Transcript Allergy PPT - University of Leicester

Mechanisms of Allergic Immunity
[email protected]
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)
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
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
IL-5
IL-10
TGF-
B cell activation and growth
IgE isotype switch.
Induction of MHC class II.
Macrophage inhibition
Eosinophil growth
IL-6
B cell growth
Acute phase protein release
Inhibits macrophage activation
Inhibits Th1 cells
Inhibits macrophage activation
Th2
Lebman & Coffman 1988 J Exp Med 168, 853-862
‘Textbook’ scheme of allergic immunity is centred
around polarised Th cells
-ve
Ig isotype switch
Th2
B
Th1
-ve
Differentiation and
development
IgE
MF
Mast cell
Eosinophil
Where do Th2 cells
come from?
Why are they so
dominant in allergic
individuals?
What are they really
for?
The discovery of Th1 and Th2 subsets
Journal of Immunology 136, 2348-2357 1986
In vitro - Th1 and
Th2 subsets
T cell clones that make
IFN-g, but not IL-4
Do not provide help to IgE
and IgG1 secreting B cells
T cell clones that make
IL-4, but not IFN-g
Provide help to IgE and
IgG1 secreting B cells
Enhances
IgE & IgG1
IFN-g
IL-4
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-g
mRNA
Irradiated BALB/c
recipient
T
Leishmania specific T cells
Resistance
Reiner & Locksley Annu. Rev. Immunol. 13, 151-177, 1995
Relevance in vivo - Infection
IFN-g / IL-12
or anti-IL-4
Pro-Th1 treatments or anti-Th2 treatments protect against infection
Leishmania resistance - mechanism
IFN-g
Th1
Inflammatory Th1
T cell
Macrophage
and Leishmania
Macrophage infected with Leishmania
kills pathogen when activated
Macrophage activation is dependent upon Th1 cells
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
Lepromatous leprosy
Low infectivity
Localised infection
Normal serum Ig
Normal T cell response
High infectivity
Disseminated infection
Hypergammaglobulinaemia
Unresponsive
Th1
Poor growth of mycobacteria
in macrophages
Th2
Florid growth of mycobacteria
in macrophages
Tuberculoid
leprosy
Lepromatous Leprosy
‘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?
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-1
Activation and
migration of dendritic
cells to site of
inflammation
Tracheal Dendritic Cells
Langerhan’s cells
In-vitro differentiated
monocyte-derived
Dendritic Cell
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- which
bind to, and trigger CCR1,
CCR2 and CCR5 respectively.
[Ca2+]i
Time (s)
Immature DC
do not respond
to the lymph
node derived
CCR7 ligand
MIP-3
[Ca2+]i
[Ca2+]i
Time (s)
Time (s)
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-
[Ca2+]i
Time (s)
Mature DC
respond to the
lymph node
derived CCR7
ligand MIP-3
[Ca2+]i
[Ca2+]i
Time (s)
Time (s)
DC – T cell interactions in the lymph node
Mempel, T.R et al Nature 427: 154-159, 2004.
Anti OVA 323-329
TcR transgenic mouse
Pulsed with Ag
OVA 323-329
DC
labelled
RED
Splenic DC
Not pulsed with Ag
T cells
labelled
GREEN
Imaging at various timepoints
-18hr
0hr
2hr Anti-L selectin Ab
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
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
T cells start to proliferate and produce cytokines 44hr after transfer
5. 44hr after injection of T cells, DCs decrease motility and become
anchored to reticular fibres, T cells rapidly migrate again
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 and 3
Signal 1 antigen &
antigen receptor
DC
Th
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?
