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MBB311 - Molecular Immunology > Partridge > Flashcards

Partridge Flashcards

1
Q

What is the role of the IS?

A
  • 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
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2
Q

What external threats are there to the IS?

A
  • viruses
  • bacteria
  • fungi
  • protozoa
  • parasites
  • prions
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3
Q

Do most MOs cause diseases?

A
  • 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
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4
Q

Why is the IS a ‘double edged sword’?

A
  • 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
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5
Q

What is active immunisation?

A
  • vaccination

- give mod form or component of pathogen to activate immune response, to gen memory so will recognise pathogen

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6
Q

What is passive immunisation?

A
  • administration of immune components from immunised source, eg. antisera, antibodies
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7
Q

What are the main differences between the innate and adaptive IS?

A

INNATE:

  • limited specificity
  • resistance not improved by repeat infection
  • rapid response (hours)

ADAPTIVE:

  • highly specific
  • resistance improved by repeat infection
  • slower response (days-weeks)
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8
Q

What leucocytes are part of the innate and adaptive IS?

A
  • innate = phagocytes, NK cells

- adaptive = B and T lymphocytes

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9
Q

What soluble factors are part of the innate and adaptive IS?

A
  • innate = lysosyme, complement, interferons etc.

- adaptive = antibody

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10
Q

Where are cells of the IS derived from?

A
  • all hematopoietic cells derived from pluripotent stem cells
  • give rise to 2 main lineages, 1 for myeloid cells and 1 for lymphoid cells
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11
Q

What cells are derived from the common myeloid progenitor?

A
  • megakaryocytes (platelets) and erythrocytes
  • granulocytes = eosinophil, neutrophil, basophil, mast cell
  • monocyte –> macrophage
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12
Q

What cells are derived from the common lymphoid progenitor?

A
  • T helper lymphocyte
  • T cytotoxic lymphocyte
  • B lymphocyte –> plasma cell
  • NK cell
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13
Q

What are the types of professional phagocytes?

A
  • neutrophils
  • mononuclear phagocytes
  • mast cells
  • dendritic cells
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14
Q

What are the characteristics of neutrophils?

A
  • main phagocytes in blood
  • short lived and fast moving
  • specialised lysosomes release enzs, H2O2 etc.
  • DIAG*
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15
Q

What are the characteristics of mononuclear phagocytes, and what diff cells are they in diff organs?

A
  • 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*
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16
Q

What professional phagocytes can act as sentinel cells?

A
  • macrophages
  • mast cells
  • dendritic cells
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17
Q

What are the characteristics of mast cells?

A
  • often underlie mucosal surfaces
  • release inflammatory mediators (eg. histamine)
  • important in responses to parasites and allergy –> express high affinity Fc receptors for IgE
  • DIAG*
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18
Q

What are the characteristics of a dendritic cells?

A
  • in skin, mucosa and lymphoid tissue
  • specialised in presenting antigen to T cells
  • related to monocytes/macrophages but v specialised function
  • DIAG*
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19
Q

What are the characteristics of NK cells?

A
  • 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
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20
Q

What are the characteristics of receptors on phagocytes and other myeloid cells?

A
  • broadly specific for large categories of pathogen

- pattern recognition receptors (PRRs) recognise pathogen assoc molecular patterns (PAMPs)

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21
Q

What types of infection are diff soluble factors involved in?

A
  • defensins = disrupt bacterial cells
  • complement = in bacterial infections
  • interferons = in viral infections
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22
Q

What is the inflammatory response?

A
  • integrated response to local infection
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23
Q

What is the difference between B and T lymphocytes, and the process by which they differentiate to fight infection?

A
  • 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
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24
Q

What are the immunoglobulin (antibody) classes and their roles?

A
  • 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
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25
Q

What are 2 subpops in cell mediated (T cell) immunity?

A
  • T helper cells (CD4 +ve)

- T cytotoxic cells (CD8 +ve)

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26
Q

What is the role of T helper cells?

A
  • help B cells make antibody
  • activate macrophages and NK cells
  • help dev of cytotoxic T cells
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27
Q

What is the role of T cytotoxic cells?

A
  • 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)

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28
Q

How do cytokines differ from hormones?

A
  • most act locally

- but can have systemic actions

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29
Q

What are cytokines and what is their role?

A
  • 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
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30
Q

What are some of the main groups of cytokines, and how are they prod?

A
  • 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
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31
Q

How is lymphoid tissue organised?

A
  • 1° = lymphocytes reach maturity

- 2° = mature lymphocytes stimulated by antigen

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32
Q

What are the characteristics of innate immunity?

A
  • 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
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33
Q

How does the innate immune response aid the adaptive?

A
  • 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)
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34
Q

What is the consequence of the delay in adaptive immune response dev?

A
  • failure to dev immunity for organisms that mutate quickly (antigenically unstable), eg. parasites, influenza, HIV
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35
Q

What are the phases in initial response to infection?

A

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

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36
Q

What are physical innate immunity barriers in the skin, GI tract, UG tract and resp tract?

A
  • in all epithelial cells joined by tight junctions

- in resp tract cilia to move mucus and in others flow of air or fluid

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37
Q

What are chemical innate immunity barriers in the skin, GI tract, UG tract and resp tract?

A
  • in skin FAs
  • in GI tract low pH and enzs (pepsin)
  • in UG and resp tracts lysozyme
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38
Q

What are microbiological innate immunity barriers in the skin, GI tract, UG tract and resp tract?

A
  • in all antibacterial peptides (defensins) and commensals (microbiota)
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39
Q

How do secreted chemicals, antimicrobials and commensals help protect from infection?

