Proefschrift aalberse by Nicole Nijhuis

Allergic disease: the diagnosis of peanut allergy and the role of heat shock proteins in chronic allergic inflammation

Joost Aalberse

Allergic disease: the diagnosis of peanut allergy and the role of heat shock proteins in chronic allergic inflammation

Joost Aalberse

Colofon Cover: Landgoed Oostbroek, Utrecht

Design: Rozemarijn Klein Heerenbrink, Photography: Joost Aalberse Print: Gildeprint, Enschede ISBN: 978-94-6233-136-5

2012 by Frontiers in Immunology, for chapter 2

2014 by Clinical and Translational Allergy, for chapter 5 2015 by J.A. Aalberse, for chapter 1 and chapter 8 Address of correspondence

J.A. Aalberse, Wilhelmina Children’s Hospital, University Medical Center Utrecht e-mail joost.aalberse@gmail.com

Allergic disease: the diagnosis of peanut allergy and

the role of heat shock proteins in chronic allergic inflammation (met een samenvatting in het Nederlands)

Proefschrift Ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, Prof. Dr. G.J. van der Zwaan ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 26 november 2015 des middags te 4.15 uur

door Joost Adriaan Aalberse geboren op 22 januari 1977 te Amsterdam

Promotor: Prof. Dr. B.J. Prakken Copromotoren: Dr. F. van Wijk

Dr. E.F. Knol

“Know Thyself” Alexander Pope Uit: Pope, Alexander; An Essay on Man, in Four Epistles. Hartford: Silas Andrus, 1824.

Know then thyself, presume not God to scan; The proper study of mankind is Man.

Placed on this isthmus of a middle state, A being darkly wise and rudely great:

With too much knowledge for the Sceptic side, With too much weakness for the Stoic’s pride, He hangs between; in doubt to act or rest, In doubt to deem himself a God or Beast, In doubt his mind or body to prefer;

Born but to die, and reasoning but to err; Alike in ignorance, his reason such

Whether he thinks too little or too much:

Chaos of thought and passion, all confused; Still by himself abused, or disabused; Created half to rise and half to fall;

Great lord of all things, yet a prey to all;

Sole judge of truth, in endless error hurled: The glory, jest, and riddle of the world!

Contents Chapter 1

General Introduction

Chapter 2

HSP; Bystander Antigen in Atopic Diseases?

Chapter 3

Cord Blood CD4+ T cells respond to self-heat shock protein 60

Chapter 4

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

Chapter 5

Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut

Chapter 6

Moving from peanut extract to peanut components: Towards validation of component-resolved IgE tests

Chapter 7

Discussion

115

Chapter 8

Lekensamenvatting (Nederlands)

131

Chapter 9

Dankwoord

138

List of Publications

143

142

Chapter 1 General Introduction

J.A. Aalberse

Chapter 1

General Introduction PART 1 A historical introduction The term ‘allergy’ was coined by Pirquet in 1906(1;2). Symptoms of allergic diseases had been

described long before, with seasonal symptoms of hay fever and adverse responses to food(3-8). Pirquet noticed an adverse response in some patients receiving a chickenpox vaccine. The pa-

tients developed not only a protective immune response, but also symptoms of hypersensitivity, such as skin rash, arthropathy and fever. Pirquet called this altered response ‘allergy’ de-

rived from the Greek words allos (other) and ergon (work). Allergy according to the definition of Pirquet included hypersensitivity syndromes such as hay fever and food allergy, but also

auto immune disease and serum sickness. It was almost 60 years later, in 1963, that Gell and Coombs made a classification for the, at that time, confusing wide variety of hypersensitivity

diseases(9). Remarkably, this classification is still being used. For this thesis I will focus on type

I hypersensitivity, which corresponds to atopic allergy. IgE, an immunoglobulin identified in 1966, is considered the cornerstone of atopy(10-12).

Atopic syndrome and allergy

The terminology of atopy and allergy can be confusing. Allergy is hypersensitivity towards a harmless environmental allergen with an immunological response. This can be a humoral

(antibody mediated) or a cellular (cell mediated) response. The humoral response in an allergic reaction typically depends on the IgE isotype.

According to the definition of the World Allergy Organization atopy can be defined as the tendency to become sensitized and produce IgE in response to exposure to allergens(14).

Atopy has a strong genetic link, with 50-70% of mothers (and in lesser extent fathers) passing their atopic tendency to their children(15). Individuals with atopy that develop allergic

disease can have symptoms with different tissue involvement, including the skin, the upper and lower respiratory system and the gut. These diseases can follow a typical pattern during life, often referred to as the atopic march(16). Quickly after birth it starts with atopic

dermatitis and food allergy, and continues in some with asthma and allergic rhinitis. These

diseases (as seen in figure 1, in bold) are typically IgE mediated atopic diseases, which corresponds also to type I hypersensitivity. Diagnosis of allergic disease

The diagnosis of IgE mediated allergic diseases is not always straightforward. Measuring of allergen-specific IgE is not sufficient for the diagnosis, as IgE can also be present in non-allergic

individuals. Moreover, it is not indicative of the organ involved and also the severity of the symptoms cannot accurately be predicted by the levels of IgE. 12

General Introduction

Figure 1 | Nomenclature of hypersensitivity. Figure adapted from Johansson et al Allergy 2001(13) illustrating the misunderstanding that can occur when atopy and allergic disease is discussed.

The diagnosis of allergic disease is foremost made on clinical observations: both on the history of the patient as on the physical examination by the doctor. Important in the history is the relation of the symptoms with exposure to the possible allergic trigger, but also other experienced

atopic diseases as well as the family history related to atopic disease. The history of the patient is however often unreliable. In food allergy it was found to be reliable only in thirty percent(17-19).

The clinical diagnosis can be supported by a skin test and laboratory findings such as IgE to relevant allergens.

During a skin test a very small amount of allergen is put into the skin, which results in cross linking

of IgE on skin mast cells causing local swelling and redness when there is an allergic sensitization.

It is an easy and quick test and therefore often used. Another way of testing an allergic sensitization is in the blood, by measuring the level of specific IgE antibodies to an extract or a specific allergen. It is important to realize that not all individuals that are sensitized also have clinical symp-

toms, which can result in misdiagnosis. Positive predictive values with high accuracy have been reported in literature(20), but these are arbitrary. Also it has been found that sensitization to some

allergens is more clinically relevant than sensitization to others. Until recently IgE was meas-

Chapter 1

ured only against an extract, but it can now also be measured against purified and recombinant

allergens. Measuring IgE only to the clinically most relevant allergens is suggested as a possible

solution to improve the correlation between the measured sensitization and the clinical response. Diagnosis of food allergy

In addition to skin testing and IgE specific antibodies, food allergy can be diagnosed via a food challenge(19). A double-blind placebo-controlled oral food challenge (DBPCFC) is the gold standard

forÂ diagnosis(21-23). During a DBPCFC the patient is exposed to the presumed trigger and the clinical

response is observed in a safe clinical environment, with proper trained personnel and ideally

direct access to intensive care facilities in case of an anaphylactic response. The children have to be prepared accurately as the psychological effect of an exposure to an allergen they have had to avoid all life, can be dramatic. At the start of the challenge a low dose is given, followed by

incrementing doses. To ensure that it is blinded the food has to be masked (which often causes difficulties with non-standardized recipes). Ideally the challenge is spread over two days, with a placebo and a verum challenge day blinded for both the performing staff as well as the patient. Standardization of the DBPCFC between various clinical centres is a current issue(21).

Figure 2 | The diagnosis of a food allergy like peanut allergy stands and falls with a well performed DBPCFC. Often the diagnosis is only made on the history of symptoms and on specific IgE. Not all allergens are known however and many extracts are incomplete, which sometimes results in false negative responses. More often false positives responses are seen, as often sensitized patients do not have clinical allergic responses. A well performed DBPCFC tests the clinical reactivity and therefore is used as the gold standard for diagnosis of food allergy.

To conclude, it has been shown that food allergy is often misdiagnosed when only the history of the patient and serum IgE or SPT is taken in account. DBPCFC are needed for a uniform

diagnosis of food allergy, not only for the proper diagnosis in patients, but also for proper classification of patients in research. Prevalence of allergic disease

The prevalence of allergic disease has increased tremendously especially in westernized countries(24-29). As mentioned above it is important to note that allergic diseases are often

misdiagnosed, which also effects research data, with i.e. too high prevalence numbers, due to

false positive tests. Ideally, prevalence data on allergic disease should be based on both the production of specific IgE (sensitization) as well as on a proven tissue involvement.

General Introduction

Presently, sensitization (IgE antibodies) to allergens in the environment is found in up to 40% of the population(12). Allergic sensitization can cause diseases in different organs including the lungs (asthma), the nose (atopic rhinitis), the gastro-intestinal tract gut (food allergy) and

the skin (atopic eczema). The latter two are the topic of this thesis. Thirteen percent of U.S. children aged 17 years and under suffers from skin allergies, including atopic eczema(30). Often

co-morbidity is present with other allergic diseases, such as food allergy. Approximately 5-9% of children in the US have food allergy, depending on the age, but also on criteria used for the

diagnosis on food allergy(30). Eight foods account for 90% of food allergic reactions in children. These eight foods are milk, eggs, peanut, tree nuts, fish, shellfish, soy and wheat.

Peanut is the most prevalent food allergy, accounting for about 1-2% of all children in Europe(31-33)

and also has the most severe clinical reactions including anaphylaxis(34). Interestingly in recent

years it has been suggested that peanut consumption during pregnancy is associated with a decreased risk of offspring with peanut allergy(35). In addition regular peanut consumption in

children that starts before age of 11 months reduces peanut allergy up to 7-fold (36). Peanut is

a common food, present in many foods. Unlike cow milk allergy which is regularly outgrown, peanut allergy is persistent in approximately 80% of the children(26;37). Plant Food Allergens

Although a large number of food proteins are consumed, only a small number is responsible for the majority of food allergies(38). It is known that some of these food proteins have special

characteristics that can contribute to allergenicity, such as a small molecular weight, stability,

water solubility and heat- and digestion resistance(39-43). The food allergens can have properties that are functionally relevant for the plant or food, such as storage and defensive proper-

ties. These characteristics contribute to their ability to resist food processing and digestion, which sometimes can even make them more allergenic. Lipid transfer proteins (LTPs) for ex-

ample are important in the defence of plants from different kinds of pathogens and environmental stress(44). Many fruits and nuts contain LTPs that are highly resistant to digestion(45-47).

Food processing can both increase and decrease the allergenicity of proteins(48;49).

A protein that is allergenic (i.e. allergen) can cause sensitization and subsequently induce an

allergic response to the protein during the next exposure. An allergen however does not always

do both. Some allergens only cause sensitization whereas others only induce a clinical response by crossreactivity. Sensitization without clinical reactivity is often seen in hazelnut and peanut sensitized subjects that are also grass or tree pollen allergic(50;51). Often this can be explained by

respectively cross-reactive carbohydrate determinants (CCD) and pathogen related 10 (PR10) proteins. CCD induces IgE antibodies to grass, but also cross reactive IgE antibodies to hazelnut and peanut. These cross reactive antibodies however do not cause a clinical response(52;53). Similarly Bet v 1, a pollen allergen of the PR-10 family, causes sensitization and clinical reactivity to

tree pollen, but often causes cross reactive sensitization to the peanut allergen Ara h 8 (a PR-10 homologue) without (severe) clinical reactivity. This cross reactivity can be explained by a lack

Chapter 1

of discrimination of IgE. The lower affinity for the cross reactive allergens might be a reason for the absence of clinical reactivity. On the other hand some allergens can cause clinical reactivity,

but are dependent on sensitization via other allergens. These allergens are called incomplete allergens. A nice example of an incomplete allergen is mal d 1, an apple allergen. This protein

rarely induces IgE antibodies, due to its instable form. Bet v 1 however is a very similar protein to Mal d 1, which does induce IgE antibodies and also causes reactivity to Mal d 1(54).

Better insight in the functions, properties and cross reactivity of allergens is important for diagnosis and in treatment. Peanut is a legume (such as beans) and taxonomically distantly related

to tree nuts. Several peanut and tree nut allergens however belong to the same protein family(55).

So far 16 peanut allergens have been described. These allergens belong to different families of seed storage proteins, pathogenesis related proteins, lipid transfer proteins, oleosins and defensins (PR)-10(56). In table 1 the characteristics of these peanut allergens are shown in more detail.

Table 1 | Peanut components Allergen

Protein Family

Related Proteins

Ara h 1

Cupin

Gly m 8

Cor a 11

Ara h 3

Cupin

Cor a 9

Gly m 9

Ara h 6

Conglutin

Cor a 14

PR-10

Bet v 1

Ara h 2 Ara h 5 Ara h 7 Ara h 8 Ara h 9

Ara h 10 Ara h 11 Ara h 12 Ara h 13 Ara h 14 Ara h 15 Ara h 16 Ara h 17

Conglutin

Profilin

Conglutin Lipid Transfer Protein

Cor a 14 Bet v 2

Cor a 14 Pru p 3

Oleosin

Cor a 12

Defensin

Gly m 2

Oleosin

Defensin

Cor a 12 Gly m 2

Oleosin

Cor a 12

Lipid Transfer Protein

Pru p 3

Oleosin

Lipid Transfer Protein

Cor a 12 Pru p 3

Gly m 8 Gly m 3 Gly m 8 Gly m 8 Mal d 1 Art v 3 Ses i 4 Ses i 4

Art v 1 Art v 1 Ses i 4 Ses i 4

Art v 3 Art v 3

The 16 peanut allergens so far known are outlined in table 1. Ara h 4 is not mentioned, as this is now recognized as being similar to Ara h 3. Knowledge of the protein family helps in understanding possible cross-reactivities and functions of the protein. Ara h: peanut allergen (Arachis hypogaea); Gly m: Soybean allergen (Glycine max): Cor a: hazelnut allergen (Corylus avellana); Bet v: birch pollen allergen (Betula verrucosa); Mal d: apple allergen (Malus domestica); Pru p: peach allergen (Prunus persica); Art v: mugwort pollen allergen (Artemisia Vulgaris); Ses i: sesame allergen (Sesamum indicum). It is likely that more allergens will be discovered in coming years. 16

General Introduction

Not all allergens are present in extracts used for diagnosis, which is underlined by false-ne-

gative IgE tests(57). In numerous studies it has been shown that extracts used for IgE tests

and SPT are often not comparable(58;59). Which proteins are present in an extract depends on

the source materials and production procedures. It is suggested that the diagnosis would be better interpretable if tests could be performed for selected, purified or recombinant allergens(60). This can be determined by molecular or component resolved diagnostics (CRD).

Mechanisms in IgE sensitization and allergy symptoms

The initial allergic response can immunologically be divided in two different phases: sensitization and elicitation. The typical allergic inflammatory response is an acute response within

two hours after exposure to the allergen. Although some food allergic patients outgrow their allergies (as seen in cowâ&#x20AC;&#x2122;s milk allergy), food allergy is often persistent, with acute symptoms upon every exposure. Acute allergic responses can result in remodelling of tissue which causes chronic inflammation independent of the trigger.

Initial allergen exposure causes an immune response that is speculated to be initiated by ep-

ithelial cell-derived cytokines such as TSLP, IL-25 and IL-33(61-66). These cytokines activate group 2 innate lymphoid cells (ILC2) and dendritic cells. Activated ILC2 cells produce large quantities of IL-5 and IL-13.The activated dendritic cells present the allergen via interaction

of MHC and the T cell receptor, which then induces Th2 cells. Th2 cells produce type 2 cytokines like IL-4, IL-5, IL-9 and IL-13 that result in differentiation, recruitment and activation

of eosinophils, basophils and mast cells(67;68). The production of IL-4 by Th2 cells results in

provision of B cell help in isotype switching to IgG1 and IgE. B cells continuously produce IgE, but specific IgE is only formed after recognition of the antigen(61;69;70). IgE binds via its

Fc-part, to its high affinity receptor on the mast cells and basophils, arming them for the next

exposure. This arming phase is the sensitization phase. After re-exposure with the allergen,

elicitation of allergic symptoms can occur. Within a few minutes after the allergen has crosslinked the high affinity IgE receptors via binding to IgE on mast cells and basophils, degranulation of these cells occurs, with the release of mediators. Histamine is an important mediator,

causing vasodilatation, increased vascular permeability and pruritus. The acute allergic re-

sponse is dominated by Th2 cytokines. The number of Th1 cytokines however increases in the chronic state of allergic diseases such as atopic dermatitis and allergic asthma, leading to tissue remodelling(71;72). It is not yet known if tissue remodelling is also seen in food allergy(73). In addition, TSLP, IL-25 and IL-33, more innate-type cytokines, also seem to contribute to the

chronic inflammation. Although these Th2 driving cytokines have an important regulatory

function maintaning the balance of immune homeostasis, uncontrolled production of these cytokines can cause chronic allergic inflammation(74).

Chapter 1

PART 2 Immune therapy Inflammation can be acute, persistent (repetitive acute inflammation) or chronic. The basics

for treatment in allergic disease are to control environmental factors and specific medicines that relieve or control symptoms. The most important step in controlling environmental factors is avoiding the triggering allergen. Avoidance of allergens is however often not possible

or very difficult (i.e. pollen in rhinitis, or low amounts of peanut which can be present in many foods). Fatalities due to allergic reactions are still reported every year(75). Therapies aimed at

relieving acute symptoms (such as anti-histamines) do not affect the persistency or chronicity

of symptoms. Corticosteroids can control symptoms of chronic inflammation, as often seen in patients with atopic dermatitis or asthma. However the disease remains and is not cured.

Various biologicals, including cytokine blockers and anti-IgE have been introduced as therapy in allergic disease(76;77). These have thus far been of limited success in specific patient sub-

groups only, which can be explained by the complexity of allergic diseases as well as the limited repertoire of biomarkers(78;79). Anti-IgE treatment for example is an approved treatment

for patients with moderate to severe persistent asthmatics. However, this is non-effective in

about a third of patients and only mildly effective in another third. The treatment response cannot be explained by differences in total IgE levels(80). Better predictors for success and

improvement of this kind of therapy are being researched.

The only long-term cure for allergic disease is seen after allergen-specific immunotherapy (SIT)(81;82). Various randomized studies have shown effective reduction of symptoms in in-

sects, pollen and dust mite allergy(83;84). Tolerance is induced by repeated and increasing doses of the causative allergen. Immunological mechanisms include the induction of Tregs, produc-

tion of IL-10 and TGFÎ˛ as well as the production of specific antibody isotypes, such as IgG1 and IgG4. SIT often induces immune tolerance with desensitization for several years. Drawbacks of immune therapy are its antigen specificity and the so far limited number of allergens available, as well as the number of side effects and the long duration of treatment.

In chronic allergic disease allergen targeted therapy is probably insufficient. Remodelling of tissue, as seen in asthma, leads to chronic inflammation which can be present independent of the original trigger(85). Antigen specific treatment will therefore not be successful in this

chronic inflammatory state.

Tolerance and immune regulation Regulatory T cells play an important role in tolerance induction and controlling inflamma-

tion(86). FOXP3+ regulatory T cells are the best known and characterized T cells subset dedicated to suppression of unwanted immunity. FOXP3 is a transcription factor that is crucial for the development and function of these Tregs. The significant role of FOXP3+ Tregs is illustrated by

General Introduction

a syndrome called IPEX (which stands for Immune-dysregulation Polyendocrinopathy Enteropathy X-linked(87)). A mutation in FOXP3 causes a defect in regulatory T cells in IPEX. Patients

with IPEX can present with auto immune diseases, atopic dermatitis and have increased levels of IgE(88;89). Although we know that FOXP3 Tregs are related with the production of IgE

and allergic symptoms the studies are limited(90-92). In general it is hypothesized that defective Tregs promote the development of allergic disease.

Regulatory T cells are both derived from the thymus (the natural FOXP3+ Tregs (nTregs)), as induced in the periphery (iTregs). iTregs can be further divided in FOXP3+ Tregs, Tr1 cells producing

IL-10 and Th3 cells producing TGFβ(86) The distinction between the different types of Tregs is not absolute. For example FOXP3 expressing Tregs are also capable of producing IL-10 and TGF-β.

IL-10 is an important cytokine for Tregs, but also plays an important role in the production of IgG4(88;89;93-96), an immunoglobulin often upregulated when patients develop tolerance(97). T cell

clones derived from children with persistent cow’s milk allergy have been shown to produce Th2 cytokines, whereas allergic control subjects that are cow’s milk tolerant produced a mixed

Th1/Th2 response, associated with significant elevated IL-10 levels(98). Children with high ex-

posure to cats that are less likely to become allergic to cats are characterized by an elevated level of T cell derived IL-10, as well as elevated levels of allergen specific IgG and IgG4(99). Similar

observations have been made in bee keepers with repeated exposure to bee venom(100). Again

the high exposure to the allergen results in an increase in allergen specific IgG. Interestingly,

reactions to the stings disappear simultaneously with the increased IL-10 production by T cells, while they reappear at the beginning of the next season. As repeated exposure to antigen can in-

crease IL-10 and IgG4, this concept is also used in some models of immune therapy as described in more detail below.

The Loeys–Dietz syndrome (LDS), like IPEX, is a syndrome that is associated with allergic diseases, high IgE and also eosinophilia(101). This syndrome however is caused by a mutation

that affects the TGFβ-receptors. TGFβ plays an important role in mediating the immunosuppressive effects of Tregs through cell-cell contact, in activating regulatory T cells, but also by

directly suppressing effector cells (102). Both IL-10 and TGFβ are also key cytokines produced by Bregs(103), and in recent years the absence or loss of regulatory B cells (Bregs) has also been

associated with allergic disease.

Above mentioned studies suggest that FOXP3+ Tregs and the cytokines IL-10 and TGFβ play

an important regulatory role in the prevention and control of allergic responses. Heat Shock Proteins

Heat shock proteins (HSP) are a family of stress induced proteins, ubiquitously expressed in

eukaryotic and prokaryotic cellular organisms. Heat shock, but also exposure to other kinds of physiological and environmental stress can lead to the expression of HSP. They are evolution-

Chapter 1

ary highly conserved both in human, as in bacteria and are immune dominant(104;105). HSP can

both trigger innate and adaptive immune responses, and HSP-specific antibodies are already

present directly after birth(104;106-108). HSP are associated with both pro- and anti-inflammatory

responses (109;110). They serve as damage associated molecular pattern molecules (DAMP) that

can bind with several receptors, including toll like receptors (TLR) (111;112). HSP can thereby ac-

tivate the innate system and induce pro-inflammatory cytokines. Anti-inflammatory responses related to HSP are associated with adaptive immune responses(113-114).The induction and

presence of HSP specific T cells has been shown to be protective in an experimental model of adjuvant arthritis and is associated with a favourable diagnosis in patients with juvenile idio-

pathic arthritis(104;115;116). Protective effects of various microbial HSP were seen in many other

experimental modes of chronic inflammation (109;117-119). So far little is known about the role

of HSP in human models of allergic chronic inflammation, as further reviewed in chapter 3. Outline of this thesis

In the first part of this thesis we have investigated the immunogenic response of HSP. In chap-

ter 2 we have reviewed the role of HSP in atopic disease. In chapter 3 and chapter 4 we have investigated the immunogenic response of HSP at birth in a disease free environment and in patients with atopic dermatitis.

In the second part of this thesis we have investigated how we can improve the diagnosis of peanut allergy. In chapter 5 we investigated if there was a different cytokine profile in

children with a clinical response to peanut compared to patients with only sensitization to peanut. In chapter 6 we have discussed the role of CRD compared to IgE testing to peanut extracts in the diagnosis of peanut allergy.

General Introduction

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General Introduction

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Smith M, Tourigny MR, Noakes P, Thornton CA, Tulic MK, Prescott SL. Children with egg allergy have evidence of reduced neonatal CD4(+)CD25(+)CD127(lo/-) regulatory T cell function. J Allergy Clin Immunol 2008; 121(6):1460-6, 1466. Sakaguchi S, Setoguchi R, Yagi H, Nomura T. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in self-tolerance and autoimmune disease. Curr Top Microbiol Immunol 2006; 305:51-66.:51-66. Palomares O, Yaman G, Azkur AK, Akkoc T, Akdis M, Akdis CA. Role of Treg in immune regulation of allergic diseases. Eur J Immunol 2010; 40(5):1232-40. Soyer OU, Akdis M, Ring J, Behrendt H, Crameri R, Lauener R et al. Mechanisms of peripheral tolerance to allergens. Allergy 2013; 68(2):161-70.

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Akdis M, Akdis CA. Mechanisms of allergen-specific immunotherapy: Multiple suppressor factors at work in immune tolerance to allergens. J Allergy Clin Immunol 2014; 133(3):621-31. Akdis CA, Blaser K. IL-10-induced anergy in peripheral T cell and reactivation by microenvironmental cytokines: two key steps in specific immunotherapy. FASEB J 1999; 13(6):603-9. van der Veen W, Stanic B, Yaman G, Wawrzyniak M, Sollner S, Akdis DG et al. IgG4 production is confined to human IL-10-producing regulatory B cells that suppress antigen-specific immune responses. J Allergy Clin Immunol 2013; 131(4):1204-12. Tiemessen MM, Van Ieperen-Van Dijk A, Bruijnzeel-Koomen CA, Garssen J, Knol EF, Van Hoffen E. Cowâ&#x20AC;&#x2122;s milk-specific T-cell reactivity of children with and without persistent cowâ&#x20AC;&#x2122;s milk allergy: key role for IL-10. J Allergy Clin Immunol 2004; 113(5):932-9. Platts-Mills TA, Woodfolk JA, Erwin EA, Aalberse R. Mechanisms of tolerance to inhalant allergens: the relevance of a modified Th2 response to allergens from domestic animals. Springer Semin Immunopathol 2004; 25(3-4):271-9.

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Merbl Y, Zucker-Toledano M, Quintana FJ, Cohen IR. Newborn humans manifest autoantibodies to defined self molecules detected by antigen microarray informatics. J Clin Invest 2007; 117(3):712-8.

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Birnbaum G, Kotilinek L, Miller SD, Raine CS, Gao YL, Lehmann PV et al. Heat shock proteins and experimental autoimmune encephalomyelitis. II: environmental infection and extra-neuraxial inflammation alter the course of chronic relapsing encephalomyelitis. J Neuroimmunol 1998; 90(2):149-61. Harats D, Yacov N, Gilburd B, Shoenfeld Y, George J. Oral tolerance with heat shock protein 65 attenuates Mycobacterium tuberculosis-induced and high-fat-diet-driven atherosclerotic lesions. J Am Coll Cardiol 2002; 40(7):1333-8.

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General Introduction

Chapter 2

HSP: Bystander Antigen in Atopic Diseases?

Joost A Aalberse; Berent J Prakken; and Berber Kapitein Front Immunol. 2012 31;3:139

Chapter 2

Abstract Over the last years insight in the complex interactions between innate and adaptive immunity

in the regulation of an inflammatory response has increased enormously. This has revived the

interest in stress proteins; proteins that are expressed during cell stress. As these proteins can attract and trigger an immunological response they can act as important mediators in this interaction. In this respect, of special interest are proteins that may act as modulators of both

innate and adaptive immunity. Heat shock proteins (HSP) are stress proteins that have these, and more, characteristics. More than two decades of studies on HSP has revealed that they are part of intrinsic, â&#x20AC;&#x153;naturalâ&#x20AC;? mechanisms that steer inflammation. This has provoked compre-

hensive explorations of the role of HSP in various human inflammatory diseases. Most studies have focused on classical autoimmune diseases. This has led to the development of clinical

studies with HSP that have shown promise in Phase II/III clinical trials. Remarkably, only very

little is yet known of the role of HSP in atopic diseases. In allergic disease a number of stud-

ies have investigated the possibility that allergen-specific regulatory T cell (Treg) function is defective in individuals with allergic diseases. This raises the question whether methods can

be identified to improve the Treg repertoire. Studies from other inflammatory diseases have suggested HSP may have such a beneficial effect on the T cell repertoire. Based on the immune mechanisms of atopic diseases, in this review we will argue that, as in other human inflam-

matory conditions, understanding immunity to HSP is likely also relevant for atopic diseases. Specifically, we will discuss why certain HSP such as HSP60 connect the immune response to

environmental antigens with regulation of the inflammatory response. Thus they provide a molecular link that may eventually even help to better understand the immune pathological basis of the hygiene hypothesis.

