Changes in CD4⁺CD25⁺FoxP3⁺ Regulatory T Cells and Serum Cytokines in Sublingual and Subcutaneous Immunotherapy in Allergic Rhinitis with or without Asthma

The prevalence of allergic rhinitis (AR) and asthma has increased dramatically in children and adults in China over recent decades [1, 2]. A recent survey in 18 major cities in mainland China revealed an average prevalence of self-reported AR of 17.6%. Concurrently, the incidence of asthma was significantly higher among individuals with AR [1]. Dermatophagoides pteronyssinus (Der-p) and Dermatophagoides farinae (Der-f) are the most common allergens in patients with AR and asthma in China [3]. Pharmacotherapy offers clinical control of the disease but does not offer long-term benefit. Discontinuation of the medications results in relapse in symptoms and reduction of lung function [4, 5].

Allergen-specific immunotherapy (AIT) is the only treatment modality with the capacity to alter the natural course of allergic diseases [6]. Subcutaneous immunotherapy (SCIT) has been validated for the treatment of both asthma and AR and clinical improvement has been shown to persist for 3–10 years after discontinuation of the treatment [7, 8]. Compared to SCIT, sublingual immunotherapy (SLIT) appears to be associated with similar efficacy but with a lower incidence of systemic reactions [9, 10]. However, there is no conclusive evidence that one route is more cost effective or clinically effective [11].

Previous data showed that specific immunoglobulin G4 (sIgG4), T-cell and cytokines induced by AIT are regarded as immunological markers of clinical tolerance [12, 13]. But the immunological mechanisms underlying the clinical effects of SCIT and SLIT still remain debated. In this study, we directly compared the clinical efficacy and immunologic responses of both SCIT and SLIT in relation to immunologic changes for Der-p-sIgG4, CD4⁺CD25⁺FoxP3⁺ Tregs and serum cytokines in patients with AR. The design was single centre, randomized, double-blinded, double-mimic, placebo controlled, 3 parallel grouped and prospectively followed for a period of 1 year.

The study included 70 patients (34 females and 36 males, 5–55 years of age) diagnosed with mild-severe AR (with or without asthma) according to ARIA and GINA guidelines [4, 5], strictly sensitized to Der-p and Der-f as confirmed by a positive skin prick test and specific immunoglobulin E (sIgE) level of ≥0.35 kU/L. The patients with a clinical history of significant symptomatic seasonal or perennial AR caused by an allergen (e.g., pollens, cat, dog, cockroach… except house dust mites [HDMs]) to which the patient is regularly exposed and sensitized were excluded. Eligible patients underwent the 2-month run-in period to evaluate their baseline clinical conditions by means of symptom and medication scores, visual analogue scale (VAS), and histamine bronchial provocation test. According to the suggestion of Ethics Committee, patients were randomized into 3 groups (SLIT, SCIT and placebo, 2:2:1) by computer-generated method. The SLIT group received active sublingual drops and placebo subcutaneous injections, and the SCIT group received active subcutaneous injections and placebo sublingual drops, while the placebo group received placebo sublingual drops and placebo subcutaneous injections. Immunological parameters including total-IgE and sIgE were evaluated before and after 1-year treatment. Levels of Der-p-sIgG4, CD4⁺CD25⁺FoxP3⁺ Tregs and serum cytokines including interferon-γ (IFN-γ), tumour necrosis factor alpha (TNF-α), interleukin (IL)-10 and IL-5 were evaluated before and 1, 6, and 12 months after the treatment (Fig. 1). The study was regi-stered in the Chinese Clinical Trial Registry (ChiCTR-OOC-15006207) and the clinicaltrials.gov (NCT01603056) and approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (GYFYY-200908).

