The molecular diagnosis of contact allergy

The molecular diagnosis of contact allergy

IDEA workshop 12th december 2018

CIRI -‐ Inserm U 1111 / Team 17

Team « Immunology of skin allergy & vaccination » CoPIs: Marc Vocanson / Jean-François Nicolas

CIRI – INSERM U1111, Lyon - France

Contact Dermatitis (CD)

Patch-tests

ALLERGY? ALLERGEN?

Dermatoallergology reference medical centre

Non-allergic Innate immunity

Allergic (ACD) Adaptive immunity

T cells

§  Objectives §  To discover diagnostic & prognostic biomarkers in

skin and blood of ACD patients

§  To develop new tools that could help for the diagnosis of contact allergy

MODAL project -‐ MOlecular Diagnosis of skin ALlergy

3

When diagnosis turns out to be problema>c?

How to differentiate irritation from allergy? §  Are they irritant or allergic?

Are true positive tests clinically relevant? §  Strong reactions: Yes §  Low reactions: Poorly

Irritant control SLS 0.25% SLS 0.5%

Allergens

Sensi4ve skin

Scoring (ICDRG)

Nega%ve' IR'irrita%ve'

Posi%ve'+' Posi%ve'++' Posi%ve'+++'

CONTACT ALLERGY

CONTACT IRRITATION

PATCH TEST INTENSITY +/- + ++ +++

false posi4ve ? Di#mar D, Contact Derma//s, 2018 Brasch J, J Am Acad Dermatol, 1994

4

How to circumvent problema>c diagnosis?

By demonstrating the existence of an allergic reaction §  Characterize the skin DTH reaction

à cell infiltration & activation using transcriptomic, immunohistochemistry…

§  Characterize circulating specific T cells à in vitro T cell reactivation using LTT, Elispot assays

How to differentiate irritation from allergy? §  Are they irritant or allergic?

Are true positive tests clinically relevant? §  Strong reactions: Yes §  Low reactions: Poorly


Allergens

Sensi4ve skin


Suspected skin

allergic pa4ent ?

Non allergic

pa4ent

Allergic pa4ent ACD asymptoma4c pa4ent / Low

ACD pa4ent /

Strong

Disease stra>fica>on / Clinical relevance

New medical device

•  Electro-‐mechanical HOLDER

+

•  FLUORORESCENT –BASED READING SYSTEM

•  LAB-‐ON-‐CHIP including ACD BIOMARKER LIGANDS

MICRO SKIN BIOPSY •  HOLLOW

mLANCET ARRAY


Allergens

Sensi4ve skin

Affymetrix chip Human Genome U133

Plus 2.0 Array > 22000 genes

Microarrays generation

72h +++, ++, +, +/-

Patch tests to induce skin allergy Clinical study

Vehicle

§  48 patients suspected for skin allergy §  Positive patch-tests (+++, ++, +, +/-, -)

§  Reference Allergens: ü  Nickel (n=10), ü  Linalool peroxyde (n=10), ü  Methylisothiazolinone (n=6) ü  Amoxicillin (n=6) ü  Diamox (n=1) ü  Tazocillin (n=1)

§  Reference irritants: ü  SLS (n=12), ü  Cantharidin (n=8) ü  Nonanoïc acid (n=4)



Bioinformatic / biostatistic analysis (collab. AltraBio, Lyon)

Clinical study




72h +++, ++, +, +/-

Patch tests to induce skin allergy

Vehicle

Clinical study Principal Component Analysis (PCA)