HSP: Bystander Antigen in Atopic Diseases?

Introduction Over the last years insight in the complex interactions between innate and adaptive immunity

in the regulation of an inflammatory response has increased enormously. This has revived the

interest in stress proteins; proteins that are expressed during cell stress. As these proteins can attract and trigger an immunological response they can act as important mediators in

this interaction. In this respect, of special interest are proteins that may act as modulators of both innate and adaptive immunity. Heat shock proteins (HSP) are stress proteins that have these, and more, characteristics. More than two decades of studies on HSP has revealed that

they are part of intrinsic, â&#x20AC;&#x153;naturalâ&#x20AC;? mechanisms that steer an inflammatory response(1;2). This

has provoked comprehensive explorations of the role of HSP in various human inflammatory diseases. Most of these studies have focused on classical autoimmune diseases such as type

I diabetes (IDDM), rheumatoid arthritis (RA), and juvenile idiopathic arthritis (JIA)(3-5). This

has led to the development of clinical studies with HSP that have shown promise in Phase II/ III clinical trials in both IDDM and RA(6-8).

Remarkably, only very little is yet known of the role of HSP in atopic diseases. Based on the immune mechanisms of atopic diseases, in this review we will argue that, as in other human

inflammatory conditions, understanding immunity to HSP is likely also relevant for atopic diseases. Specifically, we will discuss why certain HSP such as HSP60 connect the immune response to environmental antigens with regulation of the inflammatory response. Thus, they provide a molecular link that may eventually even help to better understand the immune

pathological basis of the hygiene hypothesis: an important concept relating the increase in the prevalence of atopic disease with exposure to microbes or microbial products early in life.

Chapter 2

Allergy Atopy is the predisposition to develop allergic diseases like atopic dermatitis, food allergy,

asthma, and allergic rhinitis. It is characterized by a predominant typical T helper 2 (Th2)-like immune response(9). Firstly, an environmental allergen, like an inhalant or food allergen in-

teracts with the innate immune system. After uptake by antigen-presenting cells, subsequent T cell priming leads to the stimulation of type 2 cytokines such as interleukin (IL)-4, IL-5, and IL-13. These cytokines interact with their receptors to induce a class switch toward IgE

production and to increase the number of eosinophils and mast cells. In immediate hypersen-

sitivity reactions IgE binds to its receptors and causes mast cells to degranulate. Apart from

IgE, increased levels of IgG4 are often measured in allergic individuals. IgG4 is considered as an antibody with anti-inflammatory activity(10). The levels of IgG4 are usually elevated in se-

rum from people that are repeatedly exposed to the same antigen, like beekeepers, or subjects

receiving increased doses of antigen during specific immune therapy (SIT). In addition to this

humoral immune response, allergy is characterized by a cellular response, mostly mediated by Th2 cells(11,12), but also by Th1 cells (like in atopic dermatitis) and regulatory T cells. The

combination of these humoral and cellular responses, both innate and adaptive, leads to an

allergic inflammatory reaction(13). Here we will discuss various aspects of the allergic immune responses in the context of the possible role of environmental antigens such as HSP in steer-

ing this immune response.

Allergy and the Hygiene Hypothesis The prevalence of atopic disease has increased tremendously in the second half of the twentieth century(14). Although the cause of this increase is not known though, a genetic cause

is unlikely due to the time span in which the increase took place. Therefore environmental

changes are more likely causes of this observed increase. Strachan(15) first proposed the concept of the hygiene hypothesis. This hypothesis states that the increase in the preva-

lence of atopic disease is due to a decreased exposure to microbes or microbial products early in life, especially in western societies. The concept was supported by the observation of an inverse association between both atopic dermatitis and hay fever and the number of

children in a household. Although the hygiene hypothesis and its proposed immune mechanism are still under debate, in the 1990s numerous of studies have been published supporting this hypothesis. For example, these observations relate the number of siblings, the

number of infections and exposure to endotoxins on the farm, with the incidence of atopic diseases(16-20).

Immune mechanisms of the hygiene hypothesis What could be the immunological cause of this inverse relation between exposure to environmental antigens and the incidence of allergic disease? The explanation takes us back to

the original observations in the eighties of Mosmann and Coffman who defined two main 34

HSP: Bystander Antigen in Atopic Diseases?

subtypes of helper T lymphocytes: Th1 and Th2 cells. Th2 cells are characterized by their pro-

duction of cytokines like IL-4, IL-5, IL-9, and IL-13 and chemokines like TARC and MDC. They are involved in mediating allergic responses and host defense against parasitic infection(21).

During fetal life and shortly after birth a predominant Th2 response is present(22). During life,

a shift to a predominant Th1 response occurs in non-allergic subjects, but this shift may be

incomplete in allergic individuals. As a consequence, an allergic individual will develop a Th2 response to an allergen leading to IgE production and typical type 2 cytokine production (IL4, IL-5, and IL-13) and ultimately to a clinical allergic reaction, whereas non-allergic subjects

develop a Th1 response leading to a protective IgG response and production of IFN-Îł(23). This

is the basis for the immunological explanation of the hygiene hypothesis, as exposure to mi-

crobes and microbial products would stimulate a Th1 response. Those individuals that were

not enough exposed to these stress factors would be more prone to develop predominant Th2 responses, and thus an allergic response. Obviously this is still a simplification of the immune

pathogenesis that does not explain all observations. For example, a Th1 response in aller-

gic individuals would implicate the development of late hypersensitivity responses, whereas

healthy individuals do not respond to allergens with a clinically noticeable response(24). The hygiene hypothesis also does not explain why not only the incidence of allergic diseases is increasing but also that of auto immune diseases like RA and IDDM(25). These diseases are

obviously known as typical Th1-like diseases. Triggering an allergic immune response

In order to fully assess and better appreciate the value of the hygiene hypothesis it is important to understand which cells are responsible for the initial trigger leading to the characteristic allergic immune response. First, naive T cells have to be activated, in a way that

promotes the classical Th2 like response. There is recent evidence that basophils may play a crucial role in antigen recognition and processing. When recruited to lymph nodes they are able to induce Th2 cell differentiation through the release of IL-4(26;27). Other cells also have

the capacity to process and present allergens, including mast cells, macrophages, eosinophils, and natural helper cells. Natural helper cells are innate cell populations that include

innate Th2 cells and are activated by IL-25 and IL-33 to secrete Th2 cytokines(28). There is

an increasing realization that Th2 cells are not the only cells involved in the pathophysiol-

ogy of allergic disease, as particularly in severe disease other cells than Th2 play a role in aggravating and perpetuating the immune response (TableÂ 1)(29).

Chapter 2

Table 1 | Different subtypes described to play a role in the pathogenesis of allergic diseases.

Cell

Main transcription factor

Activating cytokines

Effector cytokines

Role in allergic diseases

Th1

T-Bet

IL-12

IFNy

Th2

GATA-3

IL-4, IL-25, IL-33, TSLP

IL-4, IL-5, IL-13, IL-25

Possible role in chronic asthma or atopic dermatitis

Th17

Batf

TGFb, IL-1b IL-6, IL-21

IL-6, IL-8, IL-17; IL-22

Th9

PU-1

IL-4, TGFÎ˛, IL-10, IL-25

IL-9, IL-10

Eosinophil production; IgE induction

Mucus production, lung inflammation, dermatitis Possibly related to steroid resistant asthma; related to neutrophil production

Th22

RORyT(?)

IL-22

Tr1

FOXP3

TGFb

Tregs

FOXP3

TGFb

IL-5, IL-10, IL-13, TGFb Regulatory function on B cells by suppression of allergen-specific IgE and induction of IgG4 and IgA IL-10

Limiting airway inflammation in mice. Neg. association with Th2 cytokines

Diminished function and number in allergic individuals

First of all, whereas Th2 cytokines have a clear role in initiating an allergic response, espe-

cially Th1 cells have been implicated in more chronic severe allergic disease, both in atopic dermatitis and allergic asthma(30-32).

Secondly, in addition to the Th2 cytokines IL-4, IL-5, and IL-13, recently also IL-9 was shown to play an important role in asthma. At first IL-9 was discussed as a new Th2 cytokine. It

was shown that Th9 cells are dependent on both IL-4 and transforming growth factor-beta (TGF-Î˛) for their development. Interestingly, like Th2 cells, also Th9 cells are regulated by IL-25, predominantly seen in lung inflammation(33-36). IL-25 (like IL-33 and thymic stromal

lymphopoietin) is a type II initiating cytokine(37). In addition to the role of IL-25 in lung inflam-

mation, it is also of importance in atopic dermatitis and food allergy(38;39). Although IL-25 (also known as IL-17E) is a cytokine from the IL-17 family it has opposite effects of IL17A (IL-17).

Thirdly, Th17 cells (named after their main effector cytokine; IL-17) like Th1 cells may be associated with more severe asthma and mediate a more neutrophilic pattern of inflammation. 36

HSP: Bystander Antigen in Atopic Diseases?

Fourthly, it was recently shown that IL-22 (also produced by Th17 cells), is produced by a dis-

tinct set of CD4+ cells, named Th22-cells. These cells have been implicated as being protective

in a mouse model of allergic lung inflammation(40) and may play a role in the severity of atopic

dermatitis in humans(41).

Finally, a lot of attention has recently focused on a subset of T cells with the capacity to suppress other T cells, namely regulatory T cells (Treg). Two types of regulatory T cells will be

discussed below, namely IL-10 producing regulatory cells and FOXP3 expressing regulatory cells.

Regulatory T Cells Here we will discuss two types of regulatory T cells; FOXP3 expressing Tregs and IL-10 pro-

ducing regulatory T cells. However, it has to be emphasized that the distinction between these two types is not absolute, as for example FOXP3 expressing Tregs are also capable of producing IL-10. Moreover, many different regulatory T cells, may even act in concert in a single immune response.

IL-10 producing regulatory T cells As discussed above, exposure to an allergen leads to the development of sensitization in a susceptible individual. Upon a next encounter, a typical Th2 reaction will follow, including a

humoral (IgE and IgG4) response. In contrast, when healthy (non-allergic) individuals are ex-

posed to an allergen it was long thought that (in line with the Th1/Th2 dichotomy) instead of

a Th2 response a Th1 response would develop. However it has become clear that, in fact, this

is not the only difference between a normal and an allergic response to an allergen. Though indeed some IFN-Îł responses to allergens are present in healthy individuals, a study by Akdis

et al.(42) showed that this is a simplification of the reality. They isolated CD4 T cells specific to several food- and aeroallergens from healthy and allergic individuals according to their IL-4, IFN-Îł, and IL-10 secretion profile. Interestingly, allergen-specific T cells that belong to all three secretion profiles were detectable in both healthy and allergic individuals, with aller-

gen-specific IL-10-secreting T cells being the predominant subset in healthy individuals. They regarded these IL-10 producing CD4+ T cells as T regulatory cell type 1 (Tr1 cells). Seemingly,

low Tr1 numbers and high Th2 cell numbers resulted in an allergic response, whereas in healthy individuals a mixed Th1/Th2 response is associated with a strong IL-10 response(43).

The crucial role for IL-10 in allergy is also suggested by other studies on food and inhalant allergies. In cowâ&#x20AC;&#x2122;s milk allergy, T-cell clones derived from children that are persistently allergic

produced Th2 cytokines (IL-4, IL-13), whereas allergic control subjects that are cow milk tol-

erant produced a mixed Th1/Th2 response associated with markedly elevated IL-10 levels(44).

Similar observations of elevated IL-10 levels associated with less allergy symptoms were made in inhalation allergies. Children raised in a house with a cat are less likely to become al-

lergic to cat allergen than those who only get indirect exposure. Many of these highly exposed 37

Chapter 2

children had an IgG and IgG4 response to the major cat allergen Fel d 1 without production of specific IgE. This induction of high levels of allergen-specific IgG4 in the relative absence of IgE has been referred to as a modified Th2 response(45). Interestingly also in the peripher-

al blood of these non-allergic children, T cell response to the allergens are characterized by

an elevated level of IL-10. Comparable indications for a role for IL-10-producing Tr1 cells in the maintenance of tolerance to allergens can be found in individuals exposed to relatively

high doses of allergen like bee keepers and allergic patients undergoing immunotherapy. Bee

keepers with a repeated exposure to bee venom during the bee-keeping season demonstrate a marked increase in allergen-specific IL-10 secretion from peripheral blood T cells as the season progresses, while allergen-specific IgE is seen especially in the beginning of the sea-

son. Interestingly, reactions to stings disappear during the season, simultaneously with the increased IL-10 production by T-cells(46). Apart from IL-10, also TGF-Î˛, another cytokine that

can be produced by Tr1 cells, is reported to be induced by SIT, while both IL-10 and TGF-Î˛ are associated respectively with the blocking antibodies IgG4 and IgA(23;47).

FOXP3 regulatory T cells

Not only IL-10 producing T cells but also FOXP3+ Tregs are associated with an allergic re-

sponse. The important role of the transcription factor FOXP3 for maintaining immune tolerance stems for multiple basic studies, mainly in experimental models. This importance

of FOXP3+ Tregs in human disease was underscored by the IPEX syndrome (immunod-

ysregulation, polyendocrinopathy, and enteropathy, X-linked). In IPEX patients a genetic

mutation causes the FOXP3 transcription factor to be defective. Patients with IPEX have

symptoms that fit both generalized autoimmunity and allergy(48;49). The relevance of FOX

P3 Tregs has been extensively demonstrated in mice models, showing an inverse correlation to Tregs and diseases like RA, inflammatory bowel disease, MS, and IDDM(50-52). Not

only in autoimmune diseases but also in allergic disease various studies have suggested a possible defective suppressive function of Tregs(53;54). In addition to the bee keeper model

also the season-dependent antigen exposure during the pollen season offers a model to study responses in the same individuals with and without exposure. Whereas in several studies it was shown that both non-allergic as allergic individuals have CD4+CD25+

Tregs, in the allergic subjects, these cells still produced IL-5 and IL-13 cytokines(55;56).

Further related experiments demonstrated that both dose and type of allergen appear to

have effects on the ability of CD4+CD25+ T cells to suppress responses. At the time of these

studies CD25bÂ expression on CD4 T cells was used as a surrogate marker of FOXP3+ Tregs.

Later studies using FOXP3 as a direct marker seemed to confirm these data. During venom immunotherapy the increased allergen-specific IgG4 and reduced IgE correlated with circulating FOXP3 positive Tregs(57). A recent study even suggested that Tregs function

may be impaired in patients with allergic diseases and that this function can be enhanced by specific immunotherapy(58).

HSP: Bystander Antigen in Atopic Diseases?

Thus, altogether, there are ample suggestions that the presence of allergen-specific T cells

with a regulatory phenotype (either expressing FOXP3, and/or capable of producing IL-10 and TGF-β) may have a beneficial effect on allergic diseases. However in some atopic diseases, like atopic dermatitis and asthma, the specific trigger is often not known. This raises the

question whether methods can be identified to improve the Tregs repertoire, when the trig-

gering allergen is unknown. Studies from other inflammatory diseases, like JIA and RA, have suggested that a certain group of antigens, called HSP, are present at the site of inflammation and may have such a beneficial effect on the T cell repertoire. Heat Shock Proteins

The Tregs repertoire is both formed in the thymus (so-called natural Tregs) and generated

in the periphery upon encounter with an antigen (induced or adaptive Tregs). Both self and

non-self antigens can induce Tregs in the periphery, although the repertoire of Tregs might be biased toward self antigens(59).

In 1991, Cohen and Young proposed that a select group of self antigens is especially important

for the maintenance of peripheral tolerance(60). He described the presence of auto reactive im-

mune responses in a healthy individual to a limited set of self molecules, formed by auto reac-

tive T cells and antibodies, which he called the immunological homunculus. The self antigens of this “homunculus” are all evolutionary highly conserved between the self and the non-self

homolog of these proteins. According to Cohen, the immune system utilizes these self anti-

gens to form an immunological picture of self which is crucial for the balance of the immune system. One of these homunculus’ self proteins is human HSP60(2). HSP are indeed evolution-

arily highly conserved proteins and either present constitutively, functioning as chaperones,

or induced upon cell stress caused by, for instance, heat, oxidative stress, and hypoxia(61). Sev-

eral HSP have been identified and, according to their size, organized into six families: HSP100,

HSP90, HSP70, HSP60, HSP40, and HSP10. In this review we focus on the immune responses of HSP60 in atopic disease. It has to be stressed that as only very little data are available right now on HSP and allergic disease, and thus we need to deduce the role of HSP in atopic disorders mostly on what is known about HSP reactivity on other diseases. Immunity to HSP and inflammation

Humoral and cellular immune responses to HSP60 have been detected both in patients with

an inflammatory disease, such as autoimmune and allergic diseases, as well as in healthy subjects. After these initial discoveries the perception was that HSP’s may be involved in the

development of autoimmunity through antigenis mimicry: an immune response toward a microbial HSP could lead to a cross-reactive response to a self-HSP and thus cause autoimmun-

ity. However, after further studies it quickly became clear that the immune responses toward self HSP60 were more involved in the regulation and not in the induction of autoimmunity.

Indeed, a wealth of data obtained in the last decade both in experimental models and from observations in human diseases point to a regulatory role of immunity to self HSP60(2).

Chapter 2

The presence of self-HSP-reactive cells was first shown in animal studies by immunization of rats

with mammalian HSP60(62). Though immunization could induce self-HSP-specific antibodies and T

cells, a self-HSP-reactive immune repertoire was also shown to be present in the absence of immunization. The cause for this could be previous contact with homologous bacterial HSP, for example

in the mucosa of the gastro intestinal tract. The first report that immune responses to self HSP60 may have a regulatory role in inflammatory diseases was in mycobacteria-induced adjuvant ar-

thritis. In this model the induction of a self HSP60, cross-reactive T cells response was responsible

for the observed protection of HSP60 peptide immune therapy. After these initial findings, protective effects of various (conserved) microbial HSP were seen in many other experimental disease models, including arthritis, atherosclerosis, allergic encephalomyelitis, and allergic asthma(63-66).

In vitro experiments show that immune responses modulated by HSP can result in the induc-

tion of various cytokines like IFNγ and TNFα, as well as IL-10. HSP are strong immune mod-

ulators and are able to influence the impact and direction of immune responses(67). Moreover,

HSP60 has the capacity to steer both innate and humoral immunity.

In human diseases, antibody responses to HSP60 were previously described in skin lesions of patients with Behçets disease, whereas HSP60 has been described as a target for T-cells in au-

toimmune diseases such as IDDM and JIA(8;68;69). Data in the experimental models have pointed out that the protective effect of HSP60 was independent of an antigenic relationship (antigen

mimicry) between HSP60 and the disease-inducing antigen. Instead it seemed that HSP60 could confer protection through so-called bystander suppression(70;71). Inflammation causes

local damage and cell stress leading to upregulation of stress proteins such as HSP60. Next,

these bystander (self) antigens can become the target of subsequent, possibly suppressive immune responses. So, it is possible that immune responses to HSP could be involved in the control of human chronic inflammatory diseases that have distinct, although as-yet-unknown, initiating auto-antigens, or even allergens. HSP responses in early life

Apparently, responses to HSP60 are important for maintaining peripheral tolerance in the adult immune system. It is unknown when during live these specific T cells arise. It would

seem reasonable to expect that they are primed after birth upon encounter with homologous microbial HSP in the gut. However, there are indications that self HSP60 reactivity is present

at birth. In a study by Ramage et al. it was shown that cord blood cells proliferate in response to an in vitro challenge with the self HSP60(72). This supported the hypothesis that this reactivity is part of the normal naive immune repertoire. A more recent study by Merbl et al. put

these findings also in a different perspective(73). They found that normal cord blood contains

IgM and IgA auto-antibodies directed against a relatively uniform set of auto-antigens, such as auto-antigens related to immune regulation such as HSP60. This obviously benign autoim-

mune self reactivity, present at birth, may have a dual function. On the one hand it may provide 40

the basis for autoimmunity in later life, while on the other hand the “inborn” autoimmunity

HSP: Bystander Antigen in Atopic Diseases?

to regulatory self antigens such as HSP60 may actually serve to protect against autoimmune

disease. Intrigued by these studies, we questioned whether CD4+ T cells specific for HSP60 are already detectable at birth before exposure to the microbial flora, and if so, to what type

of immune response these “inborn” autoreactive CD4+ T cells have upon exposure to the self

antigen HSP60. We found that HSP60 specific T cells are indeed present at birth. Moreover,

stimulation of CBMC with HSP60 leads to CD4+ T cell proliferation and cytokine production,

and induces T cells with an in vitro regulatory phenotype that are functionally suppressive(74). HSP60 and allergy

Thus, HSP60 is a self-protein, recognized already at birth in healthy subjects, but also in individuals with chronic inflammatory diseases. Thereby a correlation between self-HSP reactivity and diminished disease activity is seen both in experimental as in human autoimmunity.

This has already led to the successful exploration of HSP60 immune therapy in human dis-

ease, firstly in IDDM. It is not known yet if the levels and responses to HSP60 are different in individuals developing chronic inflammatory disease later in life.

For once, priming of HSP60 T cell responsiveness takes places primarily in the gut through contact with microbial HSP60(2). Thus, priming of HSP60 immunity early in life through con-

tact with microbiota leads to more self HSP60 mediated immune regulation. This could be one of possibly multiple mechanisms that explain why individuals exposed to more microbial

triggers early in life, could be less prone to develop immune mediated diseases. Thus it may very well fit the concept of the hygiene hypothesis.

For that reason we set out to study the role of HSP60 immunity in human atopic diseases. Obviously the first pre-requisite for a potential role of self HSP60 in dermatitis is the (pref-

erably increased) expression of HSP60 at the site of inflammation. Indeed we were able to show that self HSP60 is increased expressed in the skin of patients with atopic dermatitis(75).

Moreover, in vitro stimulation with self HSP60 induced FOXP3 positive T cells, as well as T cells producing IL-10 and IFN-γ.

It has to be emphasized that this increased expression by no means is very specific for atopic diseases(76). Still it could be highly relevant for atopic disease as the characterization of an

antigen present at the site of inflammation, without being the disease-causing antigen has

therapeutic possibilities through, as previously mentioned, “antigen driven bystander suppression.” Bystander suppression as a mechanism is especially important in human autoimmune diseases, because often the immunizing trigger is unknown.

Theoretically, in allergic diseases this could also be an interesting option for intervention.

Although often at least one of the allergens triggering the disease is known, mono-sensitiza-

tion is rare which would imply multi-antigen immune therapy. Immune therapy with a single bystander antigen might undermine this issue.

Chapter 2

Novel Immune Therapeutic Possibilities in Allergic Disease Various novel immune therapeutic approaches to diminish allergic symptoms have been or are being studied. Of these new interventions, blocking of effector cytokines (such as IL-4

and IL-5) and IgE is the most important. As IL-5 is important for the priming and survival of

eosinophils and these cells play an important role in the pathophysiology of allergic disease, blocking IL-5 seemed logical. However only a highly selected patient group with severe asth-

ma, shown as sputum eosinophilia, had profit from this therapy(77). Also blocking IL-4 and IL-

13 has not yet been shown to be of clinical benefit in asthma(78). Thus, so far, the experiences with blocking antibodies to effector cytokines suggest that either the cytokines cannot be fully blocked or that not one of these cytokines is solely responsible for the allergic response.

As mentioned above in recent years Th2 induction cytokines have been described among

which IL-25. IL-25 is strongly elevated in a subgroup of peanut allergic subjects(38). Moreover,

in mouse models blocking IL-25 has a positive effect on bronchial hyper-reactivity(79). Apart

from blocking cytokines that are important in the pathophysiology of allergic disease, in the last decade various studies in asthmatic patients have been performed in which an IgE-blocker was used(80). Although the first results look promising, the effect is not present in all patients, underlining again the complexity of chronic allergic diseases.

Antigen SIT, in which an allergic individual is exposed to increasing doses of the allergen trig-

gering disease, has been used for over a century. It is one of the earliest and most effective forms of human immune therapy. Although for long the mechanisms behind this clearly ef-

fective immune intervention were not fully understood, we now know that IgG4, IL-10, and

Tregs probably play an important role. A disadvantage of this approach in which the eliciting

protein is used, is the chance of a severe anaphylactic reaction, as has been described following immune therapy in peanut allergic subjects(81).

To tackle this issue, recent work has focused on allergy peptide therapy, showing good results. Mice studies showed that after immunization with Der p 2, the house dust mite allergen,

downregulation of T cell and antibody responses was seen to Der p 2. Similar results were seen using Fel d 1 (cat allergen) and Bet v 1 (birch allergen) peptide therapy. As most patients

are not sensitized to just one allergen, it is now studied if combination immune therapy is also effective. Another approach is the use of a peptide of an antigen present at the site of inflam-

mation, without being the triggering antigen. This is the concept of the previous mentioned antigen bystander suppression model(70;71).

Based on the development of immune therapies using HSP60 for other human inflammatory diseases and encouraged by recent data showing that HSP60 expression is increased in the skin in patients with atopic dermatitis, it is tempting to suggest that HSP60 may also be a potential candidate for bystander therapy in allergic diseases.

HSP: Bystander Antigen in Atopic Diseases?

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Chapter 2

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Busse WW, Ring J, Huss-Marp J, Kahn JE. A review of treatment with mepolizumab, an anti-IL-5 mAb, in hypereosinophilic syndromes and asthma. J Allergy Clin Immunol 2010; 125(4):80313. Oh CK, Geba GP, Molfino N. Investigational therapeutics targeting the IL-4/IL-13/STAT-6 pathway for the treatment of asthma. Eur Respir Rev 2010; 19(115):46-54.

Ballantyne SJ, Barlow JL, Jolin HE, Nath P, Williams AS, Chung KF et al. Blocking IL-25 prevents airway hyperresponsiveness in allergic asthma. J Allergy Clin Immunol 2007; 120(6):1324-31. Rodrigo GJ, Neffen H, Castro-Rodriguez JA. Efficacy and safety of subcutaneous omalizumab vs placebo as add-on therapy to corticosteroids for children and adults with asthma: a systematic review. Chest 2011; 139(1):28-35. Reid MJ, Lockey RF, Turkeltaub PC, Platts-Mills TA. Survey of fatalities from skin testing and immunotherapy 1985-1989. J Allergy Clin Immunol 1993; 92(1 Pt 1):6-15.

HSP: Bystander Antigen in Atopic Diseases?

Chapter 3

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)

Joost A Aalberse; Berber Kapitein; Sytze de Roock; Mark R Klein; Wilco de Jager; Ruurd van der Zee; Maarten O Hoekstra; Femke van Wijk; and Berent J Prakken Plos One 2011; 6(9):e21119

Chapter 3

Abstract To prevent harmful autoimmunity most immune responses to self proteins are controlled by central and peripheral tolerance. T cells specific for a limited set of self-proteins such as human heat shock protein 60 (HSP60) may contribute to peripheral tolerance. It is not known

whether HSP60-specific T cells are present at birth and thus may play a role in neonatal tol-

erance. We studied whether self-HSP60 reactive T cells are present in cord blood, and if so, what phenotype these cells have.

Cord blood mononuclear cells (CBMC) of healthy, full term neonates (nâ&#x20AC;&#x160;=â&#x20AC;&#x160;21), were cultured with HSP60 and Tetanus Toxoid (TT) to study antigen specific proliferation, cytokine secretion and up-regulation of surface markers. The functional capacity of HSP60-induced T cells

was determined with in vitro suppression assays. Stimulation of CBMC with HSP60 led to CD4+ T cell proliferation and the production of various cytokines, most notably IL-10, Inter-

feron-gamma, and IL-6. HSP60-induced T cells expressed FOXP3 and suppressed effector T cell responses in vitro.

Self-reactive HSP60 specific T cells are already present at birth. Upon stimulation with self-

HSP60 these cells proliferate, produce cytokines and express FOXP3. These cells function as

suppressor cells in vitro and thus they may be involved in the regulation of neonatal immune responses.

