Expansion of circulating Foxp3+CD25bright CD4+ T cells during specific venom immunotherapy

Background Venom immunotherapy (VIT) induces long‐lasting immune tolerance to hymenoptera venom antigens, but the underlying mechanisms are not yet clarified. Regulatory T cells are thought to play an important role in allergic diseases and tolerance induction during specific immunotherapy. Aim Characterize longitudinally the impact of VIT on the pool of circulating regulatory T cells. Methods Fourteen hymenoptera venom‐allergic patients with severe reactions (grades III–IV) were studied before, 6 and 12 months after starting ultra‐rush VIT. Freshly isolated peripheral blood mononuclear cells were surface stained with a panel of markers of T cell differentiation and intracellularly for CTLA‐4 and Foxp3 and analysed by flow cytometry. foxp3 mRNA was quantified by real‐time PCR. VIT responses were assessed by measuring specific IgG4 and IgE levels. Eleven individuals with no history of insect venom allergy were studied as controls. Results VIT induces a significant progressive increase in both the proportion and the absolute numbers of regulatory T cells defined as CD25^bright and/or Foxp3⁺ CD4⁺ T cells. These changes are not related to alterations in the expression of activation markers or imbalances in the naïve/memory T cell compartments. foxp3 mRNA levels also increased significantly during VIT. Of note, the increase in circulating regulatory T cell counts significantly correlates with the venom‐specific IgG4/IgE ratio shift. Conclusion VIT is associated with a progressive expansion of circulating regulatory T cells, supporting a role for these cells in tolerance induction.     

Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom

Background: Specific immunotherapy with honeybee venom (BV) is highly effective, but allergic side effects can occur during treatment. Immunotherapy with peptides containing major T-cell epitopes of the relevant allergen or allergens provides an alternative strategy without these problems. Objective: The study investigates the immunologic mechanisms and clinical effects of immunotherapy with T-cell epitope peptides of the major BV allergen, the phospholipase A2 (PLA). Methods: Five patients with IgE-mediated systemic allergic reactions to bee stings were treated with a mixture of three T-cell epitope peptides of PLA. Ten patients allergic to BV receiving whole BV immunotherapy served as control subjects. Increasing doses of the peptide mixture, up to a maintenance dose of 100 μg, were administered subcutaneously within 2 months. The patients were then challenged with PLA and 1 week later with a bee sting. The cellular and humoral immune response was measured in vitro. Results: No allergic side effects were caused by the peptide immunotherapy, and all patients tolerated the challenge with PLA without systemic allergic symptoms. Two patients developed mild systemic allergic reactions after the bee sting challenge. After peptide immunotherapy, specific proliferative responses to PLA and the peptides in peripheral blood mononuclear cells were decreased in successfully treated patients. The production of T_H2 and T _H1 cytokines was inhibited, and B cells were not affected in their capacity to produce specific IgE and IgG4 antibodies. Their levels increased after allergen challenge in favor of IgG4. Conclusions: Immunotherapy of BV allergy with short T-cell peptides of PLA induces epitope-specific anergy in peripheral T cells and changes the specific isotype ratio in a fashion similar to that of conventional immunotherapy in successfully treated patients. (J Allergy Clin Immunol 1998;101:747-54.)     

New opportunities for allergen immunotherapy using synthetic peptide immuno-regulatory epitopes (SPIREs)

Introduction: Allergen immunotherapy (AIT) reduces allergic rhinoconjunctivitis (ARC) symptoms, but long-term efficacy requires treatment for 3–5 years. Synthetic peptide immuno-regulatory epitopes, a new class of AIT, are allergen peptides with a shorter, more convenient treatment regimen that could potentially have benefits on adherence and outcomes.

Areas covered: Phase 2 trials of therapies derived from cat, house dust mite, grass, and ragweed allergen peptides demonstrated significant reduction in ARC symptoms after short-course treatment; improvement was sustained for 18–24 months posttreatment. We conducted a PubMed literature search for clinical publications using the search terms AIT; allergen peptides; ARC; cat, grass, house dust mite, and ragweed allergy; SCIT; SLIT; and synthetic peptides.

