Consistent efficacy and safety of sublingual immunotherapy tablets across allergens and geographic regions.

BACKGROUND: The clinical development program of the SQ grass, ragweed, tree, and house dust mite (HDM) sublingual immunotherapy (SLIT)-tablets for allergicrhinitis/conjunctivitis (AR/C) included clinical trials conducted in North America,Europe, and Japan. OBJECTIVE: Data from these trials were analyzed to assess efficacy, immunologic mechanisms, and safety outcomes acrossallergens and geographic regions. METHODS: Thirteen phase III, double-blind, placebo controlled trials in the subjects with AR/C were conducted in NorthAmerica, Europe (including Russia), and Japan (N = 7763 analyzed). Trials were generally similar with respect to medicalpractice, target population, eligibility criteria, and efficacy and safety monitoring. Data were analyzed for the approved dosesin North America and Europe. Four statistical models were used to enhance comparison of the efficacy end points among the trials. RESULTS: The SLIT-tablets demonstrated consistent efficacy across allergens and regions, regardless of the statistical analysis used. Relative improvement in the primary efficacy end point compared with placebo by using the predefined protocol analysis ranged from 17.9% to 32.8%, 17.5% to 19.3%, 20.6% to 38.3%, and 39.6% with the grass, HDM, ragweed, and tree SLIT-tablets, respectively. The kinetics of specific immunoglobulin E (IgE) and IgG4 responses were similar among the allergensand regions. Local application-site reactions were the most common adverse events for all allergens and in all regions. Mosttreatment-related adverse events for all allergens and in all regions were mild in severity. The rate of systemic allergic reactions was similar across regions (0%-0.54%). CONCLUSION: Confirmatory phase III trials for SLIT-tablets in the treatment of AR/C showed consistent efficacy, immunologic,and safety outcomes across allergens and geographic regions.

Sublingual Immunotherapy for Japanese Cedar Pollinosis: Current Clinical and Research Status.

The incidence of Japanese cedar pollinosis is increasing significantly in Japan, and a recent survey suggested that about 40% of the population will develop this disease. However, spontaneous remission is rare. The increased incident rate of Japanese cedar pollinosis is a huge issue in Japan. Allergen immunotherapy is the only fundamental treatment that modifies the natural course of allergic rhinitis and provides long-term remission that cannot be induced by general drug therapy. Sublingual immunotherapy for Japanese cedar pollinosis has been developed and has been covered by health insurance since 2014 in Japan. The indication for children was expanded in 2018. Clinical trials of sublingual immunotherapy for Japanese cedar pollinosis have demonstrated its long-term efficacy and safety. It is recommended for patients who wish to undergo fundamental treatment regardless of the severity of the practical guidelines for the management of allergic rhinitis in Japan. For sublingual immunotherapy, a long-term treatment period of 3 years or longer is recommended to obtain stable therapeutic effects. In recent years, evidence based on basic research and clinical trials has demonstrated sublingual immunotherapy-induced immunological changes and efficacy in patients; however, biomarkers that objectively predict and judge these therapeutic effects need to be established.

LPCAT2 Methylation, a Novel Biomarker for the Severity of Cedar Pollen Allergic Rhinitis in Japan.

