Dermatophagoides Pteronyssinus Group Research Articles

DNA vaccine encoding Der p2 allergen down-regulates STAT6 expression in mouse model of allergen-induced allergic airway inflammation

Activation of signal transducer and activator of transcription 6 (STAT6) plays a critical role in the late phase of Th2-dependent allergy induction. STAT6 is essential to Th2 cell differentiation, recruitment, and effector function. Our previous study confirmed that DNA vaccination inhibited STAT6 expression of spleen cells induced by allergen. In the present study, we determined whether DNA vaccine encoding Dermatophagoides pteronyssinus group 2 (Der p2) could down-regulate the expression and activation of STAT6 in lung tissue from asthmatic mice. After DNA vaccine immunization, BALB/c mice were sensitized by intraperitoneal injection and challenged by intranasal instillation of rDer p2. The levels of the cytokines IL-4 and IL-13 in BAL fluid were measured by enzyme-linked immunosorbent assay. The lung tissue was assessed by immunohistochemical staining with anti-STAT6. The protein expression of STAT6 was determined by Western blot. The activation of STAT6 binding ability was analyzed with electrophoretic mobility shift assay. DNA vaccine encoding Der p2 allergen effectively decreased the levels of IL-4 and IL-13 in the asthmatic mice. Histological evidence and Western blot showed that the expression of STAT6 in the DNA treated mice was markedly attenuated. STAT6 binding to specific DNA motif in lung tissue from the gene vaccinated mice was inhibited. DNA vaccine encoding Der p2 prevents allergic pulmonary inflammation probably by inhibiting the STAT6 signaling pathway in mice with Der p2 allergen-induced allergic airway inflammation.

Inhibition of Mite-Induced Immunoglobulin E Synthesis, Airway Inflammation, and Hyperreactivity by Herbal Medicine STA-1

The availability of STA-1 in suppressing allergen-induced immunoglobulin E (IgE) synthesis, airway inflammation and hyperreactivity in a murine model was investigated. The mice were intraperitoneally sensitized with Dermatophagoides pteronyssinus group 5 allergen (Der p 5) and orally treated with 300 mg/kg of STA-1 every other day for 14 days. The Der p 5-specific immunologic responses including changes of specific immunoglobulin G and E, cells in the broncholarvage fluid, and airway hyperreactivity were measured when mice received inhalation challenge with Der p 5 after sensitization for 21 days. By comparing with sham-treated groups, the synthesis of Der p 5-specific IgE was downregulated while the influx of eosinophils and neutrophils in the airway were remarkably reduced. In addition, Der p 5-induced airway hyperreactivity also was significantly eliminated by STA-1 treatment. These results showed that STA-1 could effectively suppress the Der p 5-induced allergic reactions, and the availability of STA-1 for the treatment of allergic asthma was demonstrated in this study.

Reversal of established CD4+ type 2 T helper-mediated allergic airway inflammation and eosinophilia by therapeutic treatment with DNA vaccines limits progression towards chronic inflammation and remodelling.

Immunostimulatory DNA-based vaccines can prevent the induction of CD4(+) type 2 T helper (Th2) cell-mediated airway inflammation in experimental models, when administered before or at the time of allergen exposure. Here we demonstrate their efficacy in limiting the progression of an established response to chronic pulmonary inflammation and airway remodelling on subsequent allergen challenge. Mice exhibiting Th2-mediated airway inflammation induced following sensitization and challenge with group 1 allergen derived from Dermatophagoides pteronyssinus group species (Der p 1), a major allergen of house dust mite, were treated with pDNA vaccines. Their airways were rechallenged and the extent of inflammation assessed. In plasma DNA (pDNA)-vaccinated mice, infiltration of inflammatory cells, goblet cell hyperplasia and mucus production were reduced and subepithelial fibrosis attenuated. The reduction in eosinophil numbers correlated with a fall in levels of the profibrotic mediator transforming growth factor (TGF)-beta1 in bronchoalveolar lavage (BAL) and lung tissue. In addition to lung epithelial cells and resident alveolar macrophages, infiltrating eosinophils, the principle inflammatory cells recruited following allergen exposure, were a major source of TGF-beta1. Protection, conferred irrespective of the specificity of the pDNA construct, did not correlate with a sustained increase in systemic interferon (IFN)-gamma production but in a reduction in levels of the Th2 pro-inflammatory cytokines. Notably, there was a reduction in levels of interleukin (IL)-5 and IL-13 produced by systemic Der p 1 reactive CD4(+) Th2 cells on in vitro stimulation as well as in IL-4 and IL-5 levels in BAL fluid. These data suggest that suppression of CD4(+) Th2-mediated inflammation and eosinophilia were sufficient to attenuate progression towards airway remodelling. Immunostimulatory DNA may therefore have a therapeutic application in treatment of established allergic asthma in patients.

