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. 2017 Jan;139(1):189-201.e4.
doi: 10.1016/j.jaci.2016.03.057. Epub 2016 Jun 11.

Epicutaneous immunotherapy induces gastrointestinal LAP+ regulatory T cells and prevents food-induced anaphylaxis

Leticia Tordesillas  1 , Lucie Mondoulet  2 , Ana Belen Blazquez  1 , Pierre-Henri Benhamou  2 , Hugh A Sampson  3 , M Cecilia Berin  4
Affiliations

Epicutaneous immunotherapy induces gastrointestinal LAP+ regulatory T cells and prevents food-induced anaphylaxis

Leticia Tordesillas et al. J Allergy Clin Immunol. 2017 Jan.

Abstract

Background: The attempt to induce oral tolerance as a treatment for food allergy has been hampered by a lack of sustained clinical protection. Immunotherapy by nonoral routes, such as the skin, may be more effective for the development of maintained tolerance to food allergens.

Objective: We sought to determine the efficacy and mechanism of tolerance induced by epicutaneous immunotherapy (EPIT) in a model of food-induced anaphylaxis.

Methods: C3H/HeJ mice were sensitized to ovalbumin (OVA) orally or through the skin and treated with EPIT using OVA-Viaskin patches or oral immunotherapy using OVA. Mice were orally challenged with OVA to induce anaphylaxis. Antigen-specific regulatory T (Treg)-cell induction was assessed by flow cytometry using a transgenic T-cell transfer model.

Results: By using an adjuvant-free model of food allergy generated by epicutaneous sensitization and reactions triggered by oral allergen challenge, we found that EPIT induced sustained protection against anaphylaxis. We show that the gastrointestinal tract is deficient in de novo generation of Treg cells in allergic mice. This defect was tissue-specific, and epicutaneous application of antigen generated a population of gastrointestinal-homing LAP+Foxp3- Treg cells. The mechanism of protection was found to be a novel pathway of direct TGF-β-dependent Treg-cell suppression of mast cell activation, in the absence of modulation of T- or B-cell responses.

Conclusions: Our data highlight the immune communication between skin and gastrointestinal tract, and identifies novel mechanisms by which epicutaneous tolerance can suppress food-induced anaphylaxis.

Keywords: Epicutaneous immunotherapy; food allergy; mast cells; oral immunotherapy; regulatory T cells.

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Conflict of interest statement

Disclosure of potential conflict of interest: LT received travel support from DBV Technologies (the developer and owner of the Viaskin® patch) to present this work in part at the European Academy of Allergy and Clinical Immunology. LM is an employee of DBV Technologies, HAS is the Chief Scientific Officer of DVB Technologies and PHB is CEO of DBV Technologies. MCB has no conflict of interest.

Figures

Figure 1
Figure 1. EPIT induces sustained protection against food-induced anaphylaxis
(A) Experimental schematic. (B) Oral challenge with OVA in skin-sensitized mice at weeks 8 and 12. Body temperature 30 min after challenge is shown. (C) Oral challenge with OVA in orally-sensitized mice at weeks 8 and 12. Data are individual mice from 2 independent experiments. x = death. * p < 0.05, **p<0.01, ***p < 0.001.
Figure 2
Figure 2. OVA-specific IgG1 and IgG2a generated during EPIT do not modify basophil reactivity or protect against anaphylaxis
(A) IgE, IgG1 and IgG2a levels in serum prior to oral challenge from skin- or orally-sensitized mice at week 8 and 12 after EPIT or OIT. Data are mean ± SEM. (B) OVA-induced basophil activation measured by upregulation of CD200R. (C) Oral challenge of mice were passively sensitized with sera from naïve, untreated or EPIT-treated mice. Data show individual mice from 2 independent experiments. *p < 0.05 vs untreated mice.
Figure 3
Figure 3. Impact of sensitization on Treg generation by oral antigen
(A) Experimental schematic. (B) Gating strategy of OVA-specific Tregs. (C,D) Foxp3+ and LAP+ T cells generated in MLN (C) and spleen (D) of mice sensitized to OVA by the oral or skin route versus naive. (E) Foxp3+ and LAP+ T cells in MLN from naïve, OVA-sensitized mice or peanut-sensitized mice. Data are mean + SEM of at least 5 mice/group in 2 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.
Figure 4
Figure 4. Epicutaneous antigen expands LAP+ T cells in skin and gastrointestinal lymph nodes
(A) Experimental schematic. (B) Foxp3+ and LAP+ T cells in skin-draining lymph nodes, MLN and spleen of naïve mice. (C) Foxp3+ and LAP+ T cells in MLN of mice sensitized by the oral or skin route versus naive. (D) OVA-specific cells in lamina propria after EPIT. Data are mean + SEM of at least 6 mice/group (3 mice/group for lamina propria) in two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5
Figure 5. T cells are primed by epicutaneous antigen in brachial lymph nodes prior to migration to other tissues
(A) Proliferation of DO11.10 T cells recovered from brachial, inguinal and mesenteric lymph nodes and lamina propria 48h and 72h after application of antigen. (B) Proliferation of DO11.10 T cells in mice treated with FTY720. (C) Summary data showing the impact of FTY720 on proliferation at different sites. Data are mean + SEM of 3 mice/group.
Figure 6
Figure 6. LAP+ T cells express CCR9 and CCR6 after activation in skin-draining lymph nodes
(A) Expression of tissue-homing markers in DO11.10 T cells from brachial or mesenteric lymph nodes after epicutaneous or oral antigen, respectively. (B) Representative plots of the expression of tissue-homing markers by T cell subsets activated by epicutaneous antigen. (C) Summary data of chemokine receptor expression in sub-populations. Data are mean + SEM of 5 mice/group in 2 independent experiments. (D) Representative plots showing Treg surface markers. Minus control is staining control.
Figure 7
Figure 7. Tregs can directly suppress mast cell activation
(A) Experimental schematic. (B) Temperature measured 30 min after oral OVA challenge. (C) Experimental schematic. (D) MCPT-7 levels in serum obtained 30 min after challenge. (E) Levels of MCPT-7 in serum from mice injected with anti-TGFβ or isotype control calculated as % with respect to untreated mice.. Data are mean ± SEM of at least 6 mice/group in 3 independent experiments. * p < 0.05, **p < 0.01.
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