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Allergy Allergy Presentation Transcript

  • Mechanisms of Allergic Immunity [email_address]
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  • Normal larynx Laryngeal oedema
  • Cellular culprits of allergy: Mast cells
    • Most informative early analysis conducted in patients with asthma
    • Early studies (pre-1980) implicated mast cells and histamine as part of an archetypal immediate type I hypersensitivity
    • Provoked by allergenic and non allergenic substances
    • Explained atopic and non-atopic asthma
    • Explained why mast cell stabilising drugs worked
  • Cellular culprits of allergy: Mast cells??
    • Corticosteroid treatment worked, but had no effect on histamine release
    • Anti-histamine treatment had little effect on asthma
    • Could not explain ‘organ specificity’ of asthma
    • Could not explain the hyperresponsive airway in asymptomatic asthmatics
    • Fibreoptic bronchoscopy - immunohistology, biopsy and analysis of bronchoalveolar lavage (BAL) cells (1980’s - present)
    • The early evidence:
    • Eosinophil & mononuclear cells infiltrate the bronchi of asthmatics
    • Activated T cells elevated in the peripheral blood of severe acute asthmatics
    • Activated T cells in peripheral blood correlated with airway narrowing
    • Bronchial CD4 lymphocyte numbers correlated with eosinophil numbers
    • Elevated IL-5 expressing T cells in asthmatic bronchial mucosa and BAL
    • T cells that release IL-5 co-localise with eosinophils
    • Eosinophils cause airway hyperresponsiveness, inflammation desquamative bronchitis, mucous hypersecretion and smooth muscle contraction
    • IL-5 promotes differentiation and regulates the survival of eosinophils
    • Steroid treatment associated with a decrease in IL-5 producing cells
    Cellular culprits of allergy: T cells
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  • Cellular culprits of allergy: T cells Wider analysis of cytokines in atopy showed that BAL T cells that expressed elevated levels of IL-5, also expressed IL-4 - a profile typical of Th2 cells in mice IL-3 Growth of progenitor haemopoeitic cells GM-CSF Myelopoiesis. IL-4 B cell activation and growth IgE isotype switch. Induction of MHC class II. Macrophage inhibition IL-5 Eosinophil growth IL-6 B cell growth Acute phase protein release IL-10 Inhibits macrophage activation Inhibits Th1 cells TGF-  Inhibits macrophage activation Th2
  • Lebman & Coffman 1988 J Exp Med 168, 853-862
  • ‘ Textbook’ scheme of allergic immunity is centred around polarised Th cells Where do Th2 cells come from? Why are they so dominant in allergic individuals? What are they really for? Th2 Mast cell Eosinophil Differentiation and development Ig isotype switch B IgE Th1 -ve M  -ve
  • Journal of Immunology 136, 2348-2357 1986 The discovery of Th1 and Th2 subsets
  • In vitro - Th1 and Th2 subsets IL-4 IFN-  T cell clones that make IFN-  , but not IL-4 T cell clones that make IL-4, but not IFN-  Enhances IgE & IgG1 Do not provide help to IgE and IgG1 secreting B cells Provide help to IgE and IgG1 secreting B cells
  • Relevance in vivo - Infection Leishmania - specific T cells Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995 Non-healing BALB/c Resistant C57BL/6 Draining LN T cells express IL-4 mRNA Draining LN T cells express IFN-  mRNA T Irradiated BALB/c recipient Resistance
  • Pro-Th1 treatments or anti-Th2 treatments protect against infection Relevance in vivo - Infection IFN-  / IL-12 or anti-IL-4
  • Macrophage infected with Leishmania kills pathogen when activated Macrophage activation is dependent upon Th1 cells Leishmania resistance - mechanism Inflammatory Th1 T cell Th1 Macrophage and Leishmania IFN- 
  • Tuberculoid leprosy Low infectivity Localised infection Normal serum Ig Normal T cell response Poor growth of mycobacteria in macrophages Lepromatous leprosy High infectivity Disseminated infection Hypergammaglobulinaemia Unresponsive Florid growth of mycobacteria in macrophages Relevance of Th subsets in humans Lepromatous and tuberculoid leprosy Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses Th2 Th1
  • Tuberculoid leprosy
  • Lepromatous Leprosy
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  • ‘ Textbook’ scheme of allergic immunity is centred around polarised Th cells
    • Immunological fashions
    • 1960’s & 1970’s Immunoglobulin E
    • 1970’s & 1980’s Mast cells & Eosinophils
    • 1980’s & 1990’s Environment – ante-natal & adult, allergens, Th2 cells
    • 1990’s & 2000’s Microbial experience, Epithelium, Tregs
    • Although undoubtedly a useful model, the textbook ‘skew to Th2’ model is too simplistic to explain allergy
    • Allergy is a disease of impaired immune regulation
    • Where is the regulatory lesion?