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●HSR411

HSR507

HSR315

HSR312

HSR307HSR312

HSR457

HSR419

HSR467HSR375

HSR373

HSR371

HSR461

HSR411HSR507HSR315HSR312HSR307HSR457HSR419HSR467HSR375HSR373HSR371HSR461FJJ

FJJFJJ

DSAP

DSAP

DSAP

CMPV

CMPVCMPV

KPH

KPHKPH

DDR

DDRDDR

HSR749

HSR749

HSR750

HSR750

HSR751

HSR751

HSR752

HSR752

HSR753

HSR753

HSR848

HSR848

HSR757

HSR757

HSR758

HSR758HSR760

HSR760

HSR761

HSR761

HSR763

HSR763

HSR763

HSR764

HSR764

HSR766

HSR766

HSR766

HSR767

HSR767

HSR768

HSR768

HSR769

HSR769

HSR771

HSR771

HSR772

HSR772

HSR773

HSR773

HSR774

HSR774

HSR777HSR777 HSR779

HSR779

HSR782

HSR782

HSR783

HSR783

HSR808

HSR808HSR809HSR809

HSR819

HSR819HSR825

HSR825HSR830

HSR830

HSR836

HSR836

HSR836HSR847

HSR847

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PC2

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●HSR411

HSR507

HSR315

HSR312

HSR307HSR312

HSR457

HSR419

HSR467HSR375

HSR373

HSR371

HSR461


FJJ FJJ

DSAP

DSAP

DSAP

CMPV

CMPVCMPV

KPH

KPHKPH

DDR

DDRDDR

HSR749

HSR749

HSR750

HSR750

HSR751

HSR751

HSR752

HSR752

HSR753

HSR753

HSR848

HSR848

HSR757

HSR757

HSR758

HSR758HSR760

HSR760

HSR761

HSR761

HSR763

HSR763

HSR763

HSR764

HSR764

HSR766

HSR766

HSR766

HSR767

HSR767

HSR768

HSR768

HSR769

HSR769

HSR771

HSR771

HSR772

HSR772

HSR773

HSR773

HSR774

HSR774

HSR777HSR777 HSR779

HSR779

HSR782

HSR782

HSR783

HSR783

HSR808

HSR808HSR809HSR809

HSR819

HSR819HSR825

HSR825HSR830

HSR830

HSR836

HSR836

HSR836HSR847

HSR847

% va

rianc

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2030

4050

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Condition Control − Control Allergen − Control Irritant − Control

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PC4

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HSR411

HSR507

HSR315HSR312 HSR307

HSR312HSR457

HSR419

HSR467

HSR375

HSR373

HSR371

HSR461


FJJFJJ

DSAPDSAP

DSAP

CMPV

CMPV

CMPV

KPHKPH

KPH

DDR

DDR

DDR

HSR749HSR749

HSR750

HSR750

HSR751

HSR751

HSR752

HSR752

HSR753

HSR753

HSR848

HSR848HSR757

HSR757 HSR758HSR758HSR760HSR760

HSR761

HSR761

HSR763

HSR763

HSR763HSR764

HSR764HSR766

HSR766HSR766

HSR767

HSR767

HSR768

HSR768

HSR769

HSR769

HSR771

HSR771

HSR772

HSR772

HSR773HSR773HSR774HSR774

HSR777

HSR777

HSR779

HSR779

HSR782

HSR782

HSR783

HSR783 HSR808HSR808HSR809

HSR809

HSR819

HSR819HSR825

HSR825HSR830HSR830

HSR836HSR836HSR836HSR847

HSR847

The molecular signatures of allergens are clearly dis>nct from those of irritants

