Elevated expression of activated TH2 cells and milk-specific TH2 cells in milk-induced eosinophilic esophagitis

Abstract

Background: Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that is triggered by food allergens and characterized by progressive esophageal dysfunction. Esophageal biopsy specimens are characterized by eosinophilia and expression of T_H2 cytokines.

Objective: To ascertain whether T_H2 cells can exist in the peripheral blood in patients with milk-induced EoE.

Methods: Peripheral blood mononuclear cells from 20 children with milk-induced EoE were collected during active EoE (EoE-A) while consuming milk and inactive EoE (EoE-I) while not consuming milk, and 8 healthy patients without EoE were used as controls. The samples were analyzed for T-cell phenotype, including intracellular cytokines before and after incubation with milk antigens and assessed by flow cytometry.

Results: We found a significant increase in CD4⁺ T_H2 cells in the peripheral blood of patients with EoE-A compared with the controls. Furthermore, we observed a significant mean (SD) increase in the activation marker of CD154⁺ T cells (0.17% [0.047^%]) in patients with EoE-A compared with control patients (0.034% [0.007%]) and EoE-I (0.025% [0.008]). These CD4⁺ T cells expressed significantly increase levels of T_H2 cytokines (interleukins 4, 5, and 13) compared with the EoE-I and control groups. CD3⁺CD4⁺CD154⁺IL-5⁺ cells were significantly increased by milk antigens in both milk-induced EoE-A (0.050% [0.008%] to 0.079% [0.017%]) and EoE-I (0.0045% [0.002%] to 0.014% [0.008%]) compared with the controls (0.008% [0.003%] to 0.003% [0.001%]).

Conclusion: Our findings indicate that in EoE peripheral T cells have specific activation to milk allergens.

Figure 1

Eosinophils per high-power field (hpf). Esophagogastroduodenoscopy with biopsy was performed, and the highest eosinophil counts are shown for patients following a diet that contained milk (milk exposure) and a diet that did not contain milk (no milk exposure).

Figure 2

Esophageal expression of T_H2 cytokines. T_H2 gene expression from esophageal biopsy specimens was assessed by digital multiplexed gene expression assay for patients during active EoE eosinophilic esophagitis (EoE-A) and inactive EOE (EoE-I) as well as controls without EoE. Patients with EoE-A had a higher expression of interleukin (IL) 4 (A) and IL-13 (B). P < .05 was considered to be statistically significant (analysis of variance with Tukey post hoc test).

Figure 3

Ex vivo expression of CD4⁺ cells in patients undergoing esophagogastroduodenoscopy during active EoE eosinophilic esophagitis (EoE-A) and inactive EOE (EoEI). Expression is shown as percentage of positive cells (see eFigures for gating). Patients with milk-induced EoE-A have higher levels of T_H2 cytokine (interleukin [IL] 5) (A), IL-13 (B), and decreased T_H1 cytokine (interferon [IFN] Ɣ) (C) in CD4⁺ cells. P < .05 was considered to be statistically significant (Mann-Whitney U test).

Figure 4

Ex vivo expression of CD4⁺CD154⁺ T cells in patients undergoing esophagogastroduodenoscopy during active EoE eosinophilic esophagitis (EoE-A) and inactive EOE (EoE-I). Expression is shown as percentage of positive cells (see eFigures for gating). A, Patients with milk-induced EoE-A have increased levels CD154⁺ cells compared with patients with EoE-I or controls. In addition, patients with EoE-A have an increased T_H2 cytokine expression (interleukin [IL] 4) (B), IL-5 (C), and IL-13 (D) in CD4⁺CD154⁺ T cells compared with controls or patients with EoE-I. P < .05 is considered to be statistically significant (Mann-Whitney U test).

Figure 5

Stimulation of CD3⁺CD4⁺CD154⁺IL-5⁺ with milk antigens or α-CD3CD28. Peripheral blood mononuclear cells were cultured with milk antigens (250 mg/mL) (A and C) or stimulated with α-CD3CD28 (B and D). Flow cytometry was performed gating on activated T cells measured by CD154⁺ (A and B) or T_H2 activated T cells (CD154⁺IL-5⁺) (C and D). Patients with active eosinophilic esophagitis (EoE-A) have higher levels of CD154 activation marker with milk at baseline. Patients with inactive EoE (EoE-I) had increased markers of T-cell activation with milk, whereas T cells were activated in all patients with α-CD3CD28.