Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3)

Abstract

Background: The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance.

Objective: Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy.

Methods: In a phase 1 single-site study, we studied participants (n = 23) undergoing peanut OIT and compared them with age-matched allergic control subjects (n = 20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n = 7), whereas those who had an allergic reaction were categorized as nontolerant (NT; n = 13).

Results: Antibody and basophil activation measurements did not statistically differentiate between NT versus IT participants. However, T-cell function and demethylation of forkhead box protein 3 (FOXP3) CpG sites in antigen-induced regulatory T cells were significantly different between IT versus NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months), only 3 participants remained classified as IT participants, and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced regulatory T cells.

Conclusion: In summary, modifications at the DNA level of antigen-induced T-cell subsets might be predictive of a state of operationally defined clinical immune tolerance during peanut OIT.

Keywords: APC; Antigen-induced regulatory T; Antigen-presenting cell; CD40 ligand; CD40L; CFSE; Carboxyfluorescein succinimidyl ester; DBPCFC; DC; Dendritic cell; Double-blind, placebo-controlled food challenge; Effector CD4(+) T; FOXP3; Food allergy; Forkhead box protein 3; IT; Immune tolerant; Induced regulatory T; LAG3; Lymphocyte activation gene 3; MFI; Mean fluorescence intensity; NT; Natural regulatory T; Nonspecific regulatory T; Nontolerant; OFC; OIT; Oral food challenge; Oral immunotherapy; Regulatory T; SPT; Skin prick test; T(R)1; Teff; Treg; Type 1 regulatory T; T regulatory cells; ai-Treg; allergy; desensitization; epigenetics; forkhead box protein 3; iTreg; nTreg; ns-Treg; oral immunotherapy; peanut; tolerance.

Fig. 1

OIT-treated participants (squares, n=13, NT or triangles, n=7, IT) or peanut-allergic control participants (circles, n=20). A) Changes in mean peanut-specific IgE levels (kU_A/L +/−SD) among IT (N=7), NT (N=13) and Control (N=20) subjects (NS, P=0.17) B) Change from baseline in mean antibody levels (+/−SD) for peanut-specific IgG4 (mg_A/L). Slight increase in IT and NT compared to Control (NS, P=0.24). C) Mean (+/−SD) of peanut-specific IgG4:IgE ratios. Slight increase in IT and NT compared to Control (NS, P=0.27) D) Expression of CD203c levels on basophils stimulated with peanut allergen (lug/ml). Data are presented as mean ± SEM (*P < 0.001, CI: −1222 to −777.8) IT or NT vs. Control. IT vs. NT (NS, P> 0.99) Mg = milligrams of antigen-specific antibody; MFI=mean fluorescence intensity. E) Expression of CD203c levels on basophils stimulated with anti-IgE (NS, P >0.999). F) Significant decrease in SPT diameter starting at 12mo for IT or NT compared to Control (*P<0.001, CI: −16.06 to −5.942). No difference in NT vs. IT (NS, P=0.656).

Fig. 2

A) Representative staining of ai-Treg and ns-Treg. B) Representative staining for ns-Treg and ai-Treg in Control, NT and IT participants at 30mo C) ns-Treg (open shapes) and ai-Treg (closed shapes) absolute counts (*P< 0.002, CI: 2.636 to 141.1,NTN=7, ITN=13, ControlN=20). D) ns-Treg (white bars) and ai-Treg (black bars) at baseline and 24mo of treatment (*P< 0.0001, CI: 41.1 to 162.6, NT N=7, ITN=13, Control N=20). Changes in ns-Treg not significant (P=0.84). E) Treg suppressive activity on conventional responder CD4+ T cells (Teff) measured pre-therapy (baseline) and at 27mo—i.e., after 3mo off treatment (filled bars). Suppressed proliferation of Teff in response to peanut stimulation in NT and IT compared to baseline (*P=0.0001, CI: 30.18 to 69.82) but not other allergens or tetanus (P>0.999). F) Suppressive function of Treg from pre and post-OIT. Suppressive function of Treg collected pre-therapy (pre) and at month 27 (post) was assessed towards ai-Teff collected pre- and post-therapy (data represent mean + SEM; *P < 0.001, CI: −32.03 to −17.97).

Fig. 3

A) Chemotactic Indices of ai-Treg toward normal IECs. Indices for cells from immune tolerant (n=7) or non-tolerant (n=13) subjects were significantly higher than those for control (n=20) participants starting at 12mo (*P< 0.001, CI: 5.854 to 7.346). Values for IT subjects were significantly higher than those for NT subjects starting at 12mo (#P<0.0001, CI: 3.004 to 4.596). B and C) Expression levels of chemokine receptors (circles, CCR8; squares, CCR4; triangles CCR7) on ai-Treg populations were identified by flow cytometry and are presented as MFI (xlO) at baseline and at 24mo of treatment and at 27mo (i.e., 3mo post cessation of treatment) in IT patients (Fig. 3B) (*P < 0.001, CI: 38.64 to 48.50) and NT patients (Fig. 3C) (*P<0.001, CI: 34.69 to 48.01). An example of chemokine receptor staining is shown in Fig. 3D.

Fig. 4

(A) Intracellular Foxp3 protein expression levels in ns-Treg (white bars) and ai-Treg (black bars) Values are mean fluorescence intensity (MFI). Significant differences in ai-Treg for IT vs. NT or control (*P <0.001, CI: 90.34 to 159.7), not significant for ns-Treg (P =0.1698). B) FOXP3 mRNA in Treg isolated from IT OIT participants (triangles), NT OIT participants (squares) and peanut-allergic control participants (circles). Significant differences in IT participants at 24 or 27mo compared to baseline (*P<0.001, CI: 2.871 to 4.437). Difference for NT participants non-significant (P =0.18). C) ai-Treg from participants undergoing OIT (IT, n=7, blue triangles; NT, n=13, red squares) or untreated controls (n=20, green circles). 4/7 IT participants (connected via broken lines) were no longer “tolerant” at 30mo. Data represent mean number of methylated sites + SEM; (IT: * P < 0.001 vs. baseline CI: 12.13 to 14.16, NT: *P < 0.001 vs. baseline CI: 3.919 to 5.620). Not significant for control (P= 0.15). MFI= mean fluorescence intensity.