Oral administration of a mite allergen expressed by zucchini yellow mosaic virus in cucurbit species downregulates allergen-induced airway inflammation and IgE synthesis

Sublingual-swallow immunotherapy in house dust mite-related asthma has a good safety profile and improves respiratory function and bronchial hyperreactivity. Zucchini yellow mosaic virus (ZYMV) is envisaged as a promising viral vector for expressing large quantity of foreign proteins in cucurbit species. We sought to investigate whether oral feeding of dust mite allergen expressed by ZYMV in a cucurbit species can suppress allergen-induced inflammation and IgE synthesis. An infectious plant virus clone, p35SZYMV2-26, that contains the full-length cDNA to the genomic RNA of a Taiwan isolate of ZYMV, driven by the cauliflower mosaic virus 35S promoter, was engineered as an in vivo viral vector to express Dermatophagoides pteronyssinus group 5 allergen (Der p 5) in cucurbit species. Female BALB/c mice were intraperitoneally sensitized with Escherichia coli bacteria-expressed Der p 5 and orally treated with the virus-expressed Der p 5 (vDer p 5) extracted from the recombinant virus-infected squash plants. Der p 5-specific immunoglobulins were measured by ELISA, and bronchoalveolar lavage assays were used to measure airway inflammation. Infectivity assays and immunoblotting revealed that large quantities of free-form vDer p 5 are produced in the recombinant virus-infected squash plants. The recombinant virus carried and expressed the Der p 5 allergen in squash plants for at least 1 year after numerous passages. In animal tests, squash extract containing vDer p 5 inhibited Der p 5-specific IgE synthesis and airway inflammation. Our results suggest that oral feeding with allergen produced by the plant viral vector provides a novel approach for the therapy of allergic asthma.

CD4+CD25+ T cells lyse antigen-presenting B cells by Fas-Fas ligand interaction in an epitope-specific manner.

Suppression by regulatory T cells is now acknowledged to play a key role in the down-regulation of T cell responses to foreign and self Ags. In addition to the naturally occurring CD4(+)CD25(+) population, several subtypes of induced regulatory cells have been reported, but their mechanisms of action remain unclear. Conversely, cytotoxic CD4(+) cells that lyse cells presenting their cognate peptide have been described, but their potential role in immunoregulation remains to be delineated. A CD4(+) T cell line derived from BALB/c mice immunized with peptide 21-35, containing a major T cell epitope of a common allergen, Dermatophagoides pteronyssinus group 2 allergen, was found to lyse the Ag-presenting WEHI cell line via Fas-Fas ligand and only in the presence of the cognate peptide. Cytolytic activity was likewise shown for other T cell lines and occurred even after a single cycle of in vitro stimulation. Moreover, T cells that efficiently lysed WEHI cells were unresponsive to stimulation with their cognate Ag and were dependent on IL-2 for growth and survival, which was reflected in a constitutive expression of CD25 independently of activation status. Proliferating B cells were also killed by the CTLs. By lysing Ag-presenting B cells in an epitope-specific manner, the nonproliferating CTLs were shown to down-regulate the proliferation of bystander T cells. These data demonstrate that cytotoxic CD4(+)CD25(+) T cells that lack proliferation capacities have the potential to down-regulate an immune response by killing Ag-presenting B cells. This could represent an important and specific down-regulatory mechanism of secondary immune responses in vivo.

Different profile of CD8+ effector T cells induced in Der p 1-allergic and na�ve mice by DNA vaccination

DNA vaccination holds great promise in both prophylactic and therapeutic vaccines. Recent evidence suggests that DNA vaccines could be powerful therapies countering Th2-mediated disorders such as allergies. Here, we studied the allergen-specific CD4+ and CD8+ T cell populations induced following immunization of allergic and non-allergic mice with DNA vaccine vectors encoding discrete epitopes of the house dust mite (HDM) Dermatophagoides pteronyssinus group I (Der p 1) allergen. Specifically, mice were sensitized to Der p 1 and exhibited a strong Th2/allergic response. Sensitized and non-allergic mice were then compared for their responses to DNA immunization. Using Elispot analysis, we demonstrate that allergic/vaccinated mice generate a mixed Th1/Th2 response against the allergen with high numbers of allergen-specific CD4+ T cells secreting IFN-gamma or IL-4, whereas in non-allergic/vaccinated mice a polarized Th1 response was dominant. Allergen-specific CD8+ T cells secreting IFN-gamma were induced at equal frequencies in both allergic and non-allergic mice. However, the CD8+ T cells from allergic mice were markedly deficient in their cytotoxic potential when compared to their counterparts in non-allergic mice. These results indicate that during an ongoing Th2 response, DNA vaccination leads to the generation of a distinct population of non-cytotoxic/regulatory CD8+ T cells.