BACKGROUND: Recently, the role of the epigenome in allergies has been receiving increasing attention. Although several genes that are methylated in relation to serum immunoglobulin E (IgE) concentration have been reported by epigenome-wide association studies, little is known about the DNA methylation sites associated with the symptoms and severity of cedar pollinosis (CP). OBJECTIVE: Our aim was to analyze the association between DNA methylation and the symptoms and severity of CP in peripheral blood mononuclear cells (PBMCs) and nasal mucosa scraping cells (NMSCs). METHODS: We recruited 70 participants during the cedar pollen dispersal season. IgE levels were measured by a fluorescence enzyme immunoassay. We analyzed DNA methylation of acyl-CoA thioesterase 7 (ACOT7), mucin 4 (MUC4), schlafen 12 (SLFN12), lysophosphatidylcholine acyltransferase 2 (LPCAT2), and interleukin-4 (IL4) in PBMCs and NMSCs using bisulfite next-generation sequencing; the correlation of DNA methylation with non-specific IgE and cedar pollen-specific IgE levels in peripheral blood samples was also investigated. Symptom severity and DNA methylation were investigated in 15 untreated CP patients. RESULTS: Non-specific IgE levels showed a significant negative correlation with average IL4 methylation in PBMCs (r = -0.46, P < 0.0001) but not with methylation of ACOT7, MUC4, SLFN12, and LPCAT2. Cedar pollen-specific IgE levels showed a significant negative correlation with average IL4 and MUC4 methylation in PBMCs (r = -0.31, P = 0.01 and r = -0.241, P = 0.046, respectively) but not with methylation of ACOT7, SLFN12, and LPCAT2. The methylation of some genes in NMSCs was not significantly correlated with IgE levels. The mean methylation of LPCAT2 in NMSCs showed a decreasing trend with increasing severity of CP (P = 0.027). CONCLUSION: LPCAT2 methylation in NMSCs may reflect the severity of CP and could be used as a novel biomarker to identify suitable treatment options for CP.

Safety profile and immunological response of dual sublingual immunotherapy with house dust mite tablet and Japanese cedar pollen tablet.

BACKGROUND: There have been no studies of dual administration of sublingual immunotherapy (SLIT) tablets for perennial and seasonal allergic rhinitis. This trial (JapicCTI-184014) was conducted to investigate the safety profile and immunological response during dual therapy with SQ house dust mite (HDM) and Japanese cedar pollen (JCP) SLIT tablets. METHODS: This was a multicenter, open-label, randomized trial of 109 Japanese patients with coexisting HDM and JCP allergic rhinitis who had positive tests for HDM- and JCP specific IgE (≥0.7 kU/L). Patients were allocated to receive HDM (N = 54) or JCP (N = 55) SLIT tablets alone for 4 weeks followed by 8 weeks of dual therapy with both SLIT tablets administered within 5 min of each other. Adverse events (AEs), adverse drug reactions (ADRs), and serum IgE and IgG4 specific for HDM (Dermatophagoides farinae, Dermatophagoides pteronyssinus) and JCP were recorded. RESULTS: The percentage of subjects with AEs and ADRs was similar between the two groups and between the two periods of monotherapy and dual therapy. Most AEs and ADRs were mild in severity, and no serious events were observed. The most common ADRs were local events in the oral cavity. Levels of IgE and IgG4 specific for HDM (D. farinae, D. pteronyssinus) and JCP were increased after treatment with HDM and JCP SLIT tablets, respectively. CONCLUSIONS: Dual therapy with both SLIT tablets administered within 5 min after 4 weeks of monotherapy with HDM or JCP tablet was well tolerated and induced the expected immunological responses.

Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy.

BACKGROUND: Clinical efficacy of allergen-specific Immunotherapy (AIT) towards Japanese cedar (JC) pollen allergy is firmly established but JC pollen-specific biomarker assays are lacking. Treatment-related increase of allergen-specific antibodies is a robust biomarker of successful AIT. Allergen-specific non-IgE antibodies are believed to reduce the effects of allergen exposure by competing with IgE for allergen binding, and in-vitro assays quantifying the effects of AIT-induced IgE-blocking antibodies are advantageous. A cell-free enzyme-linked immunosorbent facilitated antigen binding (ELIFAB) assay of JC pollen was established. METHODS: Serum IgE-allergen complexes were captured by immobilized recombinant CD23, and allergen-IgE-CD23 complexes were detected by a biotin-conjugated anti-human IgE antibody. Sera from JC pollen-allergic subjects without or with subcutaneous immunotherapy (SCIT) with JC pollen extract were used (n = 11/group). RESULTS: Optimal assay conditions were established at 20 µg/mL CD23 and 0.3 µg/mL JC pollen extract, and the dependency on CD23 and IgE was verified. The data show that the JC pollen ELIFAB assay is fit for purpose and demonstrates that the IgE-blocking activity is significantly increased in the JC pollen SCIT group compared with the non-treated group. CONCLUSION: The JC pollen ELIFAB assay represents a simple, cell-free biomarker assay for monitoring the development of IgE-blocking antibody activity during JC pollen AIT.

