Mechanisms of Allergic Immunity

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Mechanisms of Allergic Immunity crah1_at_le.ac.uk
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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 (1980s - present)

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Cellular culprits of allergy T cells

• 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

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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
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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?
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The discovery of Th1 and Th2 subsets
Journal of Immunology 136, 2348-2357 1986
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In vitro - Th1 and Th2 subsets
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Relevance in vivo - Infection
Leishmania - specific T cells
Reiner Locksley Annu. Rev. Immunol. 13,
151-177, 1995
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Relevance in vivo - Infection
Pro-Th1 treatments or anti-Th2 treatments
protect against infection
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Leishmania resistance - mechanism
Macrophage infected with Leishmania kills
pathogen when activated Macrophage activation is
dependent upon Th1 cells
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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
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
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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
• 1960s 1970s Immunoglobulin E
• 1970s 1980s Mast cells Eosinophils
• 1980s 1990s Environment ante-natal adult,
  allergens, Th2 cells
• 1990s 2000s 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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Allergic immune responses are much like any other
immune response and involves the same regulators
Non self protein from allergen or pathogen
Barrier Skin, gut, lung, eye, nose etc
Inflammation inc. MIP-1a, MCP-1 MIP-1b
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Migration of immature DC to sites of
inflammation Sallusto et al., Eur. J. Immunol.
1998 28 2760-2769
Immature DC migrate into inflamed tissue in
response to MIP-1a, MCP-1 MIP1-b which bind to,
and trigger CCR1, CCR2 and CCR5 respectively.
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Migration of mature DC to 2º lymphoid
tissue Sallusto et al., Eur. J. Immunol. 1998 28
2760-2769
Mature DC stop migrating into inflamed tissue and
make no response to MIP-1a, MCP-1 MIP1-b
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DC T cell interactions in the lymph node
Mempel, T.R et al Nature 427 154-159, 2004.
Not pulsed with Ag
Imaging at various timepoints
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Early entry of DC to the lymph node Mempel, T.R
et al Nature 427 154-159, 2004.
1. DCs strategically cluster around HEV 18hr
after entering the LN
2. Distribution of Ag-loaded DCs and T cells is
ordered 4-5hr after T cells are injected
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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
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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
and 3
Signal 1 antigen antigen receptor
Signal 2 B7 - CD28 Costimulation
Signals 1 2 activate T cells to proliferation
and effector function But what tunes the
response to Th1 or Th2?
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Polarised DC subsets
The properties of the allergen, or allergen
carrier influences the DC to drive the
development of appropriate Th cells
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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

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Type 1 and 2 DC Polarising PAMPS
CD80/CD86
Th1 polarisingfactor IL-12
Th2 polarisingfactor CCL2 (MCP-1)
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Type 1 PAMPS and their PRR
Peptidoglycan (Gram bacteria) Lipoproteins Lipoa
rabinomannan (Mycobacteria) LPS (Leptospira) LPS
(Porphyromonas) Glycophosphatylinositol - (T.
Cruzi) Zymosan (Yeast)
LPS Lipotechoic acid - (Gram bacteria) RSV F
protein
Unmethylated CpG DNA
dsDNA
Low level IL-12p70 Some ligandsinduce IL-10or
IL-12p35
HighIL-12p70IFN-a
HighIL-12p70
HighIL-12p70IFN-a
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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 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 Receptor
s for opsonins Receptors for coagulation
and complement proteins
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Indirect activation of DC by modulatory tissue
factors
Allergen
Activates the expression of costimulatory
molecules on DC
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DC polarisation by modulatory tissue factors
DC polarising factorsIFN-g IFN-a IFN-b
Th0 to Th1 polarising cytokines IL-12p70 IL-27
TNF-b 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-12p70 IL-27 TNF-b
IL-18
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Sources of modulatory tissue factors
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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?
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
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Neonatal infant immune systems
Serial infections
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Delayed maturation of Th1 capacity
Few serial infections hygiene, small family
size etc
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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?
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Do infections only reduce Th2 dominance by
inducing Th1 responses?
Wheeze
Mycobacteria induced REGULATORY T cells
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Priming conditions IFNg U/ml IL-4
pg/ml Control Ab 5892 256 Anti-IFNg Ab 1534
624 IL-4 control Ab 1740 839 IL-4
anti-IFNg Ab 348 1245
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IL-4 from the innate immune system
IL-4 is not only a product of Th2 cells
IL-4, IL-5
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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
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Allergens of Dermatophagoides pteronyssinus
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
Proteinase allergens are common and
widespread Fungi, insects, plants, parasites,
drugs (butmost allergens are not proteases)
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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 Receptors
PAR Activators Inactivators PAR1 Thro
mbin, 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 ?
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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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Do protease allergens induce IL-4 release by Mast
cells
IL-4,
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Constitutive Induced CytokineExpression by
KU812 Basophils
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Der p1 Induces Cytokine Type-2 Cytokine
mRNA Expression in KU812
b-actin
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Protease Inhibitors Do Not Prevent Cytokine
mRNA Expression by KU812
516bp
IL-13
516bp
b-actin
-
-


