Allergy

Mechanisms of Allergic Immunity

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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

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

Lebman & Coffman 1988 J Exp Med 168, 853-862

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?

Journal of Immunology 136, 2348-2357 1986

The discovery of Th1 and Th2 subsets

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

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

IFN- / IL-12or anti-IL-4

Pro-Th1 treatments or anti-Th2 treatments protect against infection

Relevance in vivo - Infection

Inflammatory Th1T cell

Th1

Macrophageand Leishmania

Macrophage infected with Leishmaniakills pathogen when activated

Macrophage activation is dependent upon Th1 cells

Leishmania resistance - mechanism

IFN-

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

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?

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

Tracheal Dendritic Cells Langerhan’s cells

In-vitro differentiated monocyte-derived

Dendritic Cell

[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

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

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

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

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

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

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

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++

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-

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 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

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

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

NK

Epithelium

Mast

Fibroblast

PGE2

CCR2L

Histamine

IFN-

IFN- IL-18

VirusesFungi

ParasitesBacteria

Viruses

VirusesFungi

Parasites

Viruses

Sources of modulatory tissue factors

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?

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

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)

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?

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

Th cell polarisation

DC mediated – decision influenced by infection

Extracellular milieu - mediated

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

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

Journal of Experimental Medicine, 1992 176 1381-1386

Sequential 2m 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

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

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 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

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

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

Journal of Leukocyte Biology 2003, 73 165-171

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

-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

516bp

516bp

PMA/Ionomycin

Inhibitors- - + +

+ +--

-actin

IL-13

Protease Inhibitors Do Not Prevent Cytokine mRNAExpression 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 CytokinemRNA Expression by KU812

Der p1 induces IL-4 and IL-13 protein expression in Freshly isolated Basophils

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

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

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

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

YYY

T cell help to B cells

B

Antigen

ThTh

IL-4 and IL-13

CD40 Ligand

CD40

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

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

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

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

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

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

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

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

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

• 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

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

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?

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

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

Processing of staggered ends

GGGCTGGG CCCGACCCGACTCGACYCGACTCGACYCGA

TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT TCGAYTYNA

GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNTCCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA

End fill-in reactionsACTCGACYCGACTCGACYCGAC

Exonuclease activity

C2CC4C2C1C1C3CCS3 S1 S1 S2 S4 S S2S

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

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

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

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 TrypanosomaToxoplasma

EnterobiousAscaris 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

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

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