Dermatophagoides pteronyssinus immunotherapy changes the T-regulatory cell activity

Abstract

Subcutaneous specific immunotherapy (SCIT) has been shown to modify the Dermatophagoides pteronissinus (DP) allergic response, characterized by generation of Treg cells. However, studies have reported no changes in the proportion of Treg cells after immunotherapy, indicating that the effects may be due to modifications in their regulatory activities. We aimed to determine whether Tregs generated by DP-SCIT can switch the allergic response to tolerant and study the involvement of suppressive cytokines on it. Twenty-four DP-allergic rhinitis patients were recruited, 16 treated with DP-SCIT and 8 untreated. Treg and T effector cells were isolated before and after DP-SCIT, and cocultured in different combinations with α-IL-10, α-TGF-β blocking antibodies and nDer p 1. Treg cells after DP-SCIT increased Th1 and decreased Th2 and Th9 proliferation. Similarly, they increased IL-10 and decreased IL-4 and IL-9-producing cells. α-IL-10 affected the activity of Treg cells obtained after DP-SCIT only. Finally, DP-specific IgG4 levels, Treg percentage and IL-10 production were correlated after DP-SCIT. These results demonstrate that DP-SCIT induces Treg cells with different suppressive activities. These changes could be mediated by IL-10 production and appear to play an important role in the induction of the tolerance response leading to a clinical improvement of symptoms.

Figure 1

Determination of changes in DP-specific immunoglobulins and Treg populations. (A) DP-sIgE (Ku/L) and (B) DP-sIgG4 (mgA/L) levels for treated and untreated patients before (0 M) and after (12 M) 1-year of DP-SCIT. (C) Comparative analysis of treated (N = 16) and untreated (N = 8) patients before (0 M) and after (12 M) 1-year of treatment. Statistical Wilcoxon (related samples) and Mann-Whitney tests (non-related samples) were performed; p-values ≤ 0.05 are considered statistically significant.

Figure 2

DP-sIgG4 production after 12 months of DP-SCIT correlates with IL-10 production and iTregs. Correlations between percentage of Treg and DP-sIgG4 levels (mgA/L) at 12 months in treated (N = 16) (A) and untreated (N = 8) patients (B). Correlations between percentage of IL-10 secreting cells and DP-sIgG4 levels (mgA/L) at 12 months in treated (N = 16) (C) and untreated (N = 8) patients (D). Correlations between percentage of Treg and percentage of IL-10 secreting cells at 12 months in treated (N = 16) (E) and untreated (N = 8) patients (F). Statistical analysis was carried out using the Pearson correlation coefficient; p-values ≤ 0.05 are considered statistically significant.

Figure 3

Proliferative response of Teff after DP stimulation in absence or presence of Treg cells. Box plots represent comparative analysis of proliferation index (PI) of different T cell subsets Th1 (A), Th2 (B) and Th9 (C) from treated (N = 16) and Th1 (D), Th2 (E) and Th9 (F) from untreated (N = 8) patients in different combinations of Teff and Treg at different times: 0 or 12 months (0 M and 12 M, respectively). Statistical analysis was carried out by Wilcoxon test; p-values ≤ 0.05 are considered statistically significant. PI: proliferative index.

Figure 4

Cytokines  produced by Teff cells after DP stimulation in absence or presence of Treg cells. Box plots represent comparative analysis of percentages of Teff cells producing IL-10 (CD3⁺CD4⁺IL-10⁺) (A), IL-4 (CD3⁺CD4⁺IL-4⁺) (B) and IL-9 (CD3⁺CD4⁺IL-9⁺) (C) from treated (N = 16) and IL-10 (CD3⁺CD4⁺IL-10⁺) (D), IL-4 (CD3⁺CD4⁺IL-4⁺) (E) and IL-9 (CD3⁺CD4⁺IL-9⁺) (F) from untreated (N = 8) patients in different combinations of Teff and Treg at 0 or 12 months (0 M and 12 M, respectively). Statistical analysis was carried out by Wilcoxon test; p-values ≤ 0.05 are considered statistically significant.

Figure 5

Changes induced by α-IL-10 and α-TGF-β blocking antibodies in different subpopulations of DP-specific Teff cells from DP-SCIT treated patients (N = 16). Bars represent different comparisons of IL-10 and TGF-β inhibitions performed in absence or presence of Tregs (from 0 or 12 months) on Th1 0 (A) and 12 months(B), Th2 0 (C) and 12 months (D) and Th9 0 (E) and 12 months (F). Statistical analysis was carried out by Wilcoxon test; p-values ≤ 0.05 are considered statistically significant. PI: proliferative index; 0 M: Treg cells obtained at 0 months; 12 M: Treg cells obtained at 12 months.