  • Barrier: Skin, gut, lung, eye, nose etc Non self protein from allergen or pathogen Allergic immune responses are much like any other immune response and involves the same regulators Inflammation inc. MIP-1  , MCP-1 MIP-1  Activation and migration of dendritic cells to site of inflammation
  • Tracheal Dendritic Cells Langerhan’s cells In-vitro differentiated monocyte-derived Dendritic Cell
  • Immature DC migrate into inflamed tissue in response to MIP-1  , MCP-1 MIP1-  which bind to, and trigger CCR1, CCR2 and CCR5 respectively. Migration of immature DC to sites of inflammation Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 [Ca 2+ ] i Time (s) [Ca 2+ ] i Time (s) Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3  Time (s) [Ca 2+ ] i
  • Mature DC stop migrating into inflamed tissue and make no response to MIP-1  , MCP-1 MIP1-  Migration of mature DC to 2 º lymphoid tissue Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769 Mature DC respond to the lymph node derived CCR7 ligand MIP-3  Time (s) [Ca 2+ ] i Time (s) [Ca 2+ ] i Time (s) [Ca 2+ ] i
  • Mempel, T.R et al Nature 427 : 154-159, 2004. Not pulsed with Ag DC – T cell interactions in the lymph node Imaging at various timepoints Splenic DC Pulsed with Ag OVA 323-329 T cells labelled GREEN Anti OVA 323-329 TcR transgenic mouse DC labelled RED -18hr 0hr 2hr Anti-L selectin Ab
  • 2. Distribution of Ag-loaded DCs and T cells is ordered 4- 5hr after T cells are injected 1. DCs strategically cluster around HEV 18hr after entering the LN Early entry of DC to the lymph node Mempel, T.R et al Nature 427 : 154-159, 2004.
  • 3. DC become highly migratory & change shape ( 20hr )
  • 4. T cells cover large territories in LN
  • 6. Short, serial T cell-DC contacts of ~ 5 minutes (2- 4hrs after injection of T cells) 7. Stable T cell-DC conjugates of 30-180 minutes (8- 12hr after injection of T cells) 8. Simultaneous stable and dynamic interactions between DC and T cells
  • 5. 44hr after injection of T cells, DCs decrease motility and become anchored to reticular fibres, T cells rapidly migrate again T cells start to proliferate and produce cytokines 44hr after transfer
  • More information than is provided by the antigen is exchanged between the DC and T cell DC have a profound influence on the properties of the T cell that develops
  • Signals 1, 2 Signal 1 antigen & antigen receptor Signal 2 B7 - CD28 Costimulation and 3 Signals 1 & 2 activate T cells to proliferation and effector function But what ‘tunes’ the response to Th1 or Th2? DC Th Signal 3 - pathogen polarised DC
  • Polarised DC subsets The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells DC Th Signal 3 Th polarising signal Integration of signals from pathogen/allergen and the extracellular milieu polarise the DC to produce qualitatively different signals 3 Signal 1 Signal 2
  • Microbial Patterns Janeway & Medzhitov 2002 Ann Rev Immunol 20 197-216
    • Pathogen-associated molecular patterns (PAMPS)
    • Conserved microbial molecules shared by many pathogens
    • Include:
          • Bacterial lipopolysaccharides
          • Peptidoglycan
          • Zymosan
          • Flagellin
          • Unmethylated CpG DNA
    • Pattern Recognition Receptors (PRR)
    • Include:
    • Toll like receptors
    • Receptors for apoptotic cells
    • Receptors for opsonins
    • Receptors for coagulation and complement proteins
    • Pathogen-associated molecular patterns (PAMPS)
    • Conserved microbial molecules shared by many pathogens
    • Include:
          • Bacterial lipopolysaccharides
          • Peptidoglycan
          • Zymosan
          • Flagellin
          • Unmethylated CpG DNA
  • CD80/CD86 Type 1 and 2 DC Polarising PAMPS Th1 polarising factor IL-12 Th2 polarising factor CCL2 (MCP-1) CD40 + Type 1 PAMPS bind to PRR Class II Type 2 PAMPS bind to PRR T + +
  • Type 1 PAMPS and their PRR Peptidoglycan (Gram + bacteria) Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - ( T. Cruzi ) Zymosan (Yeast) LPS Lipotechoic acid - (Gram + bacteria) RSV F protein dsDNA Unmethylated CpG DNA Low level IL-12p70 Some ligands induce IL-10 or IL-12p35 High IL-12p70 IFN-  High IL-12p70 High IL-12p70 IFN-  CD14 TLR 4 MD-2 TLR 3 TLR 9 TLR 2 TLR 1 TLR 6 TLR 2
  • Type 2 PAMPS and their PRR ? ?