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)

Introduction The immune system possesses a range of safekeeping mechanisms that prevent the induction of a deleterious response to self-antigens. First through negative thymic selection (central

tolerance), by which T cells expressing a T cell receptor with a high affinity for self proteins are eliminated(1). Secondly, self reactive T cells that do escape central tolerance and develop

into mature T-cells are kept under control through peripheral tolerance(2). One of the most

prominent mechanisms of peripheral tolerance is mediated by regulatory T cells. Regulatory T cells (Tregs) suppress effector T cell responses in vitro and can down regulate an ongoing immune response in vivo(3). They appear essential for the homeostasis of the immune system

as deficiencies in Tregs numbers and/or function lead to inflammatory diseases both in mice

and in humans(4-6) and are already present at the start of life(7;8). Tregs are either directly de-

rived from the thymus or generated in the periphery. Although the antigen specificity of Tregs is still largely unknown, it is assumed that they can act through recognition of self-antigens in the periphery(9). This fits the premise that the immune response to certain self-antigens

is important for the regulation of immune responses(10). Cohen was the first to propose that

the response to a vested group of self-antigens is important for maintaining peripheral tol-

erance(11). He described the response against this limited set of self molecules in a healthy

individual, formed by auto reactive T cells and antibodies as the immunological homunculus. This â&#x20AC;&#x2DC;homunculusâ&#x20AC;&#x2122; of self-antigens shares various properties, among which evolutionary conservation between the self and the non-self homologue of these proteins. One of these

self proteins is human heat shock protein 60 (HSP60)(12). HSP60 is an evolutionary highly

conserved cellular protein. Also, it is a stress protein and acts as a danger signal for the immune system: the expression of HSP60 is strongly up regulated following cellular stress, e.g.

because of tissue damage during inflammation(13;14). HSP60 can activate both adaptive and innate immune responses(15;16) which underscores that HSP60 has the potential to be a regu-

lator in an inflammatory response. This is further supported by the observation that in both

animal and human in vitro studies HSP60-reactive T cells have a strong immune modulatory capacity(17-19). In experimental models these HSP60-specific T cells can effectively suppress in

vivo a variety of experimental autoimmune diseases. These findings have lead to the set up of several clinical trials using HSP peptides, e.g. in rheumatoid arthritis (RA) and diabetes type I, with promising results(20-22).

Thus, apparently, responses to HSP60 are important for maintaining peripheral tolerance in the adult immune system. It is unknown when during live these specific T cells arise. It would

seem reasonable to expect that they are primed after birth upon encounter with homologous microbial HSP in the gut(12). However, there are indications that self-HSP60 reactivity is present at birth. As early as 1991 it was shown that cord blood cells proliferate in response to an

in vitro challenge with the self antigen HSP60(23;24) and hypothesized that this reactivity is

part of the normal naĂŻve immune repertoire. A more recent study by Merbl et al(25) put these 53

Chapter 3

findings in a different perspective. They found that normal cord blood contains IgM and IgA

autoantibodies directed against a relatively uniform set of autoantigens, such as autoantigens that are associated with autoimmune disease (such as double stranded DNA) and autoantigens related to immune regulation such as HSP60. The authors of this study suggested that

this obviously benign autoimmune self reactivity, present already at birth, may have a dual

function. On the one hand it may provide the basis for autoimmunity in later life, while on the other hand the “inborn” autoimmunity to regulatory self antigens such as HSP60 may actu-

ally serve to protect against autoimmune disease. Intrigued by these studies, we questioned whether CD4+ T cells specific for HSP60 are already detectable at birth before exposure to the

microbial flora, and if so, to what type of immune response these “inborn” autoreactive CD4+ T cells have upon exposure to the self antigen HSP60. We found that HSP60 specific T cells are

indeed present at birth. Moreover, stimulation of CBMC with HSP60 leads to CD4+ T cell pro-

liferation and cytokine production, and induces T cells with an in vitro regulatory phenotype that are functionally suppressive.

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)

Results HSP60 induces CD4+T cell proliferation in cord blood To detect possible HSP60-specific T cells in cord blood, we first set out to study whether in vitro activation of CBMC with (human) HSP60 induces T cell proliferation. First, using thymidine

incorporation we found that five of ten (50%) cord blood samples showed a clear prolifera-

tive response to HSP60 (median Stimulation Index (SI): 2.0; Inter Quartile Range (IQR): 2.5). Though we used a very pure batch of human HSP60 for these experiments with undetectable

LPS levels (see methods) it is still conceivable that contamination of extremely low levels may have influenced these assays. We therefore wanted to confirm these preliminary proliferation data using two peptide epitopes from HSP60. We have shown before that these (panDR bind-

ing-) epitopes can induce T cell proliferation in a majority of patients with JIA and RA(25). Sixty

percent of the cord blood samples (three of five) responded to either one or both peptides tested (SI > 1.7). These responses were also significantly different (p=0.038) compared to the

proliferative response of TT, which was used as a negative control, assuming that the fetus has not formed a prior response to TT. No significant proliferation was seen in response to tetanus toxoid (TT) compared to background. (Figure 1a).

Figure 1 | Human HSP60 (HSP) induces cell proliferation in cord blood A) Protein-induced proliferation of cord blood mononuclear cells (CBMC). CBMC were cultured for 96 hours in the presence of Human HSP60, both the full protein as well as HSP60-derived peptide and Tetanus Toxoid (TT). Box whisker plots are shown and significant differences in responses are indicated with * (p < 0.05). Background (mean +/- SD): = 1395 +/- 1446 CPM. Data (n=10) were obtained from 4 independent experiments. B) Protein-induced proliferation of cord blood mononuclear cells (CBMC) cultured in the presence of CFSE. CBMC were cultured for 144 hours and afterwards stained for CD4. HSP60 proliferation is represented in lighter grey. Percentage indicates number of divided CD4+ T cells. Data are representative for 3 independent experiments. C) Protein-induced proliferation of cord blood mononuclear cells (CBMC) cultured in the presence of CFSE. Box whisker plots are shown. Data (n=3) are obtained from 3 independent experiments.

Chapter 3

Though this assay showed T cell proliferation to HSP60 we next wanted to confirm this with CFSE staining, which allowed us to determine which cells were dividing upon HSP60 stimulation. We found that indeed CD4+ T cells proliferated specifically after HSP60 stimulation in all samples (Figure 1b and 1c). Thus, HSP60 induced proliferation of cord blood derived CD4+ T cells.

To identify if these responses of CBMC and CD4 T cells could be the result of HSP60 acting as

a ligand for TLR2 or TLR4, as previously suggested, we also performed these proliferation

assays after incubation with a TLR2 and/or a TLR4 blocking antibody, or IgG isotype control (Supplemental figure 1). No differences in HSP60 induced proliferation were found in the presence of TLR blocking antibodies suggesting that the observed proliferation of CBMC in response to HSP60 is TLR2 and TLR4 independent.

Cytokine production in cord blood stimulated cells We next wanted to know whether stimulation with HSP60 also led to cytokine production by CBMC. Therefore, cytokine levels in culture supernatants after 96 hours of stimulation

with HSP60, TT or medium alone were measured with a multiplex immune assay. We found

that, compared to stimulation with medium, HSP60-induced production of IL-10 (p = 0.009), IL-5 (p=0,013) IL-6 (p=0.003), IL-13 (p=0.003), TNF-alpha (TNFα) (p=0.003) and IFN-gam-

ma (IFNγ) (p=0.005) were significantly increased. TT induced a significant increase of IL-6 (p=0.045) compared to stimulation with medium (Figure 2a).

To establish which cells are producing IL-10 and IFNγ, we next measured intracellular cytokine

production by FACS, again after stimulation in vitro with medium, HSP60 and TT. We found an

increased intracellular expression of mainly IL-10, and also IFNγ, by CD4+ T cells (Figure 2b and 2c), after stimulation with HSP60 only (and not with TT). Thus, stimulation of CBMC with HSP60 leads to proliferation and cytokine production (IL-10 and IFNγ) of CD4+ T cells. HSP60 -specific induction of regulatory T cells

There are indications that HSP60, and/or peptides derived from HSP60 may induce or enhance the regulatory T cell population. We therefore wanted to know whether HSP60 may also play a role in T cell regulation at this crucial period at, or even before birth. We therefore

stimulated cells again with TT, HSP60 or medium and next stained the cells for expression of CD4, CD30, CD45RO (memory T cells) and FOXP3. FOXP3 is a transcription factor that is char-

acteristic for Tregs. CD30 is an activation marker that has been suggested to be a marker for

Th2 cells, whereas we recently found that CD30 is upregulated on antigen-induced Tregs(26). As can be seen in figure 3a, following stimulation with HSP60 we found a significant increase

of CD4+CD30+ and CD4+CD45RO+ as well as an increase of CD4+FOXP3+cells, which is not seen

after stimulation with control antigen TT. The FOXP3+ expression was found in about 50% of the CD30+ and 25% of the memory T cells and not in CD45RO- T cells (Figure 3b and 3c).

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)

HSP60 induced regulatory T cells are functionally suppressive We next questioned whether these induced Tregs were functional and thus have in vitro sup-

pressive capacity. Therefore we performed CFSE dilution - suppression assays, in which we added HSP60 induced Tregs (CD4+CD25+CD127- T cells) and CFSE-labeled effector cells in dif-

ferent ratios. The HSP60-induced Tregs were able to functionally suppress the effector T cells.

Compared to the proliferating CD4+ T cells we saw a decreasing percentage of proliferating

cells (figure 4a). After adding HSP60-induced Tregs to the effector T cells in Treg:Teff ratios

of 1:2; and 1:1 the percentage of suppression of proliferating CD4+ T cells increased from respectively 41.3 to 56.6%. This indicates that HSP60 induced CD4+CD25+CD127- T cells are

functional suppressor cells in vitro.

Figure 2 | Cytokine production induced by human HSP60 (HSP) in cord blood cells A | Cells were cultured with human HSP60 or Tetanus Toxoid (TT) for 4 days. Cytokine levels in supernatant of the cultures were measured using a multiplex immunoassay. Levels of cytokines are presented in a histogram plot. HSP stimulated cells in white, and TT stimulated cells in light grey are compared to only medium cultured cells in black. The supernatants (n=7) were obtained in 3 independent experiments. Cytokine production was measured in one assay. B | Percentage IL-10+ and IFNÎł+ CD4+ T cells following stimulation with human HSP60. Box whisker plots are shown and significant differences in responses are indicated with * (p < 0.05). Data (n=4) were obtained from 2 independent experiments. C) A representative FACS-analysis of two independent experiments (n=4) showing intracellular IFNÎł and IL-10 staining of CD4+ T cells following stimulation with human HSP60. 57

Chapter 3

Figure 3 | Human HSP60 (HSP) induces CD30 and the expression of FOXP3 in cord blood CD4+ T cells after stimulation for 6 days. A FACS analysis shows a significant higher level of CD4+CD45RA+, CD4+CD45RO+ and CD4+CD30+ T cells as well as CD4+FOXP3+ T cells following stimulation with HSP60. Box-Whisker plots are shown and significant differences in responses are indicated as * (p < 0.05). Data (n=7) were obtained from 3 independent experiments. B FACS analysis of FOXP3 expression within the CD4+CD30+ and CD4+CD45RO+ populations following HSP60 stimulation. Data (n=7) were obtained from 3 independent experiments. C) FACS analysis representing data (n=7) from 3 independent experiments of CD4+ T cells expressing FOXP3 after stimulation with human HSP60, showing that most FOXP3 positive CD4 T cells also express CD45RO and half of them express CD30.

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)

Figure 4 | Human HSP60 (HSP)-induced CD4+CD25+ cord blood cells suppress proliferation of T effector cells Different amounts (0, 10.000 or 20.000) of sorted HSP60-induced regulatory T cells were cultured with 20.000 CFSE labeled effector T cells for 5 days in the presence of plate-bound anti-CD3. A | A CFSE dilution profile of effector T cells alone (open histogram) or in the presence of HSP60-induced Tregs at a ratio of 1:1 (closed histogram). This figure represents data (n=3) from 2 independent experiments. B | Percentages suppression measured by CFSE dilution. Data (n=3) were obtained from 2 independent experiments.

Chapter 3

Discussion With this study we show that in cord blood HSP60 induces CD4+ T cells that proliferate and

produce various cytokines, most notably IL-10 and IFNγ. Moreover such HSP60-induced T

cells express CD30, CD45RO and FOXP3, and act as suppressor T cells in vitro. These findings underscore the universality and thus presumed importance of the “inborn” recognition of self proteins, like HSP60, early in life.

A healthy immune system is able to initiate as well as regulate inflammation, while maintaining

tolerance for self-antigens. Immune responses to certain self proteins are hypothesized to be important for a healthy immune system(27). These “immunological homunculus” proteins, among

which stress proteins like HSP60, may serve as immune biomarkers to the body educating the immune system on the state of present inflammation. Our results would fit this hypothesis, as it suggests that an immune response to self HSP60 is part of an “inborn” mechanism present

already at birth that may help to regulate inflammation. It can be speculated that human HSP60

could be regarded as a recall antigen in the fetus, as self proteins are probably continuously

present in the developing fetus due to apoptosis of developing tissues. This may contribute to induction of a (tolerogenic) response to self proteins like HSP60 before birth and may explain why HSP60 in cord blood elicits such evident responses as seen in our study.

Interestingly, HSP60 has previously been identified as a potent regulator of inflammation in the adult

immune system. In the end of the 80’s human HSP60 was found to be recognized by the immune system during inflammation. It was then shown that HSP60-specific T cells can dampen experimental

autoimmune disorders, while in humans HSP60 is recognized by healthy individuals free of autoimmune disease or infection(28). The capacity of HSP60 to induce both innate and adaptive immunity

indicates that HSP60 has a double function: responsiveness to HSP60 can be related to inflammation, but also to a healthy immune system. Next it was shown in human studies that HSP60-spe-

cific immune responses in patients with juvenile arthritis (JIA) and RA are associated with a better prognosis and milder symptoms, again suggesting a role in regulating inflammation (29). In line with

these findings we now show that HSP60-specific T cells are present at birth which underscores the hypothesis that auto-reactive T cells are a principal feature of a healthy immune system.

In addition to the finding that in cord blood HSP60 can induce FOXP3+ regulatory T cells, that

are functionally suppressive, we have shown that HSP60 leads to the induction of IL-10 and IFNγ, suggesting the upregulation of Tr1 cells. We have also shown that HSP60 leads to the in-

duction of Th2 cytokines (IL-5 and IL-13) confirming that an immune response in cord blood

is primarily a Th2 response. Another interesting finding is the huge increase of IL-6, as well as an increase in TNFα. IL-6 has been reported as a Th2 inducer and an important anti-inflam-

matory cytokine, aiding in the protection of the fetus in utero(30). Both TNFα and IL-6 however

have also been shown to be upregulated by TLR agonists in cord blood. Our experiments 60

with HSP60 peptides and our experiments in which we block TLR2 and TLR4 indicate that

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)

stimulating with HSP60 induces an antigen specific response. In conclusion, functional HSP60

specific CD4+ cells are already present at birth before priming of the immune cells with the microbial flora in the gut. This mechanism may be important for the maintenance of prenatal and neonatal immune tolerance.

Materials and Methods Collection of cord blood Cord blood samples (n=21) were collected from healthy subjects at the department of gynecology and obstetrics. Inclusion criteria were an uncomplicated pregnancy, full-term infant

and normal vaginal delivery. No further information about the mother, pregnancy or the de-

livery was obtained. Written informed consent was obtained. This study was approved by the Medical Ethics Committee, UMC Utrecht (NL16498.041.07). Isolation of CBMC

Cord blood mononuclear cells (CBMC) were isolated from heparinized venous blood by Ficoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation. The cells were then frozen in liquid nitrogen and thawed prior the experiment. The culture was performed in RPMI-

1640 supplemented with 2mM glutamine, 100u/ml of penicillin/ streptomycin (Gibco BRL,

Gaithersburg, MD) and 10% (v/v) ABpos heat-inactivated (60 min at 56° Celsius) human serum (Sanquin, Amsterdam, the Netherlands). For measurement of the proliferative activity,

2x105 cells in 200 ml per well were cultured in triplicate in round bottomed 96-wells plates (Nunc, Roskilde, Denmark) for 96 hours at 37° Celsius in 5% CO2 with 100% relative humidity. Cells were cultured in the absence or presence of 10 mg/ ml human HSP60, both the full

protein as two peptides p2 (human HSP60 peptide 280-292) and p4 (human HSP60 peptide

242-256)(20). The highest response to either peptide was scored. The LPS-content of HSP60 was <0.1 EU /mg protein. Tetanus-toxoid (TT; 1.5 mg/ ml, RIVM, Bilthoven, the Netherlands)

was used as a negative control, assuming that the fetus has not formed a prior response to TT.

For the final 16 hours of culture, 1mCi/ well [3H] thymidine (ICN Biomedicals, Amsterdam, the Netherlands) was added to each well. Cells were harvested according to standard procedures and incorporated radioactivity was measured by a liquid scintillation counter and ex-

pressed as counts per minute (cpm). The magnitude of the proliferative response is expressed as stimulation index, which is the mean cpm of cells cultured with antigen divided by the mean cpm of cells cultured without the antigen. TLR-blocking assay.

Isolated CBMC were cultured for 96 hours in the presence or absence of 10 µg/ml HSP60 and

in the presence or absence of anti TLR4 blocking antibody (2.5 µg/ml, clone HTA125, Serotech), anti-TLR2 blocking antibody (2.5 µg/ml, clone TL2.1, Serotech), both, or IgG2a isotype control (2.5 µg/ml, Serotech). Proliferation was measured as described above.

Chapter 3

CFSE - assay To characterize which cells proliferate to HSP60, CFSE-assays were performed. For this pur-

pose 3 mM CFSE was added to 1x106 CBMC for ten minutes and washed twice with 10% FCS.

CBMC were resuspended in human AB 20% and stimulated with HSP60 for 6 days. Cells were then stained with anti-CD3, -CD4 and -CD14 and analyzed by FACS Calibur (Becton Dickinson Biosciences, San Jose, CA, USA), as described below. Multiplexed particle-based Immuno assay

Cytokine levels were measured after activation of the lymphocytes in vitro as described above.

After 96 hours the supernatants of the cell cultures were stored at -80°C. Cytokines levels of

IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IFNg and TNFa were measured with the Bio-Plex system and analysed with the Bio-Plex Manager software version 4.0 (Bio-rad Laboratories, Hercules

CA, USA) which uses the Luminex xMap technology(31). The cytokine production is presented in a color profile to visualise the different cytokines produced. FACS staining

CBMC were stimulated for 6 days with HSP60 and TT and then washed twice in PBS con-

taining 2% FCS (PBS-FCS) and blocked with the appropriate normal serum (30 min at 4°C). Subsequently, the cells were incubated in 50 µl of FACS buffer containing four appropriately diluted PE-, FITC-, PerCP-, or allophycocyanin-labeled mAbs against human CD4 (clone

SK3) (Becton Dickinson Biosciences), CD30 (clone BER-H8) (Becton Dickinson Biosciences), CD45RA (clone JS-83) (eBioscience, San Diego, CA, USA), CD45RO (clone 4CHL-1) (eBioscience, San Diego, CA, USA) and FOXP3 (PCH101) (eBioscience, San Diego, CA, USA). Stained

mononuclear cells were diluted in FACS fluid and run on a FACS Calibur (Becton Dickinson

Biosciences). CellQuest software (Becton Dickinson Biosciences) was used for analysis. Cytokine analysis by lymphocyte intracellular staining and Flowcytometry

CBMC were stimulated as described above. During the last 4 hours of culture, Golgistop (Becton

Dickinson Biosciences) was added (final concentration of 2 mM). The cells were harvested and stained for intracellular cytokine and FOXP3 analysis as described previously. For the staining, a

predetermined optimal concentration of PE-conjugated anti-IL-10 (clone JES3-19F1), FITC-conju-

gated anti-IFNg (clone 4S.B3) (Becton Dickinson Biosciences) and APC-conjugated FOXP3 (eBioscience, San Diego, CA, USA) was added to the cells. Cells were analyzed as described above. Suppression Assay

To examine the functionality of induced regulatory T cells, CBMC were thawed and CD25 positive cells were depleted. The CD25 negative cells were stimulated with HSP60 for 6 days. Ef-

fector T cells (thawed CBMC of the same donor) were stained with CFSE (3mM). With a FACS

sorter added different numbers of CD4+ CD25+CD127- (regulatory) T cells were added to the 20.000 effector T cells, depending on availability of cells. In every experiment we included at

least the Teff:Treg ratios 1:0, 2:1 and 1:1. The different ratios were incubated for 120 hours

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)

in the presence of plate-bound anti-CD3 (1mg/ml) and with the FACS diva the percentages of proliferation and suppression were measured. Statistical Analysis

Basic descriptive statistics were used to describe the patient population. A non-parametric test (Mann Whitney U test, 2 sided) and where appropriate a parametric test (T-test, 2 sid-

ed) was applied to determine significant differences between the stimuli and the controls regarding proliferative responses, cytokine production and expression of surface markers and

intracellular markers. A p-value < 0.05 was considered significant (SPSS Statistical Program, version 15.0; SPSS Inc, Chicago, Ill.)

Figure S1 | Human HSP60 (HSP60) induced cell proliferation of CBMC, with or without anti-TLR2 and/or anti-TLR4 blocking antibodies, or IgG isotype control. Shown are mean stimulation index (relative to medium conditions) +/- SEM. Data (n=6) are obtained from 2 independent experiments. NS= non-significant

Acknowledgements

We would like to thank Prof M. Heringa of the department of Obstetrics and Gynaecology for his contribution in collecting cord blood samples.

Chapter 3

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de Jager W, Prakken BJ, Bijlsma JW, Kuis W, Rijkers GT. Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies. J Immunol Methods 2005; 300(1-2):124-35.

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60)

Chapter 4

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

Berber Kapitein; Joost A. Aalberse; Mark R. Klein; Wilco de Jager; Maarten O. Hoekstra; Edward F. Knol; and Berent J. Prakken Cell Stress and Chaperones 2013;18:87-95

Chapter 4

Abstract Heat shock protein 60 (HSP60) is a highly conserved stress protein and target of self-reactive

T cells in various inflammatory diseases. Not much is known about a possible role in atopic disease. As atopic diseases are considered to be the result of a disturbance in the balance

between T helper cells type 2 and regulatory T cells, it is of interest to know whether HSP60 acts as a bystander antigen in atopic disease. Our aim was to investigate whether HSP60 is involved in the chronicity of inflammation of atopic dermatitis (AD). We studied the expres-

sion of HSP60 in skin tissue of adults with AD by immunohistochemistry. Peripheral blood mononuclear cells (PBMC) of children with AD were cultured with HSP60 and proliferative

responses, cytokine secretion, surface markers, and functional assays were compared to re-

sponses of PBMC of healthy controls (HC). HSP60 was detected more in lesional skin of AD

patients compared to nonlesional skin. Furthermore, PBMC of children with AD proliferated more strongly in response to HSP60 compared to HC. HSP60-reactive T cells of atopic children produced high levels of IFNÎł and low levels of IL-10. In vitro activation with HSP60 leads

to the induction of CD4+CD25bright T cells expressing FOXP3 in both HC as well as in atopic

children. However, despite their regulatory phenotype, HSP60-induced CD4+CD25bright CD127-

FOXP3+ T cells of AD patients were incapable of suppressing effector T cells in vitro. HSP60 is

recognized by proinflammatory (IFNÎł high, IL-10 low) T cells in atopic patients and is more present in lesional AD skin. This suggests that HSP60-specific T cell responses contribute to local inflammation in AD.

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

Introduction Atopic dermatitis (AD) is a chronic skin condition in patients with atopic disease. Atopic dis-

eases are inflammatory disorders caused by the induction of a TH2 response by an allergen.

Increasing evidence demonstrates that allergy is not a mere result of an over-reactive TH2 response, but that immune regulatory cells such as natural killer T cells and regulatory T cells

(Tregs) play a role in the regulation of this balance(1-4). These cell populations can suppress

effector T cell responses, both by cell窶田ell contact and by the secretion of regulatory cytokines as IL-10 and TGF-ﾎｲ. In active atopic diseases, several studies show that the suppressive function of Tregs is diminished(5-7). Thus, Tregs form an attractive target for therapy in allergic

diseases. Studies in various experimental models of autoimmunity have revealed that Tregs can be targeted in both an antigen-specific and an antigen-nonspecific fashion (8). Both venues of immune therapy are now currently being explored in various autoimmune diseases.

Specific targeting of Tregs with antigens has the advantage that only a subpopulation of T cells

is manipulated which lowers the risk of adverse effects. However, the main dilemma of anti-

gen-specific therapy in humans is to find an antigen which is suitable. In some inflammatory diseases, dominant disease-provoking antigens or allergens are well-known and these can be used for antigen-specific therapy. Indeed, the proof of principle of such an approach was

underscored by Alexander et al. who showed that peptides of Fel d 1 (the major cat allergen) can induce antigen-specific tolerance in cat allergic patients(9). In AD, as in many other chronic

inflammatory diseases, a single disease-triggering antigen is not known. For such a disease, an antigen-specific approach is still feasible if an antigen can be identified that fulfils at least

two conditions. First, the antigen needs to be recognized by T cells from patients and, sec-

ond, the antigen must be available at the site of inflammation, preferably in a disease-specific fashion. Heat shock proteins (HSP) could fit this profile. HSP are immunodominant antigens and a common target of T cell recognition in various inflammatory diseases(10-13). Especially

HSP60 seems to be an important regulating antigen in human inflammatory diseases, as it

is capable of enhancing the regulatory function of human Tregs and thereby dampening the inflammatory process(14;15).

Thus, as HSP60 can promote induction of (adaptive) Tregs, it is conceivable that it could play a

role not only in classical TH1 diseases but also TH2 diseases. Indeed, in asthma, a much stud-

ied TH2 disease, upregulation of several HSP has been shown not only in macrophages and circulating CD4+ T cells but also in epithelial cells(16-18) but it is unknown what the functional

consequences could be. We hypothesized that upregulation and subsequent T cell immune

recognition of self-HSP60 could as well take place in a typical TH2 disease such as AD. Therefore, our aim was to investigate whether HSP60 is a target for T cells in AD and thus could play a role in the chronicity of local inflammation.

Chapter 4

Methods Patients and control subjects Fifty-two atopic children and 30 healthy controls (HC) were included in this study. Eligible pa-

tients were children suffering from physician-diagnosed AD as defined by Hanifin and Rajka(19).

To assure underlying atopy, all children needed to have a positive radioallergosorbent test

for at least one of the three common food allergens, egg, cow’s milk, or peanut, with a history of a clinical allergic reaction after ingestion of the protein (either parental history (Sampson

score ≥ 3) or by food challenge). Patient characteristics are given in Table 1. HC were children who underwent a urological or orthopedic surgical procedure. None of them had a history of

allergy or a recent infection nor a first-degree relative with a history of allergy or asthma. Both written and oral information about the study was given to the parents and written informed consent from the parents was obtained. The study has been approved by the Medical Ethics

Committee of the University Medical Centre, Utrecht, The Netherlands. Patient samples were used for different assays and measurements based on the availability of T cells and serum. Table 1 | Patient characteristics

Atopic Patient (n=55)

Healthy Controls (n=30)

Male (%)

71%

78%

Eczema at presentation

51 (93%)

Age, mean (range) Food allergy

8.8 (1.5-17.5)

55 (100%)

Asthma

24 (44%)

Elevated IgE

55 (100%)

Skin prick test positive Positive food challenge

47 (87%) 31 (56%)

8.6 (1.2-17.3) 0 0 0 0 0

Immunohistochemistry Biopsy specimens (3 mm) were taken from four adult volunteers suffering from moderate to severe AD (for ethical reasons, only adult volunteers were used) under local anesthesia (Xylo-

caine) and snap-frozen in liquid nitrogen. The lesional biopsy was taken from an active lesion, whereas the nonlesional biopsy was taken from healthy-looking skin. Approval was given by the Medical Ethics Committee of the University Medical Centre, Utrecht, The Netherlands.