Comparison of the Allergenic Potency of House Dust Extract and House Dust Mite Allergen Extract for Subcutaneous Allergen Immunotherapy.

Subcutaneous allergen immunotherapy (SCIT) with non-standardized house dust (HD) extracts has been used in Japan since 1963 for house dust mite (HDM)-allergic patients. Since the potencies of HD extracts are unknown, the allergenic potency of HD extracts was examined by comparing with a standardized HDM allergen extracts. The major allergen content of HDM in the extracts was measured using a sandwich enzyme-linked immunosorbent assay (ELISA). The immunoglobulin E (IgE) inhibitory activities of the extracts were measured by a competitive ELISA. The extract concentrations giving 50% inhibition of IgE binding (log10 IC50) were determined from dose-response curves and defined as inhibitory activities. A linear regression line was constructed from the log10 IC50 values of the standardized HDM extract to interpolate the relative potency of the HD extract with strength of 1 : 10 w/v (HD 1 : 10). The amounts of major allergens (Der f 1, Der p 1 and Der 2) were 116.3 µg/mL in the HDM allergen extract (100000 Japanese Allergy Units [JAU]/mL) and 0.77 µg/mL in the HD 1 : 10. The inhibitory activity (log10 IC50 values) of HD 1 : 10 was 2.389 ± 0.078, indicating the allergenic potency was between 200 and 2000 JAU/mL. Based on regression analysis (R2 >0.99), the allergenic potency of HD 1 : 10 was estimated to be 842 ± 128 JAU/mL. The present study determined the major allergen content of HD extract, which contributes to its allergenic potency. The allergenic potency of HD 1 : 10 was ca. 100-fold less than that of HDM allergen extract.

Role of IL-35 in sublingual allergen immunotherapy.

BACKGROUND: Grass pollen-specific immunotherapy involves immunomodulation of allergen-specific TH2 responses and induction of IL-10+ and/or TGF-ß+CD4+CD25+ regulatory T cells (induced Treg cells). IL-35+CD4+CD25+ forkhead box protein 3-negative T (IL-35-inducible regulatory T [iTR35]) cells have been reported as a novel subset of induced Treg cells with modulatory characteristics. OBJECTIVE: We sought to investigate mechanisms underlying the induction and maintenance of immunologic tolerance induced by IL-35 and iTR35 cells. METHODS: The biological effects of IL-35 were assessed on group 2 innate lymphoid cells (ILC2s); dendritic cells primed with thymic stromal lymphopoietin, IL-25, and IL-33; and B and TH2 cells by using flow cytometry and quantitative RT-PCR. Grass pollen-driven TH2 cell proliferation and cytokine production were measured by using tritiated thymidine and Luminex MagPix, respectively. iTR35 cells were quantified in patients with grass pollen allergy (seasonal allergic rhinitis [SAR] group, n = 16), sublingual immunotherapy (SLIT)-treated patients (SLIT group, n = 16), and nonatopic control subjects (NACs; NAC group, n = 16). RESULTS: The SAR group had increased proportions of ILC2s (P = .002) and IL-5+ cells (P = .042), IL-13+ cells (P = .042), and IL-5+IL-13+ ILC2s (P = .003) compared with NACs. IL-35 inhibited IL-5 and IL-13 production by ILC2s in the presence of IL-25 or IL-33 (P = .031) and allergen-driven TH2 cytokines by effector T cells. IL-35 inhibited CD40 ligand-, IL-4-, and IL-21-mediated IgE production by B cells (P = .015), allergen-driven T-cell proliferation (P = .001), and TH2 cytokine production mediated by primed dendritic cells. iTR35 cells suppressed TH2 cell proliferation and cytokine production. In addition, allergen-driven IL-35 levels and iTR35 cell counts were increased in patients receiving SLIT (all, P < .001) and NACs (all, P < .001) compared with patients with SAR. CONCLUSION: IL-35 and iTR35 cells are potential novel immune regulators induced by SLIT. The clinical relevance of SLIT can be underscored by restoration of protective iTR35 cells.