Expert commentary: Long-term disease modification is a realistic goal of AIT. The inconvenience of conventional AIT regimens negatively impacts long-term persistence and, thus, efficacy. In comparison, SPIREs have a more convenient treatment regimen that could potentially have benefits on adherence and outcomes.     

A comparison of the T cell delayed-type hypersensitivity epitopes of the 19-kD antigens from Mycobacterium tuberculosis and Myco. intracellulare using overlapping synthetic peptides.

Mycobacterial disease remains a serious international public health concern. Improved methods to rapidly and specifically detect mycobacterial infections would greatly enhance clinical management of these diseases. To define species-specific T cell epitopes that may be useful for the immunodiagnosis of mycobacterial infections, polymerized synthetic peptides from the 19-kD Mycobacterium tuberculosis and Myco. intracellulare protein homologues were tested in guinea pig DTH assays. Five Myco. tuberculosis and eight Myco. intracellulare peptides evoked skin test responses. Although all of the active Myco. tuberculosis and seven of the Myco. intracellulare peptides elicited non-specific DTH reactions, the peptide IN13 induced a Myco. intracellulare-specific skin test reaction, and thus represents a specific Myco. intracellulare T cell DTH epitope. This result suggests that the development of monospecific peptide-based immunodiagnostic reagents may be feasible for future clinical use.     

Specific interaction of IgE antibodies with a carbohydrate epitope of honey bee venom phospholipase A2

Phospholipase A2 (E.C. 3.1.1.4.) is a major allergen of honey bee venom. It exists in a glycosylated and an unglycosylated variant. Both forms and the glycopeptide isolated after exhaustive proteolytic digestion were tested in RAST and RAST inhibition studies. IgE from 11 of 14 bee venom allergy sera exhibited significantly higher, and in two cases exclusive, affinity towards glycosylated phospholipase. In RAST inhibition experiments using phospholipase coupled to discs five of the sera were completely inhibited by glycopeptide at 0.1 mg/ml; four sera were partially inhibited and two sera could not be inhibited. Glycoasparagine, lacking all amino acids except the carbohydrate-linking asparagine, inhibits IgE-binding to glycopeptide discs up to 100%. These data clearly demonstrate that an oligosaccharide of a structural type frequently found in glycoproteins can represent an epitope which is recognized by IgE antibodies from allergic patients, which are specifically directed against the parent glycoprotein.     

Use of multiple peptides containing T cell epitopes is a feasible approach for peptide-based immunotherapy in Can f 1 allergy

We have previously shown that the major dog allergen Can f 1 contains seven T cell epitope regions, none of which was preferentially recognized. To identify the immune characteristics of Can f 1 epitopes and to verify their suitability for peptide-based allergen immunotherapy, short-term T cell lines were generated with epitope-containing peptides from peripheral blood mononuclear cells of Can f 1 skinprick test-positive allergic and healthy control subjects. The lines were examined for their proliferative capacity and cytokine production upon stimulation with the allergen peptide, a homologous peptide from human tear lipocalin (TL) and Can f 1 and TL proteins. Can f 1 peptides induced proliferation of T cells and gave rise to T cell lines with comparable efficiencies. In particular, the T cell lines of allergic subjects induced with p33-48 and p107-122 favoured the production of interferon-gamma and interleukin-10, respectively. A greater number of Can f 1-specific T cell lines were generated from allergic than from healthy individuals. Two p107-122-induced Can f 1-specific T cell lines also reacted to a homologous peptide of human TL. Our results suggest that several T cell epitope-containing peptides should be used in combination for specific immunotherapy in Can f 1 allergy.     

Immunological changes during specific immunotherapy of grass pollen allergy: reduced lymphoproliferative responses to allergen and shift from TH2 to TH1 in T-cell clones specific for Phi p 1, a major grass pollen allergen