The active ingredient of the trial allergen was a 50:50 mixture of Der-p and Der-f allergen extract administered as a glycerinated solution (SLIT, Pangramin, ALK-Abello, Spain) or Der-p extract adsorbed on aluminum hydroxide (SCIT, ALUTARDs. SQ, ALK-Abello). The SLIT was self-administered at home and included a 1-month induction phase (daily sublingual applications) followed by a maintenance phase (dose of 200 STU given 3 times a week), and the annual cumulative dose was 118.2 μg. The dose was placed under the tongue behind the teeth, for at least 3 min before swallowing. SCIT was administered in the clinic and included a 16-week induction phase (weekly subcutaneous injections) followed by a maintenance phase (dose of 100,000 Alutard SQ given every 6 weeks), and the annual cumulative dose was 81.2 μg (online suppl. Table 1; see www.karger.com/doi/10.1159/000503143 for all online suppl. material). The patients in the SLIT group received active drops and histamine dihydrochloride 0.01, 0.1, and 0.5 mg/mL for injections, patients in the SCIT group received active injection and glycerinated solution for the sublingual therapy, patients in the placebo group received both placebo injection and drops. The doctors carrying out the AIT had no knowledge of the randomization code, which was kept locked and sealed by a clinical coordinator not involved with the study. All patients were provided with rescue medication to relieve rhinitis as well as asthma symptoms during baseline or treatment period. The rescue medication was provided as open-labelled in a step-wise fashion depending on the persistency and severity of symptoms according to the patient diary card. For rhinitis symptoms: Step 1, short acting antihistamine; Step 2, nasal corticosteroid; Step 3, oral corticosteroid. For asthma symptoms: Step 1, short-acting β₂ agonists; Step 2, inhaled corticosteroid; Step 3, oral corticosteroid.

A 4-point scoring system (0: no symptoms, 1 point: mild symptoms, 2 points: moderate symptoms, 3 points: severe symptoms) was used to evaluate each rhinitis symptom (sneezing, nasal discharge, itching and nasal obstruction) and asthma symptom (wheezing, breathlessness, dyspnoea and cough). Total rhinitis/asthma scores (TRS/TAS) were defined as the sum of symptom score plus medication score [14]. Total rhinitis symptom score (TRSS) and total asthma symptom score represented all 4 rhinitis and asthma symptoms. Medication use was scored as follows: 1 point: short-acting β₂ agonists (salbutamol, 100 µg/puff); 2 points: steroids, budesonide nasal spray (64 µg/puff) or inhalation powder (200 µg/puff); 1.6 points: corticosteroid (prednisolone, 5 mg/tablet); 6 points: antihistamine (loratadine, 10 mg/tablet), and were calculated as total rhinitis medication score (TRMS) and total asthma medication score.

A 10-cm line to grade severity of symptoms from “no symptoms” (0 cm) to “severe symptoms” (10 cm) was given to patients. The scale answers the question “How have your nasal complaints been today?” Patients were asked to record VAS from 0 to 10 before and after 12 months of treatment.

Lung functions were assessed using Cosmed Microquark Spirometer (Italy), which met the standards of the American Thoracic Society and European Respiratory Society [15]. Every subject took a 15-min rest before examination. FEV₁ was tested at least 3 times according to the quality control standard of forced vital capacity, and the difference value between the best 2 tests <150 mL was acceptable. Histamine dilutions, starting from 0.24 µmol, were increased in a doubling manner during bronchial provocation test. Bronchial hyper-responsiveness positive was defined as forced expiratory volume in 1 s/predicted value ratio (FEV₁%Pred) decreasing ≥20% of its baseline level when ≤7.8 µmol of cumulative dose of histamine is administered. All patients were not allowed to use short-acting β₂ agonist in 6 h or long-acting β₂ agonist in 12 h before the lung function test.

Serum IgE was measured by using ImmunoCAP system (Pha-dia 1000, ThermoFisher Scientific Inc., USA) according to the manufacturer’s instructions and listed in kilo units per litre (kU/L). Der-p-sIgG4 was measured by a 4-layer sandwich ELISA [16]. The plate was coated with Der-p extract (ALK-Abello A/S) overnight at 4–8°C. Then it was incubated with diluted patient serum for 2 h and mouse anti-IgG4 monoclonal antibody (ALK-Abello A/S) for 1 h. Peroxidase-labelled anti-mouse IgG (KPL Inc., USA) was used for detection. The IgG4 concentration was read at 450 nm (EL340, Bio-Tek Instruments Inc., USA). The sIgG4 levels were reported in arbitrary units (AU/mL).