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●HSR411

HSR507

HSR315

HSR312

HSR307HSR312

HSR457

HSR419

HSR467HSR375

HSR373

HSR371

HSR461


FJJFJJ

DSAP

DSAP

DSAP

CMPV

CMPVCMPV

KPH

KPHKPH

DDR

DDRDDR

HSR749

HSR749

HSR750

HSR750

HSR751

HSR751

HSR753

HSR753

HSR848

HSR848

HSR757

HSR757

HSR758

HSR758HSR760

HSR760

HSR761

HSR761

HSR763

HSR763

HSR763

HSR764

HSR764

HSR766

HSR766

HSR766

HSR767

HSR767

HSR768

HSR768

HSR769

HSR769

HSR771

HSR771

HSR772

HSR772

HSR773

HSR773

HSR774

HSR774

HSR777HSR777 HSR779

HSR779

HSR782

HSR782

HSR783

HSR783

HSR808

HSR808HSR809HSR809

HSR819

HSR819HSR825

HSR825HSR830

HSR830

HSR836

HSR836

HSR836HSR847

HSR847

−50 0 50

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●

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●●

●

●

●

●●

●HSR411

HSR507

HSR315

HSR312

HSR307HSR312

HSR457

HSR419

HSR467HSR375

HSR373

HSR371

HSR461


FJJ FJJ

DSAP

DSAP

DSAP

CMPV

CMPVCMPV

KPH

KPHKPH

DDR

DDRDDR

HSR749

HSR749

HSR750

HSR750

HSR751

HSR751

HSR753

HSR753

HSR848

HSR848

HSR757

HSR757

HSR758

HSR758HSR760

HSR760

HSR761

HSR761

HSR763

HSR763

HSR763

HSR764

HSR764

HSR766

HSR766

HSR766

HSR767

HSR767

HSR768

HSR768

HSR769

HSR769

HSR771

HSR771

HSR772

HSR772

HSR773

HSR773

HSR774

HSR774

HSR777HSR777 HSR779

HSR779

HSR782

HSR782

HSR783

HSR783

HSR808

HSR808HSR809HSR809

HSR819

HSR819HSR825

HSR825HSR830

HSR830

HSR836

HSR836

HSR836HSR847

HSR847

% va

rianc

e

010

2030

4050

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Condition Control − Control Allergen − Control Irritant − Control

−50 0 50

−50

050

PC3

PC4

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●

●

● ●●●●

●

●●

●●●

● ●●●

●

HSR411

HSR507

HSR315HSR312

HSR307

HSR312 HSR457

HSR419

HSR467

HSR375

HSR373

HSR371

HSR461

HSR411HSR507HSR315HSR312HSR307HSR457HSR419HSR467HSR375HSR373HSR371HSR461

FJJ

FJJFJJ

DSAPDSAP DSAP

CMPV

CMPV

CMPV

KPHKPH

KPH

DDR

DDR

DDR

HSR749HSR749

HSR750

HSR750

HSR751

HSR751

HSR753

HSR753

HSR848

HSR848HSR757

HSR757 HSR758HSR758HSR760HSR760

HSR761

HSR761

HSR763

HSR763

HSR763HSR764

HSR764HSR766

HSR766HSR766

HSR767

HSR767

HSR768

HSR768

HSR769

HSR769

HSR771

HSR771

HSR772

HSR772

HSR773HSR773

HSR774HSR774

HSR777

HSR777

HSR779HSR779

HSR782

HSR782

HSR783

HSR783HSR808

HSR808HSR809HSR809

HSR819

HSR819HSR825

HSR825

HSR830HSR830

HSR836HSR836

HSR836HSR847

HSR847

Allergens

Irritants

Altrabio, lyon

Unsupervised analysis « Principal component analysis (PCA) »




Lefèvre MA, manuscript in prepara/on

Heatmap Over-‐expressed genes in allergen/irritant samples

Control skin

LINE= selected gene Column = 1 sample

Allergen signature

Common signature

Irritant signature

Irritants -‐ + 2+ 3+

SLS Nonanoïc acid Cantharidin Allergens +/-‐ 1+ 2+ 3+

Amoxicillin Linalol peroxyde MI Nickel Tazocillin Diamox

Weak allergen signatures in some pa>ents à pa>ent stra>fica>on?

Clinical study Principal Component Analysis (PCA)

PC2

PC1

Specific molecular signature for each molecule?


§  Reference Allergens: §  Nickel (n=10) §  Linalol peroxyde (n=10) §  Methylisothiazolinone (n=6) §  Amoxicillin (n=6) §  Diamox (n=1), Tazocillin (n=1)

§  Reference irritants: §  SLS (n=12) §  Cantharidin (n=8) §  Nonanoïc acid (n=4)

Dhingra N. JACI, 2014

Specifc signature for certain groups of molecules?

