- controversial role in some autoimmune diseases and asthma
What is the role of preformed effectors?
- ‘frontline’ of defence –> specialised cells embedded in tissues, eg. mast cells, tissue macrophages (particularly prevalent in lungs)
- act as sentinel cells –> all cells capable of some level of innate immunity
- help deal w/ early stages of infection
How do innate and adaptive receptors vary?
- innate encoded through germline genes –> passed on from gen to gen = pattern recognition receptors (PRRs)
- adaptive assembled through lymphocyte dev –> can change during lifetime, genes newly assembled for B and T cell dev
What do innate receptors recognise?
- PAMPs (pathogen-assoc mol patterns) –> shared by many MOs, distinct from self, critical for survival/function
- DAMPs (damage-assoc mol patterns) –> released during injury and cell damage
What do PAMPs and DAMPs have in common?
- both relatively invariant structures
What diff PAMPs do diff organisms have?
- bacteria = flagellins, unmeth CpG DNA, N-formylated proteins
- gram -ve bacteria = lipopolysaccharides
- gram +ve bacteria = lipoteichoic acid
- fungi = chitin, beta-glucans
- viruses = dsRNA
- protozoa = GPI-linked proteins, mannose-rich glycans
What diff DAMPs are there?
- fragments of ec matrix proteins, eg. fibronectin
- phosphatidylserine
- heat shock proteins
- mito components (if cell dying)
- uric acid –> formed by purine build up in times of stress
- DNA
- HMGB1
What classes of PRRs are there?
- soluble factors, eg. mannose-binding lectin, complement
- membrane receptors, eg. lectin, scavenger, chemotactic and toll-like receptors
- cytoplasmic receptors, eg. NOD-like receptors (NLR)
- membrane and cytoplasmic expressed by immune and non-immune cells
How do membrane receptors, and the diff types work? (PRRs)
- receptor binding may initiate uptake (phagocytosis), chemotaxis (cell movement) or signalling (changes in gene expression)
- C-type (Ca dep) lectins, eg. dectin-1, macrophage mannose receptor
- scavenger receptors, eg. CD14 (recognises LPS), recognise alt/damaged host proteins too
- chemotactic receptors recognise chemoattractants
- TLRs = sensors that signal MO presence, signal to body and cause changes in gene expression, eg. make cytokines/interferons
How do chemotactic receptors on phagocytes work?
- bind chemoattractants that guide phagocytes to sites of infection and increase efficiency of intracellular killing
- eg. f-met-leu-phe receptor, recognises N-formylated polypeptides (prod by bacteria)
- eg. C5a receptor, binds complement fragment C5a
- GPCRs –> binding signals chemotaxis, mediator release and prod of ROS and NO
What are TLRs (Toll-like receptors)?
- ancient pathogen recognition system
- 10 in humans, each recognising distinct PAMP
- cell surface receptors or endosomal
- often function as dimers
What are some eg.s of TLRs and their ligands?
- TLR-3 and dsRNA
- TLR-4 dimer and LPS
- TLR-5 and flagellin
What is the structure of TLRs?
- DIAG*
- ec domain of TLR3 has horseshoe shape, formed by leu-rich repeats, inner surface has β-sheet structure and forms ligand binding domain
What happens when TLR binds PAMP?
- signalling to nucleus to induce expression of inflamm cytokines and interferons
How do the diff types of cytoplasmic receptors work? (PRRs)
- NOD-like receptors (NLRs) –> large group that recognise bacterial components (eg. peptidoglycan, flagellin), signal expression of pro-inflamm cytokines and trigger assembly of inflammasomes
- RIG-I like receptors –> viral sensors that detect viral RNA prod w/in cell signal expression of interferons
What is the inflammasome?
- protein complex needed to process procytokines –> activates components too toxic to be active all time, by cleavage
What are the 4 classic signs of inflammation?
- redness
- swelling
- heat
- pain
What causes the classic signs of inflammation?
- release of inflamm mediators
- dilation of arterioles, venules and capillaries
- increased permeability and blood flow
- immune cell migration into inflamm focus
- endothelial cells retract slightly allowing fluid through
- sensidise nerve endings
What is the aim of inflammation?
- ensure immune cells, defence mols, coagulation factors etc. reach site of infection or tissue damage
What are inflamm mediators?
- cells already in in tissues ready to respond
- lipid mediators, eg. prostaglandins (prod inhibited by aspirin)
- chemoattractants, eg. f-met-leu-phe
- complement proteins, eg. C5a
- vasoactive amines, eg. histamine, bradykinin
- clotting factors
- small molecules, eg. ROI, RNI
What is extravasation?
- movement of leucocytes from blood to tissues
How does extravasation occur?
- cytokine signalling mols made by damaged cells stimulate endothelial cells to express adhesion mols –> 1st are selectins
- selectins capture neutrophils, which begin to roll along vessel wall
- leucocytes express integrins that can bind to adhesion mols on endothelial cells
- neutrophils squeeze between endothelial cells
What is a summary of the inflamm response?
- bacteria infect tissue
- macrophages engulf bacteria and release chemical mediators
- cytokines induce selectins on capillary endothelia that bind to neutrophils
- vasoactive factors induce integrins on neutrophils, which bind ICAM and VCAM
- bradykinin loosens junctions to allow extravasation and triggers prostaglandin synthesis
- peptides from bacteria and chem signals from infected tissues released and attract neutrophils
When is acute inflamm needed?
- generally beneficial in dealing w/ infection/injury
- avoided in some places where would be harmful, eg. brain, CNS
What is chronic inflamm?
- caused by chronic inflammation, eg. TB, autoimmune disease
- can be damaging
What is the role of inflamm in TB?
- can survive in macrophages
- chronic inflamm could be good, as bacteria encapsulated, so can’t spread to rest of body
How are cytokines classified?
- grouped into fams based on structural similarities
- but fam members may have distinct functions
What are the characteristics of cytokine receptors?
- binding of cytokine to receptor can cause changes in gene expression and cell activation (or in some cases cell inhibition), or induce cell movement
- many are dimeric enz-coupled receptors
- chemokine receptors are GPCRs
When and where are cytokines secreted?
- by macrophages and dendritic cells in early induced immune response
How is TNFα prod?
- as TM protein, released by proteolysis
- primarily by macrophages
- LPS is potent stimulus of its prod
What are the TNFα receptors and how do they work?
- TNFR1 major form
- TNF trimer cross-links 3 receptors
- 2 pathways –> cell stim/apoptosis
Why can’t TNFα be used to treat cancer?
- too toxic
What are the local effects of TNFα?
- influx of platelets –> clotting in capillaries helps prevent spread of infection
- microthrombosis –> link between inflamm and coronary thrombosis
- efflux of fluid from capillaries –> increased flow to lymph nodes and stim of adaptive immunity
What are the systemic effects of TNFα, and at what conc is this?
- <1µg/ml –> enough to activate receptors all over
- pyrexia (fever) –> acts of hypothalamus, inhibits growth of some bacteria/viruses, 39.5° optimum temp for B and T cell activation
- cachexia (muscle wasting) –> presumably protective response to infection, cancer, trauma
How does TNFα cause sepsis?
- at concs >1µg/ml
- widespread increase in vascular permeability –> hypotension
- disseminated thrombus formation –> myocardial infarction and organ damage
- consumption of clotting factors –> internal bleeding and spread of infection
- multiple organ failure
- septic shock (80% lethal)
How do interferons affect viruses?
- interfere w/ viral rep
What are the 2 types of interferons and how are they prod?
- type I = IFNα (12 genes in humans), IFNβ
- -> many cell types can be induced to make after viral infections (induced by eg. RIG-I)
- -> some cell types (eg. dendritic cells) specialised for this, express high levels of endosomal TLRs. eg. TLR3, TLR9
- type II = IFNγ
What is the interferon response?
