allergy
TRANSCRIPT
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Normal larynx Laryngeal oedema
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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
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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)
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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
Th2
IL-3 Growth of progenitor haemopoeitic cellsGM-CSF Myelopoiesis.
IL-4 B cell activation and growthIgE isotype switch.Induction of MHC class II.Macrophage inhibition
IL-5 Eosinophil growthIL-6 B cell growthAcute phase protein release
IL-10 Inhibits macrophage activationInhibits Th1 cells
TGF-Inhibits macrophage activation
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Lebman & Coffman 1988 J Exp Med 168, 853-862
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Th2
‘Textbook’ scheme of allergic immunity is centred around polarised Th cells
Mast cellEosinophil
Differentiation and development
Ig isotype switch
BIgE
Th1
-ve
M-ve
Where do Th2 cells come from?
Why are they so dominant in allergic
individuals?What are they
really for?
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Journal of Immunology 136, 2348-2357 1986
The discovery of Th1 and Th2 subsets
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IL-4IFN-
T cell clones that make IFN-, but not IL-4
T cell clones that make IL-4, but not IFN-
EnhancesIgE & IgG1
Do not provide help to IgE and IgG1 secreting B cells
Provide help to IgE and IgG1 secreting B cells
In vitro - Th1 and Th2 subsets
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Relevance in vivo - Infection
Non-healing BALB/c Resistant C57BL/6
Draining LN T cells express IL-4
mRNA
Draining LN T cells express IFN-
mRNAT
Leishmania - specific T cells
Irradiated BALB/c recipient
Resistance
Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995
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IFN- / IL-12or anti-IL-4
Pro-Th1 treatments or anti-Th2 treatments protect against infection
Relevance in vivo - Infection
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Inflammatory Th1T cell
Th1
Macrophageand Leishmania
Macrophage infected with Leishmaniakills pathogen when activated
Macrophage activation is dependent upon Th1 cells
Leishmania resistance - mechanism
IFN-
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Tuberculoid leprosy
Low infectivityLocalised infectionNormal serum IgNormal T cell response
Poor growth of mycobacteria in macrophages
Th2Th1
Lepromatous leprosy
High infectivityDisseminated infectionHypergammaglobulinaemiaUnresponsive
Florid growth of mycobacteria in macrophages
Relevance of Th subsets in humansLepromatous and tuberculoid leprosy
Infection with Mycobacterium leprae shows two main clinical forms associated with Th1 and Th2 responses
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Tuberculoid leprosy
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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?
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Barrier: Skin, gut, lung, eye, nose etc
Non self protein from allergen or pathogen
Allergic immune responses are much like any other immuneresponse and involves the same regulators
Inflammation inc.MIP-1, MCP-1 MIP-1
Activation and migration of dendritic cells to site of inflammation
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Tracheal Dendritic Cells Langerhan’s cells
In-vitro differentiated monocyte-derived
Dendritic Cell
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[Ca2+]i
Time (s)
[Ca2+]i
Time (s)
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 inflammationSallusto et al., Eur. J. Immunol. 1998 28 2760-2769
Immature DC do not respond to the lymph node derived CCR7 ligand MIP-3
Time (s)
[Ca2+]i
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Mature DC stop migrating into inflamed tissue and make no response to MIP-1, MCP-1 MIP1-
Mature DC respond to the lymph node derived CCR7 ligand MIP-3
Time (s)
[Ca2+]i
Migration of mature DC to 2º lymphoid tissueSallusto et al., Eur. J. Immunol. 1998 28 2760-2769
Time (s)
[Ca2+]i
Time (s)
[Ca2+]i
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Mempel, T.R et al Nature 427: 154-159, 2004.
Splenic DC
Pulsed with AgOVA 323-329
Not pulsed with Ag
T cells labelledGREEN
Anti OVA 323-329TcR transgenic mouse
DC labelledRED
DC – T cell interactions in the lymph node
-18hr 0hr
2hr Anti-L selectin Ab
Imaging at various timepoints
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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.
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3. DC become highly migratory & change shape (20hr)
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4. T cells cover large territories in LN
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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
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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
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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
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Signals 1, 2
DC Th
Signal 1 antigen &antigen receptor
Signal 2B7 - CD28
Costimulation
and 3
Signals 1 & 2 activate T cells to proliferation and effector functionBut what ‘tunes’ the response to Th1 or Th2?