A
  • make unfavourable env for pathogens
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40
Q

How does keratinised skin help protect from infection?

A
  • generally impermeable (unless damaged by burn/cut/insect bite etc.)
  • keratinocytes prod keratin, ebum (contains FAs and defensins
  • shedding
  • commensals
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41
Q

How do mucous membranes help protect from infection?

A
  • 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
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42
Q

What are the diff types of pre-formed effectors, and their characteristics?

A
  • 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
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43
Q

What is complement, and how was it discovered?

A
  • > 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)
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44
Q

How is complement activated?

A
  • components normally inert, but activated by presence of pathogens or antibody bound to pathogen
  • DIAG*
  • central event is cleavage of C3, by C3 convertase
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45
Q

What is the order of activation of complement in the classical pathway?

A
  • C1, 4, 2, 3, 5, 6, 7, 8, 9
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46
Q

How did complement originally evolve?

A
  • as part of innate immune response
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47
Q

What is the role of complement?

A
  • provides protection early in infection in absence of antibodies through other “older” activation pathways
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48
Q

Where are complement proteins made, and which is most abundant?

A
  • mainly made in liver

- C3

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49
Q

What are many activated complement components?

A
  • Ser proteases, act on 1 another to gen larger and smaller fragment
  • C3 –> C3b + C3a
  • b is larger and a is smaller
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50
Q

What is the result of C3 cleavage?

A
  • exposes reactive thioester in C3b, which can bind covalently to adj proteins/carbs, eg. on pathogens surface (rapidly inactivated in fluid phase
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51
Q

What are the 3 pathways of complement activation?

A
  • classical pathway
  • mannose-binding lectin (MBL) pathway
  • alt pathway
  • DIAG
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52
Q

What is the C3 convertase in each pathway for complement activation?

A
  • for classical and MBL = C4bC2a (from cleavage of C4 and C2 by C1r,s and MASP2, respectively)
    for alt = C3bBb
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53
Q

What is the role of C3b, and how it gen, in alt pathway?

A
  • 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
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54
Q

What are the later stages of complement activation?

A
  • C3 convertase + C3b –> C5 convertase –> C5 –> C5b, C6, C7, C8, C9 (membrane attack complex - MAC)
  • C5a disperses and C5b triggers rest of cascade
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55
Q

What is the role of the membrane attack complex (MAC)?

A
  • 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
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56
Q

What are the 3 major biological activities of complement, and what components do they involve?

A
  • opsonisation –> C3b
  • activation of IS –> C5a (or C3a but not as active)
  • lysis of foreign cells –> MAC, esp C9
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57
Q

What is opsonisation?

A
  • binding of complement proteins or antibodies (=OPSONINS) to surface of pathogen, so phagocytes can recognise and bind
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58
Q

How does complement cause activation of IS?

A
  • chemoattractants and anaphylatoxins

- stimulate mast cells to induce inflammation at local level

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59
Q

What occurs when allergy activates IS?

A
  • allergy causes widespread activation of mast cells throughout body
  • lots of histamine release
  • causing pressure on heart and lungs
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60
Q

How does complement cause lysis of foreign cells?

A
  • 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)
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61
Q

Why does complement need to be reg?

A
  • “double edged sword”

- to prevent damage to host

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62
Q

How is the complement reg?

A
  • 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
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63
Q

What can deficiencies in inhibitors of complement system cause?

A
  • increase risk of some diseases
  • age related macular degen (factor H)
  • paroxysmal nocturnal hemoglobinuria (CD59) –> body attacks own RBCs
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64
Q

What is the importance of the complement?

A
  • 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
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65
Q

What is the role of preformed effectors?

A
  • ‘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
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66
Q

How do innate and adaptive receptors vary?

A
  • 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
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67
Q

What do innate receptors recognise?

A
  • 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
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68
Q

What do PAMPs and DAMPs have in common?

A
  • both relatively invariant structures
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69
Q

What diff PAMPs do diff organisms have?

A
  • 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
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70
Q

What diff DAMPs are there?

A
  • 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
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71
Q

What classes of PRRs are there?

A
  • 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
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72
Q

How do membrane receptors, and the diff types work? (PRRs)

A
  • 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
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73
Q

How do chemotactic receptors on phagocytes work?

A
  • 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
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74
Q

What are TLRs (Toll-like receptors)?

A
  • ancient pathogen recognition system
  • 10 in humans, each recognising distinct PAMP
  • cell surface receptors or endosomal
  • often function as dimers
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75
Q

What are some eg.s of TLRs and their ligands?

A
  • TLR-3 and dsRNA
  • TLR-4 dimer and LPS
  • TLR-5 and flagellin
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76
Q

What is the structure of TLRs?

A
  • DIAG*
  • ec domain of TLR3 has horseshoe shape, formed by leu-rich repeats, inner surface has β-sheet structure and forms ligand binding domain
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77
Q

What happens when TLR binds PAMP?

A
  • signalling to nucleus to induce expression of inflamm cytokines and interferons
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78
Q

How do the diff types of cytoplasmic receptors work? (PRRs)

A
  • 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
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79
Q

What is the inflammasome?

A
  • protein complex needed to process procytokines –> activates components too toxic to be active all time, by cleavage
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80
Q

What are the 4 classic signs of inflammation?

A
  • redness
  • swelling
  • heat
  • pain
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81
Q

What causes the classic signs of inflammation?

A
  • 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
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82
Q

What is the aim of inflammation?

A
  • ensure immune cells, defence mols, coagulation factors etc. reach site of infection or tissue damage
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83
Q

What are inflamm mediators?

