Epigenetic modifications and improved regulatory T-cell function in subjects undergoing dual sublingual immunotherapy

Abstract

Background: Allergen-specific immunotherapy is the only mode of therapy that has been demonstrated to offer a cure in patients with IgE-mediated respiratory allergies.

Objective: We sought to demonstrate the safety and efficacy of timothy grass (TG) and dust mite (DM) dual sublingual immunotherapy (SLIT) and to begin to investigate the immune mechanisms involved in successful immunotherapy with multiple allergens.

Methods: The safety and efficacy of dual SLIT with TG and DM in children and adults with demonstrated allergies to TG and DM were investigated in a single-center, randomized, double-blind, controlled phase I study. Thirty subjects received either TG and DM dual SLIT (n= 20) or placebo (n = 10). Immune parameters were evaluated for differentiation of desensitized subjects from control subjects.

Results: Subjects treated with dual SLIT had decreased rhinoconjunctivitis scores (P < .001) and medication use scores (P < .001) and reduced responses to TG and DM allergen based on results of skin prick tests or nasal disk challenges (P < .01 and P < .001, respectively) compared with placebo-treated control subjects. An increase in TG- and DM-specific IgG(4) levels, reduced allergen-specific IgE levels, and subsequent basophil activation were observed in the active treatment group. Dual SLIT promoted allergen-specific suppressive CD4(+)CD25(high)CD127(low)CD45RO(+) forkhead box protein 3 (Foxp3)(+) memory regulatory T cells with reduced DNA methylation of CpG sites within the Foxp3 locus.

Conclusion: The results of this pilot study suggest that dual SLIT could be an effective means to treat subjects with sensitivities to a variety of allergens and that long-term tolerance might be induced by epigenetic modifications of Foxp3 in memory regulatory T cells.

FIG 1

Clinical efficacy of dual SLIT. A and B, Rhinoconjunctivitis symptom scores (RCS; Fig 1, A) and medication use scores (MS; Fig 1, B). C, Responses to skin prick test challenges. D, NDCs to TG at 18 months. The orange circle denotes tolerant subjects. **P < .01 and ***P < .0001.

FIG 2

Change from baseline in mean antibody levels for TG-specific humoral responses. Phl p 5–specific IgE (A) and IgG₄ (B) responses from serum collected at baseline and at various time points from dual SLIT–treated subjects (squares, n = 20) or placebo-treated subjects (circles, n = 10) is shown. *P < .05. GPS, Grass allergy season; mgA, milligrams of antigen-specific antibody.

FIG 3

Decreased basophil activation from subjects receiving dual immunotherapy. Expression of CD203c levels on basophils stimulated with TG, DM, and oak from subjects before therapy (Pre) and after (Post) 24 months of dual SLIT (A–C) or placebo (D–F) is shown ***P < .001. NS, Not significant: P = .08; MFI, mean fluorescence intensity.

FIG 4

Memory Treg cells from dual SLIT–treated subjects suppress allergen-specific T-cell responses. Treg cell–suppressive activity of total mixed Treg cells from dual SLIT–treated subjects (A), memory Treg cells from dual SLIT–treated subjects (B), total mixed Treg cells from placebo-treated subjects (C), or total Teff cell proliferation (D) before (pre) and after (post) treatment is shown. *P < .05. NS, Not significant.

FIG 5

Epigenetic regulation of Foxp3 in Treg cells from subjects treated with dual SLIT. A, CpG methylation of purified memory or naive Treg cells from subjects before (Pre) and 12 months after (Post) therapy. B, Analysis of Foxp3 transcript from memory Treg cells. Foxp3 transcription levels are shown as relative fold increase over expression of the housekeeping gene β-glucuronidase. *P < .05.

FIG 6

Increased memory Treg cells in tolerized subjects. Longitudinal analysis of memory CD4⁺CD25^highCD127^loCD45RO⁺CD62L⁻Foxp3⁺ Treg cells from tolerant (circles, n = 7) and desensitized (squares, n = 9) subjects after dual SLIT is shown. *P < .05. mo, Months after baseline.

FIG 7

Expression profiles of Treg cell markers in tolerant subjects. A and B, Expression of Treg cell markers on memory or naive Treg cells purified from dual SLIT–treated subjects (Fig 7, A) or placebo-treated subjects (Fig 7, B) before (pre) and 12 months after (post) treatment. *P < .05 for PD-1, IL-10, and Foxp3. C, Baseline expression levels from memory Treg cells from subjects who became tolerized (n = 7) or desensitized (n = 9). Gzm B, Granzyme B; MFI, mean fluorescence intensity.