Subsequently the biopsies were embedded in Tissuetek® (Sakura, Torrance, CA, USA) and stored at −80 °C until further handling. The antibody used as marker for immunohistochemical staining of the frozen sections was antihuman HSP60 (LK2, kind gift from P. van Kooten,

Department of Veterinary Medicine, University of Utrecht, The Netherlands) and mouse anti72

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

human CD3 (cat#347340, used 1:50; Becton Dickinson (BD) Biosciences, San Jose, CA, USA).

Single staining for HSP60 and CD3 combined with a biotinylated horse antimouse IgG (Vector Laboratories, Inc., Burlingame, CA, USA) was performed as described previously. Stainings

were performed on sequential slides to compare HSP60 and CD3 staining in comparable locations. Skin sections were examined by light microscopy. Proliferation and direct culture assays with PBMC

Peripheral blood mononuclear cells (PBMC) were isolated and cultured as described pre-

viously(20). For direct cultures, cells were cultured for 7 days in the absence or presence of

10 μg/ml low endotoxin human HSP60 (0.3 pg/μg protein; Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands). Concanavalin A (2.5 μg/ml; Calbiochem, La

Jolla, CA USA) and tetanus toxoid (1.5 μg/ml; RIVM, Bilthoven, The Netherlands) were used as positive controls. A mouse class II restricted peptide (Ova) was used as an irrelevant control.

For the proliferation assays, cells were cultured for 96 h only. For the final 16 h of culture, 1

μCi/well [3H]thymidine (ICN Biomedicals, Amsterdam, The Netherlands) was added to each

well. Cells were harvested according to standard procedures and incorporated radioactivity was measured by a liquid scintillation counter and expressed as counts per minute. The mag-

nitude of the proliferative response is expressed as the stimulation index (SI), which is the

mean counts per minute of cells cultured with antigen divided by the mean counts per minute of cells cultured without antigen. As additional control on the effect of possible lipopolysaccharide (LPS) contamination of HSP60, proliferation assays were performed in a control

group of patients after treatment of HSP60 (10 μg/ml) or LPS (10 μg/ml; Sigma-Aldrich Corp, St. Louis, MO, USA), with 10 μg/ml polymyxin B (Bio-Rad Laboratories, Hercules CA, USA) for 1 h at room temperature or heat inactivation at 95 °C for 30 min. Multiplexed particle-based immunoassay

Cytokine levels were measured after the activation of lymphocytes in vitro as described above.

After 96 h, the supernatants of the cell cultures were stored at −80 °C until analysis. Cytokines levels of IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, and IFNγ were measured with the Bio-Plex system and analyzed with the Bio-Plex Manager software version 6.0 (Bio-Rad) which uses the Luminex xMap technology(21). The peptide-specific cytokine production is calculated as the cytokine production of cells cultured with peptide subtracted with the cytokine production of cells cultured without peptide. Lymphocyte cell surface markers

At day 7 from the direct cell culture assays as described above, PBMC were harvested and

prepared for fluorescence-activated cell sorting (FACS) staining as described previously(22). The cells were incubated in 50 μl FACS buffer containing three appropriately diluted phycoerythrin (PE)-, fluorescein isothiocyanate (FITC)-, or Cy-Chrome-labeled monoclonal anti-

bodies against human CD4 (clone RPA-T4), CD25 (clones: activated protein C [APC], M-A251; PE, 2A3), CD30 (clone: Ber-H83), and CD69 (clone: FN50). Stained mononuclear cells were

Chapter 4

diluted in FACS fluid and run on a FACSCalibur (BD Biosciences). CellQuest software (BD Biosciences) was used for analysis.

Cytokine analysis by lymphocyte intracellular staining and flow cytometry Direct T cell lines were generated as described above. During the last 4 h of culture, GolgiStop (BD

Biosciences) was added (final concentration of 2 μM). The cells were harvested and stained for intracellular cytokine and FOXP3 analysis as described previously. For the staining, a predetermined optimal concentration of PE-conjugated anti-IL-10 (clone JES3-19F1), anti-IL-4 (clone MP4-

25D2), FITC-conjugated anti-IFNγ (clone 4S.B3; BD Biosciences), and APC-conjugated FOXP3 (Ebioscience, San Diego, CA, USA) was added to the cells. Cells were analyzed as described above. Functional assays

To test the induced CD4+CD25+FOXP3+ Tregs on suppressive function, PBMC were cultured

for 96 h as described above in the presence of HSP60. CD4+CD25+CD127− T cells (which were

used to quantify FOXP3+ T cells in the functional assay) and CD4+CD25− effector T cells from

the same donor were directly sorted by FACS (FACS Aria, BD Biosciences) into plate-bound anti-CD3-coated wells (OKT-3, 1 μg/ml) in different ratios (1:0, 1:0.5, 1:1, and 1:2). T cell-de-

pleted PBMC from the same donor were irradiated (3,500 Rad) and were used as antigen-presenting cells (1:10). The cells were incubated at 37 ° C for 96 h, with the last 16 h of incubation

in the presence of [3H]thymidine (1 μCi/well). The suppressive activity was determined by calculating the percentage difference in proliferative response (mean [3H]thymidine incorporation (counts per minute) of triplicate wells) between CD4+CD25− T cells cultured alone and CD4+CD25− T cells cultured in the presence of induced CD4+CD25+CD127− T cells.

Statistical analysis

Basic descriptive statistics were used to describe the patient population. A nonparametric test

(Mann–Whitney U test, two-sided) was applied to determine significant differences between the patient and control groups regarding proliferative responses, cytokine production, and

expression of cell surface markers. Where mentioned, values are noted as the mean ± standard error of the mean (SEM). A p value <0.05 was considered significant (SSPS Statistical Program, version 15.0.; SPSS Inc, Chicago, IL, USA).

Results Increased presence of HSP60 in skin biopsies of atopic dermatitis Since HSP are upregulated in inflammatory disorders, we questioned whether the presence of HSP60 is increased in the lesional AD skin(23). To demonstrate this, skin biopsies from both

nonlesional and lesional skin sites from AD patients were stained for HSP60 and CD3. The results of a representative donor are shown in Fig 1. The presence of HSP60 was seen in both

the healthy skin and the AD skin, but the pattern of HSP60 presence was strikingly different.

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

The dermis and epidermis of atopic skin was characterized by large cell infiltrates, with the presence of HSP60, which was not seen to that extent in the healthy skin. Staining for CD3 in sequential slides was indicative for the presence of HSP60 in CD3+ cells in the dermis. Howev-

er, since it was not a double staining, we cannot exclude that, next to T cells, HSP60 is also lo-

calized in other cells or extracellularly in dermal tissue, in close proximity to CD3 cells. These data show that HSP60 is highly present in inflamed tissue of patients with AD.

Figure 1 | Human HSP60 is expressed in the skin. A HSP60 staining in an active lesion of a patient with AD. B The same skin of the patient with AD, colored with anti-CD3. Both are representative pictures of skin biopsies of an active skin lesion of a patient with AD. Compared to the nonlesional skin (c and d), the thickened stratum corneum and epidermis are typical for AD. In the dermis, infiltration of cells is seen. HSP60 is mostly seen in these infiltrates and in the lower layers of the epidermis (a), whereas coloring grows less dense in the upper areas of the epidermis. The cell infiltrates express CD3 and the HSP60 expression in the dermis seems confined to these cells. c and d Nonlesional skin of a patient with AD. Compared to the lesional skin, the nonlesional skin shows less HSP60 (C) and CD3 (D). Ă&#x2014;200 (scale bar050 Îźm)

Human HSP60-specific proliferation

Next, we wanted to see whether this increased recognition of HSP60 is also seen in peripheral T cell of children with AD. To address this question, proliferation of PBMC was deter-

mined in response to HSP60. Proliferative responses of 28 patients were compared with those of 18 HC. The SI of the PBMC was significantly higher in the patient group compared

Chapter 4

to the HC group (p = 0.004). This indicates that PBMC from children with AD proliferate

more to HSP60 compared to PBMC from HC (Fig. 2a), whereas no significant difference was found on a control mitogen (Supplementary Fig. 1).

Figure 2 | Human HSP60 is recognized by PBMC of atopic infants and induce cytokine secretion. A Protein-induced T cell proliferation in atopic children and HC. PBMC were cultured for 96 h in the presence of human HSP60. Boxes show IQR for each group of data, horizontal lines show the median. *p<0.05, significant differences in responses. Light grey boxes HC, dark grey boxes atopic children. SI is indicated on a logarithmic scale. B Protein-induced cytokine secretion of IL-10, IFNγ, and IL-6 in atopic children and HC. Boxes show the mean levels of cytokine with SEM. White boxes medium, black boxes stimulation with HSP60. Cytokine levels are indicated on a logarithmic scale. C FACS analysis of CD4+ CD14− T cells from an atopic donor stimulated with HSP60 for 4 days and stained for IFNγ. Histogram shows the medium level (black) and IFNγ level (white) after stimulation with HSP60.

Human HSP60-specific cytokine induction

Given the increased T cell proliferation to HSP60 by the T cells of atopic children, we wanted to assess the quality of T cell activation. First, cytokine production by HSP60-induced PBMC was

measured in culture medium of 26 patients and 22 HC. When compared to the medium, stimulation with HSP60 induced a significantly higher level of IL-10 in the HC group (mean ± SEM,

4.4 ± 1.8 pg/ml; p = 0.012), while in the patient group, compared to the medium, production of

IL-10 was lower after stimulation with HSP60 (mean ± SEM, −14.77 ± 17.91 pg/ml; ns). A different pattern was seen for IFNγ. Following in vitro stimulation with HSP60, PBMC from both patients

(mean ± SEM, 198.1 ± 140.1) and HC (mean ± SEM, 5.58 ± 4.46) produced higher amounts of IFNγ. Taken together, these results demonstrate that stimulating PBMC from atopic patients with HSP60 76

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

leads to the induction of IFNγ but not IL-10 (Fig. 2). Of the other cytokines tested (IL-4, IL-5, IL-12, and IL-13), only IL-6 showed an increase after stimulation with HSP60 (Fig. 2b).

Although the LPS content of the HSP60 used was below the detection level and although

monocytes are not expected to survive after a 5-day culture, we wanted to verify that IFNγ measured was indeed secreted by T cells. Therefore, after culturing the cells with or without

HSP60, cells were stained for CD4, CD14, and IFNγ. Indeed, HSP60-induced production of IFNγ was produced by CD4+CD14− T cells, while virtually no monocytes (<1 %) were present.

Figure 2c shows a representative overlay histogram of CD4+CD14−IFNγ+cells.

Human HSP60-specific induction of CD4+CD25bright T cells expressing the transcription factor FOXP3 We next questioned whether in atopic children in vitro activation with HSP60 could lead to the induction of Tregs. To address this issue, we first investigated whether the FOXP3 expres-

sion in HSP60-specific CD4+ T cells was different between HC and atopic children. PBMC of

five HC and nine children with AD were cultured for 7 days in vitro with HSP60 (see Fig 3). Stimulation with HSP60 gave a significantly higher percentage of CD4+CD25bright T cells and

CD4+FOXP3+ T cells in both the HC and the atopic patients compared to the medium only (see Fig. 3a, 3b, and Table 2). Although at least a part of the FOXP3 induction is due to the activation of T cells, stimulation with HSP60 also seems to induce T cells with the phenotypical characteristics of Tregs, expressing CD25 and FOXP3.

Figure 3 | Human HSP60 induces CD4+ CD25bright T cells and the expression of FOXP3 Cells were cultured in human HSP60 for 7 days. a FACS analysis shows a significantly higher level of CD4+ CD25bright T cells and CD4+ FOXP3+ T cells in both HC as atopic individuals. White boxes medium only, grey boxes human HSP60. b FACS analysis of CD4+ T cells of both atopic individuals and HC expressing FOXP3 after stimulation with either medium or human HSP60. Percentages indicate the percentage of CD4+ FOXP3+ T cells in the total CD4+ T cell population measured.

Chapter 4

Table 2 | Populations of CD4+CD25bright and CD4+FOXP3+ T cells increase after stimulation with HSP60 HC

CD4+CD25bright

Atopic patients

CD4+CD25bright

CD4+FOXP3+ CD4+FOXP3+

Medium

Human HSP60

p value

1.69 ± 0.72 5.69 ± 1.73

5.13 ± 3.29 13.37 ± 8.65

< 0.05 < 0.05

1.67 ± 0.21 3.25 ± 1.39

4.95 ± 0.97 5.58 ± 1.78

< 0.001 < 0.05

This table shows the mean percentages +/- SEM for the different cell populations in FACS analysis after stimulation with human HSP60.

Human HSP60-induced CD4+CD25+CD127− T cells are not suppressive in vitro

Since CD4+ T cells responding to HSP60 produce IFNγ while also sharing phenotypical charac-

teristics of Tregs, we questioned whether these HSP60-induced CD4+CD25+FOXP3+ are functional Tregs, with the capacity to suppress T effector cells. To evaluate the functionality of these induced CD4+CD25+FOXP3 T cells, suppression assays were performed on PBMC of six children

with AD. Freshly sorted CD4+CD25− T effector cells were cocultured with CD4+CD25+CD127− T

cells obtained from PBMC precultured for 4 days with HSP60 (see Fig. 4).

Figure 4 | Human HSP60-induced CD4+ CD25+ CD127− T cells from atopic patients have no suppressive function. Functional assays were performed as described in the “Methods” section. The figure shows the percentage difference in proliferative response between CD4+ CD25− T cells (Teff) cultured alone and CD4+ CD25− T cells cultured in the presence of induced CD4+ CD25+ CD127− T cells (Tregs) in three different ratios. Only one patient showed suppressive function.

Despite high levels of FOXP3, the CD4+CD25+CD127− T cells from these cultures were not sup-

pressive in five of six patients. In only one patient, the cells were able to suppress effector

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

cells. In comparison, in all HC, these cells are suppressive in this classical suppression assay(24). These findings indicate that, even though HSP60, presented in an atopic environment,

induces T cells that resemble Tregs phenotypically, these cells are not capable of suppressing effector T cells in vitro.

Discussion

In the present study, we demonstrated for the first time the increased presence of HSP60 in the skin of patients with AD. Furthermore, we found increased proliferation of T cells from atopic children after in vitro stimulation. Both increased levels of HSP60 at the site of inflammation and the enhanced T cell response underline that HSP60 may act as the target of an (autoan-

tigenic) T cell response. The next question was what type of T cell response was induced by HSP60 in AD. At first glance, it seemed that HSP-induced T cells had a regulatory phenotype as

they expressed CD25bright and FOXP3. However, despite the expression of FOXP3, HSP60-induced

CD4+CD25+CD127â&#x2C6;&#x2019; cells of AD patients were not functionally suppressive in vitro. Thus, these

cells are most probably not Tregs but highly activated effector T cells. The presence of activated but not Tregs may be in line with previous studies showing that, in active atopic diseases, the suppressive function of Tregs is diminished(5;6). Interestingly, a recent study by Wehrens et al.

revealed that highly activated T effector cells are resistant to suppression through PKB/c-akt hyperactivation. This might also be an explanation why these phenotypically regulatory cells fail to suppress T effector cells in vitro and needs further investigation(25).

In atopic diseases, it has been speculated whether specific targeting of T cells could provide a novel immunomodulatory pathway for treatment. Up till now, studies have focused on the

use of synthetic peptides which contain T cell epitopes derived from known allergens, such

as Fel d 1 (cat allergy) and Api m 1 (bee venom)(26;27). However, since atopic individuals are mostly polysensitized, immunotherapy aimed at one allergen might have limited effect in the majority of the patients.

Another possibility for immunomodulation is the use of bystander antigens, in which the antigen used is not the causative antigen, but an antigen which can alter the reaction by either by-

stander activation or suppression(28). Prerequisites for bystander epitopes are expression at the site of inflammation and recognition by immune-competent cells such as T cells. For this type of antigen-specific targeting of T cells, HSP60 is a potential candidate(13;15) as HSP60 was previously

described in skin lesions of patients with BehĂ§etâ&#x20AC;&#x2122;s disease(29). HSP60 has also been described as a

target for T cells in TH1 diseases such as diabetes mellitus and juvenile idiopathic arthritis and as a target for T cells in the inflammatory process contributing to atherosclerotic lesions(30-33).

It has to be noted, though, that HSP60 primarily acts as an intracellular (and intramitochondri-

al) chaperone. Only in particular conditions, such as cell stress, HSP60 will be released into the 79

Chapter 4

extracellular environment, where it may have “extra-chaperoning” roles, including interaction

with immune system cells(34;35). This secretion of HSP60 may originate either from lymphocytes themselves, from other cutaneous cells like keratinocytes and fibroblasts, or from other sources such as nerves or vessels (36). Also, next to HSP60, other proteins involved in inflammation, including proteins from other HSP families, may be involved in AD pathogenesis(37-39).

This is the first study to demonstrate that HSP60 is present at the site of inflammation and that it induces specific proinflammatory T cell responses in children with AD. This is in line

with a previous experimental study showing that HSP60 can induce cytokines of a regulatory and Th1 phenotype in the skin of dogs with AD(40). This finding is an important condition in

investigating novel targets for immunomodulation. Further studies are needed to investigate these immunomodulating properties of HSP60 in atopic diseases.

Supplemental figure | Protein-induced T cell proliferation in atopic children and healthy controls. PBMCs were cultured for 96 hours in the presence of ConA. Boxes show IQR for each group of data, horizontal lines show the median. Light grey boxes = healthy controls, dark grey boxes = atopic children. SI is indicated on a logarithmic scale. The difference in SI between the HC and atopic children was not significant (p = 0.1) after stimulation with ConA.

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

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Pene J, Desroches A, Paradis L, Lebel B, Farce M, Nicodemus CF et al. Immunotherapy with Fel d 1 peptides decreases IL-4 release by peripheral blood T cells of patients allergic to cats. J Allergy Clin Immunol 1998; 102(4 Pt 1):571-8. Muller U, Akdis CA, Fricker M, Akdis M, Blesken T, Bettens F et al. Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J Allergy Clin Immunol 1998; 101(6 Pt 1):747-54. Larche M, Akdis CA, Valenta R. Immunological mechanisms of allergen-specific immunotherapy. Nat Rev Immunol 2006; 6(10):761-71.

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis

30. 31. 32. 33. 34. 35. 36. 37.

38. 39. 40. 41.

Ergun T, Ince U, Eksioglu-Demiralp E, Direskeneli H, Gurbuz O, Gurses L et al. HSP 60 expression in mucocutaneous lesions of Behcetâ&#x20AC;&#x2122;s disease. J Am Acad Dermatol 2001; 45(6):904-9. Albani S, Keystone EC, Nelson JL, Ollier WE, La CA, Montemayor AC et al. Positive selection in autoimmunity: abnormal immune responses to a bacterial dnaJ antigenic determinant in patients with early rheumatoid arthritis. Nat Med 1995; 1(5):448-52.

Grundtman C, Kreutmayer SB, Almanzar G, Wick MC, Wick G. Heat shock protein 60 and immune inflammatory responses in atherosclerosis. Arterioscler Thromb Vasc Biol 2011; 31(5):960-8. Prakken BJ, Samodal R, Le TD, Giannoni F, Yung GP, Scavulli J et al. Epitope-specific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis. Proc Natl Acad Sci U S A 2004; 101(12):4228-33. Raz I, Elias D, Avron A, Tamir M, Metzger M, Cohen IR. Beta-cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double-blind, phase II trial. Lancet 2001; 358(9295):1749-53.

Pockley AG, Multhoff G. Cell stress proteins in extracellular fluids: friend or foe? Novartis Found Symp 2008; 291:86-95; discussion 96-100, 137-40.:86-95. Macario AJ, Cappello F, Zummo G, Conway de Macario E. Chaperonopathies of senescence and the scrambling of interactions between the chaperoning and the immune systems. Ann N Y Acad Sci 2010; 1197:85-93. doi: 10.1111/j.1749-6632.2010.05187.x.:85-93.

De Maio A. Extracellular heat shock proteins, cellular export vesicles, and the Stress Observation System: a form of communication during injury, infection, and cell damage. It is never known how far a controversial finding will go! Dedicated to Ferruccio Ritossa. Cell Stress Chaperones 2011; 16(3):235-49. Ghoreishi M, Yokozeki H, Hua WM, Nishioka K. Expression of 27 KD, 65 KD and 72/73 KD heat shock protein in atopic dermatitis: comparison with those in normal skin and contact dermatitis. J Dermatol 2000; 27(6):370-9.

Ishibashi Y, Kato H, Asahi Y, Sugita T, Nishikawa A. Identification of the major allergen of Malassezia globosa relevant for atopic dermatitis. J Dermatol Sci 2009; 55(3):185-92. Matsumoto Y, Oshida T, Obayashi I, Imai Y, Matsui K, Yoshida NL et al. Identification of highly expressed genes in peripheral blood T cells from patients with atopic dermatitis. Int Arch Allergy Immunol 2002; 129(4):327-40.

Jassies-van der Lee A, Rutten V, van Kooten P, van der Zee R, Willemse T. Intradermal injection of HSP60 induces cytokine responses in canine atopic and healthy skin. Cell Stress Chaperones 2008; 13(3):387-91.

Chapter 5

Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut

Joost A Aalberse; Anders O van Thuijl; Yolanda Meijer; Wilco de Jager; Tjitske van der Palen-Merkus; Aline B Sprikkelman; Maarten O Hoekstra; Berent J Prakken; and Femke van Wijk. Clin Transl Allergy. 2013 Dec 2;3(1):40

Chapter 5

Abstract One of the IL-17 family members, IL-25, has been implicated with the initiation and amplifica-

tion of Th2 responses in animal models and has been associated with airway hyper-reactivity. The involvement of IL-25 and also IL-17 in food allergic disease remains to be investigated.

In this study thirty children suspected of peanut allergic disease underwent a double-blind

placebo controlled food challenge (DBPCFC) and IL-25 and IL-17 plasma levels were determined before and after challenge. IL-25 was highly elevated only in subgroup of children with

a positive DBPCFC outcome. Plasma IL-25 was absent in children with a negative DBPCFC outcome and in healthy controls.

This study shows that IL-25, an IL-17 family member, is highly elevated only in children with

a clinical response to peanut. This suggests a role for IL-25 in the pathogenesis of peanut allergy and elevated plasma IL-25 may be a sign of a severe atopic phenotype.

Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut

Findings Members of the IL-17 cytokine family are emerging as key factors in immune responses(1).

The prototypic family member, IL-17A, triggers pro-inflammatory immune responses and

contributes to neutrophilia during chronic airway inflammation. IL-17E, also known as IL-25, is the most divergent cytokine in the IL-17 family and, unlike the other members, has been identified as a central player in the initiation and amplification of Th2 responses(1). In experi-

mental mouse models IL-25 mediates early differentiation towards a Th2 phenotype and de-

velopment of airway hyper-reactivity and allergic disease(2;3). Moreover, allergen provocation

in asthmatic patients increases expression of IL-25 and its receptor(4), suggesting that IL-25 is

implicated in both sensitization and memory responses in airway hypersensitivity. Increased duodenal levels of Il-25 were found in peanut allergy in a mouse study(5). In human subjects

however, the involvement of IL-25 and IL-17 in food (peanut) allergy remains unknown. To

investigate if there is a difference in IL-25- and IL-17 expression in peanut allergic versus peanut tolerant (i.e. peanut sensitized but, not clinical reactive) we determined IL-25 and

IL-17 plasma levels, as well as the Th2 cytokines IL-4, IL-5 and IL-13, in a well-defined cohort of peanut sensitized children undergoing a double-blind placebo controlled food challenge (DBPCFC).

Thirty children suspected of peanut allergic disease (based on either elevated specific IgE to peanut (ImmunoCap >0.35 kU/L) or positive skin test to peanut) were referred to The

Wilhelmina Childrenâ&#x20AC;&#x2122;s Hospital, University Medical Center, Utrecht, The Netherlands for a

DBPCFC to obtain certainty about the diagnosis of peanut allergy (for patient characteristics see TableÂ 1). The study was approved by the local medical ethics review boards (METC, UMC

Utrecht; project no. 05/084 and METC AMC, Amsterdam; project no. 05/254) and informed consent was obtained for all subjects. The oral challenge was performed as previously de-

scribed(6). Peripheral blood samples were collected before the start of the DBPCFC, as well as

when the challenge was finished. Plasma cytokine levels were determined by Xmap technology (Luminex Austin)(7).

Chapter 5

Table 1 | Characteristics peanut sensitized patients that underwent a DBPCFC No

Sex

PeanutSPT at Clinical Age Atopic specific-IgE diag- Asthma# response (years) Dermatitis$ at diagnosis nosis to DBPCFC 10

4.0

0.43

5.9

3 4 6 7 8 9

10 11 12 13 14

M M F

M M M M M F

M M

15* F

16* M 17

18 19 21 22

M M F

23* M 24

25* F 26

27* M 28

29* M 30

16 14

<0.35

<1 3

2.0

1.1

97.0

3 4

15 8 4 5 9 6

0.6 7.3

0.6

0.8 1.1

>100

2.2

>100

3 2

4.5

56.0

3.5

6 7 9 7 7

>100

1.9

>100

55.0 2.1

3 4

4.0

15.5

4 6 5 6 6 5

6.0 4.9

21.0 5.3 2.2 NP

2 2 2 3

NP 2

+ + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + +

Resp Syst

+ * + + + + + + + + + + + + + + -

+ * + + + + + + + + + + +

+ * + + + + + + + + -

Patient no. 1â&#x20AC;&#x201C;12: non-responders to peanut challenge. Patient no. 13â&#x20AC;&#x201C;30: positive responders to peanut challenge. # Asthma diagnosed according to ATS criteria. $Atopic dermatitis diagnosed according to criteria from Hanifin and Rajka. SPT, Skin Prick Test (the number represents the wheal size ratio peanut compared to histamine). DBPCFC, Double-Blind Placebo-Controlled Food Challenge. NP, Not performed. *Children with elevated plasma IL-25 as determined in Figure 1. GI: Gastrointestinal symptoms including stomach ache, nausea, vomiting and diarrhea; Resp: respiratory symptoms including rhinorrhea, stuffy nose; Syst: systemic symptoms including shortage of breath, systemic urticaria and angioedema. 88

Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut

DBPCFC resulted in a positive (allergic) and negative (tolerant) challenge group and peanut

allergy was confirmed in 18 out of 30 patients. In the plasma samples of the negative and

positive challenge groups we found a striking difference for the type 2-related cytokine IL-

25. Plasma IL-25 was not detected in any of the children with a negative challenge response, whereas plasma IL-25 was elevated in six children of the positive challenge group (Figure 1A).

In five out of six of these children the IL-25 concentration was even extremely elevated, with levels ranging up to 13000 pg/ml (Figure 1A, B). IL-25 levels were similar in plasma samples

taken after challenge (data not shown). IL-17 was found in both the positive and negative

challenge groups (in 67% versus 83% of children respectively), but in contrast to IL-25, IL-17 levels were significantly lower in the positive challenge than in the negative challenge group (p < 0.01) (Figure 1A, B). We show that in this cohort of peanut sensitized children, the pres-

ence of high levels of IL-25 in plasma is only present in children with a positive challenge and

clinical reactivity is inversely correlated with plasma IL-17. These findings seem to be rather specific for the cohort of (peanut) allergic children since both IL-25 and IL-17 plasma levels were below the detection limit in age-matched healthy controls (n = 20) (data not shown).

We searched to explain why plasma IL-25 was elevated in this subgroup of peanut allergic

patients. No difference was found in relation to the other Th2 cytokines. Levels of IL-4 were below the detection limit and IL-5 and IL-13 levels were very low when positive. Within the clinical responder group also no difference in peanut-specific IgE levels was found between the IL-25 negative and IL-25 positive subgroups (Figure 1C).