- induce expression of endoribonuclease that degrades viral RNA and protein kinase that phosphorylates euk initiation factor 2, inhibiting protein translation
- increased MHC class I expression and antigen presentation in all cells –> so infected cells recognised by cytotoxic T cells more easily
- activate NK cells to kill virus-infected cells (uninfected cells protected by increased MHCI)
- induce expression of chemokines –> attract other cells to site of infection
- if signal goes on long enough, triggers apoptosis of neighbouring cells –> barrier to virus rep
How could interferons be used therapeutically?
- poss to make recomb or synthetic versions
- have some ability to induce apoptosis of tumour cells, so could be used for this
Where is type II interferon γ prod, and what is its role?
- made by neutrophils, NK cells, T cells
- 1° role in adaptive (also some role in innate)
- important in neuronal cells
- anti-viral response by restricted range of cells
- activation of macrophages in TH1 response
How can T helper cells be further subdivided?
- dep on cytokines they make
- TH1 (inflamm cytokines) and TH2 cells
What is the role of TH1 cells?
- prod IL-2, IFNγ, TNFα
- activate macrophages and induce B cells to make opsonising antibodies (IgG)
- in classical bacterial and viral infections
What is the role of TH2 cells?
- prod IL-4, 5, 6, 10, 13
- induce B cells to make IgE (4 and 13)
- in parasitic infections
How do cytokine receptors to diff antigens determine adaptive immune responses?
- diff antigens/pathogens induce cells of innate IS to prod diff cytokines
- act on adaptive IS to prod approp response
- selection of wrong response can lead to disease
What characterises a polymorphic granulocyte?
- many shaped nuclei
What are the diff types of polymorphic granulocytes, and how were they experimentally identified?
- neutrophils –> didn’t take up acidic or basic dye
- eosinophils –> took up red dye strongly, as v acidic
- basophils –> took up basic stain
What is the main phagocyte in blood?
- neutrophils
What are eosinophils and what is their role?
- <6% leucocytes in blood
- also in connective tissue under mucosal surfaces
- receptors for C3b, IgG, IgA (and IgE can be induced, but not naturally)
- defence against parasitic infections (too big to phagocytose)
- release toxic proteins and free radicals from granules
- synthesise cytokines (eg. IL-40) and prostaglandins
- role in allergy –> esp asthma, contrib to chronic inflamm
What are basophils and what is their role?
- <1% leucocytes in blood
- similar to tissue mast cells (but free to roam and enter tissues when needed)
- receptors for C3a, C5a, IgE (high affinity IgE)
- release heparin and histamine
- make IL-4 and IL-13
- defence against parasites, role in allergy
What does heparin do?
- stops blood clotting too quickly
What does histamine do?
- vasodilator, important in early stages of infection
What is the role of mast cells?
- restricted to tissues, protect mucosal surfaces
- receptors for C3a, C5a, IgE
- release histamine etc. and make IL-4, IL-13
- sentinel cells
- defence against parasites
- role in allergy
What are the characteristics of neutrophils and where are they found?
- 50-60% of leucocytes in blood
- huge no.s can be released from bone marrow
- last <24hrs in blood (unless infection present)
- death by apoptosis (and destroyed by macrophages in spleen etc.)
- life extended on entering tissues (extravasation in response to chemoattractants)
- found in large no.s in pus
What are the functions of neutrophils?
- phagocytosis
- release of anti-microbials, eg. lysozyme, defensins
- prod of ROIs –> kill and act as inflamm mediator
- prod of cytokines
- entrapment of MOs –> form NET (neutrophil ec trap)
What is the fate of monocytes?
- to move into tissue and differentiate into macrophages (circulate in blood approx 3 days)
What are the roles of macrophages?
- sentinel cells –> bacterial invasions, dust/particles
- express wide variety of PRRs
- high phagocytic ability = 100 bacteria/cell
- driven by scavenger, mannose or complement receptors
- prod of pro-inflamm mediators (TNF, IL-12, IL-16, IL-1β)
What are the role of macrophages in adaptive immunity?
- antigen presentation to T helper cells
- T cell activation of macrophages
- interferon γ
- antibody-dep cell med cytotoxicity (ADCC)
- receptors for IgG, IgA (antigen specific phagocytosis)
What is the seq of events that occurs during phagocytosis?
- bacteria binds to surface of phagocyte (can be aided by antibody or complement)
- pseudopods extend and engulf organism
- invagination of phagocyte membrane traps organism w/in phagosome (some bacteria learnt to escape into cyto)
- lysosome fuses and deposits enzs into phagosome, enzs cleave macromols and gen ROS, destroying the organism
What are the diff classes of mechanisms of phagocyte bactericidal agents, and what are there specific products?
- acidicification = pH 3.5-4, bacteriostatic or bacteriocidal
- toxic O derived prods = O free radicals
- toxic NOs = NO
- antimicrobial peptides = defensins and cationic proteins
- enzs = lysozyme (dissolves cell walls of some Gram +ve bacteria), acid hydrolases (further digest bacteria)
- competitors = lactoferin (binds Fc) and vit B12 binding protein
What is oxygen dep killing, and the diff ROIs?
- mechanism of killing by free radicals
- “resp burst” = transient increase in oxygen, following phagocytosis, due to activation of membrane bound NADPH oxidase
- ROI:
superoxide (.O2-)
hydrogen peroxide (H2O2)
hydroxyl radicals (.OH)
hypochlorite (OCl-)
hypochlorous acid (HOCl) = bleach
What is NO prod by, and what is its role?
- prod by iNOS2 (inducible nitric oxide synthase)
- NO can kill large variety of pathogens and precursor for other RNS, eg. NO2- (nitrite) and ONOO- (peroxynitrite)
What is the role of distinct cytoplasmic granules in NK cells?
- insert into plasma membrane via perforin
- perforin similar to C9 (last component of complement) and like C9 forms hollow cylinder, perforates target cell and enzs from granules go through and enter target cell
- inducing apoptosis of target cell
What infections are NK cells important in?
- viral infections
- but also intracellular bacteria and protozoa
What are the roles of NK cells?
- helps keep viral infections in check before adaptive IS takes over
- source of IFNγ
- can use ADCC to kill infected and cancer cells
- receptors for IgG
How do NK cell receptors work?
- activity controlled by opposing stim and inhib receptors
- programmed to kill unless get signal from cell that it is self
- stimulatory = natural cytotoxicity receptors (NCRs), ligands inc nectin fam (polio virus receptors)
- inhibitory = killer Ig-like receptors (KIRs), eg. MHCI (=self labels expressed by all nucleated cells)
Why do viruses and cancers downreg MHCI?
- to evade cytotoxic T cells, even though still killed
- as worse death than by NK cells
What are dendritic cells and what is their role?
- heterogenous pop of cells in skin and lymphoid tissues
- take up foreign material by phagocytosis/macropinocytosis
- transport antigen to lymphoid tissue
- present digested antigen to T lymphocytes
- constitutively express high levels of MHCII proteins
What is macropinocytosis, and what cells do it?
- uptake of fluid and any foreign material in it
- all cells capable, but esp done by dendritic cells
What are the key features and functions of the IS?
- recognises infection (non-self or antigen) or danger
- following 1° contact w/ antigen there are innate and weak adaptive responses
- 2° contact = enhanced adaptive responses (immunological memory)
- contains and eliminates infections through effector functions
- must be reg and tolerant of body’s own cells and mols
How is adaptive IS activated in draining lymph node?
- macrophage/dendritic cell takes up foreign material
- transported to nearest draining lymph node –> where just matured B and T cells tend to congregate
- presentation of antigen to T cells, activating them
- B cells activated and differentiate into plasma cells (make antibodies)
- antibodies and activated T cells (= effector T cells) can go out into tissues and deal w/ infection
What is the purpose of T and B memory cells?
- long lived, in some cases life long, and provide immunity
What is the clonal selection hypothesis?