Signal 3 - pathogen polarised DC
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Polarised DC subsets
DC Th
The properties of the allergen, or allergen carrier influences the DC to drive the development of appropriate Th cells
Signal 3Th polarising signal
Integration of signals from pathogen/allergenand the extracellular milieu polarise the DC toproduce qualitatively different signals 3
Signal 1
Signal 2
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Microbial PatternsJaneway & Medzhitov 2002 Ann Rev Immunol 20 197-216
Pathogen-associated molecular patterns (PAMPS)• Conserved microbial molecules shared by many pathogens• Include:
Bacterial lipopolysaccharidesPeptidoglycanZymosanFlagellinUnmethylated 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 lipopolysaccharidesPeptidoglycanZymosanFlagellinUnmethylated CpG DNA
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CD40
+CD80/CD86
Type 1 and 2 DC Polarising PAMPS
Type 1PAMPS
bind to PRRClass II
Type 2PAMPS
bind to PRR
Th1 polarisingfactor IL-12
Th2 polarisingfactor CCL2 (MCP-1)
T++
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CD
14
TLR 4
MD-2
TLR 3 TLR 9TLR 2TLR 1
TLR 6TLR 2
Type 1 PAMPS and their PRR
Peptidoglycan (Gram + bacteria)Lipoproteins
Lipoarabinomannan (Mycobacteria)LPS (Leptospira)
LPS (Porphyromonas)Glycophosphatylinositol - (T. Cruzi)
Zymosan (Yeast)
LPSLipotechoic acid -(Gram + bacteria)
RSV F proteindsDNAUnmethylated
CpG DNA
Low level IL-12p70Some ligandsinduce IL-10or IL-12p35
HighIL-12p70
IFN-
HighIL-12p70
HighIL-12p70
IFN-
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Type 2 PAMPS and their PRR
? ?
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Endogenous molecular patterns
Endogenous molecular patterns•Include:
Heat shock proteins(HSP60 HSP70 GP96)
Extracellular matrix proteins(hyaluronan, fibronectin, fibrinogen)
Immune complexesSurfactant protein ANecrotic 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 cellsReceptors for opsoninsReceptors for coagulation and complement proteins
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Indirect activation of DC by ‘modulatory tissue factors’
Direct activation by PAMP-PRR interactions
Necrotic/apoptotic cell death - neo expressionof PRR ligands
Heat shock proteins
Extracellular matrix componentsNecrotic cell lipidsCytokinesChemokinesEicosanoidsCoagulation componentsComplement components
Allergen
Activates the expression of costimulatory molecules on DC
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Could be argued that the development of Th2 cells is the default pathway
DC polarisation by modulatory tissue factors
DC polarising factorsIFN- IFN- IFN-
Th0 to Th1 polarising cytokinesIL-12p70 IL-27 TNF-IL-18
DC polarising factorsCCL7 (MCP-3), CCL13
(MCP-4), PGE2, Histamine
Th0 to Th2 polarising cytokinesCCL2 (MCP-1), ?IL-4
Lack of high level IL-12p70IL-27 TNF-IL-18
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NK
Epithelium
Mast
Fibroblast
PGE2
CCR2L
Histamine
IFN-
IFN- IL-18
VirusesFungi
ParasitesBacteria
Viruses
VirusesFungi
Parasites
Viruses
Sources of modulatory tissue factors
Th2
Th1
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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?
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Th2
Th1
Th2
BalancedTh1/Th2at ~2yr
Neonatal & infant immune systems
The intrauterine environment is powerfully Th2 – this imprints Th2 dominance upon the neonate
Serial infections
Age
Immuneresponse
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Th1
Th2
UnbalancedTh1/Th2
Th2 dominanceat ~2yr
Delayed maturation of Th1 capacity
Few serial infections – hygiene, small family size etc
Age
Immuneresponse
Longer period of time in which to make and establish Th2 responses to environmental antigens (i.e.
allergens)
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Do infections only reduce Th2 dominance by inducing Th1 responses?
Aerosolised ovalbumin (OVA)
OVA – allergic mice with asthma-like symptoms
Eosinophils in airway, dominance of OVA-specific Th2 cells, OVA-specific IgE
Wheeze
Vaccinate with
mycobacteria
No asthma-like symptoms
Wheeze
Have the Th1 cells induced by the mycobacteria downregulated the activity of the Th2 responsible for the symptoms?
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Vaccinate with
mycobacteriaW
heeze
No asthma-like symptoms
Do infections only reduce Th2 dominance by inducing Th1 responses?