Lefèvre MA, manuscript in prepara/on

MODAL project (MOlecular Diagnosis of skin ALlergy)

Suspected skin

allergic pa4ent ?

Non allergic

pa4ent

Allergic pa4ent ACD asymptoma4c pa4ent / Low

ACD pa4ent /

Strong

Disease stra>fica>on / Clinical relevance

New medical device

•  Electro-‐mechanical HOLDER

+

•  FLUORORESCENT –BASED READING SYSTEM

•  LAB-‐ON-‐CHIP including ACD BIOMARKER LIGANDS

MICRO SKIN BIOPSY •  HOLLOW

mLANCET ARRAY

Sensi4ve skin

+

• TOOLBOX WITH SOLUBILIZED ALLERGENS

New T cell assay

BLOOD COLLECTION

• T-‐CELL ASSAY including NEW BLOOD BIOMARKERS

+ • MUTLTI-‐COLOR ELISPOT DETECTION

in vitro diagnosis of ACD (T cell assay)

The encapsula>on of hydrophobic allergens improves the detec>on of specific T cells circula>ng in the blood of allergic pa>ents to fragrances

Enhanced uptake by blood cells

Poly-‐ε-‐caprolactone

Nanopar4cles + fragrance mixture

Electronic microscopy

0"

10"

20"

30"

40"

50"

60"

70"

80"

90"

Hydroxy

citronell

al.

Cinnamy

l.alcoho

l.

Cinnama

l..Euge

nol.

Isoeugen

ol.Géra

niol.

Alpha;am

ylcinnam

al.Oak

moss.

Global.

NPs" Free"form"NPs" Free""

Sol

ubili

ty (%

)

Increased solubilisa4on

Imagestream

With nanopar4cles Without nanopar4cles

Dye+CD14+cells Dye+CD14+cells Bright field Bright field

In vitro diagnosis of fragrance allergy

FMIpos PT FMIneg PT0

1

2

3

4

5

6

7

8n=20 n=11

Prolifération

Index

de st

imula

tion m

axim

alRé

pons

e prol

iférat

ive

Stim

ulat

ion

indi

ces

(T

cel

l pro

lifer

atio

n)

Allergic patients / PBMCs

A.Cortial

CTLs

Memory T cell

iNKT

Foxp3+

ICOS+Treg

20 pa4ents Pa>ent stra>fica>on?

Project MODAL Conclusions & perspec>ves

ü  To determine the predic>ve value of molecular signatures in pa>ents with highly sensi>ve skin

ü  To combine molecular signatures / T cell assays / use-‐test responses to stra>fy contact allergy pa>ents, and improve clinical relevance assessment

ü  To develop new test systems (minimally invasive, low cost)

•  Molecular analysis reveals dis>nct signatures between :

-‐  allergens and irritants

-‐  allergy and irritancy (to be confirmed!)

•  The use of NPs allows for the sensi>ve detec>on of hydrophobic chemicals/mixtures in rou>ne T

cell assays

Agnès Lavoix

14

Allergology & Clinical Immunology department, Lyon-sud hospital

Clinical research unit, Lyrec- Lyon-sud hospital

Acknowledgements

Audrey Nobaum

JF Nicolas

David Bottigioli

Marc Vocanson

JF Nicolas

Team 17 / CIRI-INSERM U1111

Frédéric Bérard

Florence Hacard

Marine Lefèvre

Allergology & Clinical Immunology department, Lyon-sud hospital

Acknowledgements

Audrey Nobaum

JF Nicolas

Marc Vocanson

JF Nicolas

Team 17 / CIRI-INSERM U1111

Frédéric Bérard

Florence Hacard

MC. FERRIER-‐LEBOUEDEC / Clermont-‐Ferrand hospital P. PRALONG / Grenoble hospital C.DZIVGA / Saint E4enne hospital

A. HERMANN & M. BAECK / Clinical University Saint Luc, Bruxelles C. Svedman/L. Ljungberg/M. Bruze / Skane, Malmoe

Marine Lefèvre

The molecular diagnosis of contact allergy

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