Bet-specific iTreg ameliorated allergic symptoms in humanized mouse models of allergen-induced airway and intestinal inflammation
Humanized mice are a remarkable investigative tool and preclinical study system, which close the gap between exclusively murine and human studies.49 Here, we used well-established and standardized humanized mouse models of type I allergy to analyze the function of iTregBet on allergic symptoms in allergen-induced intestinal and airway inflammation.41,50,51 An overview of the reconstitution and challenge protocol is visualized in Figure 5A (see also Methods and Supplementary Methods). Briefly, immunodeficient mice (NOD.CB17-Prkdcscid/Jγc-/-) were engrafted with human PBMC from birch pollen allergic donors with associated hazelnut allergy and injected with birch pollen extract +/- iTregBet as indicated.41,52 After allergen-specific (birch) rectal challenge the intestinal inflammation was evaluated by a clinical score, that revealed a significantly increased inflammatory reaction of animals serving as allergic positive controls (PBMC + birch) compared to negative controls (PBMC), thereby demonstrating the validity of the humanized model and the development of an allergic immune reaction (Figure 5B, C). Intriguingly, injection of iTregBet resulted in a significant abrogation of the allergen-specific immune reaction as shown by a reduced intestinal inflammation (Figure 5B, C).
For induction of allergic asthma, the animals were engrafted and boosted as described above and were challenged intranasally with birch pollen extract as published previously.40,51 Subsequently, the airway resistance was assessed as outcome of the allergic immune reaction.40,51 In these experiments, allergic positive controls (PBMC + birch) showed a significantly enhanced airway hyperreactivity in contrast to negative control groups (PBMC) (Figure 5D). Importantly, co-injection of iTregBet curtailed the development of allergic asthma symptoms, as the airway resistance was significantly decreased compared to allergic asthma positive control animals (Figure 5D).
We also analyzed the serum concentrations of human birch-specific IgE (Figure 5E) as typical immunological parameter of type I allergic reactions prior to allergen challenge. Compared to enhanced amounts of birch-specific IgE in allergic positive controls (PMBC + birch), treatment with iTregBet significantly reduced birch-specific IgE concentrations in vivo , confirming our data of significantly reduced clinical symptoms of allergen-induced intestinal and airway inflammation after iTregBet application.
In order to investigate the induction of cross-reactive tolerancein vivo , the immunodeficient mice were engrafted and boosted as described above, but were challenged rectally with hazelnut extract prior to assessment of the intestinal inflammation (Figure 6). In these experiments, we found a less severe allergic immune response in the hazelnut-challenged compared to the birch-challenged control group (Figure 5C), likely due to lack of in vivo booster with hazelnut extract and/or to the donors’ less severe sensitization towards the food allergen. However, even after hazelnut challenge co-injection of iTregBet resulted in a pronounced inhibition of intestinal allergic symptoms compared to control animals, which was shown with data of individual experiments (Figure 6A). In addition, we observed reduced human hazelnut-specific IgE levels after iTregBet application and hazelnut challenge compared to the allergic positive control (Figure 6B). These data indicate the induction of allergen-specific (birch) as well as cross-reactive tolerance (hazelnut) in vivo through IL-10 DC-induced iTregBet.