Background and Objective The mechanisms operative in specific immunotherapy (SIT) of Type I allergy are not completely understood. In the present study we evaluated immunological changes during SIT in pollinosis. Method Eight patients suffering from pollinosis (monosensitized to grass pollen) were treated with conventional SIT. All subjects had IgE specific for Phi p 1. a major allergen of timothy grass. In vitro changes in the immunological reactivity to grass pollen extract and to recombinant Phi p 1 were evaluated. Subjects were examined at three occasions: before, after 3 months and after I year of SIT. Results Serological analysis revealed a marked increase of grass pollen- and Phi p 1-specific IgG, titres of specific IgE did not change significantly. Lymphoproliferative responses to grass pollen extract and rPhl p 1 were reduced already after 3 months of treatment. Accordingly, the cloning efficiency for Ph1 p 1-specific T-cell clones (TCC) dropped markedly in all patients. The majority of allergen-specific TCC raised before SIT revealed a TH₂-like pattern of cytokine production. TCC established after SIT revealed TH₁ characteristics. This shift was due to a decrease in IL-4 rather than an increase in IFN-production by T cells. Investigations of the epitopes recognized by T cells before and after SIT did not reveal the outgrowth of new (ldquo;protecting”) specificities. We could not observe induction of allergen-speeific CD8⁺ lymphocytes (supressor cells). Conclusion Our data indicate that — on the level of TH lymphocytes — SIT induces tolerance to the allergen and a modulation of the cytokine pattern produced in response to allergen stimulation.     

The frequency of phospholipase A2 binding of basophilic granulocytes does not decrease during bee‐venom‐specific immunotherapy

BACKGROUND: The major allergenic component of bee venom is phospholipase A2 (PLA2). METHODS: In this study, PLA2 was used to analyze and enrich PLA2-binding cells from peripheral blood by high gradient magnetic cell sorting. RESULTS: In normal donors, the frequency of allergen (PLA2)-binding cells among peripheral blood mononuclear cells (PBMC) as determined by flow cytometry is below 0.1%, whereas in bee-venom-allergic patients, PLA2-binding cells are readily detectable at frequencies of up to 2.3%. In severely bee-venom-allergic patients, many basophilic granulocytes are present, as defined by anti-CD9, CD25, and CD38 mAb, comprising up to 95% of the PLA2-binding cells. From blood of allergic and normal donors, about equal absolute numbers of allergen-binding CD19/21-positive B cells can be enriched. Severe anaphylactic reactions (Mueller grade IV) and failure of or adverse reactions during immunotherapy are associated with high numbers of circulating allergen-binding basophils. Interestingly, in the patients studied, the number of PLA2-binding basophilic granulocytes did not markedly change during rush immunotherapy and up to 6 months of maintenance immunotherapy. CONCLUSIONS: The specific and reproducible enrichment of PLA2-binding cells provides a new tool for the analysis and monitoring of effector cells in bee-venom-allergic patients with immediate-type hypersensitivity.     

T cell epitopes of the major fraction of rye grass Lolium perenne (Lol p I) defined using overlapping peptides in vitro and in vivo. I. Isoallergen clone1A.

One hundred and fifteen overlapping synthetic peptides spanning the entire sequence of the iso-allergen clone1A of Lol p I from rye grass Lolium perenne were synthesized by the multi-pin technique. The peptides were overlapping 12mers, offset by two residues and overlapping by 10 residues. Sets of six adjacent overlapping peptides (except pool-1, 15, 20) were pooled and were used in vitro and in vivo to map the T cell epitopes on Lol p I. Six atopics who were skin test and RAST positive to rye grass showed T cell responses to L. perenne extract (LPE) and its major fraction (Lol p I). Five out of six showed T cell responses in vitro to peptide pool-17, while five non-atopics did not respond to any of the peptide pools. By testing the individual peptides of pool-17, we have located the T cell epitope on Lol p I. Interestingly, when we tested pool-17 and its single peptides in vivo by intradermal skin testing we found in one patient a typical DTH after 24-48 h to pool-17 and its peptides (peptides 3 and 4) which exactly matched the in vitro responses. By defining the T cell epitopes in this way a greater understanding of the allergic response to pollen will be obtained, and a more effective and less dangerous vaccine may be possible for treating patients with hay fever.     

