Fel d 1-derived T cell peptide therapy induces recruitment of CD4+CD25+; CD4+ interferon-γ+ T helper type 1 cells to sites of allergen-induced late-phase skin reactions in cat-allergic subjects

BACKGROUND: Specific immunotherapy with whole allergen extracts is associated with local accumulation of IFN-gamma+ and CD25+ cells indicating recruitment of activated T-helper type 1 (Th1) and/or T regulatory cells. We have studied allergen-induced, late-phase skin biopsies before and after T cell peptide therapy for evidence of alterations in the pattern of local recruitment of Th1, T-helper type 2 (Th2) and T regulatory cells. OBJECTIVE: To evaluate the effect of T cell peptide therapy on the allergen-induced cutaneous late-phase reaction. METHODS: Increasing doses of synthetic Fel d 1-derived peptides were administered (by intradermal injection) to eight cat-allergic asthmatics at 14-day intervals. Twenty-four-hour skin biopsies were taken from whole cat allergen- and diluent-injected sites, before and after treatment and studied by immunohistochemistry and in situ hybridization. RESULTS: Fel-d 1 peptides decreased airway hyper-responsiveness (P = 0.02) and inhibited the late-phase cutaneous reaction (LPCR) to whole cat allergen (P = 0.03). This was associated with significant increases (post- vs. pre-treatment) in the number of cutaneous CD4+/IFN-gamma+ (P = 0.03) and CD4+/CD25+ cells (P = 0.04), but not in CD4+/IL-10+ or CD4+/CTLA-4+ cells. CONCLUSIONS: Treatment with allergen-derived T cell peptides results in allergen-dependent recruitment to the skin of Th1, rather than T regulatory cells, to cutaneous late-phase reaction sites.     

A subset of CD8+ T cells from allergic patients produce IL-4 and stimulate IgE production in vitro

Background and Objective A subset of IL-4 producing CD8⁺ T cells was recently identified in HIV patients. Based on these findings we examined whether IL-4 producing CD8⁺ T cells would also be present in allergic patients and what would be the functional relevance of this T-cell population. Methods We investigated the role of CD8⁺ T cells in IgE production of allergic diseases by analysing the cytokine profile of individual CD4⁺ and CD8⁺ T cells. Results In allergic patients about twice as many CD4⁺ T cells and six times as many CD8⁺ T cells produced IL-4 as in non-allergic controls. In contrast the frequency of IFNγ⁺ T-cell subsets did not significantly differ between the allergic and non-allergic individuals. The frequency of 1L4⁺CD8⁺ T cells correlated with the level of serum IgE. Coculture experiments with T cells or purified CD8⁺ T cells together with autologous B cells indicated that CD8⁺ T cells enhanced IgE in vitro, but not IgM production, even when they were physically separated from B cells. This effect could be partially blocked by addition of an IL-4 binding protein, a soluble IL-4 receptor indicating that lL-4 is involved in CD8⁺ T-cell mediated IgE production. Conclusions These data indicate a positive role of IL-4 secreting CD8⁺ T cells in IgE regulation in allergic patients.     

CD4+CD25+ T cell depletion impairs tolerance induction in a murine model of asthma