A
  • 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
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84
Q

What is extravasation?

A
  • movement of leucocytes from blood to tissues
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85
Q

How does extravasation occur?

A
  • 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
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86
Q

What is a summary of the inflamm response?

A
  • 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
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87
Q

When is acute inflamm needed?

A
  • generally beneficial in dealing w/ infection/injury

- avoided in some places where would be harmful, eg. brain, CNS

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88
Q

What is chronic inflamm?

A
  • caused by chronic inflammation, eg. TB, autoimmune disease

- can be damaging

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89
Q

What is the role of inflamm in TB?

A
  • can survive in macrophages

- chronic inflamm could be good, as bacteria encapsulated, so can’t spread to rest of body

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90
Q

How are cytokines classified?

A
  • grouped into fams based on structural similarities

- but fam members may have distinct functions

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91
Q

What are the characteristics of cytokine receptors?

A
  • 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
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92
Q

When and where are cytokines secreted?

A
  • by macrophages and dendritic cells in early induced immune response
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93
Q

How is TNFα prod?

A
  • as TM protein, released by proteolysis
  • primarily by macrophages
  • LPS is potent stimulus of its prod
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94
Q

What are the TNFα receptors and how do they work?

A
  • TNFR1 major form
  • TNF trimer cross-links 3 receptors
  • 2 pathways –> cell stim/apoptosis
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95
Q

Why can’t TNFα be used to treat cancer?

A
  • too toxic
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96
Q

What are the local effects of TNFα?

A
  • 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
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97
Q

What are the systemic effects of TNFα, and at what conc is this?

A
  • <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
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98
Q

How does TNFα cause sepsis?

A
  • 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)
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99
Q

How do interferons affect viruses?

A
  • interfere w/ viral rep
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100
Q

What are the 2 types of interferons and how are they prod?

A
  • 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
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101
Q

What is the interferon response?

A
  • 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
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102
Q

How could interferons be used therapeutically?

A
  • poss to make recomb or synthetic versions

- have some ability to induce apoptosis of tumour cells, so could be used for this

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103
Q

Where is type II interferon γ prod, and what is its role?

A
  • 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
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104
Q

How can T helper cells be further subdivided?

A
  • dep on cytokines they make

- TH1 (inflamm cytokines) and TH2 cells

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105
Q

What is the role of TH1 cells?

A
  • prod IL-2, IFNγ, TNFα
  • activate macrophages and induce B cells to make opsonising antibodies (IgG)
  • in classical bacterial and viral infections
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106
Q

What is the role of TH2 cells?

A
  • prod IL-4, 5, 6, 10, 13
  • induce B cells to make IgE (4 and 13)
  • in parasitic infections
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107
Q

How do cytokine receptors to diff antigens determine adaptive immune responses?

A
  • 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
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108
Q

What characterises a polymorphic granulocyte?

A
  • many shaped nuclei
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109
Q

What are the diff types of polymorphic granulocytes, and how were they experimentally identified?

A
  • neutrophils –> didn’t take up acidic or basic dye
  • eosinophils –> took up red dye strongly, as v acidic
  • basophils –> took up basic stain
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110
Q

What is the main phagocyte in blood?

A
  • neutrophils
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111
Q

What are eosinophils and what is their role?

A
  • <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
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112
Q

What are basophils and what is their role?

A
  • <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
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113
Q

What does heparin do?

A
  • stops blood clotting too quickly
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114
Q

What does histamine do?

A
  • vasodilator, important in early stages of infection
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115
Q

What is the role of mast cells?

A
  • 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
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116
Q

What are the characteristics of neutrophils and where are they found?

A
  • 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
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117
Q

What are the functions of neutrophils?

A
  • 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)
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118
Q

What is the fate of monocytes?

A
  • to move into tissue and differentiate into macrophages (circulate in blood approx 3 days)
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119
Q

What are the roles of macrophages?

A
  • 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)
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120
Q

What are the role of macrophages in adaptive immunity?

A
  • 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)
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121
Q

What is the seq of events that occurs during phagocytosis?

A
  • 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
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122
Q

What are the diff classes of mechanisms of phagocyte bactericidal agents, and what are there specific products?

A
  • 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
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123
Q

What is oxygen dep killing, and the diff ROIs?

A
  • 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
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124
Q

What is NO prod by, and what is its role?

A
  • 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)

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125
Q

What is the role of distinct cytoplasmic granules in NK cells?

A
  • 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
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126
Q

What infections are NK cells important in?

A
  • viral infections

- but also intracellular bacteria and protozoa

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127
Q

What are the roles of NK cells?

A
  • 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
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128
Q

How do NK cell receptors work?

A
  • 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)
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129
Q

Why do viruses and cancers downreg MHCI?

A
  • to evade cytotoxic T cells, even though still killed

- as worse death than by NK cells

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130
Q

What are dendritic cells and what is their role?

A
  • 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
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131
Q

What is macropinocytosis, and what cells do it?

A
  • uptake of fluid and any foreign material in it

- all cells capable, but esp done by dendritic cells

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132
Q

What are the key features and functions of the IS?

A
  • 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
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133
Q

How is adaptive IS activated in draining lymph node?

A
  • 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
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134
Q

What is the purpose of T and B memory cells?

A
  • long lived, in some cases life long, and provide immunity
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135
Q

What is the clonal selection hypothesis?

A
  • 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
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136
Q

What are the 2 roles of antibodies in the immune response, and what regions are responsible for these?