Signal 3 - pathogen polarised DC
Polarised DC subsets
Signal 1
DC
Th
Signal 2
Signal 3
Th polarising signal
Integration of signals from pathogen/allergen
and the extracellular milieu polarise the DC to
produce qualitatively different signals 3
The properties of the allergen, or allergen carrier influences the DC to drive
the development of appropriate Th cells
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
Type 1 and 2 DC Polarising PAMPS
+
+
Type 1
PAMPS
bind to PRR
Th1 polarising
factor IL-12
CD80/CD86
Class II
CD40
Type 2
PAMPS
bind to PRR
Th2 polarising
factor CCL2 (MCP-1)
+
T
Type 1 PAMPS and their PRR
Peptidoglycan (Gram + bacteria)
Lipoproteins
Lipoarabinomannan (Mycobacteria)
LPS (Leptospira)
LPS (Porphyromonas)
Glycophosphatylinositol - (T. Cruzi)
Zymosan (Yeast)
dsDNA
LPS
Lipotechoic acid (Gram + bacteria)
RSV F protein
CD14
TLR 2
TLR 1
TLR 6
TLR 2
Low level IL-12p70
Some ligands
induce IL-10
or IL-12p35
Unmethylated
CpG DNA
MD-2
TLR 3
TLR 4
TLR 9
High
IL-12p70
IFN-a
High
IL-12p70
High
IL-12p70
IFN-a
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
Indirect activation of DC by ‘modulatory tissue factors’
Direct activation by PAMP-PRR interactions
Allergen
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
Activates the expression of costimulatory molecules on DC
DC polarisation by modulatory tissue factors
DC polarising factors
IFN-g IFN-a IFN-
Th0 to Th1 polarising cytokines
IL-12p70 IL-27 TNF- IL-18
DC polarising factors
CCL7 (MCP-3), CCL13
(MCP-4), PGE2, 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
Viruses
Fungi
Parasites
Bacteria
Epithelium
IFN-a
IL-18
Th1
Viruses
IFN-g
NK
Viruses
Fungi
Parasites
Histamine
Mast
Th2
PGE2
CCR2L
Viruses
Fibroblast
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
families. This may have resulted in more widespread clinical
young
expression of atopic disease"
..can be interpreted in terms of a failure to microbially
modulate default Th2 responses in childhood
Neonatal & infant immune systems
Serial infections
Immune
response
Th1
Th2
Th2
Age
The intrauterine environment is powerfully Th2 –
this imprints Th2 dominance upon the neonate
Balanced
Th1/Th2
at ~2yr
Delayed maturation of Th1 capacity
Few serial infections – hygiene, small family size etc
Immune
response
Th1
Th2
Age
Longer period of time in which to make and establish
Th2 responses to environmental antigens (i.e.
allergens)
Unbalanced
Th1/Th2
Th2 dominance
at ~2yr
Do infections only reduce Th2 dominance by inducing
Th1 responses?
Aerosolised
ovalbumin (OVA)
Vaccinate
with
mycobacteria
OVA – allergic mice with
asthma-like symptoms
Eosinophils in airway,
dominance of OVA-specific
Th2 cells, OVA-specific IgE
No asthma-like
symptoms
Have the Th1 cells induced by the mycobacteria downregulated
the activity of the Th2 responsible for the symptoms?
Do infections only reduce Th2 dominance by inducing
Th1 responses?
Vaccinate
with
mycobacteria
Th
No asthma-like
symptoms
CD4+ cells specific
for OVA that
produce high levels
of the
immunosuppressive
cytokines TGF and
IL-10
Mycobacteria induced REGULATORY T cells
Th cell polarisation
DC mediated – decision influenced by infection
Extracellular milieu - mediated
Journal of Immunology 1994 152 4755-4782
0
1
10
Factor increase over control
Priming conditions
Control Ab
Anti-IFNg Ab
IL-4 + control Ab
IL-4 + anti-IFNg Ab
0
1
10
Factor increase over control
IFNg U/ml IL-4 pg/ml
5892
1534
1740
348
256
624
839
1245
IL-4 from the innate immune system
IL-4 is not only a product of Th2 cells
Resting Mast cell
Degranulated mast cell
Mediators released include:
Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor,
Chymase, Tryptase, Heparin, Histamine IL-4,
IL-4, IL-5
IL-5, IL-6, IL-8, TNF-a
Tryptase
Sequential
2mm sections
from a
mucosal
biopsy of a
patient with
asthma
IL-4
Journal of Experimental Medicine, 1992 176 1381-1386
What properties and characteristics
make a substance an allergen?
How do these properties disregulate
the processes described?
L. destructor
D. pteronyssinus
D. pteronyssinus
G. domesticus
A. siro
T. putrescentiae
Allergens of Dermatophagoides pteronyssinus
Der p 1
Der p 2
Der p 3
Der p 4
Der p 5
Der p 6
Der p 7
Der p 8
Der p 9
Der p 10
Der p 14
Cysteine protease
?
Trypsin (serine protease)
Amylase
?
Chymotrypsin (serine protease)
?