PMA/Ionomycin Inhibitors
-
-


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Non-Proteolytic Antigens Do Not Induce
Cytokine mRNA Expression by KU812
516bp
IL-13
516bp
b-actin
-
-
-


Tetanus toxoid
-ve
-
-
-
-

PMA/Ionomycin
Time (hr)
1
1
4
4
4
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Der p1 induces IL-4 and IL-13 protein expression
in Freshly isolated Basophils
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Necator Americanus Proteases Induce Type-2
Cytokine Expression by KU812
516bp
IFN-g
516bp
IL-5
516bp
IL-4
516bp
IL-13
516bp
b-actin
-ve
ve
Inhibitors
- Inhibitors
0 ES
0 ES
100ng/ml ES
200ng/ml ES
100ng/ml ES
200ng/ml ES
1000ng/ml ES
1000ng/ml ES
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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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Switch regions

• Switch regions - repetitive regions of DNA that
  physically recombine
• Upstream of C regions

• The Sm consists of 150 repeats of
  (GAGCT)n(GGGGGT) where n is between 3 and 7.
• Switching is mechanistically similar to V(D)J
  recombination.

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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

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T cell help to B cells
Antigen
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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

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Soluble help via IL-4 or IL-13 from T helper cells
IL-4Ra
IL-4Ra
gC
IL-13Ra1/2
JAK1
JAK1
JAK3
TYK1
TYK2
P
P
Dimerised Stat-6 translocates to nucleus
P
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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

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Cognate help via CD40 L from T helper cells
Uninhibited NFkB translocates to the nucleus
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Activation of the Ie promoter
Activation/cytokine responsive promoter
Ie
NFkB
BSAP B cell specific activator protein. C/EBP
CCAAT/enhancer binding protein. PU.1 Spi1
equivalent in humans, ets transcription factor
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Germline IgE transcripts
DNA
Germline transcripts
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?
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What do germline transcripts do?
S region RNA hybridises with template DNA
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Mechanism of class switch recombination
1. S region in the genomic DNA melts
2. S region RNA spliced from germline RNA
transcript hybridises to single-stranded DNA
R loop
3. ssDNA R loop formed a substrate for AID -
ACTIVATION- INDUCED CYTIDINE DEAMINASE
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Activation-induced cytidine deaminase
B cell activation by antigen leads to
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
AID gene is expressed under the same conditions
as B cells induced to switch Ig isotype
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Activation-induced cytidine deaminase

• 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

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Preferred Se region target sequence for AID
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
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Activation induced cytidine deaminase
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGG
CTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACY
CGACTCGACYCGACTCGAYTYNA
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT GGGCTGGG
CTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACY
CGACTCGACYCGACTCGAYTYNA
AID mediated deamination of cytidine to Uridine
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G - U mismatch repair
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCG
ACTCGACYCGACTCGACYCGAUTCGAYTYNA
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCG
ACTCGACYCGACTCGACYCGAUTCGAYTYNA
GGGUTGA CCCGACT
Base is removed, but backbone remains intact
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G - U mismatch repair
DNA is now nicked to produce a single strand break
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Processing of staggered ends
GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCC
GACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA
GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA
TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
TCGAYTYNA
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Process occurs in two S regions simultaneously

• Activation of Im Ie promoter by Ag, IL-4/13 and
  CD40L
• Production of germline transcripts and splicing
  of Sm and Se
• Deamination of ssDNA in Sm and Se by AID
• Base excision and mismatch repair
• Blunt-ended ds breaks and synapsis of Sm to Se by
  non-homologous end joining

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Non-homologous end joining in class switch
Closely resembles another B cell Ig gene mechanism
Defects in NHEJ proteins impair class switch
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BCL-6
BCL-6 binds to the Stat-6 binding site and
represses switching

• 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

Transcription blocked
Stat6 is involved in Th2 cell differentiation,
the expression of CD23 (the low affinity IgE
receptor) and VCAM expression BCL-6 may exert
its anti/pro-allergic activities via these genes
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Additional areas to think about
Cant 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?
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Text book view
Helminth infections induce IgE, mastocytosis and
eosinophilia A classic Th2-driven response
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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
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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