A
  • antigen recognition = as integral membrane proteins on B lymphocytes, Fab regions
  • antigen elimination = as soluble proteins secreted by plasma cells, Fc region
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137
Q

What is the basic antibody structure?

A
  • 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.
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138
Q

What is the 4 chain antibody structure?

A
  • DIAG*
  • light (L) chain = 25kD
  • heavy (H) chain = 50kD
  • immunoglobulin G = L2H2 = 150kD
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139
Q

How can diff antibody fragments be prod?

A
  • 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
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140
Q

How do immunoglobulin classes differ?

A
  • AA seq of heavy chains
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141
Q

What are the light chain types of antibodies?

A
  • kappa (κ) or lambda (λ)

- not class restricted, ie can have IgGκ or IgGλ antibodies

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142
Q

Where did further info on antibody structure come from?

A
  • protein seq

- eg. of myeloma proteins (cancer of plasma cells)

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143
Q

What further discoveries were made from protein seq of antibodies?

A
  • contain constant and variable regions
  • comprised of homologous domains
  • variable region domains contain 3 hypervariable regions
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144
Q

What is the role of constant and variable regions in antibodies?

A
  • variable bind antigen, differ between antibodies w/ diff specificities
  • constant same for antibodies of given H chain class or L chain type
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145
Q

What did X-ray crystallography of Fab and Fc fragments show, and why were fragments have cleaved to look at structure?

A
  • 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
146
Q

What is the Ig fold comprised of, and in what members is it found?

A
  • C domain has 7 β strands
  • V domain has 9 β strands
  • found in all members of Ig gene superfam
147
Q

What are the Ig gene superfam involved in and what are their domains like?

A
  • 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
148
Q

How many members are there of the Ig gene superfam in the human genome, and why are antibodies an unusual member?

A
  • 765

- members usually only found on cell surface as receptors

149
Q

What is the structure of the antigen combining site?

A
  • 3x hypervariable regions (approx 7-12 AAs)

- rest is framework regions

150
Q

What are hypervariable regions, and what do they determine?

A
  • complementarity determining regions (CDRs)

- how complementary determines how specifically can interact w/ antigen

151
Q

What are the characteristic of antigen-antigen interactions?

A
  • 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
152
Q

What antibodies do B cells initially express, and why do these need to assoc w/ other proteins, what does this involve?

A
  • 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
153
Q

What is they key diff w/ T lymphocyte receptors (as opposed to B cell receptors)

A
  • only expressed on membranes, not as soluble proteins
154
Q

What are the roles of T cytotoxic and T helper cells?

A
  • cytotoxic = specifically kill infected host cells

- helper = augment immune response

155
Q

How do T cells recognise antigen?

A
  • 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)
156
Q

What is the diff between MHC class I and II, in where expressed and what they present?

A
  • 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
157
Q

What is the role of MHCI?

A
  • 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

158
Q

What is the role of MHCII?

A
  • 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)
159
Q

What is the structure of T lymphocyte receptor (TCR) similar to?

A
  • 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
160
Q

What receptors do a subset of T cells express, and how do they differ?

A
  • γδ receptors
  • in 1-5% T cells
  • found at epithelial surfaces
  • less diverse –> recognise broader range of antigens
161
Q

What do TCRs recognise?

A
  • complex of antigen and self MHC
162
Q

What do the 3 CDRs bind?

A
  • CDR3 regions of α and β chains most variable –> bind foreign peptide
  • CDR1/2 bind self MHC
163
Q

Why does TCR expression on cell surface req assoc w/ add proteins?

A
  • v short C-ter, so don’t protrude much into cyto, so can’t signal by itself, needs to assoc w/ diff membrane proteins
164
Q

What is the TCR complex made up of?

A
  • α and β subunits (TCR)

- CD3 subunits (ε, δ and γ) and ξ –> CD3 subunits contain ITAMs in their cyto regions

165
Q

What is the immune repertoire?

A
  • approx 10^14 antibody receptors and 10^18 T cell receptors
166
Q

What is the structural basis of antibody diversity?

A
  • 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
167
Q

What were some of the early hypotheses for the genetic basis of antibody diversity and why were they unpopular?

A
  • multiple genes
  • somatic mutation –> not pop to have mutations in somatic cells
  • somatic recomb –> not pop to have genes in fragments
168
Q

What was the Dreyer & Bennett hypothesis (1965)?

A
  • Igs encoded by separate C region and multiple V region genes
169
Q

What did Tonnegowa discover in 1976?

A
  • Ig genes rearranged during B cell dev –> proven through restriction digests
170
Q

How was κ light chain gene recomb demonstrated experimentally?

A
  • 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
171
Q

What are the 3 sets of Ig genes?

A
  • heavy (H) chains = chromosome 14
  • kappa (κ) chains = chromosome 2
  • lambda (λ) chains = chromosome 22
172
Q

How many V and C regions are in Ig genes?

A
  • each locus has multiple variable region genes and 1, or a few constant region genes
  • V regions encoded by 2 or more exons
173
Q

How are light chain V regions encoded?

A
  • by 2 segments of DNA
  • DIAG*
  • most encoded by V segment
  • there is also joining segment
174
Q

How are heavy chain V regions encoded?

A
  • by 3 segments of DNA
  • DIAG*
  • V segment, diversity and joining
175
Q

What chain is always expressed first?

A
  • Cμ (IgM heavy chain)
176
Q

How does rearrangement of κ light chains occur?

A
  • 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
177
Q

How does rearrangement of heavy chain occur?

A
  • D-J joining
  • then V-D-J joining
  • all extra bits removed when transcribed to mRNA
178
Q

Where are the diff CDRs encoded?