  • Endogenous molecular patterns
    • Endogenous molecular patterns
    • Include:
          • Heat shock proteins
          • (HSP60 HSP70 GP96)
          • Extracellular matrix proteins
          • (hyaluronan, fibronectin, fibrinogen)
          • Immune complexes
          • Surfactant protein A
          • Necrotic cell components
    • Pattern Recognition Receptors (PRR)
    • Include:
    • Toll like receptors
    • Receptors for apoptotic cells
    • Receptors for opsonins
    • Receptors for coagulation and complement proteins
    Receptors for apoptotic cells Receptors for opsonins Receptors for coagulation and complement proteins
  • Indirect activation of DC by ‘modulatory tissue factors’ Allergen Activates the expression of costimulatory molecules on DC Direct activation by PAMP-PRR interactions Necrotic/apoptotic cell death - neo expression of PRR ligands Heat shock proteins Extracellular matrix components Necrotic cell lipids Cytokines Chemokines Eicosanoids Coagulation components Complement components
  • DC polarisation by modulatory tissue factors DC polarising factors IFN-  IFN-  IFN-  Th0 to Th1 polarising cytokines IL-12p70 IL-27 TNF-  IL-18 DC polarising factors CCL7 (MCP-3), CCL13 (MCP-4), PGE 2, Histamine Th0 to Th2 polarising cytokines CCL2 (MCP-1), ?IL-4 Lack of high level IL-12p70 IL-27 TNF-  IL-18 Could be argued that the development of Th2 cells is the default pathway
  • Sources of modulatory tissue factors NK Epithelium Mast Fibroblast PGE 2 CCR2L Histamine IFN-  IFN-  IL-18 Viruses Fungi Parasites Bacteria Viruses Viruses Fungi Parasites Viruses Th2 Th1
  • The hygiene hypothesis (Strachan, 1989) Based upon the epidemiology of hay fever “ Declining family size, improved household amenities, and higher standards of personal cleanliness have reduced the opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease" ..can be interpreted in terms of a failure to microbially modulate default Th2 responses in childhood young families Explains how Th2 arise, but… … does not explains why some individuals are allergic and others are not and why the incidence of allergy is increasing. Reduced numbers of IL-12 producing cells? Reduced ability to produce or respond to IL-12? Reduced stimulation of IL-12 by microbial substances?
  • Neonatal & infant immune systems Serial infections Th2 Th1 Th2 Balanced Th1/Th2 at ~2yr The intrauterine environment is powerfully Th2 – this imprints Th2 dominance upon the neonate Age Immune response
  • Delayed maturation of Th1 capacity Few serial infections – hygiene, small family size etc Th1 Th2 Unbalanced Th1/Th2 Th2 dominance at ~2yr Age Immune response Longer period of time in which to make and establish Th2 responses to environmental antigens (i.e. allergens)
  • Do infections only reduce Th2 dominance by inducing Th1 responses? Wheeze Wheeze Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms? Aerosolised ovalbumin (OVA) OVA – allergic mice with asthma-like symptoms Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE Vaccinate with mycobacteria No asthma-like symptoms
  • Wheeze Do infections only reduce Th2 dominance by inducing Th1 responses? Mycobacteria induced REGULATORY T cells Vaccinate with mycobacteria No asthma-like symptoms Th CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGF  and IL-10
  • Th cell polarisation DC mediated – decision influenced by infection Extracellular milieu - mediated
  • Priming conditions IFN  U/ml IL-4 pg/ml Control Ab 5892 256 Anti-IFN  Ab 1534 624 IL-4 + control Ab 1740 839 IL-4 + anti-IFN  Ab 348 1245 0 1 10 Factor increase over control 0 1 10 Factor increase over control Journal of Immunology 1994 152 4755-4782
  • IL-4, IL-5 IL-4 is not only a product of Th2 cells IL-4 from the innate immune system Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C 4 & D 4 , Prostaglandin D 2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF- 
  • Journal of Experimental Medicine, 1992 176 1381-1386 Sequential 2  m sections from a mucosal biopsy of a patient with asthma Tryptase IL-4
  • What properties and characteristics make a substance an allergen? How do these properties disregulate the processes described?