Plasma IL-25 did not associate with total IgE levels (Figure 1D), previous exposure to peanut,

severity of food allergic symptoms, or the presence of asthma and atopic dermatitis, nor did it correlate with organ specific symptoms during challenge (Table 1). As IL-25 is often men-

tioned to be related to asthma like symptoms we reviewed IgE data to inhalant allergens. Plasma levels of IgE to either birch, house dust mite (hdm) and cat from 17 patients were

available. In general most children with a peanut sensitization were also sensitized to inhalation allergens, irrespective of the positive or negative challenge or IL-25 levels.

To extend our findings in peanut allergic children we next measured IL-17 and IL-25 in a

group of infants diagnosed with cow’s milk allergy (CMA) (confirmed by DBPCFC, n = 12). In these infants IL-25 was found in 42% of the allergic patients but at very low levels (1.5-14 pg/ ml) and IL-17 levels were below the detection level (data not shown). These data indicate that elevated IL-25 in serum is not a general phenomenon in clinical food allergy.

Peanut allergy is considered as an indication of a broad and possibly severe atopic phenotype and, unlike other food allergies (such as CMA), is infrequently outgrown8. The original

diagnosis of peanut allergy in our tested cohort was not based on an oral challenge and this

poses limitations on conclusions about the resolution of peanut allergy. However, the data do demonstrate that plasma IL-25 was only present in children with ongoing peanut allergy and,

Chapter 5

Figure 1 | Cytokine profile and antibody levels of peanut sensitized patients. A colour profile (A) and scatter plot (B) of IL-25 and IL-17 plasma levels (pg/ml) in children with a negative (n = 12) and children with a positive peanut challenge (n = 18). * p < 0.05 with a Mannâ&#x20AC;&#x201C;Whitney U test. Peanut-specific IgE (C) and total IgE (D) serum levels in the negative and positive challenge groups,* p < 0.05 with a Mannâ&#x20AC;&#x201C;Whitney U test. The positive challenge group is subdivided in IL-25 positive and IL-25 negative children.

Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut

importantly, despite a peanut-free diet for at least 6Â months. Together these data may indicate that elevated plasma IL-25 is a sign of chronic immune activation that is not induced by the provoking allergen itself but represents a risk factor for the development or persistence of

clinical reactivity to peanut. Recently it was suggested that IL-25 secretion is induced during disruption of epithelial barriers. Our data are insufficient to make a conclusion on the nature

of a IL-25 subgroup, but it can be speculated that these patients have a more severe type of food allergy. The finding that only 6 out of the 18 peanut allergic patients displayed highly elevated IL-25 levels stresses the possibility of a clinical subgroup within the group of peanut allergic children and warrants larger cohort studies.

IL-25 is expressed by a variety of innate immune cells and non-hematopoietic cells including basophils, eosinophils, epithelial and endothelial cells9 and in the gut IL-25 is predominant-

ly found in epithelial cells10. Innate lymphoid cells have been described in the human that

respond to IL-25 and provide an innate source of Th2 cytokines11;12. Together, increased pro-

duction of IL-25 triggered by environmental antigens or microbes in the gut might therefore contribute to the atopic phenotype by promoting Th2 differentiation and the maintenance of allergen specific Th2 memory cells.

In conclusion, this study is the first to show that IL-25 is highly elevated in a subgroup of pea-

nut-allergic children and suggests a role for IL-25 in the development and/or persistence of peanut allergy, in this subgroup. These findings warrant further studies in a larger cohort of patients as well as in other food allergies.

Chapter 5

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Iwakura Y, Ishigame H, Saijo S, Nakae S. Functional specialization of interleukin-17 family members. Immunity 2011; 34(2):149-62.

Angkasekwinai P, Park H, Wang YH, Wang YH, Chang SH, Corry DB et al. Interleukin 25 promotes the initiation of proallergic type 2 responses. J Exp Med 2007; 204(7):1509-17. Tamachi T, Maezawa Y, Ikeda K, Kagami S, Hatano M, Seto Y et al. IL-25 enhances allergic airway inflammation by amplifying a TH2 cell-dependent pathway in mice. J Allergy Clin Immunol 2006; 118(3):606-14.

Corrigan CJ, Wang W, Meng Q, Fang C, Eid G, Caballero MR et al. Allergen-induced expression of IL-25 and IL-25 receptor in atopic asthmatic airways and late-phase cutaneous responses. J Allergy Clin Immunol 2011; 128(1):116-24. Chu DK, Llop-Guevara A, Walker TD, Flader K, Goncharova S, Boudreau JE et al. IL-33, but not thymic stromal lymphopoietin or IL-25, is central to mite and peanut allergic sensitization. J Allergy Clin Immunol 2013; 131(1):187-200. Aalberse JA, Meijer Y, Derksen N, van der Palen-Merkus T, Knol E, Aalberse RC. Moving from peanut extract to peanut components: towards validation of component-resolved IgE tests. Allergy 2013; 68(6):748-56. de Jager W, Prakken BJ, Bijlsma JW, Kuis W, Rijkers GT. Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies. J Immunol Methods 2005; 300(1-2):124-35. Skolnick HS, Conover-Walker MK, Koerner CB, Sampson HA, Burks W, Wood RA. The natural history of peanut allergy. J Allergy Clin Immunol 2001; 107(2):367-74.

Wang YH, Liu YJ. Thymic stromal lymphopoietin, OX40-ligand, and interleukin-25 in allergic responses. Clin Exp Allergy 2009; 39(6):798-806.

Zhao A, Urban JF, Jr., Sun R, Stiltz J, Morimoto M, Notari L et al. Critical role of IL-25 in nematode infection-induced alterations in intestinal function. J Immunol 2010; 185(11):6921-9. Licona-Limon P, Kim LK, Palm NW, Flavell RA. TH2, allergy and group 2 innate lymphoid cells. Nat Immunol 2013; 14(6):536-42.

Mjosberg JM, Trifari S, Crellin NK, Peters CP, van Drunen CM, Piet B et al. Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol 2011; 12(11):1055-62.

Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut

Chapter 6

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

J.A. Aalberse; Y. Meijer; N. Derksen; T. van der Palen-Merkus; E. Knol; and R.C. Aalberse Allergy 2013; 68 748-56

Chapter 6

Abstract Replacement of peanut extracts by recombinant peanut components is an important step

in allergy serologic testing. Criteria are needed for the unbiased inclusion of patients into a study to validate such a replacement.

Plasma samples from 64 peanut-positive children (42 reactors, 22 nonreactors in a double-blind, placebo-controlled food challenge) were used to compare IgE reactivity to six re-

combinant peanut allergens with reactivity to natural peanut proteins extracted at neutral or low pH. We tested the hypothesis that poor extractability of Ara h 9 and other basic allergens at neutral pH leads to under-representation of patients with such sensitization.

IgE reactivity to the components did not fully explain IgE reactivity to peanut extract in 5 of

32 reactors with IgE to peanut extract â&#x2030;¤100Â kUA/L. IgE reactivity to components was stronger than to the extract in 11 plasma samples, which was largely due to a low Ara h 8 reactivity of the extract. IgE reactivity to Ara h 9 was much lower than reactivity to other basic proteins, some of which bound IgE well in the RAST, but lost IgE reactivity upon immunoblotting.

Conventional peanut extracts are deficient in significant IgE-binding components. The inclu-

sion of patients for a validation study should be based on serology performed with improved peanut reagents to avoid a bias against these under-represented, potentially important allergens. To judge clinical relevance of an allergen, the reagent used for inclusion of patients needs to be efficient in detecting IgE to this component.

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

Introduction It is widely appreciated that recombinant allergens are potentially superior reagents for IgE-

based diagnostic tests compared to extracts of natural source materials(1-8). We want to ad-

dress some issues related to the transition from extracts to recombinant components. This article focuses on establishing a validation protocol, that is, a procedure to substantiate a

claim that a new test protocol (a combination of recombinant allergens) is at least as sensitive as a test with a conventional allergen extract (peanut extract in our case). We selected peanut

as a suitable test case for a study of a validation protocol, because it is a complex source of important allergens, six of which were available to us in recombinant form. In several recent reports, it has been suggested that recombinant allergens may substantially improve the diagnostic accuracy of peanut allergy tests(3;6). These reports may give the impression that re-

combinant allergens could already replace peanut extract. We are concerned that this would lead to a premature disappearance of tests for IgE to peanut extract. We want to argue that it

is important to have a transition period in which the results with recombinant allergens are used to improve IgE-based serology to whole peanut extract (most likely a mixture of differ-

ent extracts, spiked with purified natural and/or recombinant allergens). In the next round

of evaluation, component-based IgE tests should then be re-evaluated using the improved allergen extract.

The clinical aspect of the relation between serology and its diagnostic consequences is not the topic of this article. The data we present in this article are intended to serve as a technical case

study on a validation procedure. The focus is on serology, particularly on the possibility and need of an improvement of its sensitivity. The aim is to be able to identify more than 95% of all sensitized subjects. One topic that will be discussed is the necessity of using an improved

peanut reagent to increase the sensitivity of the IgE test needed to include patients for a dou-

ble-blind, placebo-controlled food challenge (DBPCFC). This is an important validation issue, because the peanut reagent used for the inclusion of human subjects should contain sufficient quantities of all potentially relevant allergens.

This study is based on an extensive serological analysis of plasma samples obtained from chil-

dren suspected to have a peanut allergy and subsequently subjected to a DBPCFC. Previous studies have shown that a few recombinant allergens might suffice for diagnostic purposes(5).

Some studies indicated that measuring IgE to Ara h 2 (possibly used in combination with the closely related allergens Ara h 6 and Ara h 7) might even be sufficient for diagnosis(1;4;7). How-

ever, the relevance of the two highly abundant storage proteins Ara h 1 and Ara h 3 is still a

matter of debate(5;9). The potential contribution of lipid transfer protein (LTP, Ara h 9) is also

of interest(9), not only because this class of proteins has been found to be very important in

other food allergies, but also because of a technical issue. Whole peanut extract is traditionally prepared in saline at a pH of approximately 7, which works well for slightly acidic allergens

Chapter 6

like Ara h 1, Ara h 2, Ara h 3, Ara h 6 and Ara h 7 (isoelectric point [IEP] 5.3–6.2). However,

this is not optimal for highly basic allergens, such as lipid transfer protein (LTP, Ara h 9, IEP 9.4), which are extracted more efficiently at low pH. In the results, we will highlight these basic allergens and discuss the potential consequences of their under-representation in peanut extracts used for the inclusion of subjects for validation trials.

Based on the experiments reported in this article, we will discuss three questions: (i) How often does our panel of six recombinant allergens detect less sIgE than our peanut extracts?

(ii) How often do our peanut extracts detect less sIgE than our panel of six recombinant allergens? (iii) How should we deal with IgE reactivity to highly cross-reactive peanut allergens that have a low abundance in peanut extract such as Ara h 5 (profilin), Ara h 8 (relat-

ed to Bet v 1) and Ara h 9 (LTP)? Taking Ara h 8 as a prototypic cross-reactive allergen, we

will discuss whether it would be useful to deplete Ara h 8 from the peanut extract or spike the extract to enhance Ara h 8 activity. It is evident from published data as well from our

own results that adding Ara h 8 will increase sensitivity, but will markedly decrease speci-

ficity. This might be considered inappropriate in a diagnostic setting. However, we will ar-

gue that in the context of a validation trial of an allergen panel for serological testing, it is necessary to maximize sensitivity, because we can only judge the clinical relevance of an

allergen if the reagent used for inclusion was efficient in detecting IgE to this component.

Patients, materials and methods Study population

This serological study was performed on serum collected from children referred to our hospital for a DBPCFC because of a presumed food allergic reaction and/or atopic dermatitis. Children were clinically evaluated for a peanut DBPCFC based on a history of an allergic reaction

related to peanut ingestion or a positive skin prick test (SPT) (ALK-Abello, Nieuwegein, the Netherlands(10) and/or elevated IgE to peanut (ImmunoCAP; Thermo Fisher Scientific, Uppsala, Sweden; see Supplementary Table S1).

The DBPCFC was performed in the Wilhelmina Children’s hospital. The children were chal-

lenged in seven increasing doses from 0.01 to 10 g peanut, alternated with placebo. All chil-

dren remained under observation after the final dose for at least 2 h depending on severity of the symptoms to minimize the risk associated with these challenges. DBPCFC results were considered positive after the occurrence of objective symptoms (urticaria other than perioral,

angio-oedema, vomiting, stridor and hypotension) or after the occurrence of three subjective symptoms (nausea, oral allergy and abdominal pain).

Based on serum availability, 65 children who underwent a DBPCFC to peanut were initially selected for the clinical analysis of the study. However, one sample was serologically negative at 98

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

time of the challenge and therefore excluded from our analysis. Of the 64 remaining children,

42 were peanut allergic (i.e. Sampson score >1(11), whereas 22 were peanut sensitized but not peanut allergic (no response or only perioral urticaria after DBPCFC).

Skin prick tests were performed on the patient’s back with a standardized prick needle using

a commercial peanut extract (ALK-Abello). The area of the skin wheal was determined by computer scanning. SPT responses were standardized by dividing the wheal area of the pea-

nut prick by that obtained for the histamine control. In hindsight, we realized that we have insufficient information on these peanut extracts (skin test, serology) used for inclusion. Allergens

In this article, four different types of peanut-derived allergenic preparations have been used. A peanut extract prepared by extraction in a saline solution at neutral pH (to which we will

refer as ‘conventional extract’) was used for the skin test and in the ImmunoCAP. In addition, we used extracts prepared at low pH (acetic acid (HAc) pH 4.0–4.5) and at neutral pH with

polyvinylpolypyrrolidone (PVPP, a tannin-removing substance that improves the extractability of many proteins)(12). These two peanut preparations will be referred to as HAc and PVPP.

Furthermore, we isolated a fraction containing basic proteins by adsorption of the acetic acid extract to SP-Sepharose at pH 6 in 20 mM acetic acid/10 mM MES and elution by 0.2 M NaCl in

20 mM MES, pH 6. This preparation will be referred to as BPP (basic peanut proteins). For use

in the RAST, the latter three extracts were coupled to CNBr-activated Sepharose as described elsewhere(13).

The recombinant peanut components Ara h 1, 2, 3, 6, 8 and 9 were ImmunoCAP reagents. In addition, we tested our sera for IgE reactivity to two recombinant proteins related to peanut

allergens: grass profilin Phl p 12 (related to Ara h 5) and peach LTP Pru p 3. Phl p 12 has

been reported to be a major cause of cross-reactivity between grass and peanut(5;12;14). Pru p

3 has been reported to be a major cause of peanut reactivity in Mediterranean countries(15).

The tests for IgE reactivity to the recombinant allergens were performed in the laboratory of Thermo Fisher Scientific, Uppsala (Drs J. Lidholm and L. Elfström). Serology

Quantitative IgE assays were performed either by ImmunoCAP (Thermo Fisher Scientific) or by a Sepharose-based radioallergosorbent test (RAST) procedure as described elsewhere13. Briefly, allergen was coupled to CNBr-activated Sepharose (GE Healthcare, Uppsala, Swe-

den). A suspension of the resulting allergosorbent (corresponding to 0.25–1 mg of Sepharose powder as supplied by the manufacturer and approximately 5 μg protein per test) was

incubated with 50 μl serum on a vertical rotator in a final volume of 0.75 ml. After washing

the suspension with saline using repeated centrifugation, Sepharose-bound IgE was detected with 125I-labelled affinity-purified sheep anti-IgE (approximately 1 ng per test). IgE-binding

data were converted to the equivalent of international IgE units (kUA) using a chimeric IgE

Chapter 6

anti-Der p 2 antibody prepared at Sanquin(16). For the quantitative comparison between IgE

reactivity to whole extract and to the peanut components, we calculated a cumulative sIgE

level for the components, with the sIgE level to rPhl p 12 as a surrogate for Ara h 5. Because of

the cross-reactivity between Ara h 2 and Ara h 6, we used the highest sIgE value of these two for the calculation. In this comparison, we used for whole extract either the reactivity of the ImmunoCAP peanut test or the highest value of three extracts: ImmunoCAP, PVPP and HAc.

The qualitative analysis of IgE binding to peanut proteins was performed by immunoblotting

of peanut proteins separated by Novex 4–12% SDS-PAGE (nonreducing gel, unless stated otherwise) in a MES running buffer. Blotting was performed using iBlot® (Invitrogen Life Technologies, Breda, Netherlands) according to manufacturer’s instructions. The main blotting

experiment was performed by applying the two extracts (PVPP and HAc) as three doublets on a single gel (5 μg protein/lane), transferring the whole gel to nitrocellulose and cutting the

blot into three parts containing one allergen doublet each. After incubation of each strip with

150 μl plasma and washing, IgE binding was detected using an affinity-purified 125I-labelled sheep anti-IgE antibody as described elsewhere(13).

Results Reactivity of the conventional peanut extract compared with the component panel We analysed the ImmunoCAP results based on the assumption that the IgE-binding capacity

of the ImmunoCAP is sufficient to bind all allergen-specific IgE provided that the sIgE level is not extremely high. For this upper cut-off value, we used 100 kUA/L. Of the 42 plasma samples

from patients who reacted to the DBPCFC (serum codes with a ‘p’), 10 had an sIgE level to

peanut (ImmunoCAP) that exceeded this upper cut-off value of 100 kUA/L, which made these results unsuited for a quantitative comparison between extract and components. In Fig. 1, the cumulative IgE binding for the remaining 32 plasma samples to components is shown in comparison with IgE binding to the conventional peanut extract (ImmunoCAP) for 32 plasma

samples with a sIgE level to peanut extract ≤100 kUA/L. In 5 of the 32 plasma samples (16%), the cumulative sIgE level to the components was less than 75% of the sIgE level to peanut ex-

tract. In 11 of the 32 plasma samples (34%), the cumulative sIgE level to the components was more than 125% of the sIgE level to conventional peanut extract (Fig. 1B). As some plasma

samples reacted stronger to the PVPP and/or the HAc peanut extract than to the ImmunoCAP peanut reagent, for numerical analysis, we used the highest value of these three tests. This obviously increased the peanut sIgE level, which resulted in a decrease in the number of plasma

samples with a lower extract sIgE level from 11 to 8 (31–25%). The number of plasma sam-

ples with a lower component sIgE level increased from 5 to 9 (29%) (Fig. 1C). With N = 32, we

need to find four deviant cases to have a more than 95% probability that in more than 5% of

100

the cases, a discrepancy between whole extract and components exists.

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

Figure 1 | ImmunoCAP sIgE levels of 32 patients with a reaction upon challenge (marked 04p to 54p, ‘p’ indicates peanut allergic patients) compared to the IgE level to peanut extract and recombinant allergens. In frame A and B, the ImmunoCap extract is shown, in frame 1c the highest level of ImmunoCAP, HAc and PVPP. The recombinant allergens are Ara h 1, the maximum response of Ara h 2 or Ara h 6, Ara h 3, Ara h 8 as well as the sum of Ara h 9 and grass profilin, represented together as ‘other’. (A) Absolute cumulative IgE-binding data; (B and C) cumulative IgE scores expressed as % of peanut extract, either relative to the ImmunoCAP extract (B) or relative to the highest score of ImmunoCAP, HAc and PVPP (C). 101

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102

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

< Figure 2 | SDS-PAGE analysis.

A | Silver-stained SDS-PAGE (nonreduced) of two peanut extracts (PVPP and HAc) and two fractions obtained by cation-exchange chromatography from HAc (BPP5 and BPP6). Molecular masses of the marker proteins (in lanes marked M) are indicated in kD on the left. Expected positions of peanut proteins are indicated on the right. The prominent band at 34 kD is tentatively assumed to be a major fragment of Ara h 3. This is based on MS data, in which an Ara h 3 fragment is found composed of an N-terminal proteolytic fragment that is disulphide-linked to the basic chain ref 16. B | Immunoblot of 11 plasma samples (p: peanut allergic patients; n: nonpeanut allergic patients) each tested on a doublet of two peanut extracts: PVPP (left lane of each doublet) and HAc (right lane of each doublet). The plasma samples are ranked based on their percentage coverage by the recombinant panel compared to the highest response to three peanut extracts (see Fig. 1C). The numbers below the serum codes indicate IgE-binding classes on a factor 3 scale starting at 0.1. A score 0 corresponds to an sIgE value <0.1 kUA/L; score 1 = 0.1–0.3, 2 = 0.3–0.9, 3 = 0.9–2.7, 4 = 2.7–8.1, 5 = 8.1–24.3, 6 = 24.3–72.9 kUA/L, 7 = 72.9–219 and 8 = >219 kUA/L. The arrow pointing to a 34-kD component indicates a presumed major Ara h 3 fragment, whose staining is much stronger than expected from the ImmunoCAP results in 54p and 51p relative to the undetectable staining of Ara h 2. C Analysis of the effects of blotting pH on the transfer of protein to nitrocellulose (indicated as ‘NC’) and on IgE reactivity. Lanes on the left: BPP; lanes on the right: PVPP. Serum 54p was used for the IgE staining.

Problem 1: reactivity of the components too low

A cumulative sIgE level to the recombinant allergen panel <75% of the sIgE level to peanut

extract might reflect a structural defect of the recombinant proteins and/or to the absence of a relevant allergen. We investigated whether we could identify additional components by

immunoblotting. We tested the binding to two blotted peanut extracts (PVPP and HAc, 5 μg protein/lane, see protein spectra in Fig. 2A) of 11 plasma samples, including some plasma

samples from children with a negative provocation test (indicated in the serum codes by the letter ‘n’): 11n, 52p, 53p and 60p, which are plasma samples with too low-component reactiv-

ity; 44n and 45n, which are plasma samples with component reactivity exceeding that of the extract; and 41p, 49p, 51p, 54p and 61p, which are plasma samples with component reactivity

that matched extract reactivity. Note the discrepancies between staining >25 kD and CAP re-

activity to Ara h 1/3 and the discrepancies between staining <25 kD and CAP reactivity to Ara

h 2/6. Furthermore, note that the staining in the Ara h 1/3 area is much stronger than in the Ara h 2/6 area, which is not reflected by CAP reactivity.

High staining intensity of Ara h 1, 2, 3 and 6 in the PVPP extract made it difficult to detect additional IgE-binding components (Fig. 2B, lanes on the left of each doublet). We therefore

focus our analysis on the HAc extract (Fig. 2B, lanes on the right of each doublet), which has an excellent reactivity when coupled to CNBr-activated Sepharose (Fig. 3), but a substantially

lower content of Ara h 1, 2, 3 and 6, as is illustrated by the protein stained gels (Fig. 2A) and

confirmed by the very weak IgE staining in the Ara h 1 and 3 range (>30 kD) and the absence of IgE staining in the 15–20 kD range (using plasma samples that stained well in this range

to the blotted PVPP extract, Fig. 2B). The faint bands on the immunoblots in the Ara h 1 and

3 region are possibly due to traces of these allergens and their processing products. Upon 103

Chapter 6

removal of these contaminating allergens by cation-exchange chromatography (see Fig. 2A

for the SDS-PAGE protein spectrum of this basic peanut protein fraction (BPP)), IgE-binding

activity in the RAST dropped by 50%, but the remaining RAST activity of BPP was still substantial (Fig.3).

Figure 3 | sIgE (RAST) reactivity to PVPP (on x-axis) compared with reactivity to the HAc extract (grey squares) and BPP fraction of HAc (black diamonds), both on the y-axis.

This low staining was not due to lack of protein transferred to the nitrocellulose. As shown in Fig. 2C, the protein is more efficiently eluted from the gel at the recommended pH (7.2), but

protein binding to nitrocellulose is more efficient when the pH is adjusted to pH 9.5, particularly for Ara h 2/6. Note further that even after autoradiography for 4 days, IgE staining to BPP is very weak compared to PVPP, despite the clear presence of protein on the nitrocellulose.

From these results, we conclude that BPP contains an allergen that has a high IgE-binding

activity in the RAST, but has a very low IgE-binding activity on the immunoblot. Based on the protein spectrum, oleosins (Ara h 10, Ara h 11) are potential candidates. Problem 2: reactivity of the extract too low

The majority of the plasma samples with a high level of IgE reactive with Ara h 8 (the Bet v 1 homologue of peanut) had a low reactivity to each extract, particularly in the conventional ex-

tract, most likely due to the low content of Ara h 8 in our peanut extracts (compare Fig. 1B,C). LTP and profilin are peanut components that are known to be detectable by cross-reactive antibodies induced by other allergen sources. We tested IgE reactivity to peanut LTP (Ara h 9). Peanut profilin (Ara h 5) was not available for testing, so we instead used recombinant profi-

lin from timothy grass pollen, Phl p 12. Our data indicate that IgE to these allergens contrib104

utes less to peanut IgE reactivity than Ara h 8, at least in these Dutch peanut-reactive patients.

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

It is of note that the strongest reaction to LTP (4.4 kUA/L) was found in a patient that did not

react in the DBPCFC. All plasma samples except this one reacted weaker to Ara h 9 than to Pru p 3 (LTP of peach).

Problem 3: How should we deal with the high IgE reactivity to Ara h 8 The data relevant for this topic are presented in Fig. 4, which contains results both of patients

who reacted on DBPCFC and of nonreactors. As expected, IgE to Ara h 8 is often found in nonreactors (14/22 = 64%). Ara h 8 is positive in 21/39 = 55% of the patients positive to

Ara h 2/6. In two cases with a positive DBPCFC, IgE to Ara h 8 was the only component with reactivity above the cut-off of 0.35 kUA/L.

Figure 4 | sIgE (ImmunoCAP) reactivity to Ara h 8 in plasma samples with IgE to Ara h2/6 <0.35 kUA/L (left panel) or ≥0.35 kUA/L (right panel), for reactors and nonreactors in the DBPCFC.

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Discussion The most important conclusion of this study is that conventional peanut extracts are deficient in potentially relevant allergens, which hampers a proper evaluation of the diagnostic performance of a panel of peanut components.

When discussing the way to validate the replacement of a food extract (peanut extract in our case) by a panel of recombinant allergenic components, we have to deal with two clinical

aspects: patient inclusion and the food challenge and two immunochemical aspects: the allergen panel itself and the potency of the peanut extract needed for the validation trial. Patient inclusion

Many of the preliminary analyses can be performed on almost any serum panel with IgE to

peanut, without the need of clinical data. In the validation phase, however, clinical data are

essential, including information on sensitization to related allergens. An important issue is therefore the protocol used for the inclusion of patients. Selection of patients for validation of

a component-based protocol should be unbiased. A selection independent of the use of a pea-

nut extract eliminates bias introduced by possible deficiencies of the extract (to be discussed below). Inclusion based solely on a questionnaire15;16Â is a possible approach, but it may intro-

duce a bias in relation to the spectrum of peanut-related symptoms and the outcome would

be affected by the relative uncertainty of self-reported symptoms. For validation studies, it is important to avoid a bias towards patients with severe symptoms, because these tend to have relatively high IgE antibody levels. Broadening the scope of the questions to include the full

spectrum of symptoms has the disadvantage that more subjects need to be included, because a larger fraction of subjects will have a negative DBPCFC.

How many patients need to be included? At the most basic level, an acceptable allergen panel should be able to detect patients with an unambiguously diagnosed peanut allergy. A desira-

ble target would be a sensitivity of at least 95%, using a cut-off at which negative controls with high total IgE (>2000Â IU/L; such as a low IgE serum spiked with myeloma IgE or recombinant

IgE) are negative. For our peanut extracts, the 0.35Â kUA/Lcut-off level is appropriate. Binomial

statistics indicate that at a 95% confidence level, a panel of at least 59 DBPCFC-positive pa-

tients is needed to establish a sensitivity of better than 95%, if all plasma samples are positive.