Current status of sublingual immunotherapy for allergic rhinitis in Japan.

Japanese cedar pollen (JCP) and house dust mite (HDM) are two major allergens that cause allergic rhinitis (AR) in Japan and the prevalence of AR is increasing. Pharmacothearpy is a commonly used treatment, but the level of patient satisfaction is very low. Allergen immunotherapy (AIT) is the only therapeutic modality that provides not only symptom relief but also quality of life improvement that leads to a high rate of satisfaction. In particular, sublingual immunotherapy (SLIT) is a safe and effective treatment for AR. Here we introduce a large-scale double-blind, placebo-controlled trial of SLIT in Japanese patients using JCP droplets or HDM tablets conducted in Japan. The immediate future of SLIT in Japan is also discussed.

Establishment of Synergistic Chemoimmunotherapy for Head and Neck Cancer Using Peritumoral Immature Dendritic Cell Injections and Low-Dose Chemotherapies.

The lack of available tumor antigens with strong immunogenicity, human leukocyte antigen restriction, and immunosuppression via regulatory T-cells (Tregs) and myeloid-derived suppressor cells are limitations for dendritic cell (DC)-based immunotherapy in patients with advanced head and neck cancer (HNC). We sought to overcome these limitations and induce effective antitumor immunity in the host. The effect of low-dose docetaxel (DTX) treatment on DC maturation was examined in an ex vivo study, and a phase I clinical trial of combination therapy with direct peritumoral immature DC (iDC) injection with OK-432 and low-dose cyclophosphamide (CTX) plus DTX was designed. Low-dose DTX did not negatively affect iDC viability and instead promoted maturation and IL-12 production. Five patients with metastatic or recurrent HNC were enrolled for the trial. All patients experienced grade 1 to 3 fevers. Intriguingly, elevated CD8+ effector T-cells and reduced Tregs were observed in four patients who completed two treatment cycles. All patients were judged to have progressive disease, but tumor regressions were observed in a subset of targeted metastatic lesions in two of five patients. Our results show that the combination of direct peritumoral iDC injection with OK-432 and low-dose CTX plus DTX is well tolerated and should give rise to changing the immune profile of T-cell subsets and improvement of immunosuppression in advanced HNC patients. Additionally, our ex vivo data on the effect of low-dose DTX treatment on DC maturation may contribute to developing new combination therapies with low-dose chemotherapy and immunotherapy.

Construction of mass spectra database and diagnosis algorithm for head and neck squamous cell carcinoma.