  • L. destructor G. domesticus D. pteronyssinus D. pteronyssinus A. siro T. putrescentiae
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  • Allergens of Dermatophagoides pteronyssinus Proteinase allergens are common and widespread: Fungi, insects, plants, parasites, drugs (but…most allergens are not proteases) Der p 1 Cysteine protease Der p 2 ? Der p 3 Trypsin (serine protease) Der p 4 Amylase Der p 5 ? Der p 6 Chymotrypsin (serine protease) Der p 7 ? Der p 8 Glutathione transferase Der p 9 Collagenase (serine protease) Der p 10 Tropomyosin Der p 14 Apolipophorin like protein
  • Protease allergens can breach epithelial barriers Wan et al., Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions J Clin Invest, 1999, 104, 123-133 Leads to immune sensitisation without the ‘deliberate’ invasion and infection mechanisms of a pathogen
  • Proteases as activators of cells Protease Activated Receptors PAR Activators Inactivators PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, Plasmin Proteinase 3 PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3 PAR3 Thrombin Cathepsin G, Elastatase PAR4 Thrombin, Trypsin, Cathepsin G ? Inactivators
  • Journal of Immunology 2001 167 1014-1021
    • PAR are also involved in:
    • Induction of of epithelial cell & fibroblast proliferation
    • Induction of cytokines & chemokine expression
    • Induction of pharmacological mediator release
    • Induction of metalloproteases
    • Regulation of smooth muscle tone
  • IL-4, Do protease allergens induce IL-4 release by Mast cells Resting Mast cell Degranulated mast cell Mediators released include: Leukotriene C 4 & D 4 , Prostaglandin D 2 Platelet Activating Factor, Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF- 
  • Journal of Leukocyte Biology 2003, 73 165-171
  • Constitutive & Induced Cytokine Expression by KU812 Basophils  -actin IL-3 IL-4 IL-5 IL-6 IL-8 IL-13 IFN-  516bp 516bp Constitutive PMA/Ionomycin Induced
  •  -actin Der p1 Induces Cytokine Type-2 Cytokine mRNA Expression in KU812 516bp 516bp 516bp 516bp 516bp IL-4 IL-5 IL-13 IFN-  Der p1 Inhibited Der p1 Inhibitors 0 +ve -ve
  • 516bp 516bp PMA/Ionomycin Inhibitors - - + + + + - -  -actin IL-13 Protease Inhibitors Do Not Prevent Cytokine mRNA Expression by KU812
  • 516bp  -actin IL-13 - - - + PMA/Ionomycin Tetanus toxoid - - - + - + -ve 516bp Time (hr) 1 1 4 4 4 Non-Proteolytic Antigens Do Not Induce Cytokine mRNA Expression by KU812
  • Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils
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  • 516bp 516bp 516bp 516bp 516bp  -actin IL-4 IL-5 IL-13 IFN-  - Inhibitors + Inhibitors -ve +ve 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES 200ng/ml ES 1000ng/ml ES Necator Americanus Proteases Induce Type-2 Cytokine Expression by KU812
  • Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils
  • The switch to IgE
  • Lebman & Coffman 1988 J Exp Med 168, 853-862
  • Switch regions
    • The S  consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7.
    • Switching is mechanistically similar to V(D)J recombination.