Th
CD4+ cells specific for OVA that
produce high levels of the
immunosuppressive cytokines TGF and
IL-10
Mycobacteria induced REGULATORY T cells
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Th cell polarisation
DC mediated – decision influenced by infection
Extracellular milieu - mediated
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0 1 10Factor increase over control
0 1 10Factor increase over control
Journal of Immunology 1994 152 4755-4782
Priming conditions IFN U/ml IL-4 pg/ml
Control Ab 5892 256Anti-IFN Ab 1534 624IL-4 + control Ab 1740 839IL-4 + anti-IFN Ab 348 1245
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Resting Mast cell Degranulated mast cell
Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor,
Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-IL-4, IL-5
IL-4 is not only a product of Th2 cells
IL-4 from the innate immune system
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Journal of Experimental Medicine, 1992 176 1381-1386
Sequential 2m sections from a mucosal biopsy of a patient with asthma
Tryptase
IL-4
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What properties and characteristics make a substance an allergen?
How do these properties disregulate the processes described?
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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 proteaseDer p 2 ?Der p 3 Trypsin (serine protease)Der p 4 AmylaseDer p 5 ?Der p 6 Chymotrypsin (serine protease)Der p 7 ?Der p 8 Glutathione transferaseDer p 9 Collagenase (serine protease)Der p 10 TropomyosinDer p 14 Apolipophorin like protein
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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
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Proteases as activators of cells
Protease Activated ReceptorsPAR Activators Inactivators
PAR1 Thrombin, Trypsin Granzyme A Cathepsin G, Elastase, PlasminProteinase 3
PAR2 Trypsin, Tryptase, Factor Xa, Proconvertin Cathepsin G,, Plasmin, Proteinase 3PAR3 Thrombin Cathepsin G, ElastatasePAR4 Thrombin, Trypsin, Cathepsin G ?
Inactivators
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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
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Resting Mast cell Degranulated mast cell
Mediators released include: Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor,
Chymase, Tryptase, Heparin, Histamine IL-4, IL-5, IL-6, IL-8, TNF-IL-4,
Do protease allergens induce IL-4 release by Mast cells
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Journal of Leukocyte Biology 2003, 73 165-171
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Constitutive & Induced CytokineExpression by KU812 Basophils
-actin IL-3 IL-4 IL-5 IL-6 IL-8 IL-13 IFN-
516bp
516bp
Constitutive
PMA/Ionomycin Induced
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-actin
516bp
516bp
516bp
516bp
516bp
IL-4
IL-5
IL-13
IFN-
Der
p1
Inhi
bite
dD
er p
1
Inhi
bito
rs0
+ve-ve
Der p1 Induces Cytokine Type-2 Cytokine mRNAExpression in KU812
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516bp
516bp
PMA/Ionomycin
Inhibitors- - + +
+ +--
-actin
IL-13
Protease Inhibitors Do Not Prevent Cytokine mRNAExpression by KU812
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516bp
-actin
IL-13
--
-+
PMA/Ionomycin
Tetanus toxoid
--
-+
-+
-ve
516bp
Time (hr) 1 1 4 4 4
Non-Proteolytic Antigens Do Not Induce CytokinemRNA Expression by KU812
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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 E
S
100n
g/m
l ES
200n
g/m
l ES
1000
ng/m
l ES
0 E
S
100n
g/m
l ES
200n
g/m
l ES
1000
ng/m
l ES
Necator Americanus Proteases Induce Type-2 CytokineExpression by KU812
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Der p1 and hookworm excretory/secretory products induce IL-4 and IL-13 protein expression in KU812 Basophils
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The switch to IgE
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Lebman & Coffman 1988 J Exp Med 168, 853-862
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C2CC4C2C1C1C3CC
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.
S3 S1 S1 S2 S4 S S2S
• Switch regions - repetitive regions of DNA that physically recombine
• Upstream of C regionsC
C
C3VDJ
C3VDJ
IgG3 produced.Switch from IgM
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Switch recombination to IgE
A three signal process:
1. Antigen – controls entire process
2. Soluble help via IL-4 or IL-13 from T helper cells
3. Cognate help via CD40 L from T helper cells
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YYY
T cell help to B cells
B
Antigen
ThTh
IL-4 and IL-13
CD40 Ligand
CD40
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Switch recombination to IgE
A three signal process:
1. Antigen
2. Soluble help via IL-4 or IL-13 from T helper cells
3. Cognate help via CD40 L from T helper cells
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Stat-6P
Stat-6P
Soluble help via IL-4 or IL-13 from T helper cells
IL-4R IL-4RC IL-13R1/2
IL-13IL-4
IL-13IL-4
IL-4R IL-13R
JAK1 JAK3
TYK1
JAK1
TYK2P P
Stat-6P
Stat-6P
P P
Stat-6
P
PStat-6
P
PStat-6P
P
P
Dimerised Stat-6translocates to nucleus
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Switch recombination to IgE
A three signal process:
1. Antigen
2. Soluble help via IL-4 or IL-13 from T helper cells
3. Cognate help via CD40 L from T helper cells
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Ligation promotes aggregation in lipid rafts
Cognate help via CD40 L from T helper cells
CD40
2356
TNF receptor associated factors
IB
NF B
IB
NF B
Uninhibited NFkB translocates to the
nucleus
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Stat6
I
C1 C2 C3 C4SI
NFBC/EBP PU.1 BSAPAP-1
BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein.PU.1 – Spi1 equivalent in humans, ets transcription factor
Induced by IL-4/IL-13 and CD40 ligation
Activation of the I promoter
Activation/cytokineresponsive promoter
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C1 C2 C3 C4SI
Stat6 NFBC/EBP PU.1
Germline IgE transcripts
Transcription
Why has this mechanism evolved to transcribe just the C region?VHDHJH is needed to make a functional IgE
Why is the epsilon switch region spliced out?