A
  • CDR1 and CDR2 encoded by V segments (ie. germline)

- CDR3 corresponds to VDJ (or VJ for light chain) join

179
Q

What does recombination of chains involve?

A
  • 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
180
Q

What are RSSs and where are they found?

A
  • 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
181
Q

What is the 12-23 bp rule regarding RSSs in recomb?

A
  • 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
182
Q

What is V(D)J recombinase made up of?

A
  • 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)
183
Q

What do mutations in DNA-dep protein kinase and RAG-1/RAG-2 of VDJ recombinase cause?

A
  • severe combined immunodeficiency (SCID)
184
Q

How can SCID be cured?

A
  • bone marrow transplant

- genetic techniques

185
Q

What is the mechanism for somatic recomb?

A
  • 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)
186
Q

What is the structure of the RAG-1/RAG-2 complex?

A
  • 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
187
Q

What does antigen indep B cell differentiation involve?

A
  • 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
188
Q

What happens to B cell after antigen indep differentiation, if it never meets antigen?

A
  • apoptosis
189
Q

Where does antigen indep differentiation occur?

A
  • bone marrow
190
Q

Where does antigen dep differentiation occur?

A
  • 2° lymphoid tissue
191
Q

What are the 2 processes in antigen dep differentiation that result in differentiation?

A
  • somatic hypermutation

- class switching

192
Q

What happens during somatic hypermutation?

A
  • 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
193
Q

What is activation induced cytidine deaminase (AID)?

A
  • enz expressed only by B cells in lymphoid tissue responding to antigen
194
Q

What is the role of somatic hypermutation?

A
  • main role to improve immune response

- but can also add diversity

195
Q

What is affinity maturation?

A
  • higher affinity receptors selected as immune response proceeds –> “survival of the fittest”
196
Q

What is the rate of mutation in somatic hypermutation, and how does this compare to normal rate?

A
  • 1bp per 10^3 bp per cell division

- normal = 1bp per 10^10 bp per cell division

197
Q

What happens during class switching?

A
  • 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
198
Q

What do somatic hypermutation and class switching both req?

A
  • T cell help

- AID

199
Q

How does mechanism for class switching differ from VDJ joining?

A
  • initiated by AID acting at switch regions –> G rich tandem repeated DNA seqs found close to C region genes
200
Q

What is AIDs mechanism of action?

A
  • 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
201
Q

How can co-expression of membrane IgM and IgD occur?

A
  • 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
202
Q

How can you get prod of surface and secreted forms of Ig from same H chain?

A
  • 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
203
Q

What is the structure of TCR genes?

A
  • 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
204
Q

How does TCR gene rearrangement occur during differentiation?

A
  • somatic recomb of TCR V region genes –> same machinery as used in dev B lymphocytes
  • only diff is occurs in thymus
205
Q

What is the diversity of TCR genes?

A
  • 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
206
Q

Do V regions of TCRs undergo somatic hypermutation?

A
  • no
207
Q

What are γδ T cells?

A
  • 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”
208
Q

Where are MHC encoded in humans ?

A
  • encoded by genes of MHC chromosome 6
209
Q

What are MHC also known as in humans?

A
  • HLA = human leucocyte antigen

- eg. HLA-A, HLA-B, HLA-C

210
Q

Are MHC genes polymorphic?

A
  • 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)
211
Q

What is MHC restriction?

A
  • T lymphocytes can only recognise antigen in context of self MHC molecules
212
Q

How was MHC restriction demonstrated experimentally?

A
  • 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
213
Q

WHat 2 theories arose from experiments on inbred mice relating to MHC restriction, and what provided final proof?

A

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

214
Q

How do MHCI and MHCII structures differ?

A
  • MHCI = polymorphic TM α chain and invariant β2-microglobulin
  • MHCII = polymorphic TM α and β chains
215
Q

What similarities are there in MHCI and MHCII structures?

A
  • membrane-prox domains Ig like

- membrane distal domains bind peptides and contain polymorphisms

216
Q

What peptides are MHCI and MHCII capable of binding?

A

> > 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
217
Q

What did crystallographic studies show about MHC?

A
  • 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
218
Q

How is bound peptide an integral part of MHC structure?

A
  • 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
219
Q

How does antigen presentation occur w/ MHCI?

A
  • 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
220
Q

How does antigen presentation occur w/ MHCII?

A
  • antigen taken up by phagocytosis or endocytosis etc.
  • acidification promotes folding and proteolysis
  • peptides assoc w/ MHCII in endocytic compartment
221
Q

What is cross presentation?

A
  • process by which certain antigen presenting cells, eg. dendritic cells, present peptide assoc w/ MHCI to cytotoxic T cells
222
Q

What is req for TCR to form TCR complex?

A
  • TCR must assoc w/ other cell surface receptors (CD3 and ζ chain) to signal
223
Q

Why are co-receptors req for T cell activation, and what are they?

A
  • stabilise interaction
  • facilitate signalling
  • CD8/4 interact w/ invariant regions on MHCI/II
  • MHCI + antigen CD8
  • MHCII + antigen CD4
224
Q

What does binding of MHC ligand to TCR cause?

A
  • 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)
225
Q

How are MHC genes expressed?

A
  • co-dominantly –> increasing no. diff MHC molecules expressed per cell
  • MHCI most variable
226
Q

What are the consequences of MHC genes being polymorphic?

A
  • 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
227
Q

What did MHC polymorphism evolve in response to?

A
  • pathogens

- eg. black death/flu/HIV ??

228
Q

What is greater polymorphism of MHC assoc w/?