- millions of cells made w/ diff receptors –> gen indep of antigen
- any lymphocytes that recognise self deleted early in dev
- if get infection w/ antigen recognised by receptor, that cell undergoes clonal selection –> get 100s-1000s cells from 1 parent w/ same receptor
- some B cells differentiate into plasma cells to make antibodies
- also get gen of long lived memory cells
What are the 2 roles of antibodies in the immune response, and what regions are responsible for these?
- antigen recognition = as integral membrane proteins on B lymphocytes, Fab regions
- antigen elimination = as soluble proteins secreted by plasma cells, Fc region
What is the basic antibody structure?
- DIAG*
- Fab regions variable in seq and bind diff antigens specifically
- Fc region constant in seq, bind to complement, Fc receptors on phagocytes, NK cells etc.
What is the 4 chain antibody structure?
- DIAG*
- light (L) chain = 25kD
- heavy (H) chain = 50kD
- immunoglobulin G = L2H2 = 150kD
How can diff antibody fragments be prod?
- proteolytic cleavage w/ papain = 2 Fab fragments and 1 Fc fragment
- proteolytic cleavage w/ pepsin = 1 Fab fragment (attached via disulphide bonds) and Fc fragment extensively degraded
How do immunoglobulin classes differ?
- AA seq of heavy chains
What are the light chain types of antibodies?
- kappa (κ) or lambda (λ)
- not class restricted, ie can have IgGκ or IgGλ antibodies
Where did further info on antibody structure come from?
- protein seq
- eg. of myeloma proteins (cancer of plasma cells)
What further discoveries were made from protein seq of antibodies?
- contain constant and variable regions
- comprised of homologous domains
- variable region domains contain 3 hypervariable regions
What is the role of constant and variable regions in antibodies?
- variable bind antigen, differ between antibodies w/ diff specificities
- constant same for antibodies of given H chain class or L chain type
What did X-ray crystallography of Fab and Fc fragments show, and why were fragments have cleaved to look at structure?
- approx 110 AAs w/ intrachain S-S bridge that fold to form compact globular domains
- folding pattern known as Ig fold
- cleavage needed, as hinge region not folded into compact domains, so quite floppy
What is the Ig fold comprised of, and in what members is it found?
- C domain has 7 β strands
- V domain has 9 β strands
- found in all members of Ig gene superfam
What are the Ig gene superfam involved in and what are their domains like?
- involved in recognition, binding and adhesion
- domains can be V-like or C-like, domain structure is v stable and therefore prevalent, β strands stable and loops can vary w/o disrupting other cell structure
How many members are there of the Ig gene superfam in the human genome, and why are antibodies an unusual member?
- 765
- members usually only found on cell surface as receptors
What is the structure of the antigen combining site?
- 3x hypervariable regions (approx 7-12 AAs)
- rest is framework regions
What are hypervariable regions, and what do they determine?
- complementarity determining regions (CDRs)
- how complementary determines how specifically can interact w/ antigen
What are the characteristic of antigen-antigen interactions?
- non-covalent (electrostatic interactions, H-bonds, VdWs, hydrophobic interactions)
- indiv weak, but if many form simultaneously (ie. if antigen combining site and antigen contain many complementary residues), then interaction specific and of high affinity
What antibodies do B cells initially express, and why do these need to assoc w/ other proteins, what does this involve?
- most B cells express IgM (1st to be prod in antibody response) and/or IgD –> but all classes can serve as B cell receptor
- recognises and binds to antigen, but cant prod signal itself
- membrane bound Igs assoc w/ 2 other prots –> Igα and Igβ
- Igα and Igβ contain single ITAM (immunoreceptor Tyr activation motif) in their long cyto domains
What is they key diff w/ T lymphocyte receptors (as opposed to B cell receptors)
- only expressed on membranes, not as soluble proteins
What are the roles of T cytotoxic and T helper cells?
- cytotoxic = specifically kill infected host cells
- helper = augment immune response
How do T cells recognise antigen?
- T cell immunity for intracellular pathogens, so recognise “cell-assoc”, free, native antigens
- T cells recognise processed peptides presented to them on cell surface by major histocompatibility proteins (MHC)
What is the diff between MHC class I and II, in where expressed and what they present?
- MHCI = expressed by all nucleated cells, present peptides derived from endogenous proteins
- MHCII = expressed by certain leucocytes (dendritic cells, B cells, macrophages), present peptides derived from exogenous proteins
What is the role of MHCI?
- cytotoxic T cells recognise peptide bound to it
- virus infected cell makes virus proteins, broken down in cytosol, peptides transported to ER and bind MHCI on surface
What is the role of MHCII?
- helper T cells recognise peptide bound to it
- antigen presenting cell internalises and breaks down foreign material, peptides bind to MHCII in endosomes on cell surface (cell not infected, just needs help dealing w/ infection)
What is the structure of T lymphocyte receptor (TCR) similar to?
- Fab like –> disulphide bond holding together and some flexibility (like hinge region)
- ec domains homologous to V and C regions of Ig –> each V region has 3 hypervariable regions of CDRs
What receptors do a subset of T cells express, and how do they differ?
- γδ receptors
- in 1-5% T cells
- found at epithelial surfaces
- less diverse –> recognise broader range of antigens
What do TCRs recognise?
- complex of antigen and self MHC
What do the 3 CDRs bind?
- CDR3 regions of α and β chains most variable –> bind foreign peptide
- CDR1/2 bind self MHC
Why does TCR expression on cell surface req assoc w/ add proteins?
- v short C-ter, so don’t protrude much into cyto, so can’t signal by itself, needs to assoc w/ diff membrane proteins
What is the TCR complex made up of?
- α and β subunits (TCR)
- CD3 subunits (ε, δ and γ) and ξ –> CD3 subunits contain ITAMs in their cyto regions
What is the immune repertoire?
- approx 10^14 antibody receptors and 10^18 T cell receptors
What is the structural basis of antibody diversity?
- variation in seq and length of CDRs are main determinants of antibody diversity
- CDR3 tends to be most variable in length and seq
- heavy chain generally contributes more to antigen binding and more variable than light chain
What were some of the early hypotheses for the genetic basis of antibody diversity and why were they unpopular?
- multiple genes
- somatic mutation –> not pop to have mutations in somatic cells
- somatic recomb –> not pop to have genes in fragments
What was the Dreyer & Bennett hypothesis (1965)?
- Igs encoded by separate C region and multiple V region genes
What did Tonnegowa discover in 1976?
- Ig genes rearranged during B cell dev –> proven through restriction digests
How was κ light chain gene recomb demonstrated experimentally?
- used mouse embryo and mouse myeloma cells
- DNA extracted and digested w/ REs
- restriction fragments separated by gel electrophoresis
- V and C regions identified by hybridisation w/ radioactive probes
- in myeloma both probes bound same region –> so must have been recomb event so V and C lying close together
- embryo pattern (V and C probes bound separately) in all cells, except those of B lineage
What are the 3 sets of Ig genes?
- heavy (H) chains = chromosome 14
- kappa (κ) chains = chromosome 2
- lambda (λ) chains = chromosome 22
How many V and C regions are in Ig genes?
- each locus has multiple variable region genes and 1, or a few constant region genes
- V regions encoded by 2 or more exons
How are light chain V regions encoded?
- by 2 segments of DNA
- DIAG*
- most encoded by V segment
- there is also joining segment
How are heavy chain V regions encoded?
- by 3 segments of DNA
- DIAG*
- V segment, diversity and joining
What chain is always expressed first?
- Cμ (IgM heavy chain)
How does rearrangement of κ light chains occur?
- somatic recomb = V gene spliced to J gene and intervening DNA excised
- rearranged V promoter now close to enhancer allowing transcrip
- intervening seqs removed by RNA processing
- end up w/ mRNA corresponding to particular V and J region, and C region
How does rearrangement of heavy chain occur?
- D-J joining
- then V-D-J joining
- all extra bits removed when transcribed to mRNA
Where are the diff CDRs encoded?