OBJECTIVES: Intraoperative identification of tumor margins is essential to achieving complete tumor resection. However, the process of intraoperative pathological diagnosis involves cumbersome procedures, such as preparation of cryosections and microscopic examination, thus requiring more than 30 min. Moreover, intraoperative diagnoses made by examining cryosections are occasionally inconsistent with postoperative diagnoses made by examining paraffin-embedded sections because the former are of poorer quality. We sought to establish a more rapid accurate method of intraoperative assessment. MATERIALS AND METHODS: A diagnostic algorithm of head and neck squamous cell carcinoma (HNSCC) using machine learning was constructed by mass spectra obtained from 15 non-cancerous and 19 HNSCC specimens by probe electrospray ionization mass spectrometry (PESI-MS). The clinical validity of this system was evaluated using intraoperative specimens of HNSCC and normal mucosa. RESULTS: A total of 114 and 141 mass spectra were acquired from non-cancerous and cancerous specimens, respectively, using both positive- and negative-ion modes of PESI-MS. These data were fed into partial least squares-logistic regression (PLS-LR) to discriminate tumor-specific spectral patterns. Leave-one-patient-out cross validation of this algorithm in positive- and negative-ion modes showed accuracies in HNSCC diagnosis of 90.48% and 95.35%, respectively. In intraoperative specimens of HNSCC, this algorithm precisely defined the borders of the cancerous regions; these corresponded with those determined by examining histologic sections. The procedure took approximately 5 min. CONCLUSION: This diagnostic system, based on machine learning, enables accurate discrimination of cancerous regions and has the potential to provide rapid intraoperative assessment of HNSCC margins.

Pooled efficacy and safety data for house dust mite sublingual immunotherapy tablets in adolescents.

BACKGROUND: House dust mite (HDM) respiratory allergy is a common and burdensome disease in children and adolescents. There are few HDM allergy immunotherapy trials in children with perennial allergic rhinitis. This post hoc analysis used pooled data to evaluate efficacy and safety of the SQ HDM sublingual immunotherapy (SLIT) tablet in adolescents (12-17 years). METHODS: In two double-blind, placebo-controlled trials conducted in North America and Japan, respectively, subjects aged 12+ years with HDM allergic rhinitis were randomized to up to 1 year of treatment. The primary end-point in both trials was the average total combined rhinitis score (TCRS) during the last 8 weeks of treatment in the active group compared with placebo. Data from subjects aged 12-17 years were pooled (N=395). RESULTS: In the pooled adolescent subpopulation, average TCRS improved 22% with 12 SQ HDM vs placebo (absolute treatment difference of 1.04; P<.01). Rhinitis daily symptom score (DSS), conjunctivitis DSS and rhinitis daily medication score (DMS) were also significantly improved vs placebo in the pooled adolescent subpopulation (all P<.05). There were no new safety signals for adolescents. The frequency of adverse events was similar in adolescents and adults with the majority being mild application site-related events. CONCLUSIONS: Treatment with 12 SQ HDM appears to be effective and well tolerated in adolescents with HDM allergic rhinitis.

Questionnaire about Snoring and Daytime Sleepiness Due to Springtime Hay Fever.

Bothersome symptoms of hay fever impair not only patients' quality of life but also their labor productivity and learning efficiency. Excessive daytime sleepiness (EDS) caused by hay fever is thought to be one of the reasons for these impairments. The purpose of this study was to investigate the relationship between the severity of springtime hay fever and EDS by using a questionnaire. The questionnaire included information about age, sex, height, weight, severity of hay fever, treatment for hay fever, smoking and alcohol consumption habit, history of drug use for sleeping, existence of snoring, and Japanese version of the Epworth Sleepiness Scale. After excluding responses containing insufficient data, responses from 1,734 patients were considered as eligible. By performing logistic regression analysis, we analyzed the effect of the aforementioned parameters on the comorbidity of EDS and snoring. The odds ratio (OR) to comorbid EDS was significantly higher in the moderate and severe hay fever groups than in the asymptomatic hay fever group (moderate: OR=1.76, p=0.014, severe: OR=2.53, p<0.001). Also, OR to comorbid snoring was significantly higher in the severe hay fever group than in the asymptomatic hay fever group (severe: OR=1.90, p=0.001).

Allergen immunotherapy and tolerance.