Background Regulatory T cells (Tregs) are key players in controlling the development of airway inflammation. However, their role in the mechanisms leading to tolerance in established allergic asthma is unclear. Objective To examine the role of Tregs in tolerance induction in a murine model of asthma. Methods Ovalbumin (OVA) sensitized asthmatic mice were depleted or not of CD25⁺ T cells by anti‐CD25 PC61 monoclonal antibody (mAb) before intranasal treatment (INT) with OVA, then challenged with OVA aerosol. To further evaluate the respective regulatory activity of CD4⁺CD25⁺ and CD4⁺CD25^? T cells, both T cell subsets were transferred from tolerized or non‐tolerized animals to asthmatic recipients. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. Results Intranasal treatment with OVA led to increased levels of IL‐10, TGF‐β and IL‐17 in lung homogenates, inhibition of eosinophil recruitment into the BALF and antigen specific T cell hyporesponsiveness. CD4⁺CD25⁺Foxp3⁺ T cells were markedly upregulated in lungs and suppressed in vitro and in vivo OVA‐specific T cell responses. Depletion of CD25⁺ cells before OVA INT severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge. However, the transfer of CD4⁺CD25^? T cells not only suppressed antigen specific T cell responsiveness but also significantly reduced eosinophil recruitment as opposed to CD4⁺CD25⁺ T cells. As compared with control mice, a significantly higher proportion of CD4⁺CD25^? T cells from OVA treated mice expressed mTGF‐β. Conclusion Both CD4⁺CD25⁺ and CD4⁺CD25^? T cells appear to be essential to tolerance induction. The relationship between both subsets and the mechanisms of their regulatory activity will have to be further analyzed.     

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.     

Allergen-induced cytokine phenotypes in mice: role of CD4+ and CD8+ T cell populations

BACKGROUND: CD4+ T cells expressing type 2 cytokines have been implicated in the pathogenesis of asthma to high-molecular-weight allergens. Topical exposure of BALB/c strain mice to low-molecular-weight chemical contact and respiratory allergens stimulates type 1 and type 2 cytokine secretion phenotypes, respectively. OBJECTIVE: To examine the relative frequencies of cytokine-positive CD4+ and CD8+ T cells and their contributions to these cytokine secretion profiles. Methods Draining auricular lymph nodes were isolated 13 days after initiation of topical exposure of female BALB/c strain mice to chemical allergen, or to vehicle alone. The frequency of intracellular cytokine (IL-4 and IFN-gamma)-positive CD4+ and CD8+ lymphocytes was enumerated by flow cytometry. The relative contribution of CD4+ and CD8+ cells to cytokine secretion profiles was assessed by negative selection. RESULTS: Exposure to allergen resulted in an increased frequency of both IFN-gamma+ CD4+ and CD8+ lymphocytes, although there were no marked differences between trimellitic anhydride (TMA)- and 2,4-dinitrochlorobenzene (DNCB)-activated lymph node cells. Treatment with TMA induced approximately five times as many IL-4+ CD4+ cells as did exposure to DNCB. This pattern of cytokine staining was also observed for a further pair of contact and respiratory allergens; respectively, formalin and fluorescein isothiocyanate. CONCLUSION: These data demonstrate that the divergent immune responses induced in mice by different classes of chemical allergen are independent of changes in the frequency of IFN-gamma+ cells, but are associated with differential frequencies of IL-4-expressing CD4+ T cells.     

CD4+CD25+ regulatory T cells: I. Phenotype and physiology

The immune system protects us against foreign pathogens. However, if fine discrimination between self and non-self is not carried out properly, immunological attacks against self may be launched leading to autoimmune diseases, estimated to afflict up to 5% of the population. During the last decade it has become increasingly clear that regulatory CD4⁺CD25⁺ T cells (T_reg cells) play an important role in the maintenance of immunological self-tolerance, and that this cell subset exerts its function by suppressing the proliferation or function of autoreactive T cells. Based on human and murine observations, this review presents a characterization of the phenotype and functions of the Treg cells in vitro and in vivo . An overview of the surface molecules associated with and the cytokines produced by the Treg cells is given and the origin, activation requirements and mode of action of the Treg cells are discussed. Finally, we address the possibility that Treg cells may play a central role in immune homeostasis, regulating not only autoimmune responses, but also immune responses toward foreign antigens.     