DNA
C1 C2 C3 C4SI RNA
CISpliced
RNAGermline transcripts
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What do germline transcripts do?
CI
C1 C2 C3 C4SI RNA
SplicedRNA
S RNA
C1 C2 C3 C4SI
Stat6 NFBC/EBP PU.1
S region RNA hybridises with template DNA
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Single stranded DNA
I C1S
S5’ 3’
S
S
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
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NFBStat6
Activation-induced cytidine deaminase
gene
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
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• 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
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AID
RPA
AID
RPA
AID
RPA
AID
RPA
AID
RPA
AID
RPA
AID
RPA
AID
RPA
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNTCCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA
IgE S region
Non-template strand is G-Rich and contains RGYW (A/G G T/C A/T) motifs
Preferred S region target sequence for AID
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
AID
RPA
AID
RPA
Replication protein A (RPA) targets AID to ssDNA in R loops by binding to RGYW motifs
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GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNTCCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA
Non-template ssDNA
RNA/template DNA hybrid
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNTCCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA
Activation induced cytidine deaminase
NH2
N
N
O
Cytidine
O
N
HN
O
Uridine
AIDAID mediated deamination of cytidine to Uridine
Activation induced cytidine deaminase
AID may also deaminate C on the template strand?RNAase?
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GGGUTGACCCGACTGGGUTGACCCGACT
S region DNA now contains mismatched G – U pairs that must be repairede.g. by the base excision repair mechanism
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNTCCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA
G - U mismatch repair
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNTCCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA
P P P P P P
P P P P P P
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNTCCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA
Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region
UNG UNG UNG UNGUNGUNGUNG
UNG
Base is removed, but backbone remains intact
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GGGUTGACCCGACT
P P P P P P
P P P P P P
G - U mismatch repair
APE1
Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1)
GGGUTGACCCGACT
P P
P
P P P
P P P P P P
OH
DNA is now nicked to produce a single strand break
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNTCCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA
APE1
APE1
GGGCTGGGU TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
CCCGACCCGACTCGACYCGACTCGACYCGAU TCGAYTYNA
Similar mechanism on the template strand creates a staggered double strand break
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Processing of staggered ends
GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA
TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT TCGAYTYNA
GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNTCCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA
End fill-in reactionsACTCGACYCGACTCGACYCGAC
Exonuclease activity
C2CC4C2C1C1C3CCS3 S1 S1 S2 S4 S S2S
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C2CC4C2C1C1C3CCS3 S1 S1 S2 S4 S S2S
C
C
C3
VDJ
C1 C1
C2
C4
C C2 VDJ C C2
C
C
C3
C1 C1
C2
C4
Excised episomal circle of
intervening DNA
• 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
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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
Non-homologous end joining in class switch
V
DJ
Closely resembles another B cell Ig gene mechanism
Defects in NHEJ proteins impair class switch
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Stat6 NFBC/EBP PU.1 BSAP
BCL-6
BCL-6
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
Stat6
Transcriptionblocked
• 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
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Additional areas to think about
Can’t get over a 2.2 mark without showing evidence of outside reading in answers
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• 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?
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What are allergic immune responses really for?
Trichuris TrypanosomaToxoplasma
EnterobiousAscaris Leishmania
Schistosome
Hookworm
Plasmodium
Wuchereria
Onchocerca Taenia
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Text book view
Helminth infections induce IgE, mastocytosis and eosinophilia
A classic Th2-driven response
Eosinophils killing a schistosome egg in vitro
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Susceptible mice
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
Resistant mice
Else et al., 1994 J. Exp Med 179
347-351
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Nippostrongylus infection
Th2 cells themselves may not be needed
IL-4 from any source is sufficient to induce worm expulsion
IL-4
Urban et al., 1995 J. Immunol. 154, 4675-4684