Glutathione transferase
Collagenase (serine protease)
Tropomyosin
Apolipophorin like protein
Proteinase allergens are common and widespread:
Fungi, insects, plants, parasites, drugs
(but…most allergens are not proteases)
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
Inactivators
Protease Activated Receptors
PAR
Activators
Inactivators
PAR1
Thrombin, Trypsin Granzyme A
PAR2
PAR3
PAR4
Trypsin, Tryptase, Factor Xa, Proconvertin
Thrombin
Thrombin, Trypsin, Cathepsin G
Cathepsin G, Elastase, Plasmin
Proteinase 3
Cathepsin G,, Plasmin, Proteinase 3
Cathepsin G, Elastatase
?
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
Do protease allergens induce IL-4 release by Mast cells
Resting Mast cell
Degranulated mast cell
Mediators released include:
Leukotriene C4 & D4, Prostaglandin D2 Platelet Activating Factor,
Chymase, Tryptase, Heparin, Histamine IL-4,
IL-4, IL-5, IL-6, IL-8, TNF-a
Journal of Leukocyte Biology 2003, 73 165-171
Constitutive & Induced Cytokine
Expression by KU812 Basophils
Constitutive
516bp
PMA/Ionomycin Induced
516bp
-actin IL-3 IL-4 IL-5 IL-6 IL-8 IL-13 IFN-g
Der p1 Induces Cytokine Type-2 Cytokine mRNA
Expression in KU812
516bp
IL-4
516bp
IL-5
516bp
IL-13
516bp
IFN-g
516bp
+ve
-ve
Inhibitors
Inhibited
Der p1
Der p1
0
-actin
Protease Inhibitors Do Not Prevent Cytokine mRNA
Expression by KU812
516bp
IL-13
516bp
-actin
- - +
- + -
+
+
PMA/Ionomycin
Inhibitors
Non-Proteolytic Antigens Do Not Induce Cytokine
mRNA Expression by KU812
516bp
IL-13
516bp
-actin
Time (hr)
1
1
4
4
+
PMA/Ionomycin
- + - +
- - - -
-
Tetanus toxoid
4
-ve
Der p1 induces IL-4 and IL-13 protein
expression in Freshly isolated Basophils
Necator Americanus Proteases Induce Type-2 Cytokine
Expression by KU812
516bp
IFN-g
516bp
IL-5
516bp
IL-4
516bp
IL-13
516bp
-actin
-ve +ve
200ng/ml ES
1000ng/ml ES
+ Inhibitors
0 ES
100ng/ml ES
1000ng/ml ES
200ng/ml ES
100ng/ml ES
0 ES
- Inhibitors
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
Cm
Sm
Cd
Cg3
Sg3
Cg1
Sg1
Ca1
Sa1
Cg2
Sg2
Cg4
Ce
Sg4
Se
Ca2
Sa2
Cd
• 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.
Cm
VDJ
Cg3
VDJ
Cg3
IgG3 produced.
Switch from IgM
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
T cell help to B cells
IL-4 and IL-13
B
Th
Th
Y
Antigen
CD40
CD40 Ligand
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
Soluble help via IL-4 or IL-13 from T helper cells
IL-4R
IL-13R
IL-4
IL-13
IL-4
IL-13
gC
IL-4Ra
JAK1
JAK3
P
TYK1
IL-4Ra
JAK1
P
Stat-6
Stat-6
P
P
Stat-6
P
P
TYK2
PDimerised Stat-6
P
Stat-6 Stat-6
translocates to nucleus
P
Stat-6
P
Stat-6
P
IL-13Ra1/2
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
Cognate help via CD40 L from T helper cells
Ligation promotes aggregation in lipid rafts
CD40
2
3
5
6
TNF receptor
associated factors
IkB
Uninhibited NFkB
translocates to the
IkB
nucleus
NF kB
NF kB
Activation of the Ie promoter
Activation/cytokine
responsive promoter
Ie
C/EBP Stat6
AP-1
Se
PU.1 NFkB
Ce1 Ce2 Ce3 Ce4
BSAP
Ie
Induced by IL-4/IL-13 and CD40 ligation
BSAP – B cell specific activator protein. C/EBP CCAAT/enhancer binding protein.
PU.1 – Spi1 equivalent in humans, ets transcription factor
Germline IgE transcripts
Transcription
C/EBP
Stat6
PU.1
NFkB
Ie
Se
Ce1
Ce2
Ce3
Ce4
DNA
Ie
Se
Ce1
Ce2
Ce3
Ce4
RNA
Germline transcripts
Ie
Ce
Spliced
RNA
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?
What do germline transcripts do?