Successful allergen-specific immunotherapy (AIT) is associated with a marked decrease in symptoms on allergen exposure, a reduced requirement for 'rescue' anti-allergic drugs and improvement in patients' quality of life. These benefits persist for at least several years following discontinuation of immunotherapy - the hallmark of clinical and immunological tolerance. AIT has been shown to modulate both innate and adaptive immunological responses. Early suppression of innate effector cells of allergic inflammation (mast cells, basophils), regulation of pro-allergic T helper 2 type (Th 2) responses and IgE+ B cell responses have been shown to occur both in the tissue and in the peripheral blood during AIT. The allergen-tolerant state is associated with local and systemic induction of distinct populations of allergen-specific T regulatory cells including IL-10+ Tregs (Tr1 cells), TGF-ß+ Tregs and FoxP3+ memory T regs. B cells are switched in favour of producing IgG (particularly IgG4) antibodies and associated blocking activity for IgE-dependent events, including basophil activation and IgE-facilitated allergen binding to B cells. An induction of IL-10+ B regulatory cells and alterations in dendritic cell subsets have also recently been described. These events are followed by the induction of T regulatory cells, suppression of allergen-specific T cell proliferation and immune deviation from Th2 in favour of Th1 responses. Alternative mechanisms of tolerance include apoptosis/deletion of antigen-specific memory Th2 cells and/or a failure of co-stimulation leading to T cell anergy.

[Effect of posterior nasal neurectomy combined with the inferior turbinate surgery for the patients with perennial allergic rhinitis or vasomotor rhinitis].

INTRODUCTION: Until recently, Vidian neurectomy had been applied mainly in intractable vasomotor rhinitis and severe perennial allergic rhinitis. Although the results were excellent, the operation has not been applied recently because of the adverse events such as xerophthalmia and trigeminal neuralgia. To resolve these problems, a new surgical technique, posterior nasal neurectomy, was developed. In this report, we examined the effectiveness of posterior nasal neurectomy combined with the inferior turbinate surgery for severe perennial allergic rhinitis and intrinsic rhinitis by questionnaire. PATIENTS AND METHODS: Twenty patients who had undergone posterior nasal neurectomy combined with the inferior turbinate surgery between April in 2005 and March in 2009 were enrolled. Numeric Rating Scale was used to evaluate clinical symptoms and quality of life (QOL) of pre- and postsurgery. Frequency of medication (oral administration and nasal spray) was also evaluated. RESULTS: Questionnaires were collected from 17 patients. As for all patients but one, the surgery significantly reduced rhinorrhea, nasal obstruction, and sneezing. Furthermore, QOL for many symptoms such as sleep disorder and malaise/feebleness was also significantly improved after the surgery. Eighty-one percent of patients were satisfied with the surgery. CONCLUSION: Posterior nasal neurectomy combined with the inferior turbinate surgery is effective in alleviating clinical symptoms and improving QOL in the patients with severe perennial allergic rhinitis and intrinsic rhinitis, although a longer follow-up is needed.

A randomized control trail of stepwise treatment with fluticasone propionate nasal spray and fexofenadine hydrochloride tablet for seasonal allergic rhinitis.

BACKGROUND: In Japan, oral antihistamines are frequently used as the initial treatment for seasonal allergic rhinitis (SAR), and intranasal steroids are added when nasal symptoms worsen. This study aimed to evaluate whether starting treatment with fluticasone propionate nasal spray (FP) from the beginning of pollinosis symptoms and adding fexofenadine hydrochloride tablet (FEX) when SAR is aggravated could achieve improved amelioration of nasal symptoms throughout the pollen season in comparison with a treatment that involves starting with FEX and later adding FP. METHODS: In this pragmatic, randomized, open-label, parallel-group trial, 51 Japanese cedar pollinosis patients (age, 16-85 years) were randomly divided and administered FP 100 mcg twice daily as an initial drug with FEX 60 mg twice daily as an additional drug and the same treatment in the reverse order. Nasal symptoms were evaluated in a daily dairy using a 4-point scale. The primary outcome was area under curve of the line representing the daily total nasal symptom score in the pollen season on a graph. RESULTS: Initial treatment with FP was significantly (P = 0.0015) more effective than initial treatment with FEX in improving the primary outcome. The average daily total nasal symptom score in the initial treatment with FP group was better than that in the initial treatment with FEX group throughout the pollen season. CONCLUSIONS: Initiating treatment with FP and adding FEX might lead to improved outcomes for nasal symptoms in comparison with the same drugs administered in the reverse order.