    • Switch regions - repetitive regions of DNA that physically recombine
    • Upstream of C regions
    C  2 C  C  4 C  2 C  1 C  1 C  3 C  C  S  3 S  1 S  1 S  2 S  4 S  S  2 S  C  C  C  3 VDJ C  3 VDJ IgG3 produced. Switch from IgM
  • Switch recombination to IgE
    • A three signal process:
    • Antigen – controls entire process
    • Soluble help via IL-4 or IL-13 from T helper cells
    • Cognate help via CD40 L from T helper cells
  • T cell help to B cells Antigen Y Y Y B Th Th IL-4 and IL-13 CD40 Ligand CD40
  • Switch recombination to IgE
    • A three signal process:
    • Antigen
    • Soluble help via IL-4 or IL-13 from T helper cells
    • Cognate help via CD40 L from T helper cells
  • Soluble help via IL-4 or IL-13 from T helper cells IL-4R  IL-4R   C IL-13R  1/2 JAK1 JAK3 TYK1 JAK1 TYK2 P P P Dimerised Stat-6 translocates to nucleus Stat-6 P Stat-6 P IL-13 IL-4 IL-13 IL-4 IL-4R IL-13R Stat-6 P Stat-6 P P P Stat-6 P P Stat-6 P P Stat-6 P P
  • Switch recombination to IgE
    • A three signal process:
    • Antigen
    • Soluble help via IL-4 or IL-13 from T helper cells
    • Cognate help via CD40 L from T helper cells
  • Cognate help via CD40 L from T helper cells Uninhibited NFkB translocates to the nucleus Ligation promotes aggregation in lipid rafts CD40 2 3 5 6 TNF receptor associated factors I  B NF  B I  B NF  B
  • I  NF  B BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein. PU.1 – Spi1 equivalent in humans, ets transcription factor Activation of the I  promoter Activation/cytokine responsive promoter Stat6 I  C  1 C  2 C  3 C  4 S  C/EBP PU.1 BSAP AP-1 Induced by IL-4/IL-13 and CD40 ligation
  • Germline IgE transcripts Why has this mechanism evolved to transcribe just the C region? V H D H J H is needed to make a functional IgE Why is the epsilon switch region spliced out? DNA Germline transcripts C  1 C  2 C  3 C  4 S  I  Stat6 NF  B C/EBP PU.1 Transcription C  1 C  2 C  3 C  4 S  I  RNA C  I  Spliced RNA
  • What do germline transcripts do? S region RNA hybridises with template DNA C  I  C  1 C  2 C  3 C  4 S  I  RNA Spliced RNA S  RNA C  1 C  2 C  3 C  4 S  I  Stat6 NF  B C/EBP PU.1
  • R loop 1. S region in the genomic DNA ‘melts’ 2. S region RNA spliced from germline RNA transcript hybridises to single-stranded DNA 3. ssDNA R loop formed – a substrate for AID - ACTIVATION- INDUCED CYTIDINE DEAMINASE Mechanism of class switch recombination Single stranded DNA I  C  1 S  S  5’ 3’ S  S 
  • Activation-induced cytidine deaminase Soluble help via Th cell IL-4 or IL-13 Induces Stat 6 Cognate help via Th cell CD40 L from T helper Releases NFkB from IkB B cell activation by antigen leads to: AID gene is expressed under the same conditions as B cells induced to switch Ig isotype NF  B Stat6 Activation-induced cytidine deaminase gene
    • Expressed only in B cells
    • Involved in isotype class switching & somatic hypermutation
    • AID knockout mice do not class switch Ig isotype
    • Ectopic expression in non B cells causes class switch
    • Mutation in the AID gene can cause hyper IgM syndrome
    • Deaminates cytidine on ssDNA, i.e. substitutes U for C
    Activation-induced cytidine deaminase
  • Preferred S  region target sequence for AID GGG CT GGG CT G A G CT G R G CT G A G CT G R G CT G A G CTRARNT AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA AID RPA GGG CT GGG CT G A G CT G R G CT G A G CT G R G CT G A G CTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA IgE S region Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs AID RPA AID RPA Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs
  • GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA GGG C TGGG C TGAG C TGRG C TGAG C TGRG C TGAG C TRARNT GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA C TCGAYTYNA AID mediated deamination of cytidine to Uridine Activation induced cytidine deaminase Non-template ssDNA RNA/template DNA hybrid Activation induced cytidine deaminase NH 2 N N O Cytidine O N HN O Uridine AID AID may also deaminate C on the template strand ?RNAase?
