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Background Allergen-specific immunotherapy favours immune deviation from a Th2 to a Th1 response and increases the number of regulatory T cells (Tregs). Epicutaneous immunotherapy (EPIT) of sensitized mice decreases the clinical and the allergen-specific Th2 responses and increases local and peripheral Foxp3(+) Tregs. Objective To investigate the role of Tregs in EPIT and characterize their phenotype and maintenance following EPIT. Methods Tregs were investigated using in vivo depletion or adoptive transfer into BALB/c mice. Tregs were depleted using anti-CD25 antibody injection during EPIT, and allergen-specific responses were compared with Sham, EPIT alone and naive mice. To demonstrate that Tregs can mediate protection by their own, and to study their maintenance following the end of EPIT, CD25(+) CD4(+) Tregs isolated from mice just after or 8 weeks after EPIT were transferred into peanut-sensitized mice. Foxp3-IRES-mRFP mice were transferred with EPIT-induced Tregs to analyse the induction of host Tregs. Results The anti-CD25 antibody injection to EPIT mice abrogated the induction of Tregs in spleen and the expression of Foxp3 in oesophagus. This resulted in levels of peanut-induced eosinophilic infiltration in oesophagus similar to Sham and significantly higher than EPIT. Whereas the transfer of Tregs from Sham-treated mice demonstrated no effect, the transfer of Tregs isolated just after EPIT prevented peanut-induced eosinophil infiltration and eotaxin expression and induced Foxp3 in oesophagus. The transfer of Tregs isolated 8 weeks after EPIT suppressed allergen-specific responses as efficiently as did Tregs isolated just after EPIT and increased spleen Foxp3(+) CD25(+) CD4(+) cells similarly. The use of reporter mice demonstrated an increase in host Tregs. Conclusions These results confirm the Tregs-mediated mechanism of EPIT and demonstrate the persistence of efficient Tregs during a long period of time after treatment cessation. This suggests that EPIT induces long-term tolerance in peanut-sensitized mice.

Introduction L’immunotherapie epicutanee sur peau intacte (EPIT) est une voie therapeutique des allergies alimentaires en cours de developpement. Dans les modeles animaux, la desensibilisation obtenue par l’EPIT est maintenue apres arret du traitement (tolerance) et previent de sensibilisations a d’autres allergenes. Le but de ce travail etait d’evaluer, dans un modele de souris sensibilisees, la cinetique des modifications epigenetiques etayant l’effet therapeutique de l’EPIT et l’induction de tolerance. Methodes Des souris BALB/c femelles sensibilisees a l’arachide ont ete traitees par EPIT ou immunotherapie orale (OIT) ou non traitees pendant 8 semaines. Les prelevements de rate et sang ont ete realises aux semaines 1, 2, 4 et 8 du traitement et 8 semaines apres arret du traitement. Un autre panel de groupes de souris a ete analyse apres une phase de sensibilisation a un autre allergene, l’ovalbumine. La methylation de l’ADN a ete analysee par pyrosequencage dans les cellules CD4+ purifiees de la rate et du sang. Resultats Dans les cellules CD4+ (rate et sang), une hypermethylation des ilots CpG associes au promoteur de Gata3 apparait des la 4e semaine d’EPIT, persiste jusqu’a la fin du traitement (p Conclusion L’EPIT comparee a l’OIT conduit a une signature epigenetique unique et stable permettant une regulation negative de l’expression des genes de la voie du Th2 et une activation des cellules T regulatrices. Ces mecanismes seraient impliques dans l’induction de tolerance par EPIT.