Abundance of H-2 promiscuous T cells specific for mycobacterial determinants in H-2b/d F1 hybrid mice

A majority of immunodominant epitopes of mycobacterial antigens are known to be recognized by murine T cells in the context of several H-2 haplotypes. In this study, we established the frequency of T cells able to recognize these peptides promiscuously, i.e. in the context of allogeneic antigen-presenting cells, using hybridomas from peptide-immunized H-2 homologous and heterologous mice. The degree of promiscuity in homozygous mice varied between 4–27% between different specificities and genetic backgrounds. In particular, the results showed that promiscuity between A^b and A^d in respect to a peptide from the Mycobacterium tuberculosis 38-kDa protein (residues 350–369) was displayed by 22% of BALB/c and 4% of C57BL/10-derived hybrids, but by 42% of [BALB/c x C57BL/10] F1-derived clones. This represents a significant increase (p < 0.001) of T cell promiscuity compared to the parental haplotypes. It is noteworthy that considerably lower peptide concentrations were able to stimulate the promiscuous hybridomas compared to the H-2-restricted hybrids. This finding suggests a functional advantage of promiscuous T cells which enables them to expand preferentially in the initial stages of infections with M. tuberculosis and thus enables the host to mount a rapid protective immune response.     

Allergenic epitopes of bovine αS1-casein recognized by human IgE and IgG

B-cell epitopes of bovine α_S1-casein, one of the major allergens of cow's milk, were identified by a screening method based on synthetic peptides. According to the known amino acid sequence of α_S1-casein, a set of 188 overlapping sequential decapeptides shifted by one amino acid was manually synthesized on polyethylene pins by the 9-fluorenyl-methoxycarbonyl (Fmoc) method. Peptides were screened by an enzyme-linked immunosorbent assay (ELISA) specific for human IgE and IgG. Bound antibodies were detected by successive incubation with up to three polyclonal antibodies, the last one conjugated to horseradish peroxidase. Tested sera were from 15 patients with acute clinical reactions to cow's milk and IgE-specific reactions to bovine α-casein in the ELISA and immunoblot. Sera from 10 healthy subjects without remarkable reactions to cow's milk proteins were used as controls. All sera from allergic subjects showed reactions with three regions of α_S1-casein, corresponding to amino acids 19–30, 93–98, and 141–150. Furthermore, individual sera showed reactions with other parts of the protein. No essential differences in the epitope specificity of IgE and IgG were found. Inhibition of IgE binding to α_S1-casein with soluble synthetic peptides confirmed the results and revealed peptide CN-2 as the most inhibiting one.     

Multiple roles for CD4+ T cells in anti-tumor immune responses

Summary: Our understanding of the importance of CD4⁺ T cells in orchestrating immune responses has grown dramatically over the past decade. This lymphocyte family consists of diverse subsets ranging from interferon-γ (IFN-γ)-producing T-helper 1 (Th1) cells to transforming growth factor-β (TGF-β)-secreting T-regulatory cells, which have opposite roles in modulating immune responses to pathogens, tumor cells, and self-antigens. This review briefly addresses the various T-cell subsets within the CD4⁺ T-cell family and discusses recent research efforts aimed at elucidating the nature of the 'T-cell help' that has been shown to be essential for optimal immune function. Particular attention is paid to the role of Th cells in tumor immunotherapy. We review some of our own work in the field describing how CD4⁺ Th cells can enhance anti-tumor cytotoxic T-lymphocyte (CTL) responses by enhancing clonal expansion at the tumor site, preventing activation-induced cell death and functioning as antigen-presenting cells for CTLs to preferentially generate immune memory cells. These unconventional roles for Th lymphocytes, which require direct cell-to-cell communication with CTLs, are clear examples of how versatile these immunoregulatory cells are.     

Non-covalent presentation of sulfamethoxazole to human CD4+ T cells is independent of distinct human leucocyte antigen-bound peptides