Ie
Se
Ce1 Ce2 Ce3 Ce4
Ie
Stat6
PU.1
Spliced
RNA
Ce
RNA
Se
C/EBP
RNA
S region RNA
hybridises with
template DNA
NFkB
Ie
Se
Ce1
Ce2
Ce3
Ce4
Mechanism of class switch recombination
Single stranded DNA
1. S region in the genomic DNA ‘melts’
Se
Ie
R Se
loop
Ce1
Se
5’
Se
3’
2. S region RNA spliced from
germline RNA transcript hybridises
to single-stranded DNA
3. ssDNA R loop formed – a
substrate for AID - ACTIVATIONINDUCED CYTIDINE DEAMINASE
Activation-induced cytidine deaminase
B cell activation by antigen leads to:
Soluble help via Th cell IL-4 or
IL-13
Cognate help via Th cell
CD40 L from T helper
Induces Stat 6
Releases NFkB from IkB
Stat6
NFkB
Activation-induced
cytidine deaminase
gene
AID gene is expressed under the same conditions as B cells
induced to switch Ig isotype
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
Preferred Se region target sequence for AID
IgE S region
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA
Non-template strand is G-Rich
and contains RGYW (A/G G T/C A/T) motifs
Replication protein A (RPA) targets AID to
ssDNA in R loops by binding to RGYW motifs
AID
RPA
RPA
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
Activation induced cytidine deaminase
Activation induced cytidine deaminase
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT Non-template ssDNA
GGGCTGGGCTGAGCTGRGCTGAGCTGRGCTGAGCTRARNT RNA/template
CCCGACCCGACTCGACYCGACTCGACYCGACTCGAYTYNA DNA hybrid
NH2
HN
AID
N
O
O
N
Cytidine
O
N
Uridine
AID may also
deaminate C on the
template strand
?RNAase?
AID mediated
deamination of
cytidine to Uridine
G - U mismatch repair
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT
CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA
S region DNA now contains mismatched G – U pairs that must be repaired
e.g. by the base excision repair mechanism
Uracil-DNA glycolase (UNG) removes uracil to leave abasic sites in S region
UNG
UNG
UNG
UNG
UNG
UNG
UNG
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT
CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA
UNG
P
P
P
P
P
P
GGGUTGA
CCCGACT
Base is removed, but
backbone remains intact
P
P
P
P
P
P
G - U mismatch repair
OH
Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1)
APE1P
P
P
P
P
P
P
GGGUTGA
CCCGACT
P
P
P
P
P
P
DNA is now nicked to produce a single strand break
GGGCTGGGU
TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
APE1
CCCGACCCGACTCGACYCGACTCGACYCGAU
TCGAYTYNA
GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT
CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA
Similar mechanism on the template strand creates a staggered
double strand break
APE1
Processing of staggered ends
GGGCTGGG
TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
CCCGACCCGACTCGACYCGACTCGACYCGA
TCGAYTYNA
GGGCTGGG
Exonuclease activity
CCCGACCCGACTCGACYCGACTCGACYCGA
End fill-in reactions
Cm
Sm
Cd
TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
ACTCGACYCGACTCGACYCGAC TCGAYTYNA
Cg3
Sg3
Cg1
Sg1
Ca1
Sa1
Cg2
Sg2
Cg4
Sg4
Ce
Se
Ca2
Sa2
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
Cm
Cd
Sm
Cg3
Sg3
Cg1
Sg1
Ca1
Sa1
Cg2
Sg2
Sg4
Ce
Se
Ca2
Sa2
Cg1 Ca1
Cg1 Ca1
Cg3
Cg2
Cd
Cg4
Excised episomal
circle of
intervening DNA
Cg3
Cg2
Cd
Cg4
Cm
Cm
VDJ
Cg4
Ce
Ca2
VDJ
Ce
Ca2
Non-homologous end joining in class switch
Closely resembles another B cell Ig gene mechanism
Ig gene recombination
7
23
9 12 7
V
9
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.
Defects in NHEJ proteins impair class switch
D J
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
Stat6
BCL-6
Transcription
blocked
C/EBP Stat6
BCL-6 PU.1 NFkB
BSAP
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
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?
Ascaris
Onchocerca
Toxoplasma
Enterobious
Plasmodium
Trichuris
Hookworm
Schistosome
Trypanosoma
Leishmania
Taenia
Wuchereria
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
Susceptible mice
Resistant mice
Else et al., 1994
J. Exp Med 179
347-351
Th2 cells themselves may not be needed
Nippostrongylus infection
IL-4
IL-4 from any source is sufficient to induce worm expulsion
Urban et al., 1995 J. Immunol. 154, 4675-4684