For the flow cytometric analysis, peripheral blood mononuclear cells were incubated to block non-specific binding and stained with each antibody. Cells were stained with fluorescein-isothiocyanate-conjugated anti-CD4 and phycoerythrin-conjugated anti-CD25 (all from BD Biosciences), then fixed with Foxp3 fixation concentrate and permeabilized with permeabilization buffer, prior to incubation for 30 min at 4°C with anti-Foxp3-PE. An EPICS XL flow cytometer (Beckman Coulter Inc., USA) was used for analysis. The percentage of CD4⁺CD25⁺FoxP3⁺ Tregs in CD4⁺ T cells was analysed by the gating strategy (online suppl. Fig. 1).

Cytokines in serum were detected by the suspension chip method. A standard curve was set up and 50 μL MicroBeads was added to 96-well micro culture plates for 2 min and washed twice. The serum and standard sample (50 μL) were incubated for 40 min (washed with buffer 3 times) and then with 25 μL of test anti-body at 37°C for 30 min. The streptavidin-PE was then added and incubated for 15 min. After washing, assay buffer (125 μL) was added, and the plates examined for cytokine levels (IL-5, IL-10, IFN-γ and TNF-α) by Lumine Bio-Plex 200 (Bio-Rad Laboratories, Inc., USA).

Values are presented as mean ± SD or median (range). Comparisons for quantitative variables were performed by non-parametrical analysis, Mann-Whitney and Kruskal-Wallis tests for independent samples. Comparisons at 2 different time points were carried out using the Wilcoxon’s test for related samples. Pearson’s correlations were used to assess the relationships between clinical improvement and serum parameters. Significance was set at p < 0.05. SPSS 16.0 (SPSS Inc., USA) was used for analysis.

Sixty-seven enrolled patients (3 dropped out) only sensitized to Der-p and Der-f were randomized (26 to SCIT, 27 to SLIT, 14 to the placebo group). The baseline levels of sIgE, symptom and medication scores and the wheal diameter of skin prick test were similar between the 3 groups, and no significant differences were observed in regard to demographic characteristics (Table 1).

After 1 year of treatment, a significant improvement of TRS, TRMS and VAS occurred in both SLIT and SCIT compared with baseline (p < 0.05). TRS (p = 0.045) and TAS (p = 0.042) were significantly reduced in SLIT when compared with the placebo group (Table 2). As for SCIT, the TRS (p = 0.024), TRMS (p = 0.026) and TAS (p = 0.024) were all significantly reduced when compared with the placebo group. A significant reduction of TRMS within the SCIT group was observed when directly compared with the SLIT group (p = 0.026), while other scores were no different (Table 2).

No difference was found for FEV₁%Pred among 3 groups at 12 months after treatment (p > 0.05). Compared with baseline (Table 1), significant increases in Der-p and Der-f sIgE levels were observed in all 3 groups after 12 months of treatment (Placebo, 57.70 ± 34.00 kU/L, p = 0.020, 60.59 ± 35.37 kU/L, p = 0.032; SLIT, 57.18 ± 35.79 kU/L, p = 0.033, 62.23 ± 33.34 kU/L, p = 0.009; SCIT, 60.28 ± 36.45 kU/L, p = 0.026, 63.45 ± 35.67 kU/L, p = 0.010), while no difference was found among the 3 groups.

A significant increase over baseline in Der-p sIgG4 in SCIT (p = 0.016) and SLIT (p = 0.022) groups after 1-year of treatment was observed (Fig. 2; online suppl. Fig. 2). The mean level of Der-p IgG4 in the SCIT group was almost 30 times higher than that of the SLIT group after therapy (p < 0.05; Fig. 2, 4). But no correlation was found between clinical improvement and the increase of Der-p sIgG4 in the SLIT or the SCIT group (Fig. 3).

A significant increase in percentage of CD4⁺CD25⁺FoxP3⁺ Tregs in CD4⁺ T cells occurred in subjects treated with SLIT (p = 0.012) and SCIT (p = 0.027) after 1 year when compared with the baseline (Fig. 2; online suppl. Fig. 2). Also, the percentage in ether SLIT or SCIT was significantly higher than the placebo group at 1 year (Fig. 4). The change of CD4⁺CD25⁺FoxP3⁺ Tregs inversely correlated with TRS (p = 0.024) only in the SLIT group (Fig. 3).