A panel of 93 patients is required if a negative test with a single serum is to be accommodated. The DBPCFC

The criteria for a validation study are not necessarily the same as for a diagnostic procedure. This is reflected in the type of symptoms needed to score a test as positive, in particular how to deal with exclusively (peri) oral symptoms. Another issue is the top test dose. The clinical

relevance of a positive test at a 10-gram dose may be lower than one at a 10-mg dose, but the requirement of a high dose may reflect a potent allergen with a low abundance in peanut, 106

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

which might be considered a relevant component in an allergen panel. A third issue is the validation of the masking procedure used for the food challenge material. The potential instability of newly discovered allergens should be taken into consideration. The allergen panel

As mentioned above, the panel should be positive in >95% of challenge-positive patients. An additional criterion that should be considered is whether the panel covers all the relevant

allergens found in an optimal peanut extract (which will be specified below). One approach is to compare the summed component levels with the level for the optimal peanut extract

(Fig.1), preferably using a serological protocol in which closely related allergens such as Ara

h 2, 6 and 7 are combined into a single test. This procedure could be improved by removing IgE reactivity to allergens of uncertain relevance, for example, absorbing out IgE to Bet v 1 to

eliminate cross-reactive IgE responses to Ara h 8. An even more informative approach would be to absorb out all panel-specific IgE from serum and test the depleted serum for remaining IgE reactivity to peanut extract, on an immunoblot and/or RAST. A limitation of the immunob-

lot in this context will be discussed below. The discussion on the number of plasma samples

required and the way these plasma samples should be selected is similar to the discussion concerning patient selection.

Insufficiency of a panel of recombinant allergens may reflect either incompleteness of the panel or qualitative differences between the natural and recombinant allergen, particularly regarding folding and post-translational processing(17). The peanut extract

When we performed the studies reported in this article, we did not appreciate the critical role of the peanut extracts, that is, the extracts used for skin testing and serology. Our data indi-

cated that our extracts were deficient in potentially relevant allergens. Only then we realized that this might have biased our patient selection, because one of the inclusion criteria was a

positive skin test and/or peanut IgE test. In addition, the serological comparison between the component panel and peanut extract shown in Fig.Â 1B is biased towards the well-established allergens Ara h 1, 2, 3 and 6, because these allergens are abundantly represented in conven-

tional peanut extracts. When other extracts (PVPP, HAc) are included in the comparison, some gaps in the allergen representation became more evident (Fig.Â 1C).

If the deficient (i.e. missing or defective) allergens are known components (e.g. Ara h 8), this can be taken into account during the inclusion phase. However, our results with the acidic ex-

tract indicate that peanut contains at least one hitherto unidentified major allergen. Since we found high IgE binding to the HAc extract, the (almost) negative results of the immunoblots

with the HAc preparation were surprising. For the total HAc extract, it can be hypothesized that trace amounts of the traditional peanut allergens (Ara h 1, 2, 3, 6), too low to show well on protein staining of the gel and on immunoblot, might still be coupled in high enough quan-

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tities to CNBr-activated Sepharose to allow substantial IgE binding. However, this becomes less likely with the more purified BPP, which is expected to have a substantially lower content

of these allergens, but still reacted almost as well in the Sepharose test (Fig. 3). One possible

explanation could be a major contribution of proteolytic fragments derived from Ara h 118 or

Ara h 319. Our preliminary data obtained for the N-terminal peptide of Ara h 1 support such an

explanation. This is unlikely to explain all discrepancies, because some of the plasma samples

that reacted well with BPP were negative for Ara h 1 and Ara h 3 (e.g. serum 04p, 34p). We

initially assumed that our blot procedure, which was performed at pH 7.2, was inefficient for proteins with an isoelectric point well above this pH. Protein staining of the nitrocellulose blots (Fig. 2C) indicated that this was not the explanation. These results rather suggest that

binding to nitrocellulose leads to a major loss in IgE binding. One possibility would be that

oleosins are involved. The oleosins form a protein family of basic small (10–20 kD) triglyc-

eride-binding proteins, including Ara h 10 and Ara h 11 from peanut. Their presence in BPP would be unexpected, as they are generally assumed to be unstable in the absence of lipids or detergents(20). It is known that oleosins form oligomers(21), which might be more stable in

aqueous buffers. Upon binding to nitrocellulose, they may unfold and thus largely lose their IgE-binding potential.

We conclude that in a validation protocol, it is important to use an in vitro test based on an

improved peanut extract (most likely a blend of different extracts, enriched fractions and recombinant allergens) for the selection of subjects. Ara h 8 as a prototypic problematic allergen

Many of the issues raised by the availability of recombinant allergens relate to their usefulness in the clinical sensitivity/specificity balance. The concentration of Ara h 8 in current

peanut extracts is low, so spiking the extract with recombinant protein is an obvious possi-

bility to increase sensitivity. While this would result in an unacceptably frequent detection of

birch pollen sensitized but peanut tolerant individuals (i.e. a loss of clinical specificity), it is important to note that IgE to Ara h 8 was present in 23/42 reactors and as the only detected sensitization in two of these 23 subjects.

Why does the clinical relevance of IgE seem to vary? The allergen inducing the IgE antibod-

ies reactive with Ara h 8 may be relevant. If the IgE is induced by the birch allergen Bet v 1,

nonsymptomatic cross-reactivity is common(22). This is presumably due to instability of Ara

h 8 during roasting and digestion. However, IgE induction via exposure to Gly m 4 (the soy equivalent of Ara h 8) by oral consumption of a yoghurt-like soy drink(13;23) may be more likely

to result in symptomatic cross-reactivity. We do not have information on soy exposure in our patient population, but it is unlikely to be the full explanation of the high prevalence of IgE

to Ara h 8 in the symptomatic group. This may indicate either that some birch-induced IgE

responses are composed such that they do cause symptoms or that other foods may induce 108

cross-reactive antibodies more likely to elicit an allergic reaction or that peanut itself may in-

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

duce clinically relevant mast cell-activating IgE antibodies to Ara h 8. Similarly, IgE reactivity due to cross-reactivity without clinical sensitivity has been reported for Ara h 5(5;14).

In our study population, two of 7 Ara h 8 sensitized patients without IgE to Ara h 1, 2, 3, 6 or

9 reacted to peanut in DBPCFC with more than oral symptoms. This finding suggests that it is

too simplistic to assume that all patients with such a sensitization pattern will tolerate peanut or have only oral symptoms and that it may be problematic to extrapolate results from a

population-based study(22)Â to patients specifically selected on the suspicion of peanut allergy.

It will be interesting to investigate the importance of Ara h 8 in geographical areas with low exposure to birch pollen.

Including other sources of PR10 proteins (Bet v 1, Gly m 4) to the allergen panel to more

precisely define the specificity of the Ara h 8 antibodies may help to improve the specificity of serology. However, it is unlikely that data obtained by serology will fully predict the clinical

relevance of IgE antibodies to Ara h 8. Statistical algorithms more sophisticated than ROC

analyses will be needed to achieve optimal information from serological data. Using a test with suboptimal sensitivity for Ara h 8 may be an acceptable interim solution, but we should be able to do better in the near future.

Conclusion

As long as the extracts used for inclusion of patients for test validation analysis are inefficient in

detecting IgE to an allergenic component, we cannot judge the clinical relevance of this allergen.

Acknowledgements

We thank Arjen Brenkman (UMC, Utrecht) for his work on characterizing the low MW compo-

nent in our peanut extract. We thank Jonas Lidholm and Lisa ElfstrĂśm (Uppsala, Sweden) for the ImmunoCAP analyses and stimulating discussions. We thank Karin Hoffmann-Sommergruber (Vienna, Austria), Arndt Petersen (Borstel, Germany), Angela Simpson (Manchester, UK), Anna Pomes (Charlottesville, Virginia) and Steven Stapel, Laurian Zuidmeer and Jaap

Akkerdaas (Amsterdam, NL) for stimulating discussions. We thank Intersnack (Doetinchem, NL) for generously providing raw peanuts.

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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Asarnoj A, Moverare R, Ostblom E, Poorafshar M, Lilja G, Hedlin G et al. IgE to peanut allergen components: relation to peanut symptoms and pollen sensitization in 8-year-olds. Allergy 2010; 65(9):1189-95. Astier C, Morisset M, Roitel O, Codreanu F, Jacquenet S, Franck P et al. Predictive value of skin prick tests using recombinant allergens for diagnosis of peanut allergy. J Allergy Clin Immunol 2006; 118(1):250-6.

Dang TD, Tang M, Choo S, Licciardi PV, Koplin JJ, Martin PE et al. Increasing the accuracy of peanut allergy diagnosis by using Ara h 2. J Allergy Clin Immunol 2012; 129(4):1056-63.

Flinterman AE, Van Hoffen E, den Hartog Jager CF, Koppelman S, Pasmans SG, Hoekstra MO et al. Children with peanut allergy recognize predominantly Ara h2 and Ara h6, which remains stable over time. Clin Exp Allergy 2007; 37(8):1221-8. Kleber-Janke T, Crameri R, Appenzeller U, Schlaak M, Becker WM. Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology. Int Arch Allergy Immunol 1999; 119(4):265-74. Maloney JM, Rudengren M, Ahlstedt S, Bock SA, Sampson HA. The use of serum-specific IgE measurements for the diagnosis of peanut, tree nut, and seed allergy. J Allergy Clin Immunol 2008; 122(1):145-51.

Nicolaou N, Murray C, Belgrave D, Poorafshar M, Simpson A, Custovic A. Quantification of specific IgE to whole peanut extract and peanut components in prediction of peanut allergy. J Allergy Clin Immunol 2011; 127(3):684-5. Peeters KA, Koppelman SJ, Van Hoffen E, van der Tas CW, den Hartog Jager CF, Penninks AH et al. Does skin prick test reactivity to purified allergens correlate with clinical severity of peanut allergy? Clin Exp Allergy 2007; 37(1):108-15.

Vereda A, van Hage M, Ahlstedt S, Ibanez MD, Cuesta-Herranz J, van Odijk J et al. Peanut allergy: Clinical and immunologic differences among patients from 3 different geographic regions. J Allergy Clin Immunol 2011; 127(3):603-7. Flinterman AE, Pasmans SG, Hoekstra MO, Meijer Y, Van Hoffen E, Knol EF et al. Determination of no-observed-adverse-effect levels and eliciting doses in a representative group of peanutsensitized children. J Allergy Clin Immunol 2006; 117(2):448-54. Sampson HA. Anaphylaxis and emergency treatment. Pediatrics 2003; 111(6 Pt 3):1601-8.

van der Veen W, van Ree R, Aalberse RC, Akkerdaas J, Koppelman SJ, Jansen HM et al. Poor biologic activity of cross-reactive IgE directed to carbohydrate determinants of glycoproteins. J Allergy Clin Immunol 1997; 100(3):327-34. Mittag D, Akkerdaas J, Ballmer-Weber BK, Vogel L, Wensing M, Becker WM et al. Ara h 8, a Bet v 1-homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy. J Allergy Clin Immunol 2004; 114(6):1410-7.

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

14. 15. 16.

17. 18. 19. 20. 21. 22. 23.

Cabanos C, Tandang-Silvas MR, Odijk V, Brostedt P, Tanaka A, Utsumi S et al. Expression, purification, cross-reactivity and homology modeling of peanut profilin. Protein Expr Purif 2010; 73(1):36-45. Javaloyes G, Goikoetxea MJ, Garcia N, I, Aranda A, Sanz ML, Blanca M et al. Pru p 3 acts as a strong sensitizer for peanut allergy in Spain. J Allergy Clin Immunol 2012; 130(6):1432-4.

Schuurman J, Perdok GJ, Lourens TE, Parren PW, Chapman MD, Aalberse RC. Production of a mouse/human chimeric IgE monoclonal antibody to the house dust mite allergen Der p 2 and its use for the absolute quantification of allergen-specific IgE. J Allergy Clin Immunol 1997; 99(4):545-50. Koppelman SJ, Knol EF, Vlooswijk RA, Wensing M, Knulst AC, Hefle SL et al. Peanut allergen Ara h 3: isolation from peanuts and biochemical characterization. Allergy 2003; 58(11):1144-51.

Wichers HJ, De Beijer T, Savelkoul HF, Van Amerongen A. The major peanut allergen Ara h 1 and its cleaved-off N-terminal peptide; possible implications for peanut allergen detection. J Agric Food Chem 2004; 52(15):4903-7. Piersma SR, Gaspari M, Hefle SL, Koppelman SJ. Proteolytic processing of the peanut allergen Ara h 3. Mol Nutr Food Res 2005; 49(8):744-55.

Pons L, Chery C, Romano A, Namour F, Artesani MC, Gueant JL. The 18 kDa peanut oleosin is a candidate allergen for IgE-mediated reactions to peanuts. Allergy 2002; 57 Suppl 72:8893.:88-93. Lee K, Ratnayake C, Huang AH. Genetic dissection of the co-expression of genes encoding the two isoforms of oleosins in the oil bodies of maize kernel. Plant J 1995; 7(4):603-11.

Asarnoj A, Nilsson C, Lidholm J, Glaumann S, Ostblom E, Hedlin G et al. Peanut component Ara h 8 sensitization and tolerance to peanut. J Allergy Clin Immunol 2012; 130(2):468-72.

Mittag D, Vieths S, Vogel L, Becker WM, Rihs HP, Helbling A et al. Soybean allergy in patients allergic to birch pollen: clinical investigation and molecular characterization of allergens. J Allergy Clin Immunol 2004; 113(1):148-54.

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Supplementary Table A ID

112

chalcomavoidatopy SPT sIgE lenge plaint ance

Eliciting ImmunoCAP ImmunoCAP ImmunoCAP ImmunoCAP dose peanut rAra h 1 rAra h 2 rAra h 3

kUA/L kUA/L kUA/L grams kUA/L 01n 0 E E + + none N10 0,10 0,10 0,10 0,10 02n 1 E F + strict N10 0,10 0,10 0,10 0,10 03p 2 ER E + + strict 1 0,10 0,10 0,10 0,10 04p 2 EA F + + strict 1 0,21 0,10 0,10 0,10 05n 0 E E + + strict N10 0,25 0,10 0,19 0,10 06n 0 EA E + strict N10 0,26 0,10 0,10 0,10 07n 0 EAR E + + relaxed N10 0,65 0,10 0,10 0,10 08n 0 EA E + + none N10 0,67 0,10 0,72 0,10 09n 0 EAR P + strict N10 0,79 0,10 0,10 0,10 10p 3 EAR P + + strict 10 0,79 0,10 0,23 0,10 11n 0 ER F + strict N10 1,12 0,10 0,10 0,10 12n 0 EAR E + + strict N10 1,24 0,10 0,10 0,10 13n 0 EAR P + strict N10 1,42 0,11 0,10 0,10 14p 3 EA P nt + strict 1 1,72 0,10 2,12 0,10 15n 0 EAR E + strict N10 2,69 0,10 0,10 0,10 16p 2 E P + + strict 10 2,82 1,37 0,72 0,10 17p 2 A F + + strict 0.3 2,97 0,89 2,54 0,10 18p 2 EAR F + + strict 1 2,99 0,22 1,53 0,10 19p 2 E F + + strict 1 3,07 0,10 0,91 0,10 20n 0 EAR E + + relaxed N10 3,65 0,10 0,10 0,10 21p 3 EA F + + strict 3 3,66 0,10 3,87 0,10 22p 2 EAR P + + strict 0.3 3,82 0,10 2,06 0,10 23p 2 E F + + strict 0.3 4,13 0,10 1,36 0,10 24p 2 EAR P + + strict 0.3 4,87 0,10 7,97 0,10 25p 2 EAR F + + strict 10 5,14 0,10 3,66 0,10 26n 1 E P + + strict N10 5,32 0,20 2,14 0,10 27p 2 EA P + + strict 10 5,45 0,10 3,55 0,10 28p 2 any? P + + strict 0.3 6,37 2,59 6,97 0,10 29p 2 EA F + + strict 0.1 7,30 0,10 0,19 0,11 + + strict 0.3 7,43 0,30 2,67 0,10 30p 2 E F 31n 0 EAR E + + strict N10 7,90 0,10 2,86 0,74 32p 2 ER P + + strict 1 8,30 2,18 3,57 0,10 33p 2 E P + + strict 10 9,90 0,44 11,9 0,10 34p 3 EA F + + strict 10 11,1 0,16 10,4 0,10 35p 3 EAR E + + strict 10 11,8 0,16 5,90 0,21 36n 0 EA P + strict N10 12,0 0,17 0,22 0,10 37p 2 EAR F + + strict 10 13,1 0,73 6,18 4,04 38n 0 ER E + + strict N10 15,2 0,10 3,67 1,24 39n 0 E E + strict N10 17,9 0,19 0,19 0,23 40p 2 EAR E + + strict 0.3 20,2 0,40 0,10 0,32 41p 4 EA E + + relaxed 0.3 21,7 4,62 7,29 0,10 42n 0 EA F + strict N10 22,8 0,22 0,56 0,40 43n 0 E E nt + relaxed N10 31,0 0,10 0,10 0,28 44n 1 EA P + + strict N10 33,0 0,76 15,2 0,92 45n 0 EA E + + strict N10 38,7 17,1 0,11 1,34 46p 3 E E + + relaxed 0.3 46,5 0,11 13,1 11,82 47p 2 EAR E nt + strict 0.01 57,8 16,16 25,4 0,84 48p 2 E P* + + relaxed 3 58,0 0,10 45,9 0,11 49p 3 EAR F + + strict 0.3 62,5 11,0 46,0 8,05 50p 2 EAR P + + strict 1 62,9 1,11 75,2 0,10 51p 2 E P + + strict 0.1 74,7 54,4 10,9 1,72 52p 3 any? P nt + strict 10 80,8 35,9 7,9 9,07 53p 2 any? P + + strict 3 84,4 8,16 78,2 0,18 54p 3 EAR P + + strict 0.1 98,5 67,9 17,3 6,06 55p 4 EA F + + strict 1 100 46,4 54,9 6,52 56p 2 EAR F + + relaxed 0.1 100 14,5 98,6 2,13 57p 2 EAR P + + strict 0.3 100 69,8 83,0 28,2 58p 2 EAR P + + strict 0.3 100 10,0 100 4,92 59n 0 EAR P* + + relaxed N10 100 100 100 85,6 60p 3 E P + + strict 10 100 94,6 100 79,1 61p 2 EAR E + + strict 1 100 95,0 100 14,7 62p 4 A P + + strict 0.3 100 100 100 94,5 100 100 100 60,1 63p 3 E P nt + strict 10 64p 4 A p + + strict 0.3 100 100 100 16,5 65p 2 E E + + strict 10 100 100 100 12,7 Patient characteristics Legend to the Columns. A: Patient code (n=negative challenge, p=positive challenge). B: Challenge score (Sampson score(11)). C: Atopic history other than food allergy (E: eczema; A: asthma; R: rhinitis). D: The reason of an allergy test (E: eczema; P: response to peanut; P*: 2 patients previously had a positive response to a peanut challenge; F: response to other foods). E-F:Allergy test prior to challenge (SPT; serum IgE to peanut).

Moving from peanut extract to peanut components: towards validation of component resolved IgE test

ImmunoCAP ImmunoCAP ImmunoCAP ImmunoCAP ImmunoCAP Sepharose Sepharose Sepharose Sepharose rAra h 6 rAra h 8 rAra h 9 rPru p 3 rPhl p 12 PVPP HAc BPP total IgE kUA/L kUA/L kUA/L kUA/L kUA/L kUA/L kUA/L IU/L kUA/L 0,10 0,10 0,10 0,10 0,10 0,10 0,10 0,10 51 0,10 0,10 0,10 0,10 0,10 0,10 0,17 0,10 30 0,10 0,10 0,10 0,10 0,10 1,35 nt 1470 0,10 13,48 0,10 0,10 0,10 14,4 16,6 10,2 202 0,20 0,63 0,10 0,10 0,10 0,31 0,50 0,29 37 0,30 0,10 0,10 0,10 0,10 0,12 0,43 0,26 53 0,14 38,7 0,10 0,17 0,10 2,92 0,47 0,11 1060 nt 0,10 0,10 0,19 0,10 0,64 1,09 0,53 256 0,36 1,52 0,12 0,10 0,10 1,40 nt 876 0,29 14,18 0,10 0,10 0,10 0,73 2,03 0,28 2860 0,10 0,16 0,10 0,10 0,10 6,36 3,56 0,14 116 1,09 11,75 0,10 0,83 0,10 2,17 1,31 1,26 2300 0,10 0,19 0,10 3,93 0,18 2,31 3,27 0,93 2300 1,18 1,59 0,10 0,10 0,10 1,10 2,61 1,12 250 0,23 66,0 0,10 0,10 3,03 0,59 3,18 0,10 583 0,42 0,10 0,10 0,27 0,10 1,02 0,78 0,34 183 1,39 17,4 0,10 0,10 0,10 1,82 4,02 1,01 245 2,81 2,64 0,10 0,10 0,10 2,10 3,66 1,97 558 1,43 1,16 0,10 0,10 0,11 2,19 3,34 1,62 90 0,10 28,6 0,10 0,10 3,35 3,34 nt 354 3,92 0,10 0,10 0,10 0,10 2,90 4,91 4,77 252 1,23 64,7 0,10 0,12 0,10 2,32 4,49 0,71 450 3,93 0,35 0,10 0,10 0,10 8,00 nt nt 4,01 4,48 0,10 0,18 nt 3,97 nt 191 1,79 9,83 0,10 0,10 0,10 2,79 6,39 1,40 480 2,37 1,86 0,10 0,10 0,13 5,00 5,44 1,79 864 4,05 0,73 0,10 0,10 0,10 8,85 6,74 1,68 600 1,27 0,10 0,10 0,10 0,10 6,43 9,07 3,66 35 5,26 0,10 0,10 0,48 0,10 4,37 3,95 5,39 901 6,43 0,21 3,12 3,76 1,13 8,73 10,9 7,40 305 0,71 31,52 0,14 0,15 0,22 4,60 9,90 1,18 2920 2,99 0,25 0,10 0,10 0,10 6,92 7,61 2,02 181 4,94 0,10 0,10 0,10 0,10 63,3 68,6 5,82 30 8,54 0,10 0,10 0,10 0,10 8,49 12,3 9,85 353 4,05 31,16 0,33 5,65 0,22 16,3 15,3 4,79 3350 0,13 58,9 0,10 1,28 0,10 5,43 18,2 2,54 1600 1,93 2,66 0,12 0,12 0,14 9,79 8,73 2,11 1520 3,33 0,10 4,43 1,91 0,16 16,3 9,86 3,28 800 0,59 17,0 0,34 0,42 9,85 9,93 nt 3470 0,48 8,37 35,2 28,7 10,9 1100 12,6 0,10 0,10 12,2 0,17 0,10 0,21 0,10 17,5 21,9 16,3 165 0,40 100 0,29 0,60 0,30 11,4 30,5 16,8 4360 1,19 0,94 0,10 0,10 0,10 23,5 5,28 1,18 705 9,43 40,5 0,37 2,36 0,18 34,0 21,5 8,18 2300 0,34 71,7 0,31 5,77 0,19 62,5 51,9 7,49 3350 2,65 10,4 0,24 0,97 11,50 22,6 33,1 3,33 2300 22,1 0,10 0,10 0,10 0,10 37,9 39,5 32,5 325 40,5 4,85 0,52 9,25 0,14 68,4 nt 2340 30,2 0,10 0,10 0,11 4,86 81,2 45,1 19,4 737 48,9 2,09 0,10 0,10 0,10 52,6 65,8 47,0 719 8,07 2,10 0,10 0,44 0,10 46,7 17,3 10,9 217 7,07 0,10 0,10 0,10 0,22 51,3 17,3 18,6 511 39,3 0,15 0,10 0,16 0,64 240 168 45,7 1610 30,9 0,10 0,10 0,10 0,10 61,3 43,8 44,2 79 2,05 nt 0,20 0,79 nt 136 115 56,5 2200 49,9 9,38 0,10 0,13 0,17 86,4 114 47,1 2300 41,3 10,6 0,13 0,12 0,10 161 129 50,0 508 60,5 53,7 0,14 0,93 5,59 500 500 89,3 2136 73,5 0,11 0,15 0,17 0,26 1000 413 220 1060 85,7 5,77 0,40 2,25 34,5 1000 1000 315 2300 84,2 10,5 0,30 0,42 0,10 236 202 188 1000 97,3 0,11 0,12 0,21 0,20 1000 355 255 2040 80,7 0,10 0,10 0,10 0,10 297 272 127 808 58,3 2,91 0,10 0,10 0,10 479 1000 116 437 93,4 1,80 0,10 0,10 0,10 500 500 87,7 950 G: Exposure to peanut prior to challenge (0: strict avoidance; 1: relaxed avoidance; 2: no avoidance). 01n had no response to an open challenge performed at home. H: Eliciting dose in grams. N10: no response to the maximum dose of 10 grams. I-Q: ImmunoCAP serology: IgE to peanut and single allergens components (kUA/L). nt: not tested. R-U: Sanquin serology: total IgE (kIU/L) and peanut IgE to PVPP extract, HAc extract and BPP(kUA/L). BPP = Basic peanut protein fraction obtained from the HAc extract. Due to limited plasma availability some Sepharoses were not tested (nt) or tested only as a combination (PVPP + HAc).

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Chapter 7 Discussion

Partly published in J Allergy Clin Immunol Pract 2014 635-6 Aalberse JA & Aalberse RC

Chapter 7

Food allergy: Current and future diagnosis and implications for treatment In this chapter I will discuss the difficulties of allergen specific diagnosis and the role of alarmins in diagnosis and treatment. PART 1

The clinical relevance of sensitization In chapter 6 we have discussed the difficulties of the diagnosis with a serum IgE test. We have shown that regularly used IgE tests for peanut sensitization are deficient of certain allergens and have discussed the importance of an improved test to increase the sensitivity of the test.

It is both important to have a sensitive test and to know what allergens are clinically relevant.

The first is especially important for general practitioners, the latter is more important for more specialized doctors in the hospital.

The current guideline states that an IgE test should not be performed by a GP, because of low

sensitivity and specificity. As will be discussed below, these guideline need to be reconsidered. The diagnosis of food allergy is based upon the history of the patient, a skin prick test and/

or serum IgE tests, the latter two combined referred to as sIgE test. The sIgE test is often false

positive. In an unselected population up to 10% of people are found to have an SPT positive to

peanut(1). As only 0.5-1% is found to be truly allergic in the general population, up to 90-95%

of positively tested individuals for peanut do not have a clinical response. Even in a selected patient group suspected for peanut allergy on history of an allergic response upon exposure,

50% is sensitized without a clinical response(2;3). As discussed below new tests show better predicting outcomes, but adjustments of the test are clearly needed.

The gold standard for the diagnosis of peanut allergy, like other food allergies, is the double blind placebo controlled food challenge(4). This is an academic clinical diagnosis, but not

a workable diagnosis for general practitioners and smaller hospitals. Even in academic research it is often not included in food allergy diagnosis. The search for tests that can more accurately predict a clinical response is therefore important.

As we have discussed in chapter 6, many conventional extracts are deficient in some sig-

nificant IgE binding components, which can result in a low sensitivity of the test. Also some components are clinically irrelevant, causing a low specificity of the test.

The accuracy of a test can be described with several parameters of which sensitivity and spec-

ificity are most often used, but also positive predictive value (PPV) and negative predictive value (NPV) can aid in interpretation of a test. These parameters often cause confusion, but can be very helpful to understand the value of a test(5). 116

Discussion

Sensitivity relates to the testâ&#x20AC;&#x2122;s ability to identify a condition correctly. In other words how often is the food specific IgE test positive when you are food allergic. Specificity relates to the

testâ&#x20AC;&#x2122;s ability to exclude a condition correctly. In other words how often is the IgE test found negative if a subject is not food allergic.

These tests are independent of the population they are used for. However PPV and NPV describe the proportion of the test that is truly positive or negative, respectively, which can be

different in a general practice population and in a patient population visiting a specialized

physician in a secondary or tertiary referral clinic. The NPV is especially of importance in a GP

practice. A high NPV can rule out food allergy with high certainty. Remaining patients can then

be referred to the specialized physician who needs a high PPV lowering the need for DBPCFC. As can be seen in figure 1 the NPV and PPV are dependent on the sensitivity and specificity of the test, but also on the expected prevalence of the disease, which is underlined by the differ-

ence in NPV and PPV in the population of a general practice and the secondary and tertiary referral clinic.