BACKGROUND: It has been shown that drugs comprise a group of non-peptide antigens that can be recognized by human T cells in the context of HLA class II and that this recognition is involved in allergic reactions. Recent studies have demonstrated a MHC-restricted but processing- and metabolism-independent pathway for the presentation of allergenic drugs such as lidocaine and sulfamethoxazole (SMX) to drug-specific T cells. However, there is little information so far on the precise molecular mechanisms of this non-covalent drug presentation. OBJECTIVE: The aim of this study was to evaluate the requirements for a specific peptide occupying the groove of the MHC class II molecule for the efficient presentation of non-covalently bound drugs to CD4+ T cells. METHODS: We analysed the effect of coincubation or prepulse of antigen presenting cells (APC) with different peptides on the proliferative responses of SMX-specific CD4+ T cell clones. In a second series of experiments, we eluted HLA-bound peptides from the surface of antigen presenting cells by mild acid treatment. Successful removal of peptides was tested directly using labelled peptides and functionally by monitoring activation and proliferation of peptide-specific T cell clones. Finally, the presentation of SMX to SMX-specific T cell clones before and after elution of MHC class II bound peptides was tested. RESULTS: We found that neither peptide coincubation nor peptide prepulse of APC altered the proliferative response of SMX-specific T cells. APC treated with the acid for a short time retained cell viability, MHC class II expression and antigen presenting cell function. However, defined peptides could be eluted from surface MHC class II molecules nearly quantitatively. Nevertheless, the chemically non-reactive drug SMX could still be presented to specific T cells independent of the presence of distinct self-peptides. CONCLUSION: Our data suggest that small molecules like drugs can bind to a multitude of HLA-bound peptides or that, similar to superantigens, they might bind directly to HLA.     

The in vitro generation of multi-tumor antigen-specific cytotoxic T cell clones: Candidates for leukemia adoptive immunotherapy following allogeneic stem cell transplantation

Adoptive T-cell immunotherapy is a promising approach to manage and maintain relapse-free survival of leukemia patients, especially following allogeneic stem cell transplantation. Post-transplant adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) of the donor origin provide graft-versus-tumor effects, with or without graft–versus-host disease. Myeloid leukemias express immunogenic leukemia associated antigens (LAAs); such as WT-1, PRAME, MAGE, h-TERT and others, most of them are able to induce specific T cell responses whenever associated with the proper co-stimulation. We investigated the ability of a LAA-expressing hybridoma cell line to induce CTL clones in PBMCs of HLA-matched healthy donors in vitro. The CTL clones were induced by repetitive co-culture with LAAs-expressing, HLA-A*0201⁺ hybrid cell line, generated by fusion of leukemia blasts to human immortalized APC (EBV-sensitized B-lymphoblastoid cell line; HMy2). The induced cytotoxic T cell clones were phenotypically and functionally characterized by pentamer analysis, IFN-γ release ELISPOT and cellular cytotoxicity assays. All T cell lines showed robust peptide recognition and functional activity when sensitized with HLA-A*0201-restricted WT-1_235–243, hTERT_615–624 or PRAME_100–108 peptides-pulsed T2 cells, in addition to partially HLA-matched leukemia blasts. This study demonstrates the feasibility of developing multi-tumor antigen-specific T cell lines in allogeneic PBMCs in vitro, using LAA-expressing tumor/HMy2 hybrid cell line model, for potential use in leukemia adoptive immunotherapy in partially matched donor-recipient setting.     

Oral tolerance, food allergy, and immunotherapy: implications for future treatment

The lumen of the gastrointestinal tract is exposed daily to an array of dietary proteins. The vast majority of proteins are tolerated through suppression of cellular or humoral responses, a process known as oral tolerance. However, in approximately 6% of children and 4% of adults in the United States, tolerance to a given dietary antigen either is not established or breaks down, resulting in food hypersensitivity. Although food allergies can result in sudden and life-threatening symptoms, their prevalence is remarkably low considering the complexities of the gut-associated mucosal system. Suppression involves signaling by an array of nonprofessional antigen-presenting cells, dendritic cells, and regulatory T cells, as well as lymphocyte anergy or deletion. Several factors, including antigen properties, route of exposure, and genetics and age of the host, contribute to the development of oral tolerance. Although the current standard of care for patients with food allergies is based on avoidance of the trigger, increased understanding of the mechanisms involved in tolerance has shifted focus of treatment and prevention toward inducing tolerance. Data from early-phase clinical trials suggest both sublingual and oral immunotherapy are effective in reducing sensitivity to allergens. In this article we review the mechanisms of tolerance, discuss aberrations in oral tolerance, and provide information on novel prevention and treatment paradigms for food allergy.     

Inhibition of T cell proliferation specific for acetylcholine receptor epitopes related to myasthenia gravis with antibody to T cell receptor or with competitive synthetic polymers.

