Oral immunotherapy with omalizumab reverses the Th2 cell-like programme of regulatory T cells and restores their function

Abstract

Background: Oral immunotherapy (OIT) successfully desensitizes patients with food allergies, but the immune mechanisms mediating its efficacy remain obscure.

Objectives: We tested the hypothesis that allergen-specific regulatory T (Treg) cell function is impaired in food allergy and is restored by anti-IgE antibody (omalizumab)-supplemented OIT.

Methods: Peanut-specific T effector (Teff) and Treg cell proliferative responses, activation markers and cytokine expression were analysed by flow cytometry in 13 peanut-allergic subjects before the start of omalizumab-supplemented OIT and periodically in some subjects thereafter for up to 2 years. Peripheral blood regulatory T cells (Treg cells) were analysed for their peanut-specific suppressor function before and at 1 year following OIT. This study was registered on ClinicalTrials.gov (NCT01290913).

Results: Proliferation of allergen-specific Teff and Treg cells precipitously declined following the initiation of omalizumab therapy prior to OIT, followed by partial recovery after the initiation of OIT. At baseline, peanut-specific Treg cells exhibited a Th2 cell-like phenotype, characterized by increased IL-4 expression, which progressively reversed upon OIT. Peanut-specific Treg cell suppressor activity was absent at the start of omalizumab/OIT therapy but became robust following OIT. Absent peanut-specific Treg cell function could also be recovered by the acute blockade of IL-4/IL-4R receptor signalling in Treg cells, which inhibited their IL-4 production.

Conclusions and clinical relevance: OIT supplemented by omalizumab promotes allergen desensitization through an initial omalizumab-dependent step that acutely depletes allergen-reactive T cells, followed by an increase in allergen-specific Treg cell activity due to the reversal of their Th2 cell-like programme. Improved Treg cell function may be a key mechanism by which OIT ameliorates food allergy.

Keywords: FOXP3; food allergy; omalizumab; oral immunotherapy; regulatory T cells.

Fig 1. Peanut-specific CD4 T cell proliferation is reduced during rush desensitization

A, Gating strategy for identifying proliferating Violet^lowCD4⁺CD3⁺ Treg cells in PBMC cultures stimulated with anti-CD3+anti-CD28 mAb beads. Treg cells were first identified by gating on CD25^highCD127^low cells among Violet^lowCD4⁺CD3⁺ T cells, followed by gating on CD4⁺FOXP3⁺ cells. The same gating strategy was used for peanut protein-stimulated cultures. B–E, Flow cytometric analysis and enumeration of proliferating Violet^low total CD3⁺CD4⁺ cells loaded with CTV proliferation dye and cultured with peanut protein (Fig 1, B and C) or with anti-CD3+anti-CD28 mAb beads (Fig 1, D and E). Graph represents average data for all patients normalized to day 0 ±SEM over the course of the study. ****p<0.0001 versus baseline determined using paired t-test. F–I, Representative plots at 4 weeks 0, 12, 52, 104 of % peanut-specific Violet^low CD4⁺ CD3⁺ FOXP3⁺ cells. G and I, Graphs representing the average data for all patients normalized to day 0 ±SEM over the course of the study. *p<0.05, **p<0.01, ****p<0.0001 by one way ANOVA with post test analysis with comparisons made to week 12 values.