In the current protocol used by GPs in food allergy it is suggested to refrain from measuring

IgE because the test result has insufficient specificity and sensitivity(6). With the increased

technical knowledge on peanut allergy diagnosis as well as based on the different prevalence

numbers in a general practice compared to secondary and tertiary referral clinics, I would suggest that the serum IgE test for peanut is included in the test protocol for GPâ&#x20AC;&#x2122;s to be able

to rule out peanut allergy. However when the patient tests positive for a food, it is very important that the patient is well informed on the high chances of false positive values. Component resolved diagnostics and an ongoing need of food extracts

In recent years technical progress has been made that enables the measurement of binding

of IgE to multiple single allergens instead of crude extracts via so-called component resolved diagnostics (CRD). As we have shown in chapter 6 the development of CRD can be of help to achieve a better diagnosis. It has been suggested that components can replace extract based

testing. For peanut it has been shown that the measurement of solely Ara h 2 and Ara h 6 is

sufficient for diagnosis. Often inclusion criteria are however based on deficient extract, which could bias the importance of certain allergens. We have discussed in an editorial on hazelnut

allergy that CRD is a helpful tool to improve IgE diagnosis but cannot yet replace extract-based

testing. We have argued that we can learn from component-based tests how to improve our extract-based testing until we can definitely use only CRD (see textbox).

117

118

IgE test

food allergy total B PT D NT ND T

86% 74% sensitivity specificity

(Academical Center) food allergy + 3600 1500 + 600 4300 total 4200 5800

86% 74% sensitivity specificity

total 5100 4900 10000

(5 times an average practice = 10000 patients) food allergy + total 600 2400 3000 + 100 6900 7000 total 700 9300 10000

A/PD D/ND sensitivity specificity

+ A - C total PD

42% pretest probability

71% PPV 88% NPV

7% pretest probability

20% PPV 98.6% NPV

PD/T pretest probability

IgE test

Specialist

IgE test

+ total

food allergy + total 665 2400 3065 35 6900 6935 700 9300 10000

95% 74% sensitivity specificity

food allergy + 4000 1500 5500 200 4300 4500 4200 5800 10000

95% 74% sensitivity specificity

Figure 1c; increased sensitvity General Practice

A/PT positive predictive value (PPV) D/NT negative predictive value (NPV)

42% pretest probability

73% PPV 96% NPV

7% pretest probability

22% PPV 99.5% NPV

IgE test

Specialist + total

95% 95% sensitivity specificity

food allergy + total 4000 300 4300 200 5500 5700 4200 5800 10000

95% 95% sensitivity specificity

Figure 1d: increased specificity General Practice food allergy + total 665 435 1100 + IgE test 35 8865 8900 total 700 9300 10000

42% pretest probability

93% PPV 96% NPV

7% pretest probability

60% PPV 99.6% NPV

and specificity. This is of important for the is interpretation of these tests in either a general practice orvalues in a hospital. The key message this figure that the negative and positive predictive can be different depending on the population it is tested in, despite the same sensitivity and specificity. This is important for the interpretation of these tests in either a general practice or in a hospital.

The key message of this figure is that the negative and positive predictive values can be different depending on the population it is tested in, despite the same sensitivity

Legend to figure 1 Legend to figure 1 This figure illustrates the concepts of sensitivity, specificity, positive predictive value and negative predictive value. This figure illustrates the concepts of sensitivity, specificity, positive predictive value and negative predictive value. Figure 1a| Formulas | Formulas the 2x2A=table. A=ofnumber of correct positive tests; B= number ofnumber false positive tests; tests; C= number ofoffalse negative Figure 1a of theof 2x2 table. number correct positive tests; B= number of false positive tests; C= of false negative D= number correct nega- tests; D= number of correct negative tests. number of positive tests; NT= number of negative PD= number of disease NDof number of nopresent; disease present. T: total number of tests. present. T: total number of tests. tive tests.PT= PT= number of positive tests; NT= numbertests; of negative tests; PD=present; number disease ND number of no disease Figure 1b-d | In these figures the numbers chosen are not related to a true situation but give estimates assuming a pre-test probability of 7% in general practice and 42% Figure 1b-d | In these figures the numbers chosen are not related to a true situation but give estimates assuming a pre-test probability of 7% in general practice and 42% at the specialist. at the specialist. Figure 1b1b | This box indicates the baseline Figure | This box indicates thesituation. baseline situation. Figure 1c1c | In| the box the box sensitivity is increased. Sensitivity can be increased by adding missing allergens by withadding better extracts or viaallergens CRD, but also by decreasing Figure In second the second the sensitivity is increased. Sensitivity can be increased missing with better extracts or via CRD, but also by the cut off value for a positive test. Both interventions will cause an increase in sensitivity. decreasing the cut off value for a positive test. Both interventions will cause an increase in sensitivity. Figure 1d | In the final box on the right also the specificity in increased. This can be achieved by removing clinical irrelevant allergens. Figure 1d | n the final box on the right also the specificity in increased. This can be achieved by removing clinical irrelevant allergens.

Specialist

IgE test

Figure 1b; baseline General Practice

IgE test

Figure 1a Formulas

Chapter 7

Discussion

The point is that patients with a very high or a very low likelihood of sensitization are not informative for deciding how useful it is to add another test to a diagnostic protocol and that it is not statistically helpful to increase numbers by mixing groups of subjects with very different pretest likelihoods. Although subjects with a moderate pretest likelihood are most informative, subjects with a very low or a very high likelihood can help us to decide which components might be useful in diagnostic protocols. Tolerant subjects are informative in 2 ways. First, to establish which allergens are least likely to be clinically relevant. Second, to establish which characteristics* make subjects likely to be food sensitized without symptoms on oral exposure. Sera from patients who are evidently symptomatic are important to identify the spectrum of allergenic proteins and to check whether all these components are adequately present in diagnostic extracts. If not, we need to decide whether these deficient components (such as Cor a 1, a prominent allergen in the majority of the patients who are provocation positive) are useful to add to our diagnostic protocol. The proposed “additional studies”** require oral provocation. To recruit patients for provocation studies, most protocols rely on tests with extracts for inclusion. We have discussed this potential bias elsewhere in relation to peanut components.5 Our conclusion was that it is impossible to judge the clinical relevance of an allergic component if the extract used for inclusion of patients is inefficient in detecting sensitization to this component (such a Cor a 1 in hazelnut extracts). Another issue is how to deal with potential cross-reactivity, for example, between the birch pollen allergen Bet v 1 and Cor a 1. Should patients with pollen sensitization be excluded from these studies, and, if so, how? IgE induced by exposure to birch pollen often reacts with Cor a 1.01 from hazel pollen and Cor a 1.04 from hazelnut.6 Because many patients who are allergic to birch tolerate hazelnuts despite such cross-reactive antibodies, it is tempting to conclude that IgE to hazelnut with a patient who is sensitized to birch is clinically irrelevant. However, 85% of the clinically reactive patients in the article by Kattan et al are Cor a 1 positive***. The effect of sensitization to cross-reactive pollen components will be an important aspect of the future studies. Similarly, the effect of sensitization to fruit-derived lipid transfer protein (eg, peach) should be taken into account.(7,8) 7. Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-70.

8. Schocker F, Lüttkopf D, Scheurer S, Petersen A, Cisteró-Bahima A, Enrique E, et al. Recombinant lipid transfer protein Cor a 8 from hazelnut: a new tool for in vitro diagnosis of potentially severe hazelnut allergy. J Allergy Clin Immunol 2004;113:141-7

This textbox is a citation from an Editorial by Aalberse and Aalberse; A lesson from component resolved testing: we need better extracts. Journal of Allergy and Clinical Immunology in Practice 2014 SepOct;2(5):635-6. doi: 10.1016/j.jaip.2014.07.006. * Characteristics of allergens. Another subject dependent characteristic is IgG

** The need for additional studies was proposed by Kattan et al (J Allergy Clin Immunol Pract. 2014 Sep-Oct;2(5):633-4), in response to whom this Editorial was written. *** In contrast to the situation in paediatric hazelnut allergy, pollen allergy is very often found in genuine adult peanut allergy. In the latter situation, measuring IgE to Ara h 8 is not informative and the diagnosis is much more dependent on IgE to Ara h 2 or Ara h 6.

119

Chapter 7

The IgE responses to hazelnut allergens show many similarities compared to peanut allergens, however some aspects are clearly different. Both in peanut- and in hazelnut allergy subjects are often also pollen allergic. Cor a1 and Ara h8 are allergens that show cross-reactivity in pollen allergic subjects with hazelnut and peanut allergy, respectively. In hazelnut allergy IgE to just Cor a1

is often seen. However in symptomatic peanut allergy IgE to only Ara h8 is uncommon(7). IgE to

Ara h2 and Ara h6 have shown to be important components in peanut allergy diagnosis(8). Future

studies will have to evaluate if also other allergens are needed for diagnosis of peanut allergy. Predicting severity of a food allergic response

Next to the difficulties in relation to sensitization, another diagnostic problem is that when a

positive diagnosis is made, it is hard to predict severity (i.e. the threshold dose needed for a response and local versus systemic complaints)(9). Skin prick testing and measuring serum-spe-

cific IgE are not consistent and reliable in terms of predicting theÂ severityÂ of reactions. Also DB-

PCFC are valuable to diagnose the food allergic response, but the dose response thresholds are not reproducible(10). Recently it was suggested that the BAT test (basophil activation test) can

predict severity, but the data is still insufficient to draw conclusions(11). Severe allergic reactions

are more likely to occur in older patients and those with underlying asthma(12). Other data sug-

gest the role of biological mediators in allergic reactions such as platelet-activating factor. These may provide a future additional tool in predicting those at risk of severe reactions(13).

Phenotypes and endotypes

Beyond the problem of insufficient food extracts or clinically irrelevant allergens, it is likely that other factors also play a role in the difficulties of diagnosis. The diagnosis of asthma

and rhinitis were similarly difficult several years ago, but this has gradually changed. It has

become clear that asthma and allergic rhinitis are not single entities but syndromes(14;15). It is plausible that food allergy is also a syndrome with different subgroups.

A phenotype can be defined as a feature or a cluster of features that can discriminate that

subgroup from the general population. An endotype is a phenotype with a pathophysiologic

character. Little is known so far on phenotypes and endotypes in food allergy. Classifications based on phenotypes and endotypes in rhinitis, but especially in asthma have promising implications on the management(16). In the future a similar classification could result in a more

uniform diagnosis and better response to treatment also in food allergy. Phenotypes and endotypes in asthma and rhinitis.

A group of experts proposed distinct endotypes based on seven parameters: clinical characteristics, biomarkers, airway physiology, genetics, histopathology, epidemiology and treat-

ment response(15). This has lead to practical allergy classifications (PRACTALL) in asthma and

rhinitis. Another convincing example of the success of endotypes is the IL-13 based endo-

types, which has been shown to have a clear response to inhaled corticosteroids(17). In a phase

II study it was shown that a subgroup of patients with high levels of periostin, a surrogate

120

Discussion

marker for IL-13 activity, improved significantly upon treatment with anti IL-13. Discriminat-

ing endotypes aids in diagnosis and treatment in asthma and rhinitis, but a practical allergy classification has not yet been made for food allergy. Phenotypes and endotypes in food allergy

It is likely that food allergy is also a syndrome, including distinct phenotypes. In the last few years a small number of studies have described different phenotypes. It is a challenge to formulate endotypes in food allergy that may help to establish new treatment possibilities.

A large prospective cohort identified distinct phenotypes in children in their first year of life with wheeze, eczema or food sensitization(18). The five phenotypes found included no allergic disease, non-food sensitized eczema, egg allergy, multiple food allergies (predominantly pea-

nut) and multiple food allergies (predominantly egg). Eosinophilic oesophagitis and food pro-

tein induced enterocolitis syndrome have recently been suggested as phenotypes linked to food allergy(19). Additional endotypes can be formulated by discriminating patients within the groups

that are proven peanut allergic or by comparing differences between peanut allergic to only

peanut sensitized individuals. Tryptase has been associated with a severe acute allergic response (anaphylaxis) with a higher basal serum level of tryptase in anaphylactic patients com-

pared to patients without anaphylaxis. We and others have compared peanut allergic to just

peanut sensitized children and compared cytokine responses. In 2012 Xie et al found clearly higher IL-9 production after in vitro peanut stimulation in the peanut allergic group compared to the peanut tolerant individuals(20;21). In our study in chapter 5 we have shown that IL-25,

an IL-17 family member, is highly elevated in plasma only in some children with a clinical response to peanut: a subgroup in the peanut allergic patients. This suggests a role for IL-25 in

the pathogenesis of peanut allergy and elevated plasma IL-25 may be a sign of a chronic severe atopic phenotype(22). Overproduction of the uric acid (UA) alarmin in the local microenviron-

ment plays a critical role in the induction of peanut-allergic sensitization, likely due to its ability

to activate DCs. These findings suggest that cellular damage or tissue injury may be an essential

requisite for the development of allergic sensitization to foods(23). In chapter 2, 3 and 4 we have

discussed the potential role of another alarmin, HSP (heat shock proteins), in atopic disease (see also part 2 in this chapter).

For the food allergic syndrome, endotypes have only just started to be formulated and large cohorts are needed to discriminate between the endotypes. The above mentioned seven se-

lected parameters, with clinical characteristics (food sensitized, but tolerant versus clinically

reactive), biomarkers (UA, IL-25, IL-9, tryptase), mouth/gut physiology (locally activated DC’s and mast cells), genetics, histopathology, epidemiology (including age of onset), and treat-

ment response have great potential to formulate new distinct endotypes in food allergy with better diagnostic possibilities and improved treatment results.

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Chapter 7

PART 2 Heat shock proteins and alarmins in allergic disease. Immunogenic responses to heat shock proteins This thesis shows that HSP is immunogenic both in a disease free environment as in patients with atopic dermatitis(24;25). At the start of our research data primarily suggested that HSP is an in-

teresting protein because of its potential of inducing regulatory T cells(26). Therefore when our

research was initiated this had our prime interest. Latter research, including our own, additionally showed that HSP not only induces regulatory T cells, but also activated, non-regulatory T cells,

depending on the environment in which the immune response takes place(27-29). In chapter 3 we show that immunogenic regulatory responses to HSP are already present in cord blood(24). The

immune cells found in cord blood are taken from a disease free, relatively sterile environment, directly after birth. This supports the hypothesis that responses to HSP can be a healthy immu-

nogenic response. In chapter 4 we show that immune responses to HSP are stronger in children

with atopic dermatitis than in non-atopic children(25). These responses have a pro-inflammatory

character indicating that responses towards HSP are dependent on the environment. Alarmins in allergic inflammation

HSP are endogenous proteins that have an important chaperone function in the cell, upregulated dur-

ing stress, that are present in abundance(30). These endogenous HSP have a molecular mimicry with

microbes(31). Interestingly, HSP have an effect both on the innate as the adaptive immune system(32). Endogenous proteins, like HSP, that are released upon tissue damage and that can activate the innate immune system have been called damage associated molecular patterns (DAMPs) or alarmins(33-35).

Recent data have suggested that alarmins contribute to the dysregulated processes seen in chronic

inflammation. Research in mice has shown the presence of the proteins early in the inflammation, including in a contact dermatitis model(36). In this thesis we show that HSP is found more in lesional

skin than in non-lesional skin of atopic dermatitis patients(25). Other research has shown a role for

IL-33 as an important alarmin in epithelia and lymphoid organs in allergy(37). Just recently a study

was published on the role of uric acid and its involvement in peanut allergy(23). The authors showed

that when in mice uric acid is blocked peanut sensitization could be blocked. Elevated levels of uric acid were seen in peanut allergic children compared to healthy controls. Together these data suggest that in allergic settings triggers of the innate immune system such as alarmins may play a role in sustaining the (partly) allergen-independent chronic inflammation that is present in the tissues. A very

interesting observation from this thesis (as discussed in chapter 5) is that a subgroup of children that all had a positive DBPCFC to peanut without exposure to peanut for years, showed highly increased circulating levels of IL-25. Since gut epithelial cells are a major source of IL-25 this suggest that also

in a (sub)group of peanut allergic children there may be chronic tissue inflammation involvement(22). The role of endogenous alarmins in the initiation and maintenance of tissue inflammation in

allergic disease and their role as potential therapeutic targets is one of the interesting and 122

important avenues for future research, as partly reviewed in chapter 2(38).

Discussion

PART 3 Management of peanut allergy Peanut allergy management It can be concluded that there is need for improvements in the management of food allergy

by better sensitization tests, but also by identifying different phenotypes and endotypes. Im-

proved diagnosis is likely to result in more targeted therapy including appropriate diets. As discussed below immunotherapy is the only curative therapy in allergic rhinitis and possibly also in food allergy. Combining food allergy immunotherapy with anti IgE treatment might

reduce the risk of side effects while allowing for more rapid desensitization schemes. On the other hand, it is plausible that alarmins can be a potential target for therapy in chronic allergic inflammation, including food allergy. Allergen Specific Management

Allergen specific immunotherapy is the only curative therapy, mostly applied in tree- and grass pollen-allergic patient(39). Specific immunotherapy induces clinical tolerance by applying in-

creasing doses of the allergen(40). The effect seen with immunotherapy in venom or aero aller-

gies has different pathophysiologic mechanisms compared to immunotherapy in food allergies. Whereas in immunotherapy for allergic rhinitis the altered T cells responses and decreased IgE production with an increase in IgG4 clearly plays an important role, in food allergy desensitiza-

tion of mast cells and basophils seems to contribute most to the effect of immunotherapy(41-43). A small number of trials in peanut allergy have shown a reduced risk of allergic symptoms after

therapy(44;45). The down side of immunotherapy in general is its duration, and in peanut specif-

ic immunotherapy also the high rate of adverse reactions, and waning of the protection after stopping the active therapy(46). As in other allergens, an improved effectiveness with less side-ef-

fects is suggested via T-cell-reactive peptides and chemically modified allergens in peanut aller-

gy(47-49). Currently, peanut specific immunotherapy is not in the stage that it can be applied in clinic. A different approach is to prevent peanut allergy. Recently it was published that peanut

allergy can be prevented by early exposure to peanut(50;51). Oral exposure to peanut early in life

was even found to diminish already existing allergic complaints. This is an exciting study, but needs to be repeated and further evaluated to draw clear conclusions. Immunotherapy based on endotypes

As discussed above new endotypes are designed to be used for therapy. It is conceivable that

biologicals specifically aimed at an endotype and its immunopathomechanism can be used also in food allergy in the future(21;52). Anti-IgE is the first biological used in peanut allergy(53;54). Both in adults and in children with high-risk peanut allergy, treatment with omalizumab (an-

ti-IgE) facilitated rapid oral desensitization and qualitatively improved the desensitization process(55;56). As seen in asthma, it is likely that with the introduction of new biologicals such as anti-IL-13 and anti-IL-25, new subtypes or endotypes will be demonstrated that will improve the diagnosis and treatment responses(57-60).

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Chapter 7

Immunotherapy based on alarmins Alarmins such as HSP, clearly play a role in (local) inflammation. They may therefore be interesting targets for therapy. As discussed by Chan et al., it first needs to be defined which alarmins are important in a specific type of inflammation, and if this depends on the different phas-

es of an inflammatory response(61). A big challenge concerns the issue of balance. The same

alarmins can be both harmful and beneficial within the same disease context, depending on

the disease activity and the environment (chapters 2,3, and 4) (24;25;38). While dampening of

inflammation may be desirable in certain pathological contexts, total abolition of the host defense is disadvantageous, leaving the patient at risk to opportunistic infections and malig-

nancies as well as impairing repair and remodelling pathways.Â Therefore further research is needed to evaluate the overall effect of targeting alarmins.

In conclusion, it is to be expected that a combination of allergen specific immunotherapy, bi-

ologicals and alarmin-related therapy will be needed to tackle specific clinical manifestation and progression of peanut allergy and other food allergies. Improved diagnostics, including

newly discovered allergens, as well as better-defined endotypes, will contribute to a better understanding of the disease with better management of peanut allergy.

124

Discussion

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Steurer J, Fischer JE, Bachmann LM, Koller M, ter Riet G. Communicating accuracy of tests to general practitioners: a controlled study. BMJ 2002; 324(7341):824-6. NHG-Standaard Voedselovergevoeligheid (Eerste herziening). Ref Type: Generic

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van Erp FC, Lagrouw J. KACMYvdECKKEF. The diagnostic value of the basophil activation test using Ara h 2 and Ara h 6 in children with suspected peanut allergy. [EAACI abstracts]. 2015. Ref Type: Generic van Erp FC, Knulst AC, Kentie PA, Pasmans SG, van der Ent CK, Meijer Y. Can we predict severe reactions during peanut challenges in children? Pediatr Allergy Immunol 2013; 24(6):596-602.

Glaumann S, Nopp A, Johansson SG, Borres MP, Nilsson C. Oral peanut challenge identifies an allergy but the peanut allergen threshold sensitivity is not reproducible. PLoS One 2013; 8(1):e53465. Santos AF, Du Toit G, Douiri A, Radulovic S, Stephens A, Turcanu V et al. Distinct parameters of the basophil activation test reflect the severity and threshold of allergic reactions to peanut. J Allergy Clin Immunol 2015; 135(1):179-86.

van der Zee T, Dubois A, Kerkhof M, van der Heide S, Vlieg-Boerstra B. The eliciting dose of peanut in double-blind, placebo-controlled food challenges decreases with increasing age and specific IgE level in children and young adults. J Allergy Clin Immunol 2011; 128(5):1031-6. Flinn A, Hourihane JO. Allergic reaction to peanuts: can we predict reaction severity in the wild? Curr Allergy Asthma Rep 2013; 13(6):645-50. Papadopoulos NG, Bernstein JA, Demoly P, Dykewicz M, Fokkens W, Hellings PW et al. Phenotypes and endotypes of rhinitis and their impact on management: a PRACTALL report. Allergy 2015;10.

Lotvall J, Akdis CA, Bacharier LB, Bjermer L, Casale TB, Custovic A et al. Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome. J Allergy Clin Immunol 2011; 127(2):355-60. 125

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Corren J. Asthma phenotypes and endotypes: an evolving paradigm for classification. Discov Med 2013; 15(83):243-9.

Woodruff PG, Modrek B, Choy DF, Jia G, Abbas AR, Ellwanger A et al. T-helper type 2-driven inflammation defines major subphenotypes of asthma. Am J Respir Crit Care Med 2009; 180(5):388-95.

Peters RL, Allen KJ, Dharmage SC, Lodge CJ, Koplin JJ, Ponsonby A et al. Differential factors associated with challenge-proven food allergy phenotypes in a population cohort of infants: a latent class analysis. Clin Exp Allergy 2014;10. Nowak-Wegrzyn A, Katz Y, Mehr SS, Koletzko S. Non-IgE-mediated gastrointestinal food allergy. J Allergy Clin Immunol 2015; 135(5):1114-24.

Brough HA, Cousins DJ, Munteanu A, Wong YF, Sudra A, Makinson K et al. IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy. J Allergy Clin Immunol 2014; 134(6):1329-38.

Xie J, Lotoski LC, Chooniedass R, Su RC, Simons FE, Liem J et al. Elevated antigen-driven IL-9 responses are prominent in peanut allergic humans. PLoS One 2012; 7(10):e45377. Aalberse JA, van Thuijl AO, Meijer Y, de Jager W, van der Palen-Merkus T, Sprikkelman AB et al. Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut. Clin Transl Allergy 2013; 3(1):40-3.

Kong J, Chalcraft K, Mandur TS, Jimenez-Saiz R, Walker TD, Goncharova S et al. Comprehensive metabolomics identifies the alarmin uric acid as a critical signal for the induction of peanut allergy. Allergy 2015;10.

Aalberse JA, Kapitein B, de Roock S, Klein MR, de Jager W, van der Zee R et al. Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60). PLoS One 2011; 6(9):e24119. Kapitein B, Aalberse JA, Klein MR, de Jager W, Hoekstra MO, Knol EF et al. Recognition of selfheat shock protein 60 by T cells from patients with atopic dermatitis. Cell Stress Chaperones 2013; 18(1):87-95. van Eden W, van der Zee R, Prakken B. Heat-shock proteins induce T-cell regulation of chronic inflammation. Nat Rev Immunol 2005; 5(4):318-30.

de Kleer I, Vercoulen Y, Klein M, Meerding J, Albani S, van der Zee, R et al. CD30 discriminates heat shock protein 60-induced FOXP3+ CD4+ T cells with a regulatory phenotype. J Immunol 2010; 185(4):2071-9. van Wijk F, Prakken B. Heat shock proteins: Darwinistic immune modulation on dangerous grounds. J Leukoc Biol 2010; 88(3):431-4. Henderson B, Pockley AG. Molecular chaperones and protein-folding catalysts as intercellular signaling regulators in immunity and inflammation. J Leukoc Biol 2010; 88(3):445-62.

Quintana FJ, Cohen IR. Heat shock proteins as endogenous adjuvants in sterile and septic inflammation. J Immunol 2005; 175(5):2777-82.

van Eden W. Heat-shock proteins as immunogenic bacterial antigens with the potential to induce and regulate autoimmune arthritis. Immunol Rev 1991; 121:5-28.:5-28.

Discussion

32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

van Eden W, Koets A, van Kooten P, Prakken B, van der Zee R. Immunopotentiating heat shock proteins: negotiators between innate danger and control of autoimmunity. Vaccine 2003; 21(9-10):897-901. Bianchi ME. DAMPs, PAMPs and alarmins: all we need to know about danger. J Leukoc Biol 2007; 81(1):1-5.

Medzhitov R. Origin and physiological roles of inflammation. Nature 2008; 454(7203):428-35. Oppenheim JJ, Yang D. Alarmins: chemotactic activators of immune responses. Curr Opin Immunol 2005; 17(4):359-65.

Vogl T, Eisenblatter M, Voller T, Zenker S, Hermann S, van Lent P et al. Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity. Nat Commun 2014; 5:4593. doi: 10.1038/ncomms5593.:4593.

Cayrol C, Girard JP. IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy. Curr Opin Immunol 2014; 31:31-7. doi: 10.1016/j.coi.2014.09.004. Epub;%2014 Sep 29.:31-7. Aalberse JA, Prakken BJ, Kapitein B. HSP: Bystander Antigen in Atopic Diseases? Front Immunol 2012; 3:139. doi: 10.3389/fimmu.2012.00139. eCollection;%2012.:139.

Holgate ST, Polosa R. Treatment strategies for allergy and asthma. Nat Rev Immunol 2008; 8(3):218-30. Bousquet J, Lockey R, Malling HJ. Allergen immunotherapy: therapeutic vaccines for allergic diseases. A WHO position paper. J Allergy Clin Immunol 1998; 102(4 Pt 1):558-62.

Radulovic S, Calderon MA, Wilson D, Durham S. Sublingual immunotherapy for allergic rhinitis. Cochrane Database Syst Rev 2010;(12):CD002893. Thyagarajan A, Jones SM, Calatroni A, Pons L, Kulis M, Woo CS et al. Evidence of pathwayspecific basophil anergy induced by peanut oral immunotherapy in peanut-allergic children. Clin Exp Allergy 2012; 42(8):1197-205.

Gorelik M, Narisety SD, Guerrerio AL, Chichester KL, Keet CA, Bieneman AP et al. Suppression of the immunologic response to peanut during immunotherapy is often transient. J Allergy Clin Immunol 2014;(14):10. Nurmatov U, Venderbosch I, Devereux G, Simons FE, Sheikh A. Allergen-specific oral immunotherapy for peanut allergy. Cochrane Database Syst Rev 2012; 9:CD009014. doi: 10.1002/14651858.CD009014.pub2.:CD009014. Anagnostou K, Clark A. Peanut immunotherapy. Clin Transl Allergy 2014; 4:30. doi: 10.1186/2045-7022-4-30. eCollection;%2014.:30-4. Jones SM, Pons L, Roberts JL, Scurlock AM, Perry TT, Kulis M et al. Clinical efficacy and immune regulation with peanut oral immunotherapy. J Allergy Clin Immunol 2009; 124(2):292-300, 300. Larche M. Update on the current status of peptide immunotherapy. J Allergy Clin Immunol 2007; 119(4):906-9.

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48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61.