- CDR1 and CDR2 encoded by V segments (ie. germline)
- CDR3 corresponds to VDJ (or VJ for light chain) join
What does recombination of chains involve?
- involves lymphocyte specific recombinases and conserved recognition signal seqs (RSSs)
- RSSs guide recomb, make sure correct order and don’t get misjoining
- 12-23 bp rule
What are RSSs and where are they found?
- conserved heptamer (7bp) and nonamer (9bp) separated by 12 or 23 random nucleotides
- found directly adj to coding seq of V, D or J gene segments
What is the 12-23 bp rule regarding RSSs in recomb?
- gene segment w/ 12bp spacer only joins w/ gene segment w/ 23bp spacer
- 12bp and 23bp spacer correspond to 1 and 2 turns of DNA helix
What is V(D)J recombinase made up of?
- complex of several enz req for somatic V region gene recomb –> normal DNA cleavage/repair enzs, inc DNA-dep protein kinase
- products of Rag-1 and RAG-2 genes (recombination activation genes)
- terminal deoxynucleotide transferase (TdT)
What do mutations in DNA-dep protein kinase and RAG-1/RAG-2 of VDJ recombinase cause?
- severe combined immunodeficiency (SCID)
How can SCID be cured?
- bone marrow transplant
- genetic techniques
What is the mechanism for somatic recomb?
- RAG-1/RAG-2 complex recognises and aligns RSSs adj to gene segments to be joined (germline DNA folded)
- 2 ssDNA breaks made close to RSSs
- free 3’ OH attacks phosphodiester bond on other strand DNA to create hairpin at segments to be joined and flush ds break at RSS boundary
- other proteins bind to repair joints, but imprecise, w/ nts added/subtracted
- -> DNA hairpins claved at random, symmetrically or asymmetrically
- -> for VDJ joining of H chain, nts can be added in a template indep, by TdT
- -> unpaired overhangs filled in by DNA pol or may be excised by endonuclease
- DNA ligase joins nicked and repaired hairpins to form “coding joint” (blunt ends ligated to form “signal joint” and typically excised)
What is the structure of the RAG-1/RAG-2 complex?
- looks a bit like a pair of scissors
- hinge region and flexible pair of domain that bind nonamer => NBD (nonamer binding domain)
- structure tilts, so if bound to RSS w/ 12bp spacer, will now bind 23bp spacer
What does antigen indep B cell differentiation involve?
- heavy chain rearrangement (D-J, then V-D-J) –> μ heavy chain
- light chain gene rearrangement (V-J) –> expresses membrane IgM
- selection against self recognising cells
- in 2° lymphoid tissue naive B cell expresses membrane IgM or IgM and IgD
What happens to B cell after antigen indep differentiation, if it never meets antigen?
- apoptosis
Where does antigen indep differentiation occur?
- bone marrow
Where does antigen dep differentiation occur?
- 2° lymphoid tissue
What are the 2 processes in antigen dep differentiation that result in differentiation?
- somatic hypermutation
- class switching
What happens during somatic hypermutation?
- point mutations introd in rearranged V regions
- mechanism is activation induced cytidine deaminase (AID) –> mutations introd t/o V regions, but in mature B cells mutations clustered in CDRs
What is activation induced cytidine deaminase (AID)?
- enz expressed only by B cells in lymphoid tissue responding to antigen
What is the role of somatic hypermutation?
- main role to improve immune response
- but can also add diversity
What is affinity maturation?
- higher affinity receptors selected as immune response proceeds –> “survival of the fittest”
What is the rate of mutation in somatic hypermutation, and how does this compare to normal rate?
- 1bp per 10^3 bp per cell division
- normal = 1bp per 10^10 bp per cell division
What happens during class switching?
- IgM –> IgG, IgA etc.
- same recombined V region assoc w/ diff C region genes
- antigen specifically retained, but diff localisation/effector functions induced –> flexible response to pathogens
- by DNA recomb between switch regions –> irreversible and intervening DNA lot
- can get sequential switchin
What do somatic hypermutation and class switching both req?
- T cell help
- AID
How does mechanism for class switching differ from VDJ joining?
- initiated by AID acting at switch regions –> G rich tandem repeated DNA seqs found close to C region genes
What is AIDs mechanism of action?
- DIAG* –> deaminated C to form U
- AID expressed in activated B lymphocytes, only active on ssDNA
- activity triggers DNA repair pathways (mismatch repair, base excision repair)
- ie. not intro of U itself that causes mutation, its the repair pathways
- repair pathways in B cells error prone, leading to diff mutational outcomes
- -> mismatch/base excision = somatic hypermutation
- -> ss nicks to ds nicks (common in G-rich tandem repeat switch regions) = class switching
How can co-expression of membrane IgM and IgD occur?
- prior to class switching B cells may express diff classes by differential transcript processing and splicing
- allows simultaneous membrane expression of 2 classes –> usually IgM and IgD (but can get IgM w/ any other class)
- reversible, as no change in DNA
How can you get prod of surface and secreted forms of Ig from same H chain?
- differential processing of 1° transcript
- at C-ter of IgM 2 diff exons –> 1 encodes hydrophobic residue, and other a hydrophilic residue
- dep on which polyadenylation site used, decides which –> so whether can sit on lipid bilayer (hydrophobic) or exported and secreted (hydrophilic)
- as B cells become activated, secreted form predominates
What is the structure of TCR genes?
- look like antibody genes
- chromosome 14 has 1 C region for α chain
- chromosome 7 has 2 C regions for β chains –> v homologous, don’t know function of having both
How does TCR gene rearrangement occur during differentiation?
- somatic recomb of TCR V region genes –> same machinery as used in dev B lymphocytes
- only diff is occurs in thymus
What is the diversity of TCR genes?
- mutliple copies of V region gene segment (Vn x Jn / Vn x Dn x Jn) –> α, β, γ (α-like) and δ (β-like)
- α x β chain conformation (Vα x Jα) x (Vβ x Dβ x Jβ) = approx 6 x 10^6
- junctional diversity = approx 2 x 10^11
- -> conc in CDR3 of TCR α and β chains
- -> total diversity = approx 10^18
Do V regions of TCRs undergo somatic hypermutation?
- no
What are γδ T cells?
- 1-5% of T cells
- also gen by gene rearrangement
- fewer V region gene segments, junctional variability (focussed at CDR3) may compensate to some extent –> but less diverse, recognise broader range of antigens, inc lipids
- do not appear to req processing or presentation by MHC –> receptors more “antibody-like”
Where are MHC encoded in humans ?
- encoded by genes of MHC chromosome 6
What are MHC also known as in humans?
- HLA = human leucocyte antigen
- eg. HLA-A, HLA-B, HLA-C
Are MHC genes polymorphic?
- v polymorphic
- eg. >1400 alleles of HLA-B locus
- alleles may differ by up to 20 AA substitution
- polymorphisms clustered in distal peptide binding domains (variability inherited, 1 person only has few diff variants of these alleles)
What is MHC restriction?
- T lymphocytes can only recognise antigen in context of self MHC molecules
How was MHC restriction demonstrated experimentally?
- via experiments w/ inbred mice (identical MHC alleles) and virally infected cells (in vitro)
- cytotoxic T cells from mouse A could kill infected cells
- cytotoxic T cells from mouse B (even though from mouse immune to virus) couldn’t kill cells taken from mouse A
WHat 2 theories arose from experiments on inbred mice relating to MHC restriction, and what provided final proof?
1) 2 receptors on T cells - 1 (TCR) for antigen and 1 for MHC
2) 1 receptor on T cells (TCR) - recognises antigen and MHC
- -> proof from X-ray crystallography
How do MHCI and MHCII structures differ?
- MHCI = polymorphic TM α chain and invariant β2-microglobulin
- MHCII = polymorphic TM α and β chains
What similarities are there in MHCI and MHCII structures?