The lifestyle of naturally occurring CD4+CD25+Foxp3+ regulatory T cells

Summary: Numerous studies over the past 10 years have demonstrated the importance of naturally occurring CD4⁺CD25⁺Foxp3⁺ regulatory T cells (nTregs) in immune regulation. We analyzed the mechanism of action of nTregs in a well‐characterized model of autoimmune gastritis and demonstrated that nTregs act at an early stage of disease progression to inhibit the differentiation of naïve T cells to pathogenic T‐helper 1 effectors. The effects of nTregs in this model are not antigen‐specific but are mediated by activation of the nTregs by ubiquitous self‐peptide major histocompatibility complex class II complexes together with cytokines released by activated effector cells. Studies in vitro confirmed that some nTregs exist in an activated state in vivo and can be activated to exert non‐specific suppressor effector function by stimulation with interleukin‐2 in the absence of engagement of their T‐cell receptor. Natural Tregs can differentiate in vitro to exhibit potent granzyme B‐dependent, partially perforin‐independent cytotoxic cells that are capable of specifically killing antigen‐presenting B cells. Natural Treg‐mediated killing of antigen‐presenting cells may represent one pathway by which they can induce long‐lasting suppression of autoimmune disease.     

Defective suppression of Th2 cytokines by CD4+CD25+ regulatory T cells in birch allergics during birch pollen season

BACKGROUND: CD4(+)CD25+ regulatory T cells suppress proliferation and cytokine production by human T cells both to self-antigens and exogenous antigens. Absence of these cells in human newborns leads to multiple autoimmune and inflammatory disorders together with elevated IgE levels. However, their role in human allergic disease is still unclear. OBJECTIVE: This study aimed to evaluate the capacity of CD4(+)CD25+ regulatory T cells to suppress proliferation and cytokine production outside and during birch-pollen season in birch-allergic patients relative to non-allergic controls. METHODS: CD4+ cells were obtained from blood of 13 birch-allergic patients and six non-allergic controls outside pollen season and from 10 birch-allergic patients and 10 non-allergic controls during birch-pollen season. CD25+ and CD25- fractions were purified with magnetic beads and cell fractions, alone or together in various ratios, were cultured with antigen-presenting cells and birch-pollen extract or anti-CD3 antibody. Proliferation and levels of IFN-gamma, IL-13, IL-5 and IL-10 were measured by thymidin incorporation and ELISA, respectively. Numbers of CD25+ cells were analysed by flow cytometry. RESULTS: CD4(+)CD25+ regulatory T cells from both allergics and non-allergics potently suppressed T cell proliferation to birch allergen both outside and during birch-pollen season. However, during season CD4(+)CD25+ regulatory T cells from allergic patients but not from non-allergic controls were defective in down-regulating birch pollen induced IL-13 and IL-5 production, while their capacity to suppress IFN-gamma production was retained. In contrast, outside pollen season the regulatory cells of both allergics and non-allergic controls were able to inhibit T-helper 2 cytokine production. CONCLUSION: This is the first study to show differential suppression of Th1 and Th2 cytokines, with CD4(+)CD25+ regulatory T cells from birch-pollen-allergic patients being unable to down-regulate Th2, but not Th1 responses during birch-pollen season.     

Cyclic nucleotide phosphodiesterases from purified human CD4+ and CD8+ T lymphocytes

