Mechanisms of sensitization, disease development and desensitization:towards novel approaches for prevention and therapy

Ronald van ReeAcademic Medical Center, Amsterdam

Immunology: B-cells and antibodies

Immunotherapy: mechanisms and monitoring

Atopic march: eczema → asthma → rhinitis → asthma?

Priorities for allergy/asthma research

Exposure to allergen results in:

no response?

protective response (“active tolerance”)?

sensitization?

+/- symptoms?

Interaction between allergen and IgE is atthe basis of allergy and (extrinsic) asthma.

How is the process of B-cell activity towards or away fromallergic inflammation regulated?

B-cells and antibodies

Ig?B-cell

DC

Th1 Th2

naïveTh

IgMB-cell

Th1

Th2

no allergy

IL4/IL13IFNγTh1

Th2

allergy

IgEB-cell

genetic factorsexogenous factors

(environment incl. allergens infections, diet, lifestyle)

Ig?B-cell

DC

Th1 Th2

naïveTh

IgMB-cell

Th1

Th2

no allergy

IL4/IL13IFNγTh1

Th2

allergy

IgEB-cell

genetic factorsexogenous factors

(environment incl. allergens infections, diet, lifestyle)

Scheme of a simple Th1-Th2 disbalance to distinguishbetween allergic and non-allergic has actually been

dismissed as being too simple.

IgG/A?B-cell

DC

Th1and/orTreg

Th2

naïveTh

IgMB-cell

Th1 Th2

no allergy

IL4/IL13IFNγTh1

Th2

allergy

IgEB-cell

genetic factorsexogenous factors

(environment incl. allergens infections, diet, lifestyle)

Treg

no antibody response?IgG? IgA?

?

healthy non-atopic?

IL10TGFβ

IgG/A?B-cell

DC

Th1and/orTreg

Th2

naïveTh

IgMB-cell

Th1 Th2

no allergy

IL4/IL13IFNγTh1

Th2

allergy

IgEB-cell

genetic factorsexogenous factors

(environment incl. allergens infections, diet, lifestyle)

Treg

no antibody response?IgG? IgA?

?

healthy non-atopic?

IL10TGFβ

Questions and Discrepancies

• healthy immune response against allergens: Treg/IL10/TGFβ or Th1/IFNγ or both?• do B-cells of non-atopic individuals ignore allergens or produce IgG/IgA antibodies?• Is this different for low-exposure allergens (e.g. pollen and mite) and high- exposure allergens (e.g. cat, bee venom and occupational allergens)?

IgG1B-cell

DC

Th2

naïveTh

IgMB-cell

no allergy

IL4/IL13

IL10

Th1

Th2

allergy

IgEB-cell

genetic factorsexogenous factors

timing/dose/context of allergen exposure

modTh2

IgG4B-cell

Th1

modTh2

IgE?B-cell

healthy atopic (or atopic treated by immunotherapy) and non-atopic?

IgG1B-cell

DC

Th2

naïveTh

IgMB-cell

no allergy

IL4/IL13

IL10

Th1

Th2

allergy

IgEB-cell

genetic factorsexogenous factors

timing/dose/context of allergen exposure

modTh2

IgG4B-cell

Th1

modTh2

IgE?B-cell

healthy atopic (or atopic treated by immunotherapy) and non-atopic?

Questions and Discrepancies• is a modified Th2 response in atopic individuals IgG4 without IgE or with (little/harmless) IgE?• is there an early-life window of opportunity for inducing the protective modified Th2?• why are IgG4 responses to allergens higher in atopics than in non-atopics?• atopic: predisposition to produce IgE and high IgG4? non-atopic: no tendency to develop IgE and only low IgG4?• atopic background seems to be less important for IgE/IgG4 production in case of high exposures: cats, bee venom, occupational allergens, parasites.

Increased exposure to cat allergen

Allergy (sensitization)IgG (exposure / protection)

Lancet 2001 ; Tom Platts-Mills et al.

High early exposure to cat allergen is protective

Why are IgE responses always so low compared to IgG responses?

Half-life of IgE is very short but this can not explain the 1000-folddifference in serum titers.

A major cause most likely is the poor generation of memory B-cellsfor IgE caused by inefficient processing of mRNA for membrane IgE.

Circulating IgE is derived from long-lived plasma cells hiding in survivalniches like the bone-marrow and inflammatory sites.

Two situations:• low allergen exposure (e.g. pollen/mite) i.e. a weak Th2 response that will not effectively induce a germinal center necessary for induction of memory B-cells

• high allergen exposure (e.g. cat, bee venom) i.e. a strong Th2 response that will a generate mature germinal center facilitating induction of memory

Low exposure situation: no memory only plasma cells

plasma cells

Atopics respond with all three but IgG is not protective under these conditionsNon-atopics only make a little IgG / are hypo-responsive overall compared toatopics.