  • GGG U TGA CCCGACT GGG U TGGG U TGAG U TGRG U TGAG U TGRG U TGAG U TRARNT CCCGACCCGACTCGACYCGACTCGACYCGA U TCGAYTYNA G - U mismatch repair GGG U TGGG U TGAG U TGRG U TGAG U TGRG U TGAG U TRARNT CCCGACCCGACTCGACYCGACTCGACYCGA U TCGAYTYNA Base is removed, but backbone remains intact GGGUTGA CCCGACT S region DNA now contains mismatched G – U pairs that must be repaired e.g. by the base excision repair mechanism P P P P P P P P P P P P GGG U TGGG U TGAG U TGRG U TGAG U TGRG U TGAG U TRARNT CCCGACCCGACTCGACYCGACTCGACYCGA U TCGAYTYNA Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region UNG UNG UNG UNG UNG UNG UNG UNG
  • G - U mismatch repair DNA is now nicked to produce a single strand break GGG U TGA CCCGACT P P P P P P P P P P P P APE1 Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1) GGG U TGA CCCGACT P P P P P P P P P P P P OH GGGUTGGG U TGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA U TCGAYTYNA APE1 APE1 GGGCTGGG U TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA U TCGAYTYNA Similar mechanism on the template strand creates a staggered double strand break
  • Processing of staggered ends GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT TCGAYTYNA GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA End fill-in reactions ACTCGACYCGACTCGACYCGAC Exonuclease activity C  2 C  C  4 C  2 C  1 C  1 C  3 C  C  S  3 S  1 S  1 S  2 S  4 S  S  2 S 
    • Activation of I  & I  promoter by Ag, IL-4/13 and CD40L
    • Production of germline transcripts and splicing of S  and S 
    • Deamination of ssDNA in S  and S  by AID
    • Base excision and mismatch repair
    • Blunt-ended ds breaks and synapsis of S  to S  by non-homologous end joining
    Process occurs in two S regions simultaneously C  2 C  C  4 C  2 C  1 C  1 C  3 C  C  S  3 S  1 S  1 S  2 S  4 S  S  2 S  C  C  C  3 VDJ C  1 C  1 C  2 C  4 C  C  2 VDJ C  C  2 C  C  C  3 C  1 C  1 C  2 C  4 Excised episomal circle of intervening DNA
  • Non-homologous end joining in class switch Closely resembles another B cell Ig gene mechanism Defects in NHEJ proteins impair class switch After N and P nucleotides have been inserted, several other proteins, (Ku70:Ku80, XRCC4 and DNA dependent protein kinases,ARTEMIS exonuclease, DNA ligase IV) bind to the hairpins and the heptamer ends. Ig gene recombination 7 23 9 7 12 9 V D J
  • BCL-6 BCL-6 binds to the Stat-6 binding site and represses switching Stat6 is involved in Th2 cell differentiation, the expression of CD23 (the low affinity IgE receptor) and VCAM expression BCL-6 may exert it’s anti/pro-allergic activities via these genes Transcription blocked
    • BCL-6 -/- mice have enhanced IgE isotype switching
    • BCL-6 -/- Stat6 -/- mice have no IgE
    • An RFLP has been mapped to the first intron of the BCL-6 gene that is significantly associated with atopy - but not IgE levels
    Stat6 NF  B C/EBP PU.1 BSAP BCL-6 BCL-6 Stat6
  • Additional areas to think about Can’t get over a 2.2 mark without showing evidence of outside reading in answers
    • Relationship between isotype switch, somatic hypermutation and proliferation of B cells in the germinal centre
    • What is the relationship between the deliberately mutagenic mechanisms of isotype switch and somatic hypermutation in B cells and the propensity of B cells to form tumours
    • Where are the holes in the ‘skew to Th2’ model of allergy?
    • What are allergic responses really for?
  • What are allergic immune responses really for? Trichuris Trypanosoma Toxoplasma Enterobious Ascaris Leishmania Schistosome Hookworm Plasmodium Wuchereria Onchocerca Taenia
  • Text book view Helminth infections induce IgE, mastocytosis and eosinophilia A classic Th2-driven response Eosinophils killing a schistosome egg in vitro
  • However…….. Heavily parasitised individuals exist - despite Th2 responses and eosinophilia. Scarce in vivo evidence of eosinophil and IgE control of helminth infection Yet IL-4 may be involved - Trichuris muris model Else et al., 1994 J. Exp Med 179 347-351 Susceptible mice Resistant mice
  • Th2 cells themselves may not be needed IL-4 from any source is sufficient to induce worm expulsion Urban et al., 1995 J. Immunol. 154, 4675-4684 Nippostrongylus infection IL-4