A
  • greater social interaction

- and therefore chance of passing on diseases

229
Q

What are the functions of MHC protein?

A
  • 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?
230
Q

In terms of MHC proteins, what do individuals want in terms of a mate?

A
  • someone w/ MHC proteins as diff as poss
231
Q

How does thymic selection occur?

A
  • 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+)
232
Q

What is the consequence of the fact that antibodies are flexible adaptors?

A
  • diff Ig classes have diff biological roles

- way they deal w/ pathogens is dep on class

233
Q

What is the structure of IgG (γ chain), and what subclasses are there?

A
  • 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
234
Q

What is the role of IgG (γ chain)?

A
  • 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
235
Q

What is the structure of IgM (μ chain)?

A
  • DIAG*
  • pentamer –> 5 antibody units and J chain
  • mw = 970,000d
  • no defined hinge = “functional hinge”, so not as much flex
236
Q

What is the role of IgM (μ chain)?

A
  • 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)
237
Q

What is the structure of IgA (α chain), and what are the subclasses?

A
  • 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
238
Q

What is the role of the secretory component in IgA?

A
  • helps protect from digestion and can bind mucin to anchor IgA to mucus
239
Q

What is the role of IgA (α chain)?

A
  • 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
240
Q

What specialised transport mechanism exists for IgA?

A
  • 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
241
Q

What is the structure of IgD (δ chain)?

A
  • DIAG*
  • monomeric
  • mw = 184,000
  • long extended hinge, heavily glycosylated
242
Q

What is the role of IgD (δ chain)?

A
  • <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
243
Q

What is the structure of IgE (ε chain)?

A
  • DIAG*
  • monomeric
  • mw = 190,000d
  • no defined hinge –> “functional hinge” like IgM
244
Q

What is the role of IgE (ε chain)?

A
  • 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
245
Q

What is the biological role of Igs (ie. methods of action), and which classes are responsible for these?

A
  • 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
246
Q

Can antibodies directly kill bacteria?

A
  • if bind to transporter, but usually need to react w/ complement/leucocyte to kill
247
Q

What are the functions of Fc effectors?

A
  • invoke destruction of labelled pathogens
  • activate complement (IgM, IgG)
  • bind Fc receptors on leucocyte surfaces (IgG, IgA, IgE)
248
Q

What are the Fc effector mechanisms that operate dep on?

A
  • site and type of infection

- stage of immune response (1° or 2°)

249
Q

What is req for complement activation through the classical pathway?

A
  • 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
250
Q

Why is IgM a much more potent activator of complement than IgG?

A
  • IgM pentameric, so C1q bind 1 molecule, rather than at least 2 of IgG
251
Q

How does IgM activate complement?

A
  • 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)
252
Q

How does IgG activate complement?

A
  • 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)
253
Q

What does complement activation result in?

A
  • inflam
  • activation of leucocytes (eg. for chemotaxis)
  • opsonisation
  • lysis of foreign cells
254
Q

What is the importance of complement activation?

A
  • immune defence against bacteria (and viruses), clearance of immune complexes
  • inducer of inflam, can cause pathology
255
Q

What is the effect of antibody and complement on clearance of bacteria from blood?

A
  • DIAG*

- when Ab and C3b coated best recognised, as phagocytes have receptors for both

256
Q

Where are Fc receptors (FcR) expressed?

A
  • variety of effector cells

- inc granulocytes, mononuclear phagocytes, NK cells

257
Q

How do Fc receptors function?

A
  • most function as part of a multi-subunit complex

- α chains bind Fc and assoc chains facilitate cell surface expression/signalling

258
Q

How are Fc receptors activated?

A
  • 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)
259
Q

How are Fc receptors inhibited?

A
  • inhibitory receptors contain immunoreceptor Tyr inhibitory motifs (ITIMs)
260
Q

Where do diff isoforms of Fcγ receptors occur?

A
  • differ in cytoplasmic/membrane spanning regions
261
Q

What is the role of Fc receptors on phagocytes?

A
  • uptake of immune complexes
  • opsonisation
  • cell activation
  • resp burst (release of ROI)
  • release lysosomal contents (“frustrated phagocytes”)
262
Q

What Fc receptors are found on phagocytes?

A
  • IgG (IgG1 = IgG3 > IgG4)

- IgA

263
Q

What Fc receptors are found on NK cells?

A
  • IgG (IgG = IgG3)
264
Q

What is the role of receptors on NK cells?

A
  • 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
265
Q

What is TRIM21 and what is its role?

A
  • 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
266
Q

What is FcRn and where is it present?

A
  • neonatal receptor for IgG (IgG1 > IgG3 > IgG2 > IgG4) and present in neonatal gut
  • also in adults in gut, liver and endothelial cells
267
Q

What is the role of FcRn?

A
  • placental receptor of IgG (on trophoblast), protects fetus and newborn
  • in adults binds and recycles IgG, preventing excretion (improves half life)
268
Q

What Fc receptors are found on mast cells and basophils?

A
  • IgE
269
Q

What is the role of Fc receptors on mast cells and basophils?

A
  • med allergy/defence against large parasites

- mast cells secrete inflam mediators and cytokines

270
Q

What happens in mast cells if someone is allergic to something?

A
  • 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
271
Q

What are “nude” mice?

A
  • have no thymus, so no T cell responses

- survive reasonably well as have innate and some B cell response

272
Q

What are thymus indep antigens and what is there effect?

A
  • eg. bacterial polysaccharides
  • induce more rapid response and prod of IgM antibodies
  • memory cells not gen
273
Q

What are thymus dep antigens and what is their effect?