Background CD4⁺ and CD8⁺ T-lymphocytes are suggested to differentially affect airway inflammation in asthma. Agents which increase intracellular cAMP levels, such as PDE inhibitors, have been shown to diminish lymphocyte growth and differentiation, and to affect cytokine expression. Differences in the PDE isoenzyme profile between CD4⁺ and CD8⁺ cells might form a basis to differentially modify their functions by PDE inhibitors. Objective The study investigates and compares the PDE isoenzyme activity profiles of human peripheral blood CD4⁺ and CD8⁺ T-lymphocytes. Methods CD4⁺ and CD8⁺ T-lymphocytes were purified (>98%) from peripheral blood mononuclear cells by negative selection. PDE isoenzyme activity profiles were investigated using PDE isoenzyme selective inhibitors and activators. Results In CD4⁺ and CD8+ T-lymphocyte homogenates, PDE IV and PDE III activities were the predominant PDE isoenzyme activities at 0.5μM cyclic nucleotide substrate concentrations. PDE IV was localized in the soluble fraction whereas PDE III was membrane bound. Low PDE I, II and V activities were detected. About 20% of total eAMP hydrolysing capacity at 0.5 μM cAMP was insensitive to PDE isoenzyme selective inhibitors and activators and therefore could not be assigned to PDE I-IV. The PDE isoenzyme pattern was not different between CD4⁺ and CDS⁺ T-lymphocytes. Moreover, representative inhibitors of PDE HI and IV activity inhibited cAMP hydrolysis in soluble fractions of both T-lymphocyte subsets with similar potency. Enzyme kinetic analysis similarly did not reveal differences between CD4^h and CD8⁺ T-lymphocytes. Conclusion Normal CD4⁺ and CD8⁺ T-lymphocytes are likely to be equally sensitive targets for the effects of PDE inhibitors.     

Wasp venom immunotherapy induces a shift from IL-4-producing towards interferon-gamma-producing CD4+ and CD8+ T lymphocytes

BACKGROUND: Venom immunotherapy (VIT) has proven to be very effective in hymenoptera venom anaphylaxis. However, the underlying immunoregulatory mechanisms of venom immunotherapy remain poorly understood. Recent studies measured the total amount of cytokine in culture supernatans, suggesting a shift in cytokine production from Th2 to a Th1 cytokine profile during VIT. We wanted to examine the contribution of specific T lymphocyte subpopulations, which is impossible using an extracellular method to determine cytokines in supernatants. OBJECTIVE: The present study was designed to evaluate the effect of VIT on the percentages of type 1 (IL-2, interferon-gamma (IFN-gamma)) and type 2 (IL-4) CD4+ and CD8+ T lymphocytes of patients with wasp venom anaphylaxis during immunotherapy. METHODS: Peripheral blood mononuclear cells of 20 individuals with a history of wasp sting anaphylaxis and a positive serum wasp venom specific IgE were isolated and in vitro stimulated with phorbol-12-myristate-13-acetate and ionomycin before VIT, at the end of a 5-day semirush VIT and at 6 months during VIT. Three-colour flow cytometric analysis was used for intracellular cytokine (IL-2, IFN-gamma, IL-4) detection in CD4+ (CD3+CD8-) T lymphocytes and CD8+ (CD3+CD8+) T lymphocytes. RESULTS: At the end of a 5-day semirush VIT, there was a significant decrease in percentage of IL-4-producing CD4+ and CD8+ T cells, compared with cytokine-producing cells before VIT (P = 0.0002 and 0.004). After 6 months of VIT, patients showed an increased number of IL-2-producing stimulated CD4+ and CD8+ lymphocytes compared with values before VIT (P = 0.002 and P = 0.0003). A higher amount of IFN-gamma-producing stimulated CD4+ and CD8+ cells was found after 6 months of VIT (P = 0.001 and P = 0.0006). There was no correlation between cytokine-producing cells and specific IgE for wasp. CONCLUSION: Venom immunotherapy induced a shift from IL-4-producing towards IFN-gamma-producing CD4+ as well as CD8+ T lymphocytes.     

CD4+CD25+ regulatory T cells: II. Origin, disease models and clinical aspects

Autoimmune diseases afflict approximately 5% of the population and reflect a failure in the immune system to discriminate between self and non-self resulting in the breakdown of self-tolerance. Regulatory CD4⁺CD25⁺ T cells (T_reg cells) have been shown to play an important role in the maintenance of immune homeostasis and self-tolerance by counteracting the development and effector functions of potentially autoreactive T cells. We have in the previous APMIS review described the phenotype and physiology of T_reg cells. The present overview deals with the thymic origin of T_reg cells and their role in disease models such as autoimmune gastritis and inflammatory bowel disease. Finally, we will consider some aspects of the therapeutic potential of T_reg cells.     

