Circulating Food Allergen-Specific Antibodies, Beyond IgG4, Are Elevated in Eosinophilic Esophagitis

Abstract

Introduction: Eosinophilic esophagitis (EoE) is a chronic inflammatory condition with an incompletely understood immuno-pathogenesis involving a T2 response. EoE is triggered by food allergens although, unlike IgE-mediated allergies, it exhibits high IgG4 levels in oesophageal biopsies and in circulation. We investigated whether other antibody isotypes specific for food allergens are elevated in EoE and vary with disease activity.

Methods: Plasma samples from patients with active EoE (n = 51), inactive EoE (n = 82) and non-EoE controls (n = 14) were analysed for food-specific IgG and IgA subclasses against casein, whey, wheat, egg and individual cow's milk allergens by ELISA. α-lactalbumin (Bos d 4)- and β-lactoglobulin (Bos d 5)-specific B cells were measured by flow cytometry in a subset of patients.

Results: Food allergen-specific antibodies in the plasma varied across EoE subgroups and non-EoE controls. Elevated IgG4 in EoE patients confirmed a strong antibody response to food allergens, including casein, wheat and egg. α_S1-casein (Bos d 9)-specific IgG, IgG2, IgG4, IgA1 and IgA2 differed between EoE and non-EoE controls and between active and inactive EoE. β-casein (Bos d 11, A1 variant) measurements showed higher levels of specific IgG2 and IgG4 in both EoE groups, whereas whey-derived allergens showed opposing responses: Bos d 4 responses favoured IgG4, and Bos d 5 responses were elevated across multiple IgG and IgA subclasses in EoE. Allergen-specific B cells could not be isolated from the circulation.

Conclusion: Our findings reveal distinct antibody profiles in EoE plasma, with elevated IgG and IgA subclasses beyond IgG4, highlighting a complex immune response to food allergens. Differential antibody responses support their clinical relevance in dietary management strategies, while the absence of allergen-specific B cells in circulation likely restricts antibody production to the inflamed oesophagus. Future research should explore whether these antibody profiles can guide personalised treatment and novel therapeutic targets in EoE.

Keywords: allergen‐specific antibodies; eosinophilic esophagitis; humoural response.

FIGURE 1

Food extract‐specific IgG and IgG4. (A) Specific IgG and IgG4 levels against casein, whey, wheat and egg extracts in EoE patients and healthy controls. Statistical significance was calculated using the Kruskal‐Wallis test and the Dunn's multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (B) Correlation between the food‐extract specific IgG and IgG4 measurements. Both point colour and size represent correlation strength: Red indicates stronger negative correlations, whereas blue indicates stronger positive correlations, with larger points corresponding to stronger correlations. The numerical value inside each point represents the p value. S, specific.

FIGURE 2

Individual casein‐derived allergen‐specific antibodies in EoE patients and non‐EoE controls. (A) Bos d 9 total specific IgG, specific IgG1‐4 and specific IgA1‐2. (B) Bos d 11/A1 β‐casein variant‐specific IgG, specific IgG1‐4 and specific IgA1 levels. (C, D) Total specific IgG and specific IgG4 were measured against Bos d 11/A2 and I β‐casein variants. Statistical significance was calculated using the Kruskal–Wallis test and the Dunn's multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. S, specific.

FIGURE 3

Individual whey‐derived allergen‐specific antibodies in EoE patients and non‐EoE controls. (A) Bos d 4 and (B) Bos d 5 total specific IgG, specific IgG1‐4 and specific IgA1‐2. Statistical significance was calculated using the Kruskal–Wallis test and the Dunn's multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. S, specific. [Correction added on 19 September 2025, after first online publication: The Figure 3 caption has been corrected.]

FIGURE 4

Heatmaps of individual cow's milk protein‐specific antibodies of (A) the IgG subclasses and (B) of the IgA subclasses. [Correction added on 19 September 2025, after first online publication: The Figure 4 legend has been corrected.]

FIGURE 5

Method used to isolate and expand allergen‐specific B cells. Class‐switched B cells (CD19⁺IgD⁻IgM⁻) were isolated and immortalised as described by Kwakkenbos et al. Cells were expanded in IMDM and stimulated with IL‐21 and CD40L‐L cells at 37°C and 5% CO₂. Allergen‐specific B cells were isolated after being stained with allergen‐PE and allergen‐AF647 and expanded again. Specificity was confirmed by flow cytometry and ELISA on the supernatant of cell cultures.

FIGURE 6

Isolation of Bos d 4‐ and Bos d 5‐specific B cells. (A) Flow cytometry gating strategy for live B cells. (B) Representative flow cytometry plots showing the isolation of Bos d 4 and Bos d 5‐specific B cells before and after expansion. Staining with Strep‐AF647 and Strep‐PE was used as a negative control.