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Lund L, Henmar H, Wurtzen PA, Lund G, Hjortskov N, Larsen JN. Comparison of allergenicity and immunogenicity of an intact allergen vaccine and commercially available allergoid products for birch pollen immunotherapy. Clin Exp Allergy 2007; 37(4):564-71. Van Hoffen E, van der Kleij HP, den Hartog Jager CF, van Doorn WA, Knol EF, Opstelten DJ et al. Chemical modification of peanut conglutin reduces IgE reactivity but not T cell reactivity in peanut-allergic patients. Clin Exp Allergy 2014; 44(12):1558-66.

Du Toit, G, Roberts G, Sayre PH, Bahnson HT, Radulovic S, Santos AF et al. Randomized trial of peanut consumption in infants at risk for peanut allergy. N Engl J Med 2015; 372(9):803-13.

Gruchalla RS, Sampson HA. Preventing peanut allergy through early consumption--ready for prime time? N Engl J Med 2015; 372(9):875-7. Keet CA, Wood RA. Emerging therapies for food allergy. J Clin Invest 2014; 124(5):1880-6.

Bublin M, Breiteneder H. Developing therapies for peanut allergy. Int Arch Allergy Immunol 2014; 165(3):179-94.

Bauer RN, Manohar M, Singh AM, Jay DC, Nadeau KC. The future of biologics: applications for food allergy. J Allergy Clin Immunol 2015; 135(2):312-23. Schneider LC, Rachid R, LeBovidge J, Blood E, Mittal M, Umetsu DT. A pilot study of omalizumab to facilitate rapid oral desensitization in high-risk peanut-allergic patients. J Allergy Clin Immunol 2013; 132(6):1368-74.

Sampson HA, Leung DY, Burks AW, Lack G, Bahna SL, Jones SM et al. A phase II, randomized, doubleblind, parallelgroup, placebocontrolled oral food challenge trial of Xolair (omalizumab) in peanut allergy. J Allergy Clin Immunol 2011; 127(5):1309-10. De Boever EH, Ashman C, Cahn AP, Locantore NW, Overend P, Pouliquen IJ et al. Efficacy and safety of an anti-IL-13 mAb in patients with severe asthma: a randomized trial. J Allergy Clin Immunol 2014; 133(4):989-96. Hilvering B, Pavord ID. What goes up must come down: biomarkers and novel biologicals in severe asthma. Clin Exp Allergy 2015;10.

Ballantyne SJ, Barlow JL, Jolin HE, Nath P, Williams AS, Chung KF et al. Blocking IL-25 prevents airway hyperresponsiveness in allergic asthma. J Allergy Clin Immunol 2007; 120(6):1324-31. Shin HW, Kim DK, Park MH, Eun KM, Lee M, So D et al. IL-25 as a novel therapeutic target in nasal polyps of patients with chronic rhinosinusitis. J Allergy Clin Immunol 2015;(15):10. Chan JK, Roth J, Oppenheim JJ, Tracey KJ, Vogl T, Feldmann M et al. Alarmins: awaiting a clinical response. J Clin Invest 2012; 122(8):2711-9.

Discussion

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Chapter 8 Lekensamenvatting

J.A. Aalberse

Chapter 8

Introductie Dit proefschrift beschrijft de diagnose van pinda allergie en de rol van alarmins, zoals heat shock eiwitten, bij allergische ziekten.

Allergie is een immunologische ziekte waarbij je reageert op stofjes, bijvoorbeeld in de voe-

ding of uit de lucht, waar mensen zonder die allergie niet op reageren. Zo kan je allergisch

reageren op huisstof, waardoor je bijvoorbeeld gaat niezen of reageren op de kat en daar benauwd van worden. Ook zijn er mensen die allergisch zijn voor voedselcomponenten. Iemand

met pinda allergie krijgt na het eten van pinda bijvoorbeeld uitslag, maar kan ook een heftige reactie krijgen met shock verschijnselen, dit heet een anaphylactische shock.

De basis van een allergische ziekte lijkt zo simpel. Als je allergisch bent maak je namelijk een antistof, IgE, aan tegen stofjes in bijvoorbeeld de lucht of in het voedsel. Deze stofjes, meestal

eiwitten, noemen we dan allergenen. Mensen die niet allergisch zijn maken geen IgE tegen deze stofjes. Door de binding van het IgE met een allergeen ontstaat er een reactie bij bepaalde cellen in het lichaam. Door deze reactie kan iemand fysieke klachten ervaren, zoals jeuk.

Kinderen die allergisch zijn tegen pinda hebben inderdaad IgE tegen pinda. Maar helaas

wordt het dan ingewikkelder. Niet alle kinderen die IgE tegen pinda hebben in het bloed, hebben daadwerkelijk een allergische reactie tegen pinda. Mensen met IgE tegen pinda noemen we gesensibiliseerd (gevoelig) voor pinda. Je bent echter pas echt pinda-allergisch als je een pinda sensibilisatie hebt met een allergische reactie na blootstelling aan pinda.

De diagnose van pinda allergie wordt meestal gedaan op basis van het verhaal van de patiënt of de ouders, een huidtest en/of een bloedtest. Ten slotte is een dubbel blinde placebo

gecontroleerde provocatie test nodig. Deze provocatie test is de gouden standaard en maakt

het onderscheid tussen kinderen die alleen een gevoeligheid hebben voor pinda en kinderen die daadwerkelijk ook een reactie hebben als ze worden bloodgesteld aan pinda. Dubbelblind

placebo gecontroleerd betekent dat noch de patiënt noch de dokter weten of de patiënt place-

bo (een hapje zonder pinda) of pinda toegediend krijgt. Zo kan je zo veel mogelijk voorkomen dat er een bevooroordeelde observatie ontstaat bij de test.

Handig zou zijn als je die provocatietest niet nodig zou hebben. Het is namelijk best spannend

voor de patiënt en de ouders, daarnaast is het intensief en kost het veel tijd van patiënten, verpleegkundigen, dokters en ziekenhuizen.

Helaas ontbreekt de provocatietest om deze reden nu al vaak. Veel kinderen hebben daardoor

mogelijk een onterechte diagnose van pinda allergie met alle ingrijpende gevolgen van dien. Ook in veel wetenschappelijke artikelen is geen gebruik gemaakt van provocatie testen en gaat het dus eigenlijk over gesensibiliseerde patiënten maar is het onbekend of ze ook daar132

werkelijk pinda allergisch zijn. Ze worden wel beschreven als pinda allergisch. Deze soms

Lekensamenvatting

onterechte conclusie geeft veel problemen bij de interpretatie van wetenschappelijke studies. Je kunt je afvragen wat de waarde is van een studie van het aantal patiënten dat over zijn

pinda allergie heen is gegroeid als je niet met zekerheid weet of iemand ooit pinda allergisch

is geweest. Wat is het nut van een voorspellende waarde als je eigenlijk slechts sensibiliteit voorspelt, maar geen allergische ziekte.

In onze studies beschreven in dit proefschrift hebben we wel gebruik gemaakt van provocatie testen om de diagnose te bevestigen of te ontkrachten. In deze patiënten populatie hebben

we gekeken of we de voorspellende waarde van de bloed test die het IgE tegen allergenen meet kunnen verbeteren of kunnen aanvullen. Voor de bloedtest met IgE zijn twee aspecten

belangrijk. Ten eerste moet het een test zijn waarin alle allergenen aanwezig zijn, waarvoor je allergisch bent. Ten tweede is het belangrijk om te weten of al die allergenen ook daadwerkelijk klinisch relevant zijn.

Hoewel dit beide logisch klinkt is het nog niet zo vanzelfsprekend. In hoofdstuk 6 laten we zien dat de manieren van extraheren (een methode om allergenen uit een pinda te halen) erg

verschillen, en ook verschillend succesvol zijn. Dit heeft veel te maken met de extractie buffer (de vloeistof die gebruikt wordt om de eiwitten uit de pinda te halen). Het blijkt dat als je een

zure buffer gebruikt, zoals azijnzuur, je bijvoorbeeld meer basische allergenen kan extraheren, die je mist als je alleen water gebruikt als extractie buffer. Met meer allergenen is de kans dat je iets mist kleiner. Helaas wordt de kans dat de diagnose goed is niet evenredig beter,

en zelfs soms slechter. Dit komt omdat sommige allergenen bij de meeste mensen niet leidt tot allergische klachten. Deze allergenen zijn maar bij enkele patiënten klinisch relevant. De uitdaging van de bloedtest is om aan de ene kant zoveel mogelijk eiwitten in de test te hebben en aan de andere kant de test zo efficient mogelijk te maken.

We hebben ook gekeken wat het verschil is tussen patiënten met een IgE tegen pinda die alleen gesensibiliseerd zijn, en de patiënten die ook echt allergische symptomen hebben na

blootstelling aan pinda. Onze onderzoeksvraag was of we aan de hand van in het bloed gemeten cytokines (bepaalde eiwitten die processen aanzetten voor de ontstekingsreactie) kunnen voorspellen wie wel en wie niet gaat reageren op de pinda. Uit ons onderzoek, beschreven in hoofdstuk 5, blijkt dat IL-25 alleen te vinden is bij patiënten die ook daadwerkelijk een pinda

allergische reactie vertoonden bij de provocatie. Het is echter niet te vinden bij alle patiënten met een positieve reactie. Dit suggereert dat IL-25 mogelijk een bepaalde subgroep identificeert. Dit zou bijvoorbeeld een meer chronische groep patiënten kunnen zijn.

Il-25 is een alarmin. Een alarmin is een endogeen eiwit, dit betekent dat het wordt gemaakt

in de cel, verder komt een alarmin vrij bij schade en kan het het (aangeboren) immuunsysteem aanzetten. Meerdere alarmins lijken een rol te spelen bij allergische ziekten, zoals ook urinezuur en IL-33.

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Deze alarmins helpen niet alleen in de diagnose of bij een beter begrip van de ziekte, maar zou ook een aanknopingspunt kunnen zijn voor therapie. Een mogelijkheid in de toekomst

zou bijvoorbeeld zijn om met behulp van antilichamen IL-25 te blokkeren om op de manier de chronische ontsteking te onderdrukken.

Het andere deel van dit proefschrift gaat over heat shock eiwitten (HSP = heat shock proteins). Ook deze eiwitten zijn alarmins en worden in verband gebracht met therapie, bij bij-

voorbeeld auto immuunziekten. In dit proefschrift hebben we gekeken wat de rol is HSP bij allergische ziekten.

HSP zijn endogenen eiwitten. De HSP in de cellen van de mens lijken erg op de HSP van planten en bacterieën wat er op duidt dat de eiwitten weinig zijn veranderd tijdens de evolutie.

Een belangrijke functie van HSP is dat ze chaperonen zijn: ze helpen bij het transport van

eiwitten van binnen naar buiten de cel. HSP blijken echter nog meer functies te hebben. Ze zijn namelijk immunogeen, ze triggeren het immuunsysteem. Bij hitte (vandaar de naam heat

shock) of andere vorm van stress welke leidt tot schade aan het weefsel, worden deze eiwitten extra aangemaakt. In hoofdstuk 2 van dit proefschrift hebben we op een rij gezet wat er tot dan toe bekend was over de rol van HSP bij allergische ziekten.

In hoofdstuk 3 en hoofdstuk 4 hebben we laten zien dat HSP immunogeen is zowel in een

ziektevrije omgeving (direct na de geboorte) als in patiënten met eczeem. Dit ondersteunt de hypothese dat reacties op hsp een gezonde immunogene respons kan zijn, maar dat het in een immuun actieve omgeving ook juist een reactie kan geven die bijdraagt aan de onsteking. De rol van endogene alarmins bij het begin van de ontsteking maar ook bij het in stand hou-

den van weefsel ontsteking bij allergische aandoeningen alsook hun rol als potentiële the-

rapeutische targets is een van de interessante en belangrijke invalshoeken voor toekomstig allergie onderzoek.

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135

Chapter 9

Dankwoord & Curriculum Vitea

Chapter 9

DANKWOORD Promoveren bestaat uit veel stapjes. Terugkijkend zijn het voornamelijk leuke stapjes, maar

natuurlijk ook frustrerende en te kleine stapjes, of stapjes terug. Zelfs stapjes in de hele an-

dere richting. Onderweg ben ik veel mensen tegengekomen die me hebben gestimuleerd en geholpen om te komen waar ik nu ben. Ook de mensen die voor welkome afleiding hebben gezorgd of de humor kende van de frustratie zijn erg het bedanken waard.

In de eerste plaats: het onderzoek in dit proefschrift had nooit kunnen plaatsvinden zonder de (net bevallen) moeders, vaders en hun kinderen die meededen aan het onderzoek. Heel hartelijk dank. Beste Berent,

Jij gunt je promovendi het maken van eigen stappen. Stappen out of the box, daardoor ont-

stond bijvoorbeeld het stuk over hsp in cordblood. Je luistert met een glimlach, je stuurt onopvallend en bovenal weet je altijd weer het goede gevoel boven te halen, hoe diep de dip ook lijkt. Erg veel dank voor je aanhoudende vertrouwen en de bijzondere gesprekken. Beste Femke,

De eerste stapjes zette ik zonder jou, ik kwam overal. Dankzij jou kreeg mijn onderzoek nog

duidelijker focus. Je geduld, je oordeelloos vermogen om telkens weer opnieuw in te gaan op die vraag die ik toch nog net niet begreep. Inhoudelijke discussies en leuke gesprekken. Ik vind je een bijzonder kundige onderzoeker en ben erg blij dat jij mijn co-promotor bent. Beste Edward,

Een bijzondere verlengde stap. Ik mag promoveren bij jou, nadat mijn vader eerder bij jouw

promotieonderzoek betrokken was. Dank dat jij steeds meer betrokken bent geraakt bij mijn

onderzoek. Jouw enthousiasme is aanstekelijk. Dank voor al je tijd en inzet om mij te helpen de promotie af te ronden. Het is gelukt! Beste Maarten,

Zonder jou had ik de eerste stappen niet kunnen maken. We kenden elkaar al vanaf het AMC

waar ik als student meedeed aan een ander mooi allergie onderzoek van Trinette Steenhuis.

Ik ben je stappen gevolgd naar het WKZ waar ik mee mocht denken over allergie onderzoek en heat shock eiwitten. Met je vele interesses op vele terreinen en mijn wat langere traject

sluiten we het alleen officieus samen af. Dank voor de kans die je mij gunde om dit onderzoek op te zetten. Beste Erica,

Wat een bijzondere secretaresse ben jij. Niet mijn secretaresse natuurlijk, maar zo liet je het

soms wel lijken. Je was er altijd. Je had altijd tijd, terwijl je het enorm druk had. Zelfs pro-ac138

tief maakte je voor mij afspraken met Berent. Je bent bijzonder in je betrokkenheid. Stijn en

Dankwoord & Curriculum Vitea

Roos vonden de ballon prachtig. Dank ook aan Heleen, Angela en Mirjam in mijn weg naar de verdediging van mijn proefschrift.

Lieve Marloes en Yasser, lieve paranimfen

Marloes als vriendin en ex-roomie, Yasser als vriend en ex-buur, dank dat jullie mijn paranimfen willen zijn. Het geeft rust dat jullie daar straks in het zweetkamertje zitten en mij willen

vergezellen met ijsberende stappen, met jullie heerlijke grappen, net niet onder de gordel. Dank voor jullie steun en voor het al een paar jaar vrijhouden van de agenda voor deze verdediging.

Beste kamergenoten,

De stappen tijdens een promotietraject, de frustraties en de pret, worden het best begrepen door mede onderzoekers. Dank aan mijn lieve kamergenoten, arash, joost s, korneel, lianne,

maja, marieke, marije, marloes, nan en titia. Ik denk dan meteen aan fietsen, mooie excel programma’s, “zeg nee tegen ….”, het altijd veranderlijke weer, het dubbele proefschrift, de trap

onder de kont, de tranen van commerciele belangen, paranymfen, Lelystad, Rome, liedjes, en lief gemene opmerkingen, maar vooral de altijd aanwezige lach en steun, door dik en dun. Heel veel dank!

Binnenstappen in een lab is als arts niet vanzelfsprekend. De genen had ik mee, ietsepiets er-

varing, maar meer ook niet. Mark maakte het mogelijk. Moest er nou 1 of 2 ml bij. Mark weet het. En ook bij de tiende keer legde Mark het weer heel rustig uit alsof je het voor de eerste keer vroeg. Het lab was gebouwd op jou als steunpilaar, en daar heb ik veel aan gehad. Dank

voor de tent-lessen in het Eiffel gebied! Dank Wilco en Jenny voor jullie hulp bij de expirementen en het meten van de cytokines. Zo kwamen de publicaties dan echt.

En dan de andere prakkers en WKZ’ers: in de eerste plaats Berber. Gezamenlijke frustraties, er tegen op boksen. We konden er wel om lachen. Tja en natuurlijk de eindeloze soap met elke keer weer onverwachte nieuwe wendingen. Hopelijk heb je nu wat meer rust en kan je met zijn tweeen aan de weg timmeren! Alma, Alvin, Annemarie, Annick, Astrid, Arjan, Bas, Ellen, Eva, Evelien, Evelien, Henk, Huib, Isme, Joris, Lieke, Lise, Pleun, Ruud, Sanne, Sarah, Selma,

Sylvia, Theo en Yvonne, en erg belangrijk Sytze! Jasr, stoelendans, koffieautomaat, lunchstap-

jes door het natuurgebied achter het WKZ (inspiratie voor de omslag van dit proefschrift), serieuze gesprekken, ezelsbruggetjes van de spar en de den. Je leert veel tijdens een promotie-traject.

En dan de stappen in de kelder van het WKZ, het militair hospitaal naar de dagbehandeling

bij Pauw, en verder door naar de operatiekamers. Dank nogmaals aan alle kinderen en ouders die mee wilden doen aan het onderzoek, zowel de kinderen verdacht van pinda allergie, als de kinderen die juist geen allergie hadden en bloed afstonden voor de controle samples. Daar-

naast veel dank aan de specialisten die de logistiek mogelijk maakten: Maarten Hoekstra en 139

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Yolanda Meijer, en ook de anesthesisten op de OK, en zeker ook alle artsen en verpleegkundigen werkzaam op Pauw, die zorg droegen voor de bloedafnames. Niet in de laatste plaats dank aan Petra Kentie. Het onderzoek begon zonder jou, maar floreerde dankzij jou.

Een groot deel van dit onderzoek vond ook plaats in Amsterdam, met het AMC, maar voorna-

melijk op het CLB, op het lab van Rob. Het is bijzonder om zo welkom te zijn op een plek waar

ik mijn hele leven om uiteenlopende reden elke keer weer mijn stappen zet. Het monnikenwerk van het pinda pellen hebben de meeste indruk gemaakt. Veel dank aan Tjitske, Ninotska, Astrid, Jolanda, Ellen, Ingrid, Kim, Theo, Pleun, Theresa, Henk, Meriam, Tom, Marianne, Janny, Ronald, Bouke, Fatima, Kaoutar, Laurian, Jaap, Erica, Ed, Anders, Steven en Rob.

Natuurlijk zijn de Coffers, de 3e verdieping en de diergeneeskunde erg belangrijk geweest

voor het meedenken, meedoen en om tegen te voetballen, vooral dank aan Jorg, Jeffrey, Paul, Nathalie, Vanessa, Ger A, en Ger R, Koos, Pirrko en Willem.

Ik heb tijdens die tien jaar ook nog stappen buiten gemaakt, alhoewel de vrienden soms het onderspit dolven omdat ik weer dacht dat ik de promotie nu echt ging afronden… Dank aan

de posse Aswin, Bas, Daan, Edin, Guido, Tom, Tim de V, dank Arjen en Tim L, dank TP’ers voor het elke keer opnieuw vragen of ik dan toch mee ga stappen in de bergen… Yasser, Andreas, Bas, en ook dank Jessie, natuurlijk Fonnet voor onze onderzoeksweddenschap, Jenneke, Niels voor het delen van onderzoeksperikelen en Marieke en Arash voor de fietstocht naar Rome.

Andere stappen heb ik gemaakt in Haarlem bij de kindergeneeskunde met de gezellige ver-

pleging, leuke kinderartsen en aniossen, met de aiossen, docenten en opleiders bij de huis-

artsenopleiding, in Oosterblokker en Osdorp, en nu verder in onder andere Amsterdam en

Hoorn. Speciale dank aan Nynke, Annemarie en Monique voor de aanhoudende steun en kritische noot.

A special thanks to the Kagens and to Tom Platts-Mills. The Kagens, Steffi, Tommy, Mike, Missy,

Gayle and Steve have made me feel at home many steps away from Amsterdam. The round

table discussions with Marv, Steve, Charlie and Mike have contributed to my steps to become

a doctor and a researcher. I have fond memories of the lab with Mutthiah. Also special thanks to Tom and his lab and the doctors in Charlottesville who have shown how serious research can be combined with fun in the lab. I have fond memories of the mountain walks to clear the mind. With the latest walk during the WIRM in over three feet high snow. Big steps!

Lieve familie en schoonfamilie, Pauline, Rob, Suzanne, Jeroen, Pauline en Floor, Tanneke, Lodewijk, Fenna, Arthur, Steven en Taeke, Laurien, Marius en Willem, Noor, Paul, Lucas en Philine. Al-

tijd vroegen jullie lief naar mijn promotie, hoe het ging, zonder irritant te zijn. Altijd geinteres-

seerd, elke keer weer luisteren en doorvragen. Dankzij jullie bleef elke stap toch weer bijzonder. Beste Tonny dank voor je kritische blik en je inspanningen om het proefschrift te begrijpen. 140

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Lieve Rob, dank je wel voor jouw meerdere rollen in dit onderzoek. Je liet me mijn eigen stap-

pen maken, je liet me zwemmen, maar zorgde er ongemerkt voor dat ik niet verdronk. Je was er op de achtergrond, altijd aanwezig voor de discussie, altijd met heel veel geduld. Jij bent

vaak al vele stappen verder, maar kan ook weer zien waar ik nog sta. Ik ben er ontzettend trots op dat wij samen een gedeelte van dit onderzoek hebben mogen doen met prachtige artikelen

als resultaat. Lieve Pauline, dank je wel voor de ruimte die jij schiep voor de tafeldiscussies met Rob over onze pinda-stukken. Veel dank voor je lieve mailtjes met vrolijke noot en je steun. Heel fijn.

Lieve Stijn en Roos, jullie stapjes zijn de mooiste. Het is een groot geluk. Lieve Maaike, dank voor alle liefde en steun, het volhouden en relativeren. Met jou is dit boekje gelukt.

141

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Curriculum Vitea Joost Aalberse werd geboren op 22 januari 1977 te Amsterdam. In 1986 heeft hij in Charlottesville, Virginia gewoond. Hij is getrouwd met Maaike van Dijk. Zij hebben samen twee kinderen, Stijn en Roos. In 1996 behaalde hij het VWO diploma aan het Fons Vitae lyceum te Amsterdam. Datzelfde jaar

begon hij met de studie Geneeskunde aan de Universiteit van Amsterdam. Tijdens de opleiding heeft hij een extracurriculair vak gevolgd bij de faculteit Geesteswetenschappen: “Introductie

tot de filosofie”. Daarnaast heeft hij verschillende cursussen gevolgd waaronder Muziekgeschiedenis bij CREA, Expirementele Oncologie aan het NKI en Medische Ethiek aan de VU.

Als student assistent was hij betrokken bij een onderzoek over obstipatie bij kinderen onder supervisie van Dr. R. van Ginkel en Dr. M. Benninga. Daarna was hij een aantal jaar betrokken bij

onderzoek van Trinette Steenhuis en Dr. M. Hoekstra, waarbij onderzoek werd gedaan naar een mogelijk vaccin tegen allergie.

Hij heeft tijdens zijn studie onderzoek gedaan in Charlottesville, Virginia, terug op de plek waar

hij eerder naar de basisschool ging. Dit onderzoek richtte zich op IgG anti-lichamen tegen tetanus toxoid, zowel in moederlijk- als in navelstrengbloed. Het onderzoek vond plaats bij Prof. Dr. T. Platts-Mills , waarbij in het AMC ook Prof. Dr. J. van der Zee betrokken was. Vervolgens werd

dat onderzoek voortgezet in het CLB (nu Sanquin), te Amsterdam bij Dr. S. Stapel en Prof. Dr. R. Aalberse. Op dat lab heeft hij ook onderzoek gedaan naar kruisreactiviteit tussen hond en koe-allergenen, en naar de rol van IgG4 bij kinderen met een allergie voor katten. Bij de presen-

tatie van enkele van deze resultaten op een summerschool in Cagliari, Italie, werd hij uitgeno-

digd door Prof. Dr. S. Bonini om een studenten-congres voor allergie te initiëren in Europa voor Europese studenten. Dit heeft geresulteerd in het Chiron Project, welke hij vier jaar lang organi-

seerde samen met Claudio d’Ambrosio, met congressen in Napels, Berlijn, Parijs en Amsterdam. Na het behalen van het doctoraal examen in 2002 heeft hij onderzoek gedaan in Australie bij Prof. Dr. D.

Lehmann en Dr. C. Jeffries. Dit onderzoek richtte zich op de oorzaken van middenoorontsteking bij Aboriginals. Deze tijd heeft hem ook veel kennis gegeven over de geschiedenis en cultuur van Aboriginals.

Hierna is hij gestart met zijn coschappen, met onderzoek naar Henoch Schonlein purpura bij

Prof. Dr. J. Davin van de afdeling kinder-nierziekten. In 2005 heeft hij zijn coschappen afgerond. Direct aansluitend is hij gestart met het promotieonderzoek. Het onderzoek is aanvankelijk gestart onder supervisie van Prof. Dr. B. Prakken en Dr. M. Hoekstra. Vlot daarna is ook Dr. F. van

Wijk copromotor geworden bij de studie. Vanwege meerdere interesses van Dr. M. Hoekstra en werkzaamheden elders, is Dr. E. Knol halverwege het traject copromotor geworden in plaats van Dr. M. Hoekstra. Het promotieonderzoek heeft geleid tot dit proefschrift.

Tijdens de promotie heeft hij lesgegeven en als vrijwilliger gewerkt op een huisartsenpost voor onverzekerden. Sinds 2014 werkt hij op verschillende plekken in Noord Holland als huisarts. 142

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List of publications Molecular allergen-specific IgE assays as a complement to allergen extract based sensitization assessment.

Aalberse RC, Aalberse JA. J Allergy Clin Immunol Practice 2015 accepted for publicationÂ A lesson from component-resolved testing: we need better extracts.

Aalberse JA, Aalberse RC. J Allergy Clin Immunol Pract. 2014 Sep-Oct;2(5):635-6

Plasma IL-25 is elevated in a subgroup of patients with clinical reactivity to peanut. Aalberse JA. et al. Clin Transl Allergy. 2013 Dec 2;3(1):40

Moving from peanut extract to peanut components: towards validation of component-resolved IgE tests.

Aalberse JA, et al. Allergy. 2013 Jun;68(6):748-56

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis. Kapitein B, Aalberse JA, et al. Cell Stress Chaperones. 2013 Jan;18(1):87-95.

Association between early bacterial carriage and otitis media in Aboriginal and non-Aboriginal children in a semi-arid area of Western Australia: a cohort study. Sun W. et al. BMC Infect Dis. 2012 Dec 21;12:366. HSP: Bystander Antigen in Atopic Diseases?

Aalberse JA. et al. Front Immunol. 2012 May 31;3:139

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60). Aalberse JA. et al. PLoS One. 2011;6(9):e24119

The interaction between respiratory viruses and pathogenic bacteria in the upper respiratory tract of asymptomatic Aboriginal and non-Aboriginal children. Moore HC, et al. Pediatr Infect Dis J. 2010 Jun;29(6):540-5.

Absent otoacoustic emissions predict otitis media in young Aboriginal children: a birth cohort study in Aboriginal and non-Aboriginal children in an arid zone of Western Australia. Lehmann D. et al. BMC Pediatr. 2008 Aug 28;8:32

Henoch Schonlein purpura in children: an epidemiological study among Dutch paediatricians on incidence and diagnostic criteria.

Aalberse J. et al. Ann Rheum Dis. 2007 Dec;66(12):1648-50.

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