Differential capacity of CD8α+ or CD8α− dendritic cell subsets to prime for eosinophilic airway inflammation in the T-helper type 2-prone milieu of the lung

BACKGROUND: Different subsets of dendritic cells (DCs), identified in mouse spleen by their differential expression of CD8 alpha, can induce different T-helper (Th) responses after systemic administration. CD8 alpha(-) DCs have been shown to preferentially induce Th type 2 (Th2) responses whereas CD8 alpha(+) DCs induce Th1 responses. OBJECTIVE: To study if these DC subsets can still induce different Th responses in the Th2-prone milieu of the lung and differentially prime for eosinophilic airway inflammation, typical of asthma. METHODS: Donor mice first received daily Flt3L injections to expand DC numbers. Purified CD8 alpha(+) or CD8 alpha(-) splenic DCs were pulsed with ovalbumin (OVA) or phosphate-buffered saline and injected intratracheally into recipient mice in which carboxyfluorescein diacetate succinimidyl ester-labelled OVA-specific T cell receptor transgenic T cells had been injected intravenously 2 days earlier. T cell proliferation and cytokine production of Ag-specific T cells were evaluated in the mediastinal lymph nodes (MLNs) 4 days later. The capacity of both subsets of DCs, to prime for eosinophilic airway inflammation was determined by challenging the mice with OVA aerosol 10 days later. RESULTS: CD8 alpha(-) DCs migrated to the MLN and induced a vigorous proliferative T cell response accompanied by high-level production of IL-4, IL-5, IL-10 and also IFN-gamma during the primary response and during challenge with aerosol, leading to eosinophilic airway inflammation. In the absence of migration to the MLN, CD8 alpha(+) DCs still induced a proliferative response with identical levels of IFN-gamma but reduced Th2 cytokines compared with CD8 alpha(-) DCs, which led to weak eosinophilic airway inflammation upon OVA aerosol challenge. Unpulsed DCs did not induce proliferation or cytokine production in Ag-specific T cells. CONCLUSION: CD8 alpha(-) DCs are superior compared with CD8 alpha(+) DCs in inducing Th2 responses and eosinophilic airway inflammation in the Th2-prone environment of the lung.     

Interferon-gamma-dependent inhibition of late allergic airway responses and eosinophilia by CD8+ gammadelta T cells

We have previously shown that CD8(+)gammadelta T cells decrease late allergic airway responses, airway eosinophilia, T helper 2 cytokine expression and increase interferon-gamma (IFN-gamma) expression. We hypothesized that the effects of CD8(+)gammadelta T cells were IFN-gamma mediated. Brown Norway rats were sensitized to ovalbumin on day 1. Cervical lymph node CD8(+)gammadelta T cells from sensitized animals were treated with antisense oligodeoxynucleotide (5 micromol/l) to inhibit IFN-gamma synthesis or control oligodeoxynucleotide and 3.5 x 10(4) CD8(+)gammadelta T cells were injected intraperitoneally into sensitized recipients on day 13. Rats were challenged with aerosolized ovalbumin on day 15 and lung resistance was monitored over an 8 hr period, after which bronchoalveolar lavage was performed. Control oligodeoxynucleotide treated gammadelta T cells decreased late airway responses and eosinophilia in bronchoalveolar lavage. There was a complete recovery of late airway responses and a partial recovery of airway eosinophilia in recipients of antisense oligodeoxynucleotide treated cells. Macrophage ingestion of eosinophils was frequent in rats administered gammadeltaT cells but reduced in recipients of antisense oligodeoxynucleotide treated cells. These results indicate that CD8(+)gammadelta T cells inhibit late airway responses and airway eosinophilia through the secretion of IFN-gamma. Defective or altered gammadelta T-cell function may account for some forms of allergic asthma.     