A significant increase in serum level of IFN-γ was observed in SCIT after 1-, 6- and 12-month treatment (p = 0.008, p = 0.007, p = 0.008, respectively) compared with the placebo group (Fig. 4). Also, the change of IFN-γ inversely correlated with TRS (p = 0.032) only in the SCIT group (Fig. 3). Compared with the placebo group, significant increases in TNF-α were observed in SCIT (p = 0.009) at 12 months (Fig. 4), while no difference was found when compared with baseline (Fig. 2; online suppl. Fig. 2). No significant increase was found for serum IL-5 and IL-10 among 3 groups after 1-year treatment (Fig. 2; online suppl. Fig. 2).

Our study revealed that SCIT and SLIT had similar clinical efficacy and there was a trend towards the upregulation of CD4⁺CD25⁺FoxP3⁺ Tregs in both SCIT and SLIT, while the Tregs correlated with clinical improvement only in SLIT. IFN-γ was induced at the early stage of treatment and correlated with clinical improvement in SCIT. And no significant changes of other cytokines such as IL-5, IL-10 and TNF-α were found among groups.

Both SCIT and SLIT have been accepted as treatment for mite-AR and asthma; however, some studies have yielded variable results [17-21]. Although direct comparative trials of 2 modes for efficacy are lacking, some data suggest that SCIT might have a slightly better efficacy profile [17, 22]. According to our direct comparative study, SCIT provided a little better outcome of TRMS than SLIT. But it was not enough to state that SCIT works better than SLIT. These 2 modes had similar clinical efficacy, while the total annual cumulative dosages of Der-p were different (SLIT, 118.2 μg; SCIT, 81.2 μg) according to pharmaceutical instructions. It was not clear whether the cumulative dosage was the main cause that affected clinical outcomes. The differences in clinical efficacy of our and other studies may result from different immune-mechanisms involving immunological parameters responses and methodological differences between the 2 therapeutic modes.

In this comparative study, an increase in Der-p and Der-f sIgE was observed in all 3 groups after 1-year therapy, which was in line with our previous study that levels of sIgE increased during 1-year treatment in SCIT [23]. Furthermore, perennial HDM allergens were detected in very high levels in household dust all year round in Guangzhou city [24], where all 3 groups of our patients exposed during the treatment could also explain their higher sIgE level. This finding is also consistent with some other studies [16, 25, 26]. However, other studies reported that the sIgE levels decreased the first year following AIT [27, 28]. The inconformity in sIgE levels of the studies may be due to the differences of geographical environments and immunologic responses in subjects of the studies.

Previous data demonstrated that increases in sIgG4 level correlated with the clinical improvement in SLIT [29] and the high level of sIgG4 in SCIT linked with an increase in serum inhibitory activity for sIgE binding to B cells [30, 31]. Also, one concept was that the counter-IgG responses induced by SCTI could lead to steric hindrance of IgE by the reactivity in the vicinity of IgE epitopes or the occupation of certain amino acid interacted with IgE [32]. As for our study, the increases in allergen-specific IgG4 level were observed in both SCIT and SLIT, and SCIT had a higher magnitude of sIgG4 than SLIT, but there was no correlation observed between sIgG4 and clinical improvement, which was consistent with some reports [27, 33]. AIT did not always induce cross-protective IgG antibodies [34] and our previous study also demonstrated that sIgG4 was not correlated with clinical efficacy in the early phase of SCIT [23]. According to our findings together with the findings of others, we suggested that the level of sIgG4 is just an immunological phenomenon, often not directly correlated with clinical outcomes. However, its absolute quantity in sera reflects the ability of immune-reactive but not functional levels in AIT.