Background Allergy to cow's milk increases the risk of sensitization to other foods in young children. Objectives We sought to evaluate the effect of early epicutaneous immunotherapy (EPIT) on further sensitization to peanut or house dust mite (HDM) in a murine model of sensitization to cow's milk. Methods BALB/c mice orally sensitized to milk were epicutaneously treated with a Viaskin patch (DBV Technologies) loaded with milk proteins for 8 weeks. Mice were then sensitized to peanut or HDM. After sensitization to peanut, mice were exposed to a peanut regimen known to induce eosinophilic esophageal inflammation. After sensitization to HDM, mice were challenged with aerosols to HDM, and airway hyperresponsiveness was evaluated by using plethysmography. Humoral response was also analyzed. The role of regulatory T (Treg) cells was evaluated by adoptively transferring Treg cells from milk EPIT–treated mice to naive mice before sensitization to peanut. Protection against anaphylaxis was also investigated. Methylation of the promoter region of transcription factors was analyzed by using PCR assays. Results In milk-sensitized mice specific EPIT prevented further sensitization to peanut or HDM. EPIT significantly modified the humoral response, reduced T H 2 cytokine levels, decreased eosinophilic esophageal infiltration, and suppressed airway hyperresponsiveness. The protective effect was sustained over 2 months. Moreover, the adoptive transfer of milk EPIT Treg cells completely prevented sensitization to peanut and peanut-induced anaphylaxis. Milk EPIT enhanced methylation of the GATA-3 promoter region. Conclusions Our results showed that EPIT influences the natural history of allergy and reduces the risk of further sensitization through a Treg cell–dependent mechanism.

Introduction L’immunotherapie epicutanee sur peau saine (EPIT®) constitue une voie therapeutique nouvelle des allergies alimentaires. Ses mecanismes d’actions ont ete mis en evidence dans plusieurs modeles precliniques et sont largement lies aux cellules T regulatrices (Tregs). Par son profil d’efficacite et sa securite d’emploi, elle est adaptee au traitement precoce des allergies. Le but de ce travail est d’evaluer, dans un modele de souris sensibilisees, l’impact du traitement precoce par EPIT sur l’evolution naturelle des allergies. Methodes Des souris BALB/c femelles sensibilisees au lait ont ete traitees par EPIT ou non traitees (NT) pendant 8 semaines avant d’etre sensibilisees a l’arachide. Les animaux ont ensuite ete testes par injection intraveineuse (IV) d’arachide afin de declencher une anaphylaxie evaluee par la baisse de temperature et la liberation de mMCP-1dans le plasma. Des souris uniquement sensibilisees a l’arachide et des souris naives constituent respectivement les groupes controles positifs et negatifs. Dans un deuxieme temps, des Tregs provenant de souris sensibilisees au lait EPIT ou NT ont ete transferees a des souris naives avant sensibilisation a l’arachide. La reponse a l’injection IV d’arachide a ete etudiee dans les memes conditions. Resultats Les souris NT sensibilisees successivement au lait puis a l’arachide presentaient, comme les souris sensibilisees a l’arachide, une forte chute de la temperature corporelle apres le challenge IV (4,1 °C ; p Conclusion Le traitement precoce par EPIT pourrait prevenir les sensibilisations ulterieures par un mecanisme dependant des Tregs. Des etudes cliniques devront confirmer cet effet protecteur chez l’Homme.

Background Eosinophilia is often linked to allergic gastrointestinal disorders linked to food allergy. EPIT using Viaskin® device has been described as a therapeutic method in food allergy. We developed a model of mice sensitized to peanut, exhibiting EoE and AE after exclusive feeding with peanut protein extracts (PPE). This study was conducted in order to evaluate the efficacy of EPIT. Methods After oral sensitization with PPE and cholera toxin, 30 BALB/c mice were treated weekly during 8 weeks by PPE skin applications (EPIT), 20 mice were not treated (Sham) and 10 mice constituted the control group (C). Mice were then exclusively fed with PPE. Specific IgE, IgG1 and IgG2a were monitored during immunotherapy. Esophageal and jejunal samples were taken for histological analyses. Results sIgE increased after oral sensitization, respectively 0.207 ±0.03 and 0.214±0.04 μg/ml, in EPIT and Sham, with undetectable values in C. Following EPIT, sIgE decreased and sIgG2a increased, respectively 0.139±0.01 vs 0.166±0.01 μg/ml (EPIT vs Sham, p