A
  • 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
274
Q

What can T cell subsets and cytokines they prod influence?

A
  • can influence slass/subclass of antibody prod in humoral response
275
Q

How areneffector (/”primed”/memory) T cells activated?

A
  • recognition of MHC + peptide + co-receptor
  • causes activation (signal 1)
  • TCR +CD3 + CD4/CD8 MHCII/I + peptide
276
Q

How are “naive” T cells activated?

A
  • recognition of MHC + peptide + co-receptor (CD4/CD8) –> signal 1
  • recognition of co-stimulatory molecules –> signal 2
277
Q

What cells are able to deliver signal 1 and 2, and why?

A
  • 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
278
Q

What happens if signal 1 prod in absence of signal 2?

A
  • T cells become unresponsive/tolerised
279
Q

What is the best characterised co-stimulatory molecule in T cell prod, and what does it do?

A
  • 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)
280
Q

By what cells is signal 3 prod, and what does it differ dep on?

A
  • dendritic cells and other innate cells

- varies dep on type of pathogen

281
Q

What signal 3 prod TH1 cells and what is their role?

A
  • 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
282
Q

What signal 3 prod TH2 cells and what is their role?

A
  • 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)
283
Q

How does no. TH2 cells differ in people w/ allergy?

A
  • more than would expect
284
Q

What signal 3 prod TH17 CD4 T cells, and what is their role?

A
  • 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?
285
Q

What signal 3 prod TFH CD4 T cells (follicular helper T cells), and what is their role?

A
  • 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
286
Q

What are the 2 types of T regulatory cells (TREGs)?

A
  • natural

- induced

287
Q

How do natural and induced TREGS vary in terms of where they dev?

A
  • natural = thymus

- induced = periphery (mucosal lymphoid tissue)

288
Q

What is the phenotype of natural and induced TREGs

A
  • both CD4+, CD25+, FoxP3+
289
Q

How do natural and induced TREGS vary in terms of what they recognise?

A
  • natural = MHC and self peptide

- induced = MHC and non-self peptide

290
Q

How do natural and induced TREGS vary in terms of suppression?

A
  • natural = contact dep, IL-10, TGFβ

- induced = IL-10, TGFβ

291
Q

How do natural and induced TREGS vary in terms of target?

A
  • natural = dendritic cells, effector T cells

- induced = effector T cells

292
Q

How do natural and induced TREGS vary in terms of role?

A
  • natural = suppression of autoreactive T cells

- induced = downreg of mucosal immunity (dampens down immune response once pathogen destroyed), inflam responses

293
Q

Why do natural TREGs recognise MHC and self pepide?

A
  • if finds, makes cytokines to downreg immune response and stop any autoimmune response
294
Q

What is the overall role of CD4 TREGs?

A
  • 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)
295
Q

What is the role of CD8 cytotoxic cells?

A
  • 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)
296
Q

How do cytotoxic T cells kill?

A
  • 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
297
Q

How can the killing by cytotoxic T cells be decribed?

A
  • specific
  • efficient
  • “clean” –> no release of enz/toxins etc, and corpse removed by macrophages
298
Q

Apart from killing, what are the other functions of cytotoxic T cells?

A
  • 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/
299
Q

What is the role of IFNγ?

A
  • antiviral effects –> induces MHCI, enhances activity of immunoproteasome
  • recruits macrophages
300
Q

What is allergy?

A
  • disease following immune response to innocuous antigen (=allergen)
301
Q

How does IgE med allergy/hypersensitivity?

A
  • 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
302
Q

What do inflam mediators and cytokines cause in allergy?

A
  • 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)
303
Q

What is the hygiene hypothesis?

A
  • 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
304
Q

What suggested that the hygiene hypothesis was not true?

A
  • -ve correlation between helminth infections and allergic disease
  • would expect people responding to pathogens to be more prone to allergy, but opp true
305
Q

What is the counter regulation or “old friends” hypothesis?

A
  • 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)
306
Q

How are immune responses terminated?

A
  • 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)
307
Q

How does engagement of CD28 on naive T cells w/ B7 provide co-stim signal for activation?

A
  • 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
308
Q

How are antibodies used in research?

A
  • used widely, as diagnostics and increasingly as new class therapeutic drugs
  • generally used to identify and label molecules in complex mixtures (blood/urine etc.)
309
Q

What are the properties of antibodies that make them useful in research and medicine?

A
  • 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
310
Q

In what form are antibodies used in research?

A
  • prod antisera (serum containing high levels of antibodies to target antigen)
  • can be purified and labelled w/ detectable tag
311
Q

What are the diff types of label that can be added to antibodies, and what techniques are they used for?

A
  • 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
312
Q

Why do antibodies req carrier proteins to be used?

A
  • as mol size <1000da

- as generally wouldn’t stay in body long enough to cause response otherwise

313
Q

What must be considered when gen antibodies?

A
  • 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
314
Q

How are polyclonal antibodies gen?

A
  • immunise animal
  • prod 2° response
  • collect serum
315
Q

What are polyclonal antibodies?

A
  • 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
316
Q

What are epitopes?

A
  • part of antigen where antibody binds
317
Q

What are the adv of polyclonal antisera?

A
  • cheap
  • robust (may recognise partially denatured/unfolded antigen)
  • form immune complexes well (good at cross-linking)
318
Q

What are the disadv of polyclonal antisera?

A
  • poly-specific
  • need pure antigen to immunise
  • can be difficult to standardise
319
Q

What are the uses of polyclonal antisera?