Long-term T cell lines and clones of C3H.SW origin specific to synthetic peptides representing immunogenic epitopes of the human aetylcholine receptor alpha-subunit were established. Using these lines and clones, it was possible to characterize the T cell recognition process of myasthenic epitopes. Testing a panel of N-and/or C-terminal truncated peptides it could be demonstrated that the deletion of the two C-terminal amino acids of peptides p195-212 and p259-271 resulted in a loss or reduction of the stimulatory capacity of the peptides towards the specific T cell lines. In contrast, no substantial effect on the stimulation of the line could be observed by shortening peptide p195-212 by up to five amino acids at the N-terminal end. The proliferation of T cell lines and clones specific to peptide p195-212 was inhibited by a mAb directed against the V beta 8 region of the T cell receptor. Furthermore, it was possible to block the peptide-specific proliferative responses of the lines and clones by the I-Ab restricted synthetic polypeptide (T, G)-A--L but not by the I-Ak restricted polypeptide antigen (H,G)-A--L. Similarly, p195-212 inhibited the proliferative response of the TCSW259-271 T cell line and p259-271 inhibited the specific proliferative response of the TCSW195-212 line. Moreover, the C-terminal shortened peptides inhibited significantly the in vitro stimulation of the T cell lines by the immunogenic peptides p195-212 and p259-271. The inhibition by the synthetic peptides or by (T,G)-A--L may be due to competitive blockade of the MHC binding site for the T cell line stimulating AChR peptides.     

Detection of ABCB5 tumour antigen‐specific CD8+ T cells in melanoma patients and implications for immunotherapy

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour‐initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti‐ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5‐reactive T cells are present in human melanoma patients and tested the applicability of ABCB5‐derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co‐cultured with ABCB5 antigen‐loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5‐reactive CD8⁺ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART‐1)‐reactive CD8⁺ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART‐1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide‐loaded autologous DC. As ABCB5‐reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5‐specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.     

Induction of T ''regulatory'' cells by standardized house dust mite immunotherapy: an increase in CD4+CD25+ interleukin-10+ T cells expressing peripheral tissue trafficking markers

BACKGROUND: Clinically effective subcutaneous allergen-specific immunotherapy (SIT) is associated with altered circulating T cell cytokine production and altered local cytokine responses with increased IL-10 following allergen challenge in target organs. OBJECTIVE: This study aimed to elucidate mechanisms for these T cell changes, by examining surface expression of markers for peripheral tissue trafficking on circulating cytokine-positive T cells following standardized house dust mite- (HDM-) SIT. METHODS: A randomized conventional HDM immunotherapy study was performed on a panel of 12 HDM-allergic subjects. Nine subjects received treatment with conventional HDM immunotherapy using a standardized extract and three subjects were treated by standard pharmacotherapy alone. Symptom and medication scores and allergen-induced cutaneous late-phase responses were assessed before and 9 months after institution of therapy. Before and at 3 and 9 months of SIT, peripheral blood mononuclear cells were cultured for 14 days with HDM extract and CD4+ and CD8+ T cell expression of CD62L, CD49d and CCR5 and production of IL-10, IFN-gamma and IL-4 were analysed by flow cytometry. Allergen-specific T cell proliferation was assessed by 3H-thymidine incorporation. RESULTS: At 9 months, all SIT-treated patients showed reduced symptom scores and late-phase cutaneous responses to HDM compared with baseline levels. The proportions of CD4+ T cells which were IL-10+ were increased (P < 0.01), and the proportions of CD4+ and CD8+ T cells which were IL-4+ decreased (P < 0.05) compared with baseline. CD4+ and CD8+ T cell IFN-gamma production, expression of surface markers for peripheral tissue trafficking and allergen-specific proliferation remained unchanged during SIT treatment. However, increased proportions of CD4+CD62L(-), CD4+CD49d(hi), CD4+CCR5+ T cells expressing IL-10 were detected at 9 months of SIT compared with baseline (P < 0.05). IL-10 staining co-localized with CD4+CD25+ T cells. CONCLUSION: Clinically effective subcutaneous immunotherapy with a standardized HDM Dermatophagoides pteronyssinus preparation results in decreased numbers of IL-4+ T cells and expansion of CD4+IL-10+ T cells expressing a peripheral tissue trafficking phenotype. The co-localization of IL-10+ staining to CD4+CD25+ T cells is consistent with the induction of a T regulatory cell population by SIT.