- membrane-prox domains Ig like
- membrane distal domains bind peptides and contain polymorphisms
What peptides are MHCI and MHCII capable of binding?
> > particular MHC molecule can bind wide range of related peptides
MHCI:
- bind peptides 8-10 AA long
- N and C-ter bind to invariant sites at end of groove
- 2/3 anchor residues on peptide bind to specificity pockets formed by polymorphic residues
MHCII:
- bind peptides 13-25 AA long
- peptide backbone interacts w/ conserved residues that line groove
- side chains (anchor residues) interact specifically w/ specificity pockets formed by polymorphic residues along peptide binding groove
What did crystallographic studies show about MHC?
- demonstrated MHC binds peptide
- that TCR recognises complex of peptide and self MHC
- MHC molecules have broad specificity for peptides (degenerate specificity)
- bound peptide is integral part of MHC structure
How is bound peptide an integral part of MHC structure?
- dissoc v slowly
- if not bound, doesn’t fold correctly and not transported to cell membrane correctly
- important in immune response, so want it to be displayed as long as poss
How does antigen presentation occur w/ MHCI?
- peptides transported to ER by ATP hydrolysis driven transporter, TAP (transporter assoc w/ antigen presentation)
- peptides loaded onto MHCI in ER (req chaperones)
- peptide binding essential for MHCI cel surface expression
How does antigen presentation occur w/ MHCII?
- antigen taken up by phagocytosis or endocytosis etc.
- acidification promotes folding and proteolysis
- peptides assoc w/ MHCII in endocytic compartment
What is cross presentation?
- process by which certain antigen presenting cells, eg. dendritic cells, present peptide assoc w/ MHCI to cytotoxic T cells
What is req for TCR to form TCR complex?
- TCR must assoc w/ other cell surface receptors (CD3 and ζ chain) to signal
Why are co-receptors req for T cell activation, and what are they?
- stabilise interaction
- facilitate signalling
- CD8/4 interact w/ invariant regions on MHCI/II
- MHCI + antigen CD8
- MHCII + antigen CD4
What does binding of MHC ligand to TCR cause?
- phosphorylation of ITAMs by receptor assoc kinase
- when co-receptor binds to MHC ligand, ZAP-70 binds to phosphorylated ζ chain ITAMs and phosphorylated by Lck (Y kinase)
How are MHC genes expressed?
- co-dominantly –> increasing no. diff MHC molecules expressed per cell
- MHCI most variable
What are the consequences of MHC genes being polymorphic?
- graft rejection (recognised as foreign, unless identical twin)
- ensures wide recognition of foreign peptides
- but variability of MHC mols small compared to that of TCR
- T cell responses determined by individuals MHC type (MHC restriction) –> responders and nonresponders (may get pathogen that can’t prod any peptides which bind MHC, happens in inbred mice strains, but rare in humans as not inbred)
- each MHC allele can bind restricted range of related peptides
What did MHC polymorphism evolve in response to?
- pathogens
- eg. black death/flu/HIV ??
What is greater polymorphism of MHC assoc w/?
- greater social interaction
- and therefore chance of passing on diseases
What are the functions of MHC protein?
- graft rejections
- T cell activation
- antigen presentation to T cells
- self/non self recognition (NK cell KIR = killing inhibitory receptors)
- assoc w/ certain autoimmune diseases –> eg. MS, Graves disease
- dev of T cell repertoire/tolerance in thymus
- choice of mate?
In terms of MHC proteins, what do individuals want in terms of a mate?
- someone w/ MHC proteins as diff as poss
How does thymic selection occur?
- DIAG*
- bone marrow stem cell is double -ve (CD4-/CD8-)
- rearrangement of TCR genes (γδ or αβ)
- get TCR w/ double +ve (CD4+/CD8+)
- MHC selection (αβ)
- +ve selection and any cells that don’t bind self MHC rejected and undergo apoptosis
- -ve selection and any cells that bind self MHC and self peptide strongly rejected and undergo apoptosis
- AIRE (autoimmune regulator) allows expression of proteins in thymus usually expressed elsewhere
- result is single +ve (CD4+ OR CD8+)
What is the consequence of the fact that antibodies are flexible adaptors?
- diff Ig classes have diff biological roles
- way they deal w/ pathogens is dep on class
What is the structure of IgG (γ chain), and what subclasses are there?
- DIAG*
- monomeric
- mw = 150,000d
- subclasses = IgG1, IgG2, IgG3, IgG4 –> differ mainly in length and no. disulphides of hinge region (rest is homologous)
- C2 faces covered by carb so not touching
- extended hinge region –> v flex and susceptible to proteolysis
What is the role of IgG (γ chain)?
- main antibody in tissues and blood
- can activate complement
- bind Fc receptors on phagocytes and NK cells –> IgG1 and IgG3 most active in this and complement activation, due to residues and longer hinge region
- crosses placenta –> binds FcRn on trophoblast
- long serum half life
- important in 2° or “memory” responses
What is the structure of IgM (μ chain)?
- DIAG*
- pentamer –> 5 antibody units and J chain
- mw = 970,000d
- no defined hinge = “functional hinge”, so not as much flex
What is the role of IgM (μ chain)?
- usually serum restricted (unless inflam and vessels get leaky)
- high valency –> can usually only bind 5 antigens at once, but good agglutinator of particular antigen
- most efficient class at activating complement
- important in 1° antibody response –> doesn’t undergo somatic mutation so lower affinity (but higher avidity)
What is the structure of IgA (α chain), and what are the subclasses?
- DIAG*
- monomer in serum and usually dimer in secretions
- subclasses = IgA1 and IgA2
- in secretions and at mucosal surfaces forms secretory IgA = IgA dimer + J chain + secretory component
What is the role of the secretory component in IgA?
- helps protect from digestion and can bind mucin to anchor IgA to mucus
What is the role of IgA (α chain)?
- high valency (binds 4 antigens at once, so good at clumping)
- rapid catabolism
- present in milk –> role in protecting newborn
- does NOT activate complement –> exposed to foreign material in gut all the time, don’t want big inflam response, just clumps bacteria together
- binds Fc receptors on phagocytes
What specialised transport mechanism exists for IgA?
- mucosal lymphoid tissue prod 5g IgA per day
- poly-Ig receptor binds polymeric IgA/IgM (normally IgA)
- allows secretion of IgA (and IgM) into lumen
- bacteria that penetrate mucosa can be transported back to lumen
What is the structure of IgD (δ chain)?
- DIAG*
- monomeric
- mw = 184,000
- long extended hinge, heavily glycosylated
What is the role of IgD (δ chain)?
- <1% serum Ig
- function still not entirely understood, but v conserved in evo
- present as antigen receptor on many B lymphocytes, w/ IgM (may help B cells recognise antigen, as better hinge and flex than IgM)
- prod by B cells/plasma cells in upper resp tract, interacts w/ receptors on basophils, inducing antimicrobial, inflam and B cell stimulatory factors
What is the structure of IgE (ε chain)?
- DIAG*
- monomeric
- mw = 190,000d
- no defined hinge –> “functional hinge” like IgM
What is the role of IgE (ε chain)?
- trace in serum (0.0003% Ig)
- binds to high affinity FcR on mast cells and basophils
- important in allergy
- role in immune defence against large ec parasites, eg. helminths
What is the biological role of Igs (ie. methods of action), and which classes are responsible for these?
- label pathogens –> elimination/destruction
- specific binding/multivalency –> at least divalent
- neutralise toxins (IgG, IgA)
- immobilise pathogens (IgM)
- prevent binding of pathogens to host cells (IgG, IgA)
- agglutinate particles, eg. bacteria (IgM, IgA) –> so more easily wafted through body and less mobile
- form “immune complexes” w/ soluble antigen
Can antibodies directly kill bacteria?
- if bind to transporter, but usually need to react w/ complement/leucocyte to kill
What are the functions of Fc effectors?