A
  • 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
320
Q

What are monoclonal antibodies?

A
  • have single specificity and derived from single B lymphocyte
321
Q

How are monoclonal antibodies prod?

A
  • 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
322
Q

What are the advs of monoclonal antibodies?

A
  • highly specific
  • can be standardised
  • pure antigen not needed for immunisation
323
Q

What are the disadvs of monoclonal antibodies?

A
  • often conformation sensitive
  • less good at complex formation
  • expensive (tissue culture equipment and expertise)
324
Q

How are monoclonal antibodies used in diagnostis?

A
  • detect/quantitate diagnostically important molecules in clinical samples
  • eg. pregnancy/fertility testing, Down’s syndrome testing
325
Q

How are monoclonal antibodies used for definition of cell surface molecules?

A
  • 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
326
Q

How could monoclonal antibodies be use as magic bullets for cancer?

A
  • if could use to recognise protein only found on cancer cells
327
Q

Why did passive immunisation stop being so popular?

A
  • dev of antibiotics
328
Q

What is the issue w/ using rodent antibodies to induce immune responses in human patients, eg. HAMA?

A
  • HAMA = human anti mouse antibody
  • at best monoclonal antibody not effective
  • at worst causes serum sickness
329
Q

What are 2 methods of antibody engineering?

A
  • antibody chimeras

- “humanised” antibodies

330
Q

How have antibody chimeras been engineered, and how successful has this been?

A
  • mouse V regions and human C regions (C region most immunogenic)
  • mouse V regions still immunogenic
  • reasonably successful
331
Q

How have “humanised” antibodies been engineered?

A
  • 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
332
Q

What are the problems w/ “humanised” antibodies?

A
  • may lose affinity/specificity (as framework regions can also be important in binding antigen)
  • time consuming
333
Q

How can antibodies be used to form gene libraries?

A
  • 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”
334
Q

What do phagemid vectors do?

A
  • most common vectors

- express soluble protein in bacteria or on surface of filamentous phage particles

335
Q

How are fully human antibodies gen from a gene library by phage display?

A
  • 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
336
Q

What does synthetic mean in the context of a human antibody gene lib?

A
  • genes optimised for expression in bacteria

- not real human genes, as codons swapped

337
Q

What did the chemistry nobel prize 2018 involve research on?

A
  • 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
338
Q

How can human antibodies be gen from SCID mice?

A
  • 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
339
Q

How can human antibodies be gen from transgenic mice?

A
  • 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.
340
Q

What diff formats of monoclonal antibody can be used for therapy?

A
  • fully mouse (-omab)
  • chimeric (-ximab)
  • humanised (-zumab)
  • fully human (-umab)
341
Q

What is unusual about the antibodies in the camelidae family?

A
  • 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
342
Q

What are the characteristic of Ablynx’s nanobody?

A
  • 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
343
Q

How can antibodies used for passive immunisation?

A
  • neutralise toxins –> eg. anti-tetanus (pooled IgG), anti-snake venom
  • prevent/treat infection –> eg. resp syncytial virus (RSV)
344
Q

How can antibodies be used to treat cancer, and what are 2 eg.s?

A
  • 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)
345
Q

What are problems w/ using antibodies for cancer?

A
  • antigen specificity (finding something only on cancer cells)
  • antigen shedding
  • tumour cells inaccessible, esp if large
  • HAMA responses
  • other side effects
346
Q

How can antibodies be used to modulate immune responses, and in what cases would this be useful?

A
  • 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
347
Q

Why do many cancers induce immunosuppression?

A
  • to inhibit immune checkpoints, to avoid death
348
Q

How do cancers induce immunoexpression?

A
  • 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
349
Q

What can reverse immunosuppression?

A
  • antibodies that block inhibitory immune checkpoints
350
Q

What was the 2018 nobel prize for physoilogy and medicine awarded for?

A
  • characterisation of immune checkpoints

- cancer therapy by inhibiting -ve immune cell regulation

351
Q

In general how are antibodies used for immunotherapy?

A
  • exploit antibody effector functions/binding

- label antibody w/ toxin/drug/prodrug/radionuclide

352
Q

How does mechanism differ when antibody labeled in diff ways?

A
  • 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
353
Q

What toxins can be used to label antibodies for immunotherapy, and how are they used?

A
  • immunotoxins –> retain antigen specificity, tag or replace Fc w/ toxin
  • eg. ricin, diphtheria toxin or BFL1
354
Q

How can antibodies be improved for therapy?

A
  • 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
355
Q

What is an eg. of a mouse monoclonal antibody used therapeutically?

A
  • OTK3
  • specificity = CD3
  • for transplant rejection
356
Q

What is an eg. of a mouse/human chimeric antibody used therapeutically?

A
  • infliximab
  • specificity = TNF-α
  • for Crohn’s disease, rheumatoid arthritis (inflam)
357
Q

What is an eg. of a humanised antibody used therapeutically?

A
  • herceptin (trastuzumab)
  • specificity = HER2
  • for breast cancer
358
Q

What is an eg. of a human antibody used therapeutically?

A
  • nivolumab
  • specificity = PD-1
  • for metastatic melanoma, non-small cell lung cancer
359
Q

What are CAR-T cells?

A
  • CAR = chimeric antigen receptor

- T cells engineered to recognise tumour antigen

360
Q

How are CAR-T cells prod?

A
  • 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
361
Q

Why is using CAR-T cells so expensive?

A
  • individualised medicine
362
Q

Why don’t V regions of TCRs undergo somatic mutation?

A
  • 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

MBB311 - Molecular Immunology (2 decks)

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