Fro

m:

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t al

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2004

May

;113

(5):

983

-6.

Poor expression of membrane form of IgEfavors apoptosis resulting in poor memory

High exposure situation: memory generation for IgG but not for IgE

plasma cells

This also reflects the situation during allergen-specific immunotherapy

Same specif

icity

Fro

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Aal

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e R

C e

t al

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Alle

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Clin

Im

mun

ol.

2004

May

;113

(5):

983

-6.

For primary prevention it is of the greatest importance toinvestigate the dose-response relation between allergenand (the quality) of the immune response.

The window of opportunity is of great importance.

A high-dose protective effect as observed for cat hasso far not been found for house dust mite. For foodallergens this is even more debated.

The outcome has very significant public health impact.

“Promote cats and peanut butter sandwiches early on or not?”

What is needed to study the dose-response relationbetween allergen exposure and (the quality of) theimmune response?

• Analysis of (existing) birth cohort samples for IgG and IgA responses. In most cases only IgE has been measured. Multiple time points in first year of life are required. Multiplex systems now allow in detailed analysis with limited sample.

• Analysis of (existing) birth cohort samples for mRNA profiling

• In vitro cellular analyses to study the process of immune skewing, preferably in an allergen specific fashion.

• Mouse models that go beyond the ovalbumin model by using real allergens.

plasmaB-cellmemory

B-cell

Breg

immatureB-cell

exogenous factors

(environment incl. allergens infections, diet, lifestyle)

genetic factors

Regulatory B-cells, a missing link?

CD1dhiCD5+

major source of IL10promote Treg development

In several mouse models of auto-immune diseases an anti-inflammatory (protective)role has been established for regulatoryB-cells. Do they play a protective role inallergy/asthma. If so, how can they bepromoted?

The quality of IgE antibodies

Do they always translate into clinical allergy? No

There are several examples of IgE responsesthat are without clinical relevance, i.e. the onesobserved during parasite infections, the onesdirected to plant glycans and the ones observedduring successful immunotherapy.

Tregs or Th1 cells can not explain the lack of biological activity.

The explanation can also not (always) simply be found ina protective effect of IgG4 (“blocking antibodies”).

How is the biological activity of IgE assessed?

lactic acid treatment:removal of IgE

incubation with serum: sensitization with IgE

basophils fromnon-atopic donor

stripped basophils fromnon-atopic donor

basophils re-sensitizedwith IgE under study

Stripped basophil protocol

specific IgE

0

5

10

15

20

25

30

35

40

me

an

(S

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of

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ti-m

ite I

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U/m

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Gabonese Dutch

Gabonese (pink) vs Dutch (green) (n=16) (n=7)

0

20

40

60

80

0.1 10 1000

ng/ml

% h

ista

min

e r

ele

ase

(S

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a-IgE

mite

a-IgE

mite

Allergen-specific IgE from parasite-infected children lacks activity.This is not explained by high allergen-specific IgG4 titers

What explains the poor biological activity?

0

20

40

60

0.0001 0.01 1 100 10000

concentration (ug/ml)

% h

ista

min

rele

ase transgenic human

lactoferrin

native humanlactoferrin

rPhl p 5

IgE antibodies against plant N-glycans have the samepoor biological activity

An explanation needs to be found why some IgE antibodies arepoor inducers of mediator release, i.e. without clinical relevance.

What is different about these type of IgE antibodies?

Lower affinity? Something else?

Is this also observed during immunotherapy where IgE antibodiesare persistent but skin reactivity clearly decreases.Are these IgE antibodies the same as before the start of therapyor are they (partly) newly formed IgE antibodies.

B-cells and antibodies have not received the attentionthey deserve compared to T-cells. They do howeverproduce the antibodies that cause the disease!!

Reasons to study IgE/IgG/IgA antibody responses:

• They are a good read-out of the nature of the immune response• It needs to be established when/why they are clinically relevant, i.e. either disease-inducing (IgE) or protective (IgG/IgA).• Is induction of IgG/IgA before IgE a protection against future sensitization?• What is the difference between IgE from direct (μ to ε) and from indirect (μ to ε via γ4)?

Reasons for studying B-cells:

• What determines the quality of IgE antibodies?• What is the role of Bregs in allergy/asthma?• How could we target persistent plasma B-cells for IgE?

Immunotherapy: mechanism and monitoring

Many of the same questions need to be answered.

Blocking IgG4 antibodies?What is the difference between IgE before and after therapy?Th1 skewing?Tregs?Or perhaps Bregs?

Expression profiling / proteomics for biomarker search

Investigator-initiated trials needed. Companies are too small toset up trials that include in depth immunological analyses. Betterinsight in mechanism is a prerequisite for improvement of therapy.

Primary outcome (natural exposure) is an ill-controlled disaster!Towards pollen chamber monitoring in season.