Analysis of helper T cell responses to Cry j 1-derived peptides in patients with nasal allergy: candidate for peptide-based immunotherapy of Japanese cedar pollinosis.

BACKGROUND: Allergen specific immunotherapy is highly effective, but adverse events may occur during treatment. Peptide-based immunotherapy has been proposed as one of new strategies for reduction of allergic adverse reactions. We examined the possibility of candidate peptides for the development of peptide-based immunotherapy for Japanese cedar pollinosis. METHODS: Twelve Cry j 1-specific T-cell lines were established from peripheral blood mononuclear cells (PBMC) of 12 patients with Japanese cedar pollinosis. Using these T-cell lines, 37 Cry j 1-derived overlapping peptides were assessed for their proliferative responses and cytokine production. RESULTS: Four peptides corresponding to the Cry j 1 sequence were able to induce proliferative responses to more than one T-cell line: p61-80 (3/12; 25.0%); p115-132 (2/12; 16.6%); p206-225 (4/12; 33.3%); and p337-353 (5/12; 41.7%). Furthermore, T-cell lines generated from 11 of 12 donors (91.7%) responded to at least one of these four peptides. On the other hand, the pattern of cytokine production from Cry j 1-specific T-cell lines varied. Moreover, cytokine production patterns by stimulation with Cry j 1 peptide did not reflect those by stimulation with Cry j 1 protein. CONCLUSIONS: Our results suggest four Cry j 1-derived peptides (p61-80, p115-132, p206-225 and p337-353) may be considered to be the immunodominant T-cell epitopes of the Cry j 1 molecule, and can be useful for the design of peptide-based immunotherapy for the management of Japanese cedar pollinosis.

Analysis of T-helper responses and FOXP3 gene expression in patients with Japanese cedar pollinosis.

BACKGROUND: Evidence has been accumulated indicating that regulatory T (T-reg) cells play a crucial role in the maintenance of peripheral T-cell tolerance to allergens. To explore the role of FOXP3, which is required for the development of T-reg cells, in allergen-specific immune responses, we examined the relationship between the alteration of FOXP3 gene expression and in vitro immune responses against allergens. METHODS: Peripheral blood mononuclear cells obtained from 19 human histocompatibility leukocyte antigens (HLA)-DPB1*0501 donors, including patients with Japanese cedar pollinosis and nonallergic healthy donors, were stimulated with Cry j 1 p61-75 peptide. On day 7, T cells were tested for peptide-specific reactivity in IFN-gamma and interleukin (IL)-5 enzyme-linked immunospot (ELISPOT) assays. Real-time quantitative RT-PCR was performed to assess relative change of FOXP3 gene expression before and after in vitro stimulation. Neutralization assays using anti-glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and anti-IL-10 monoclonal antibody were also performed. RESULTS: Of 14 patients with allergic pollinosis tested, 10 responders displayed T-helper type 2 (Th2)-polarized reactivity to Cry j 1 p61-75, and 2 donors showed Th0 responses. Notably, the change of FOXP3 gene expression in donors showing peptide-specific T-helper responses was significantly lower than that in nonresponders, regardless of allergic pollinosis. CONCLUSION: Our data indicate that FOXP3 is functional in nonallergic healthy donors as well as allergic patients, and FOXP3-expressing T cells may be responsible for the down-regulation of allergen-specific T-helper responses in individuals. A better understanding of the nature and specificity of FOXP3-expressing T cells in a suppressive mechanism is necessary to develop new immunotherapies against allergic rhinitis.