- invoke destruction of labelled pathogens
- activate complement (IgM, IgG)
- bind Fc receptors on leucocyte surfaces (IgG, IgA, IgE)
What are the Fc effector mechanisms that operate dep on?
- site and type of infection
- stage of immune response (1° or 2°)
What is req for complement activation through the classical pathway?
- antigen-antibody complex
- 2x IgG molecules bound to bacterial cell surface, so C1q can interact w/ CH2
- C1q must interact w/ 2 Fc regions
- need 2x C1q to bind to activate complement
Why is IgM a much more potent activator of complement than IgG?
- IgM pentameric, so C1q bind 1 molecule, rather than at least 2 of IgG
How does IgM activate complement?
- pentameric IgM bind to antigens on bacterial surface and adopt ‘stable’ form (arms dislocate so no longer in same plane)
- C1q binds to 1 bound IgM
- binding of C1q activates C1r, which cleaves and activates C1s (both Ser proteases)
How does IgG activate complement?
- bind to antigen on bacterial surface
- C1q binds to at least 2 IgG molecules
- binding activates C1r, which cleaves and activates C1s (both Ser proteases)
What does complement activation result in?
- inflam
- activation of leucocytes (eg. for chemotaxis)
- opsonisation
- lysis of foreign cells
What is the importance of complement activation?
- immune defence against bacteria (and viruses), clearance of immune complexes
- inducer of inflam, can cause pathology
What is the effect of antibody and complement on clearance of bacteria from blood?
- DIAG*
- when Ab and C3b coated best recognised, as phagocytes have receptors for both
Where are Fc receptors (FcR) expressed?
- variety of effector cells
- inc granulocytes, mononuclear phagocytes, NK cells
How do Fc receptors function?
- most function as part of a multi-subunit complex
- α chains bind Fc and assoc chains facilitate cell surface expression/signalling
How are Fc receptors activated?
- activation dep on FcR bound antibodies binding to antigen –> generally receptors quite low affinity, only activated when cross-linked
- involves immunoreceptor Tyr activation motifs (ITAMs)
How are Fc receptors inhibited?
- inhibitory receptors contain immunoreceptor Tyr inhibitory motifs (ITIMs)
Where do diff isoforms of Fcγ receptors occur?
- differ in cytoplasmic/membrane spanning regions
What is the role of Fc receptors on phagocytes?
- uptake of immune complexes
- opsonisation
- cell activation
- resp burst (release of ROI)
- release lysosomal contents (“frustrated phagocytes”)
What Fc receptors are found on phagocytes?
- IgG (IgG1 = IgG3 > IgG4)
- IgA
What Fc receptors are found on NK cells?
- IgG (IgG = IgG3)
What is the role of receptors on NK cells?
- med ADCC (antigen dep cell-med cytotoxicity)
- antibody binds antigens on surface of target cells
- Fc receptors on NK cells recognise bound antibody
- cross-linking of Fc receptors signals NK cell to kill target cell
- target cell dies by apoptosis
- occurs through binding of IgG coated target cells to FcγRIII –> NK cells release enzs and perforin from cyto granules
What is TRIM21 and what is its role?
- intracellular receptor for IgG, IgM and IgA
- meds humoral immunity
- binds Ig w/ high affinity
- recruits to internalised antibody bound virus and targets it to proteasome –> recognises viruses more efficiently
- shown to neutralise virus infection in vivo
What is FcRn and where is it present?
- neonatal receptor for IgG (IgG1 > IgG3 > IgG2 > IgG4) and present in neonatal gut
- also in adults in gut, liver and endothelial cells
What is the role of FcRn?
- placental receptor of IgG (on trophoblast), protects fetus and newborn
- in adults binds and recycles IgG, preventing excretion (improves half life)
What Fc receptors are found on mast cells and basophils?
- IgE
What is the role of Fc receptors on mast cells and basophils?
- med allergy/defence against large parasites
- mast cells secrete inflam mediators and cytokines
What happens in mast cells if someone is allergic to something?
- make IgE instead of IgG
- IgE binds to receptors on mast cells
- if come across allergen again get cross linking of receptors ad v rapidly (5-10 mins) mast cell releases granule contents
What are “nude” mice?
- have no thymus, so no T cell responses
- survive reasonably well as have innate and some B cell response
What are thymus indep antigens and what is there effect?
- eg. bacterial polysaccharides
- induce more rapid response and prod of IgM antibodies
- memory cells not gen
What are thymus dep antigens and what is their effect?
- eg. proteins
- for most antigens differentiation of B cells into plasma cells (or memory cells) us T cell dep
- responses involve somatic hypermutation (leading to affinity maturation) and class switching following AID expression
What can T cell subsets and cytokines they prod influence?
- can influence slass/subclass of antibody prod in humoral response
How areneffector (/”primed”/memory) T cells activated?
- recognition of MHC + peptide + co-receptor
- causes activation (signal 1)
- TCR +CD3 + CD4/CD8 MHCII/I + peptide
How are “naive” T cells activated?
- recognition of MHC + peptide + co-receptor (CD4/CD8) –> signal 1
- recognition of co-stimulatory molecules –> signal 2
What cells are able to deliver signal 1 and 2, and why?
- only dendritic cells, macrophages and B cells express co-stim molecules req to deliver 2 signals
- dendritic prob most important in stim naive T cells
What happens if signal 1 prod in absence of signal 2?
- T cells become unresponsive/tolerised
What is the best characterised co-stimulatory molecule in T cell prod, and what does it do?
- B7
- expressed by dendritic cells, macrophages, B cells
- interacts w/ CD28, inducing expression of IL-2 and IL-2 receptor
- IL-2 acts in autocrine manner on T helper cells, also req for cytotoxic T cell activation
- other cytokines direct T-cell differentiation into diff subsets of T effector cells (signal 3)
By what cells is signal 3 prod, and what does it differ dep on?
- dendritic cells and other innate cells
- varies dep on type of pathogen
What signal 3 prod TH1 cells and what is their role?
- signal 3 = IL-12, IFNγ
- prod IL-2, IFNγ, TNFβ
- activate macrophages, causing inflam (classic cell-med immunity)
- induce B cells to make more IgG1 and IgG3 (opsonising) antibodies
- important for dev of cytotoxic T cells
What signal 3 prod TH2 cells and what is their role?
- signal 3 = IL-4
- prod Il-4, 5, 6, 10, 13
- activate eosinophils and mast cells
- induce B cells to make IgE (promotes mast cell degranulation)
How does no. TH2 cells differ in people w/ allergy?
- more than would expect
What signal 3 prod TH17 CD4 T cells, and what is their role?
- signal 3 = TGFβ, IL-6
- prod IL-17, IL-22
- activates epithelial cells, fibroblasts
- proinflam, esp at mucosal surfaces
- recruit neutrophils to sites of infection, early in response
- important in fungal and ec bacterial infections –> role in autoimmune disease?
What signal 3 prod TFH CD4 T cells (follicular helper T cells), and what is their role?
- signal 3 = IL-6
- found in lymphoid follicles
- help B cells differentiate into plasma cells and memory cells
- promote somatic hypermutation
- prod cytokines which prod AID (important for class switching and affinity maturation)
- contact dep –> some go into germinal centres and physically interact w/ B cells
- also prod IL-21 and other cytokines
What are the 2 types of T regulatory cells (TREGs)?
- natural
- induced
How do natural and induced TREGS vary in terms of where they dev?
- natural = thymus
- induced = periphery (mucosal lymphoid tissue)
What is the phenotype of natural and induced TREGs
- both CD4+, CD25+, FoxP3+
How do natural and induced TREGS vary in terms of what they recognise?
- natural = MHC and self peptide
- induced = MHC and non-self peptide
How do natural and induced TREGS vary in terms of suppression?
- natural = contact dep, IL-10, TGFβ
- induced = IL-10, TGFβ
How do natural and induced TREGS vary in terms of target?