Figure 2. Reversal of Th2 Cytokine skewing during peanut desensitization

A, Gating strategy for analysis of Treg and Teff cells. Violet^lowCD3⁺CD4⁺ T cells were further gated on CD4⁺CD25⁺CD127^low (Treg cells) and CD4⁺CD25⁻CD127^high (Teff) then analyzed for FOXP3 expression. B, Representative FACS plots of IL-4 and IFN-γ expression in Violet^low (peanut-specific) CD4⁺ Teff cells. C–F, percentages (Fig 2, C and D) and absolute numbers (Fig 2, E and F) of IL-4 and IFN-γ expression in peanut-responsive (Violet^low) CD4⁺ Teff cells in all 10 patients responding to OIT treatment measured at weeks 0, 12, 24, 52 and 104. G, Representative FACS plots of IL-4 and IFN-γ expression in Violet^low CD3⁺CD4⁺FOXP3⁺ Treg cells. H–K, percentages (Fig 2, H and I) and absolute numbers (Fig 2, J and K) of IL-4 and IFN-γ expression in peanut-responsive (Violet^low) CD4⁺FOXP3⁺ Treg cells in the same subjects as panels C–F at weeks 0, 12, 24, 52, and 104. Results represent means ±SEM. *p<0.05, **p<0.01, ***<0.001, ****<0.0001 by one way ANOVA with post test analysis with comparisons made to week 0 values.

Figure 3. OIT therapy results in the contraction of the peanut-specific Treg cell TEM response

A, Representative FACS plots showing the gating strategy for identifying CD3⁺CD4⁺Violet^low Teff and Treg cells as shown by FOXP3 expression. B, Schematic representation of the strategy for the identification of T cell memory subsets by flow cytometry based on CD45RA and CCR7 expression. D–F, Flow cytometric analysis of CD45RA and CCR7 expression on peanut-responsive CD3⁺CD4⁺Violet^low Teff (Fig 3, C and D) and Treg cells (Fig 3, E and F) at weeks 0 and 104. Results represent means ±SEM. *p<0.05, **p<0.01 by Student’s two tailed paired t test analysis.

Figure 4. Peanut desensitization restores peanut-specific Treg cell suppression

A, Schematic diagram of the suppression assay set-up. CD3⁺CD4⁺CD25⁻ Teff cells, and CD3⁺CD4⁺CD25⁺CD127^low Treg cells were purified by FACS and cultured with peanut protein and CD3⁻ antigen-presenting cells in four different combinations: 1- Week 0 Teff with Week 0 Treg cells. 2- Week 0 Teff with Week 52 Treg cells. 3- Week 52 Teff with Week 0 Treg cells. 4- Week 52 Teff with Week 52 Treg cells. As controls, Week 0 and Week 52 Teff cells were cultured absent Treg cells. Teff cells were labeled with CTV proliferative dye at the start of the cultures B, FOXP3 CNS2 methylation analysis in FACS purified Teff and Treg isolated from Weeks 0 and Week 52 samples C–E, Representative histograms showing CTV dye dilution in CD3⁺CD4⁺ T cells of the indicate cultured cell combinations. F, Percentages of proliferating CD3⁺CD4⁺Violet^low Teff cells for all 4 patients. Results represent means ±SEM of studies on weeks 0 and week 52 samples, as indicated, derived from 4 subjects. *p<0.05, ***<0.001 by two way ANOVA with post-test analysis.

Figure 5. IL4R blockade rescues peanut-specific Treg suppressor function

A. In vitro modulation of peanut-specific Treg suppression assays by anti-IL-4Rα mAb treatment. Representative flow cytometric analysis of CTV-loaded Teff cells derived from treatment-naïve peanut-allergic subjects and co-cultured for 5 days with APCs in the absence or presence of peanut protein. Cell cultures were treated with an isotype control or an anti-IL-4Rα mAb, and Treg cells were added at a Teff:Treg cell ratios of 1:0, 2:1 and 4:1, as indicated. Teff cells were then analyzed for CTV dye dilution. B, Percentages of CD3⁺CD4⁺Violet^low cells in the respective cultures. C and D, Representative histograms showing Teff and Treg cell production of IL4 respectively. E and F, Percentages of Teff and Treg cell IL4 production. Results shown in panels B, E and F represent means ±SEM of data derived from cultures of 5 peanut allergic subjects. *p<0.05, **<0.01, ***<0.001 by paired one way ANOVA with post-test analysis.