Peripheral blood CD4+ and CD8+ T cell type 1 and type 2 cytokine production in atopic asthmatic and normal subjects

BACKGROUND: Increased production of IL-4 and IL-5 and decreased production of IFN-gamma by CD4+ T cells has been implicated in asthma pathogenesis. However, CD8+ T cells also produce type 1 and type 2 cytokines and the relative roles of CD4+ and CD8+ T cell cytokine production in asthma have not been previously studied. OBJECTIVE: To determine the production of the type 1 and type 2 cytokines by CD4+ and CD8+ T cell subsets in asthmatic and normal subjects. METHODS: Intracellular cytokine staining for IL-4, -5, -10, -13 and IFN-gamma was analysed in peripheral blood CD4+ and CD8+ T cells from 24 atopic asthmatic and 20 normal subjects. RESULTS: Both subsets of T cells produced all cytokines studied and there were no significant differences between CD4+ and CD8+ T cells in their capacity to produce either type 1 or type 2 cytokines. There were significantly increased frequencies of IFN-gamma-positive CD4+ (13.1 +/- 2.4%, vs. 7.3 +/- 1.4%) and CD8+ (20.0 +/- 2.9%, vs. 9.6 +/- 2.1%) T cells in asthmatic subjects compared with normal subjects (P < 0.05), but not in frequencies of CD4+ or CD8+ T cells staining positively for IL-4, -5, -10 or -13. CONCLUSION: The frequencies of peripheral blood CD8+ T cells producing type 1 and type 2 cytokines are comparable with the frequencies of CD4+ T cells. There was an increased frequency of IFN-gamma producing CD4+ and CD8+ T cells in asthmatic compared with normal subjects. Further studies investigating T cells derived from the airways and investigating various stages within the disease process are required to further elucidate the importance of type 2 and type 1 T cell cytokine production in the pathogenesis of human allergic disease.     

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4+, CD8+, and CD19+ cell populations derived from allergic and healthy donors

The effect of disodium cromoglycate (DSCG) on in vitro proliferation of CD4⁺ and CD8⁺ T cells and CD19⁺ B cells, positively selected by immunomagnetic separation, was investigated. The cells were obtained from allergic patients with moderate serum IgE levels and mild to moderate atopic dermatitis, and healthy controls. The different cell subfractions were stimulated with mitogens or specific allergens, as well as cell supernatants from the lymphoblastoid B- (RPMI 8866) and T-hybridoma (166 A₂) cell lines. Proliferative responses of T- and B-cell subsets stimulated with mitogens together with recombinant interleukin-2 (rIL-2) or accessory cells (AC) could be inhibited by DSCG. In allergic individuals, significant allergen-specific stimulation could be observed in the CD8-depleted peripheral blood mononuclear cell (PBMC) fractions. Isolated CD4⁺ T cells, without AC or IL-2, could also be stimulated with specific allergen, but the responses were rather low. DSCG inhibited, concentration dependently, all allergen-induced responses. Interestingly, only atopic derived CD4⁺ and CD8⁺ T cells were stimulated by soluble low-affinity IgE receptor (Fc?RII/sCD23) and IgE binding factor (IgEBF), including IgE enhancing factor, present in culture supernatants from RPMI 8866 and 166 A₂, respectively. These responses were also inhibited by DSCG. This was in contrast to the amplifying effect of DSCG on spontaneously proliferating RPMI 8866 and 166 A₂ cells, cultured in fresh cRPMI 1640 medium without sCD23 and IgE enhancing factor. Our results show that DSCG delivers an inhibitory signal or signals to PBMC subpopulations expressing Fc?RII/sCD23, either upregulated by phytohemagglutinin in normal and atopic cells, or by allergens or sCD23 in atopic cells. The findings suggest that sCD23 in supernatants or in serum may reverse the general inhibitory mode of DSCG.     

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.