- natural = dendritic cells, effector T cells
- induced = effector T cells
How do natural and induced TREGS vary in terms of role?
- natural = suppression of autoreactive T cells
- induced = downreg of mucosal immunity (dampens down immune response once pathogen destroyed), inflam responses
Why do natural TREGs recognise MHC and self pepide?
- if finds, makes cytokines to downreg immune response and stop any autoimmune response
What is the overall role of CD4 TREGs?
- suppress immune response
- selective prod of diff CD4 effector subsets can have sig consequences for type of immune response induced (influence Ig class, type of immune response or can downreg response)
What is the role of CD8 cytotoxic cells?
- once activated, bind specifically to infected target cells and induce them to undergo apoptosis
- naive cytotoxic T cells usually req co-stim from both dendritic cells (cross presentation) and effector T cells (TH1)
How do cytotoxic T cells kill?
- release proteases (granzymes) that enter target cell via perforin channel
- fas ligand induces clustering of fas (“death receptor”) on target cell –> clustering causes activation of caspase cascade which induces apoptosis
How can the killing by cytotoxic T cells be decribed?
- specific
- efficient
- “clean” –> no release of enz/toxins etc, and corpse removed by macrophages
Apart from killing, what are the other functions of cytotoxic T cells?
- can prod some cytokines, eg. IFNγ and TNFα –> can synergise in activating macrophages
- killing by fas pathway may be important in downreg immune response –> ie. may have suppressor function once immune threat dealt w/
What is the role of IFNγ?
- antiviral effects –> induces MHCI, enhances activity of immunoproteasome
- recruits macrophages
What is allergy?
- disease following immune response to innocuous antigen (=allergen)
How does IgE med allergy/hypersensitivity?
- individual sensitised (2-3 wks)
- -> IgE binds tightly to IgE FcR on mast cells (skin and mucus surfaces), basophils (blood) and activated eosinophils
- -> no symptoms at this stage
- immediate hypersensitivity reaction
- -> 5-10 mins after re-exposure to allergen
- -> cross linking of IgE by allergen on mast cell surface triggers release of inflam mediators
What do inflam mediators and cytokines cause in allergy?
- smooth muscle contraction
- increased vascular permeability
- mucus secretion
- platelet activation
- stim of nerve endings
- recruitment and activation of eosinophils
(symptoms vary dep on where encounter antigen on body)
What is the hygiene hypothesis?
- insufficient exposure to certain types of infection (“dirt”) skew TH1/TH2 balance towards TH2
- makes sense if look at interaction between TH1 and TH2 cells
What suggested that the hygiene hypothesis was not true?
- -ve correlation between helminth infections and allergic disease
- would expect people responding to pathogens to be more prone to allergy, but opp true
What is the counter regulation or “old friends” hypothesis?
- infection w/ MOs or parasites plays critical role in driving immunoreg
- human IS and “old friends” co-evolved
- infection protects against atopy by promoting IL-10 and TGFβ prod (TREG increase, and decrease in TH1/2)
- so if not exposed to enough pathogen during IS dev, then don’t get T suppressor cells
- may also explain rise in autoimmune disease (TH1/2 driven)
How are immune responses terminated?
- once antigen eliminated or infection cleared, 99% of activated and effector cells die
- mechanisms of downreg = TREGs, cytotoxic T cells
- inhibitory “immune checkpoints” expressed on lymphocytes, eg. CTLA-4 (induced on activated T cells) –> inhibits T cell activation
- lymphocyte receptors w/ ITIMs
- -> FcγRIIb on B lymphocytes (binds IgG and response switched off
- -> PD-1 on activated B and T lymphocytes interacts w/ PD ligand (downreg of B and T cells as triggers phosphorylation of ITIM)
How does engagement of CD28 on naive T cells w/ B7 provide co-stim signal for activation?
- 1 dimer of CD28 can only bind 1 B7 dimer (lower affinity/avidity)
- CTLA-4 can interact w/ lots of B7 dimers at once, providing high affinity clustering
How are antibodies used in research?
- used widely, as diagnostics and increasingly as new class therapeutic drugs
- generally used to identify and label molecules in complex mixtures (blood/urine etc.)
What are the properties of antibodies that make them useful in research and medicine?
- diverse (>10^9 specificities)
- specific, high affinity (Kd 10^-8 - 10^-9M –> higher than most enzs for sub)
- domain structure –> stable, facilitates engineering
- multivalent –> increases avidity, cross-linking can be useful
- effector properties –> useful in some techniques, therapeutics
In what form are antibodies used in research?
- prod antisera (serum containing high levels of antibodies to target antigen)
- can be purified and labelled w/ detectable tag
What are the diff types of label that can be added to antibodies, and what techniques are they used for?
- fluorescent (eg. fluorescin) = immunofluorescence microscopy, FACS (fluorescence activated cell sorting)
- enz –> coloured product = ELISA (enz linked immunoabsorbent assay), immunoblotting, immunohistology
- radioisotope = radioimmunoassay, imaging of eg. tumours
- gold particles = immuno-electron microscope
- sepharose = affinity purification, immunoprecipitation
Why do antibodies req carrier proteins to be used?
- as mol size <1000da
- as generally wouldn’t stay in body long enough to cause response otherwise
What must be considered when gen antibodies?
- immunogenecity (=ability to induce immune response)
- foreignness (=seq homology between antigen and equivalent protein in recipient)
- mol size –> need carrier proteins
- chemical composition –> aromatic groups, charged residues, some non-covalent interactions stronger than others
- ability to provoke T cell responses (need carrier proteins) –> consider genotype of recipient (esp important in inbred mice - MHC)
- use of adjuvants
How are polyclonal antibodies gen?
- immunise animal
- prod 2° response
- collect serum
What are polyclonal antibodies?
- product of several/many B cell clones (/lineages)
- mix of antibodies specific to diff epitopes
- instances where epitopes shared between antigens,s o could affect more than 1 antigen
What are epitopes?
- part of antigen where antibody binds
What are the adv of polyclonal antisera?
- cheap
- robust (may recognise partially denatured/unfolded antigen)
- form immune complexes well (good at cross-linking)
What are the disadv of polyclonal antisera?
- poly-specific
- need pure antigen to immunise
- can be difficult to standardise
What are the uses of polyclonal antisera?
- 2° antibodies for immunoassays (may use monoclonal as 1°)
- can cross react w/ diff mols, so see relationship between mols (eg. antibody isotypes)
- identification of gene products, eg. dystrophin gene in Duchenne MD (ie. gene seq –> protein seq –> peptide – > antibody
What are monoclonal antibodies?
- have single specificity and derived from single B lymphocyte
How are monoclonal antibodies prod?
- B cells from mouse immunised w/ antigen fused w/ myeloma cells
- grow in drug-containing medium –> selection against unfused hybridoma cells as only hybrid cells survive (myeloma line deficient in enz for purine biosynthesis)
- select for antigen specific hybridoma
- clone selected hybridoma cells
What are the advs of monoclonal antibodies?
- highly specific
- can be standardised
- pure antigen not needed for immunisation
What are the disadvs of monoclonal antibodies?
- often conformation sensitive
- less good at complex formation
- expensive (tissue culture equipment and expertise)
How are monoclonal antibodies used in diagnostis?
- detect/quantitate diagnostically important molecules in clinical samples
- eg. pregnancy/fertility testing, Down’s syndrome testing
How are monoclonal antibodies used for definition of cell surface molecules?
- eg. human leucocytes have CD (cluster of differentiation) classification system
- identify cell types, eg. T cells (CD3) and subpops (eg. CD4, CD8) and stage of differentiation
- functional studies –> antibody binding may inhibit, or activate (mimic natural ligand binding)
- gene cloning –> identify gene products, screen expression libs
How could monoclonal antibodies be use as magic bullets for cancer?
- if could use to recognise protein only found on cancer cells
Why did passive immunisation stop being so popular?
