Mechanisms of Allergic Immunity

crah1@le.ac.uk

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 (1980s - 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

Ig isotype switch

-ve Th2
Th1

B
IgE

-ve
Differentiation and development Mast cell Eosinophil Where do Th2 cells come from? Why are they so dominant in allergic individuals? What are they really for?
MF

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

Enhances IgE & IgG1

IFN-g

IL-4

T cell clones that make IL-4, but not IFN-g

Provide help to IgE and IgG1 secreting B cells

Relevance in vivo - Infection

Non-healing BALB/c Resistant C57BL/6

Draining LN T cells express IL-4 mRNA

Irradiated BALB/c recipient

T
Leishmania specific T cells

Draining LN T cells express IFN-g mRNA

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
Low infectivity Localised infection Normal serum Ig Normal T cell response

Lepromatous leprosy
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
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?

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

Langerhans 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

[Ca2+]i

Immature DC migrate into inflamed tissue in response to MIP-1a, MCP-1 MIP1- which bind to, and trigger CCR1, CCR2 and CCR5 respectively.

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 2 lymphoid tissue

Sallusto et al., Eur. J. Immunol. 1998 28 2760-2769

[Ca2+]i

Mature DC stop migrating into inflamed tissue and make no response to MIP-1a, MCP-1 MIP1-

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

Splenic DC
Not pulsed with Ag

DC labelled RED

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
Signal 2 B7 - CD28 Costimulation

Th

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
Signal 2

Th
Signal 3 Th polarising signal
Integration of signals from pathogen/allergen and the extracellular milieu polarise the DC to produce qualitatively different signals 3

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
CD80/CD86

Class II CD40

Th1 polarising factor IL-12 Th2 polarising factor CCL2 (MCP-1)

Type 2 PAMPS bind to PRR

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

Unmethylated CpG DNA

CD14

MD-2

TLR 2 TLR 1

TLR 6 TLR 2

TLR 3

TLR 4

TLR 9

Low level IL-12p70 Some ligands induce IL-10 or IL-12p35

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 IFN-a IL-18

Epithelium

Th1
Viruses
NK Viruses Fungi Parasites Mast

IFN-g

Histamine

Th2
Viruses Fibroblast PGE2 CCR2L

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. 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

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?

Vaccinate with mycobacteria

Aerosolised ovalbumin (OVA)

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
CD4+ cells specific for OVA that produce high levels of the immunosuppressive cytokines TGF and IL-10

No asthma-like symptoms

Mycobacteria induced REGULATORY T cells

Th cell polarisation
DC mediated decision influenced by infection Extracellular milieu - mediated

Journal of Immunology 1994 152 4755-4782

1 10 Factor increase over control

1 10 Factor increase over control

Priming conditions
Control Ab Anti-IFNg Ab IL-4 + control Ab IL-4 + anti-IFNg Ab

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-5 IL-6, IL-8, TNF-a IL-4, IL-5,

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?

D. pteronyssinus

L. destructor

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
(butmost 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
PAR1 PAR2 PAR3 PAR4

Activators
Thrombin, Trypsin Granzyme A Trypsin, Tryptase, Factor Xa, Proconvertin Thrombin Thrombin, Trypsin, Cathepsin G

Inactivators
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-5, IL-6, IL-8, TNF-a IL-4,

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 -actin 0 Inhibited Der p1 Der p1 Inhibitors -ve

+ve

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

Tetanus toxoid PMA/Ionomycin Time (hr)

+
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 516bp

IFN-g
IL-5 IL-4

516bp
516bp IL-13 516bp -ve +ve - Inhibitors 1000ng/ml ES 0 ES + Inhibitors 200ng/ml ES 1000ng/ml ES 0 ES 100ng/ml ES -actin

200ng/ml ES

100ng/ml ES

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
Sg3

Cg3
Sg1

Cg1
Sa1

Ca1
Sg2

Cg2
Sg4

Cg4
Se

Ce
Sa2 Cd

Ca2

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
Antigen

Th Th
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-4 IL-4 IL-13 IL-13

IL-13R

IL-4Ra

gC

IL-4Ra

IL-13Ra1/2

JAK1

JAK3 TYK1

JAK1

P
Stat-6

P
Stat-6

TYK2

P
P P P
Stat-6 Stat-6 Stat-6

P P P PDimerised Stat-6 P
Stat-6 Stat-6 translocates 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

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 NF kB

Uninhibited NFkB translocates to the IkB nucleus

NF kB

Activation of the Ie promoter

Activation/cytokine responsive promoter Ie Se Ce1 Ce2 Ce3 Ce4

C/EBP Stat6 AP-1

PU.1 NFkB

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

RNA

Ie

Ce

Spliced RNA

Se

RNA
S region RNA hybridises with template DNA

C/EBP

Stat6

PU.1

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 Se

Ce1 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

Se

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

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

AID may also deaminate C on the template strand ?RNAase?

NH2 N AID HN

N Cytidine

N Uridine

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

GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA
UNG P P P P P P

UNG

UNG

UNG

UNG

UNG

UNG

UNG

GGGUTGA CCCGACT
P P P P P P

Base is removed, but backbone remains intact

G - U mismatch repair
Abasic site is processed by the apurinic/apyrimidimic endonuclease 1 (APE1)
OH
P P

APE1 P
P P P P

GGGUTGA CCCGACT
DNA is now nicked to produce a single strand break

GGGCTGGGU

TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT
APE1

CCCGACCCGACTCGACYCGACTCGACYCGAU TCGAYTYNA GGGUTGGGUTGAGUTGRGUTGAGUTGRGUTGAGUTRARNT

Similar mechanism on the template strand creates a staggered double strand break APE1

CCCGACCCGACTCGACYCGACTCGACYCGAUTCGAYTYNA

Processing of staggered ends

GGGCTGGG TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT CCCGACCCGACTCGACYCGACTCGACYCGA TCGAYTYNA GGGCTGGG Exonuclease activity CCCGACCCGACTCGACYCGACTCGACYCGA End fill-in reactions TGAGCTGRGCTGAGCTGRGCTGAGCTRARNT ACTCGACYCGACTCGACYCGAC TCGAYTYNA

Cm
Sm

Cd
Sg3

Cg3
Sg1

Cg1
Sa1

Ca1
Sg2

Cg2
Sg4

Cg4
Se

Ce
Sa2

Ca2

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
Sm

Cd
Sg3

Cg3
Sg1

Cg1
Sa1

Ca1
Sg2

Cg2
Sg4

Cg4
Se

Ce
Sa2

Ca2

Cg1 Ca1 Cg3 Cg2 Cg4 Cm VDJ Ce Ca2

Cg1 Ca1

Excised episomal circle of intervening DNA

Cg3
Cd

Cg2 Cg4

Cd

Cm
VDJ Ce Ca2

Non-homologous end joining in class switch

Closely resembles another B cell Ig gene mechanism
Ig gene recombination

23

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

9 12 7

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 PU.1 NFkB BCL-6

BSAP

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

Additional areas to think about

Cant 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

Enterobious

Hookworm

Leishmania

Onchocerca

Plasmodium

Schistosome

Taenia

Toxoplasma

Trichuris

Trypanosoma

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