Epicutaneous immunotherapy onto intact skin has proved to be an efficient and safe alternative treatment of allergy in an animal model with various allergens and in children for cow’s milk allergy. The aim of this study was to analyze the different steps of the immunological handling of the allergen when deposited on intact skin using an epicutaneous delivery system and its immune consequences in sensitized BALB/c mice. As expected, when applied on intact skin, OVA exhibits neither a passive passage through the skin nor any detectable systemic delivery. The current study demonstrates that, after a prolonged application on intact skin, OVA is taken up by dendritic cells in the superficial layers of the stratum corneum and transported, after internalization, to the draining lymph nodes, with variations according to the previous level of sensitization of the mice. When OVA is applied with the epicutaneous delivery system repeatedly, specific local and systemic responses are down-modulated in association with the induction of regulatory T cells. Besides providing new insights into skin function in the presence of allergens, this study indicates that the skin might have a tolerogenic role, at least when kept intact.

Introduction Les cellules T regulatrices (Tregs) jouent un role primordial dans les mecanismes d’action de l’immunotherapie specifique. La capacite des Tregs a migrer vers les organes cibles est liee a l’expression de recepteurs specifiques. Le but de cette etude est la caracterisation de l’expression des recepteurs de migration vers la peau (CLA) et le tube digestif (CCR9) par les Tregs induit par l’immunotherapie epicutanee (EPIT) qui constitue une voie therapeutique nouvelle pour le traitement des allergies alimentaires. Methodes Des souris BALB/c femelles sensibilisees a l’arachide par voie orale ont ete traitees par EPIT ou non traitees (NT). Apres 8 semaines de traitement, les souris ont ete sacrifiees afin de prelever la rate, les ganglions mesenteriques (mLNs) et les ganglions inguinaux (iLNs) drainant la peau. Apres extractions des cellules, la proportion de Tregs et l’expression des recepteurs de migration specifique CLA + CCR9– (peau), CLA-CCR9+ (tube digestif) et CLA + CCR9+ (double tropisme) ont ete analyses par cytometrie en flux. Resultats Apres 8 semaines de traitement, la proportion de cellules Tregs CD4 + CD25 + Foxp3+ dans la rate et les iLNs est augmentee par EPIT par rapport au NT ( p p p p Discussion Les Tregs Foxp3+ jouent un role essentiel au cours du traitement de l’allergie alimentaire. Le tropisme a la fois digestif et cutane des Tregs induits par l’EPIT aussi bien dans les ganglions drainant la peau que le tube digestif, fait de cette methode un traitement de choix de l’allergie alimentaire.

Results: In spleen, expression of CCR4 increased on Tregs induced by all 3 therapies whereas the expression of CXCR3, CCR6 and CCR8 increased on EPIT-induced Tregs only. Skin homing receptor (CLA) increased on EPITbut not SLITor OIT-induced Tregs. Gut homing receptor CCR9 increased on EPITand OITbut not SLIT-induced Tregs. Interestingly, 81% of the CCR9+ Tregs induced by EPIT expressed CLA whereas only 43% did after OIT. In iLN, Tregs level increased only after EPIT with induction of CLA+CCR9-(22%) and CLA+CCR9+ (12%) Tregs. In mLN, EPIT and OIT significantly induced CCR9+ Tregs, 25% and 18% of them expressing also CLA in EPIT and OIT respectively.

Results: In splenocytes, a significant hypermethylation in GATA-3 CpG islands was induced by EPIT versus Sham, starting from the 4 of treatment (p

Methods: After sensitization to milk, mice were treated by EPIT or Sham, and 2 weeks later, submitted to a peanut-sensitization procedure. Mice were then intravenously challenged with peanut. In a second experiment, CD4+CD25+ T cells were isolated from spleen of milk-sensitized mice after EPIT or Sham, and transferred into naive mice. Recipient mice were submitted to peanut sensitization and intravenously challenged with peanut. In a third experiment, Tregs were obtained from Foxp3+-gfp mice sensitized to peanut and EPIT-treated, then adoptively transferred. Recipient mice were submitted or not to peanut sensitization before transfer, then all sensitized and IV challenged to ovalbumin. Outcome tools were rectal temperature, hypersensitivity reactions and blood mouse mast cell protease-1 (mMCP1).