- dev of antibiotics
What is the issue w/ using rodent antibodies to induce immune responses in human patients, eg. HAMA?
- HAMA = human anti mouse antibody
- at best monoclonal antibody not effective
- at worst causes serum sickness
What are 2 methods of antibody engineering?
- antibody chimeras
- “humanised” antibodies
How have antibody chimeras been engineered, and how successful has this been?
- mouse V regions and human C regions (C region most immunogenic)
- mouse V regions still immunogenic
- reasonably successful
How have “humanised” antibodies been engineered?
- human framework regions and mouse CDR regions –> aka CDR grafting (CDR most important in binding antigen)
- facilitated by Ig domain structure
- overall folding pattern not disrupted
What are the problems w/ “humanised” antibodies?
- may lose affinity/specificity (as framework regions can also be important in binding antigen)
- time consuming
How can antibodies be used to form gene libraries?
- iso mRNA from antibody prod cells –> blood, lymphoid tissue, bone marrow
- amplify Fab of Fv cDNA by PCR (part that recognises antigen)
- clone and express in bacteria/phage (phage display)
- screen antibody phage display lib vs solid phase antigen “panning”
What do phagemid vectors do?
- most common vectors
- express soluble protein in bacteria or on surface of filamentous phage particles
How are fully human antibodies gen from a gene library by phage display?
- iso pop of genes encoding antibody V regions
- construct fusion protein of V region w/ bacteriophage coat protein
- cloning random pop of V regions gives rise to mix of bacteriophage = phage display lib
- select phage w/ desired V regions by specific binding to antigens (“panning”)
- following selection, phage used to re-infect bacteria and process repeated to enrich for antigen binders
What does synthetic mean in the context of a human antibody gene lib?
- genes optimised for expression in bacteria
- not real human genes, as codons swapped
What did the chemistry nobel prize 2018 involve research on?
- synthetic/semi-synthetic human antibody gene lib created
- human V region genes and “randomised” CDR regions
- > 10^9 members
- CDRs can be further mutated to improve specificity/affinity
How can human antibodies be gen from SCID mice?
- no adaptive I, can tolerate human leucocytes
- so reconstituted w/ human lymphocytes using hybridoma techniques
- can be immunised to gen human antibodies, but repertoire limited to that of human cells used
How can human antibodies be gen from transgenic mice?
- mouse antibody genes replaced by human antibody genes (“xenomouse”)
- these genes introd using YACs
- mice can be immunised t gen human antibodies by conventional monoclonal or phage display techniques
- their IS works w/ human antibodies (seem healthy), but may have some problems as some effector sites diff etc.
What diff formats of monoclonal antibody can be used for therapy?
- fully mouse (-omab)
- chimeric (-ximab)
- humanised (-zumab)
- fully human (-umab)
What is unusual about the antibodies in the camelidae family?
- 2 forms
- conventional –> H and L chains, both req for antigen stability and binding
- and H chain antibody –> only H chains, full antigen binding capacity and v stable DIAG
What are the characteristic of Ablynx’s nanobody?
- single domain antibody (VHH)
- small (1/10 size of monoclonal antibody)
- flexible formatting
- highly potent, robust and stable
- broad target applicability
- multiple administration routes
- ease of manufacture
- speed of discovery
How can antibodies used for passive immunisation?
- neutralise toxins –> eg. anti-tetanus (pooled IgG), anti-snake venom
- prevent/treat infection –> eg. resp syncytial virus (RSV)
How can antibodies be used to treat cancer, and what are 2 eg.s?
- magic bullets to target cancer cells
- eg. anti-CD52 antibodies (CAMPATH antibodies) –> recognises leucocytes, good activator of complement and ADCC, use in leukemias and lymphomas
- eg. anti-HER2 antibodies (Herceptin) –> recognise HER2 (RTK expressed in high levels in ≈25% breast cancers)
What are problems w/ using antibodies for cancer?
- antigen specificity (finding something only on cancer cells)
- antigen shedding
- tumour cells inaccessible, esp if large
- HAMA responses
- other side effects
How can antibodies be used to modulate immune responses, and in what cases would this be useful?
- depletion of leucocytes (eg. antibodies to CD52, CD3, CD4) –> organ transplantation, graft vs host disease, autoimmune disease
- blocking of cytokines, cytokine receptors, soluble mediators (eg. antibodies to TNF-α, IL-1, IL-6, C5 or their receptors) –> inflam/autoimmune disease, allergy (antibodies to IgE, cytokines)
- immune checkpoint inhibitors (eg. antibodies to CTLA-4, PD-1) –> dampen down IS after response
Why do many cancers induce immunosuppression?
- to inhibit immune checkpoints, to avoid death
How do cancers induce immunoexpression?
- prod cytokine that recruit TREGs
- express PD-L
- CTLA-4 induced on activated T cells, expressed constitutively on TREGs, higher avidity for B7, so switched T cells off (usually only towards end of immune response)
- PD-1 (transiently expressed on activated T cells) interaction w/ PD-L also induces inhibition
What can reverse immunosuppression?
- antibodies that block inhibitory immune checkpoints
What was the 2018 nobel prize for physoilogy and medicine awarded for?
- characterisation of immune checkpoints
- cancer therapy by inhibiting -ve immune cell regulation
In general how are antibodies used for immunotherapy?
- exploit antibody effector functions/binding
- label antibody w/ toxin/drug/prodrug/radionuclide
How does mechanism differ when antibody labeled in diff ways?
- tumour specific antibody –> antibodies bind tumour cell –> NK cells w/ FcR activated to kill tumour cells
- tumour specific antibody (or fragment) conjugated to toxin –> antibody-toxin conjugates bind to tumour cell –> conjugates internalised, killing the cell
- tumour specific antibody (or fragment) conjugated to radionuclide –> radioactive antibody binds tumour cell –> radiation kills tumour cell and neighbouring tumour cells
What toxins can be used to label antibodies for immunotherapy, and how are they used?
- immunotoxins –> retain antigen specificity, tag or replace Fc w/ toxin
- eg. ricin, diphtheria toxin or BFL1
How can antibodies be improved for therapy?
- sites for eg. C1q binding, FcR binding “mapped” to AA residues on Fc
- antibody engineering to improve half life an effector functions (eg. ADCC, complement activation)
- “glycoengineering” –> removal of fructose improves interaction w/ FcγRIII and therfore ADCC
What is an eg. of a mouse monoclonal antibody used therapeutically?
- OTK3
- specificity = CD3
- for transplant rejection
What is an eg. of a mouse/human chimeric antibody used therapeutically?
- infliximab
- specificity = TNF-α
- for Crohn’s disease, rheumatoid arthritis (inflam)
What is an eg. of a humanised antibody used therapeutically?
- herceptin (trastuzumab)
- specificity = HER2
- for breast cancer
What is an eg. of a human antibody used therapeutically?
- nivolumab
- specificity = PD-1
- for metastatic melanoma, non-small cell lung cancer
What are CAR-T cells?
- CAR = chimeric antigen receptor
- T cells engineered to recognise tumour antigen
How are CAR-T cells prod?
- T cells harvested from blood of patient w/ B cell tumour
- CD19 is antigen expressed by acute lymphoma leukemia
- retrovirus encoding anti-CD19 CAR infects T cells that are activated w/ antibodies to CD3 and CD28
- infected T cells express anti-CD19 CAR, fused to signalling domain
- CAR expression and T cell activation in vitro overcomes req for MHC recognition
Why is using CAR-T cells so expensive?
- individualised medicine
Why don’t V regions of TCRs undergo somatic mutation?
- if T cells could mutate in tissues, then danger of getting T cells that recognise self –> doesn’t matter as much if B cells do, as req T cell help anyway
- would no longer be able to recognise self MHC
- TCRs don’t need v high affinity, as always recognise antigen in context of self MHC –> whereas B cells prob soluble Ig which does need to bind tightly