There was a trend towards an increased percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in peripheral blood during peanut or pollen SLIT [35, 36]. Furthermore, our data showed a significant increase in CD4⁺CD25⁺FoxP3⁺ Tregs in both HDMs SLIT and SCIT, which indeed correlated with clinical improvement only in SLIT. Though the Treg cells’ response in SLIT has not been fully elucidated, the mechanism may be different from SCIT. It has been found that the percentage in CD4⁺CD25⁺Foxp3⁺ Tregs did not change after 1 year of SCIT [37], while there was a significant reduction of follicular helper T cells in SCIT, which might relate to IL-2 production from allergen-specific T-cell responses [38]. It is also reported that SCIT may induce Th1 responses and reduce synthesis of Th2 cytokines [39], whereas in SLIT, induction of oral mucosal immune tolerance may be more important and oral dendritic cells are able to induce T-regulatory cells [40]. From these findings confirmed by us and others, we can speculate that SLIT and SCIT have different mechanisms for treatment and CD4⁺CD25⁺Foxp3⁺ Tregs may play an important role in SLIT. However, we still need to further determine the details of follicular helper T, follicular regulatory T cells, subtypes of Tregs such as induced T-regulatory cells, Tr1 and transforming growth factor-β-producing CD4⁺ T cells (Th3) in the 2 modes in a large sample size.

The roles of cytokines in AIT are under dispute. It has been reported that the level of serum IL-5 was significantly lower in SLIT compared with placebo, and no significant difference was found in IL-10 or IFN-γ levels in serum [17, 25]. In another randomized controlled trial, IL-10 significantly increased in pollen SLIT, whereas no changes were observed for IL-5 and IFN-γ [18]. The seeming inconsistencies might result from methodological differences. In our double-blind randomized study, a significant increase in IFN-γ was observed, only in SCIT, after 1, 6, and 12 months of treatment. This is supported by a prior study, in which SCIT induced the increase in IFN-γ production in the peripheral blood [41]. As a Th1 cytokine, IFN-γ can lead to immune deviation from a Th2 to a Th1-driven response and promote IgM-producing B cells to class switch to IgG-producing B cells. Our results also showed that clinical improvement correlates with increased IFN-γ production in SCIT. According to our study and other studies, we considered that SCIT offers an earlier Th1 response, which leads to clinical improvements with earlier induction of IFN-γ than SLIT.

The limitations of the study were the unmatched sample size in the placebo group and the presence of different components of allergens between SCIT and SLIT. It was due to the 1-year duration of the study that the Ethics Committee of the hospital suggested that the protocol be changed to the manner of 2:2:1 to avoid non-active treatment in placebo patients for a comparatively longer period of time. However, we believed that the sample size of 14 in the placebo group still had enough power to give significant statistical analysis. Although there were 2 species of HDM (Der-p and Der-f) with 1:1 concentration for SLIT drops but only Der-p in SCIT injection, both allergens share similarity in allergenicity and immunogenicity and would be considered to induce similar clinical efficacy and immunologic responses [42].

Our study shows that despite the similar clinical efficacy, SCIT and SLIT have different immunological mechanisms with heterogeneous changes of CD4⁺CD25⁺Foxp3⁺ Tregs, sIgG4 and cytokines. SCIT offers an earlier Th1 response with induction of IFN-γ than SLIT, while CD4⁺CD25⁺Foxp3⁺ Tregs associated with oral mucosal immune tolerance leads to clinical improvement during the early stage of SLIT. Further randomized comparative studies with a large-size population are needed to determine the long-term changes and the immunologic biomarkers with relevance to clinical improvement in these 2 modes of immunotherapy.

The authors thank Zhaowei Yang for statistical assistance and Shiyan Fu for technical assistance in this study.

The study was registered in the Chinese Clinical Trial Reg-istry (ChiCTR-OOC-15006207) and the clinicaltrials.gov (NCT01603056) and approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (GYFYY-200908). Written consent was obtained from all adult patients or parents of children.

The authors declare that they have no conflicts of interest.

This work was supported by the Precision Medicine Research Program of national key research and development project of China (2016YFC0905800), the General Program (30972808, 81370129) and Breeding Program of Major Research Plan (91542104) of the National Natural Science Foundation of China.

J.L. mainly designed the study. M.X. performed the survey, collected the data, performed the statistical analysis and drafted the manuscript. M.F., Y.D., N.W., and Q.S. mainly performed the clinical tests and collected the data.

Edited by: H.-U. Simon, Bern.