House dust mite allergen levels in carpeted sleeping accommodation are higher in private houses than public places.

To compare mite allergen levels in carpeted sleeping accommodation in private dwellings and public places. The concentration of Dermatophagoides pteronyssinus Group 1 allergen in house dust was measured in mat-tresses and bedroom floors in 12 homes, 5 hotels, 11 child care centres and a university hall of residence. Indoor temperature and relative humidity were also measured. A questionnaire clarified details regarding the age of the building, age of the carpet, method and frequency of cleaning, frequency of room use and use of air-conditioning. Median allergen levels in mattresses and carpets in private homes (21.1 and 20.6 micro g/g dust, respectively) were significantly higher than in public places (2.5 and 3.1 micro g/g, respectively; P < 0.0001). Mean relative humidity was significantly higher in private houses (68.5%; 95% CI 67.2-69.3%) than in public places (56.4%; 95% CI 52.7-60.1%; P < 0.0001). Carpeted sleeping accommodation in public places has lower house dust mite allergen levels than private houses. Lower levels of relative humidity may be an important component of the explanation.

The Dermatophagoides pteronyssinus group 2 allergen contains a universally immunogenic T cell epitope.

The use of T cell epitope-containing peptides for the induction of anergy in allergen sensitization is limited by genetic restriction that could be circumvented by using universally immunogenic epitopes. We attempted to identify such epitopes on Dermatophagoides pteronyssinus group 2 allergen (Der p 2), a major allergen of D. pteronyssinus T cells from BALB/c (H-2(d)), C57BL/6 (H-2(b)), C3H (H-2(k)), and SJL (H-2(s)) mice that were immunized with rDer p 2, recognized an immunodominant region encompassing residues 21-35. A synthetic 21-35 peptide (p21-35) induced strong dose-dependent in vitro T cell proliferation with cells of the four mouse strains and required processing for MHC class II presentation. Substitution of Ile(28) with Ala resulted in reduction of T cell proliferation in each strain. Ile(28) could represent an important MHC class II anchoring residue for T cell response to p21-35. An immunodominant T cell epitope of Der p 2 therefore behaves as a universal epitope and could be a suitable candidate for T cell anergy induction.

Helper T-cell responses elicited by Der p 1–pulsed dendritic cells and recombinant IL-12 in atopic and healthy subjects

Background: Environmental allergens, such as Dermatophagoides pteronyssinus group 1 antigen (Der p 1), induce TH2-type responses in atopic patients, whereas healthy individuals have TH1-type responses to the same antigens. Because of their efficient synthesis of IL-12, dendritic cells (DCs) are potent inducers of TH1-type immune responses. Objective: We sought to determine whether DCs would skew allergen-specific TH2-type responses from atopic individuals. Methods: Purified CD4+ T cells from healthy donors or atopic individuals were cultured in the absence or presence of recombinant (r)IL-12 with DCs derived from PBMCs and pulsed with Der p 1. Supernatants of DC-T cell cocultures were assayed by ELISA for IL-5 and IFN-γ. Results: A TH1-type response developed in purified CD4+ T cells from healthy donors in response to Der p 1–pulsed DCs, as indicated by high levels of IFN-γ in culture supernatants. In contrast, CD4+ T cells from atopic donors displayed a TH2-type profile characterized by high levels of IL-5 and low levels of IFN-γ. The addition of rIL-12 (10 ng/mL) to DC-T cell cocultures resulted in the induction of IFN-γ secretion by Der p 1–specific CD4+ T cells from atopic patients, whereas their production of IL-5 was not inhibited. Using flow cytometry after intracytoplasmic staining, we found that IFN-γ and IL-5 were secreted by distinct CD4+ T-cell subpopulations. Conclusion: The cytokine profile of Der p 1–specific TH2-like cells from atopic individuals is maintained when the allergen is presented by DCs, even in the presence of exogenous rIL-12. (J Allergy Clin Immunol 2000;105:346-52.)

Phosphorothioate Oligodeoxynucleotides Promote the In Vitro Development of Human Allergen-Specific CD4+ T Cells into Th1 Effectors

DNA vaccination is an effective approach in inducing the switch of murine immune responses from a Th2 to a Th1 profile of cytokine production that has been related to the activity of unmethylated CpG motifs present in bacterial, but not mammalian, DNA. We report here that some synthetic phosphorothioate, but not phosphodiester, oligodeoxynucleotides (ODNs) were able to induce B cell proliferation and to shift the in vitro differentiation of Dermatophagoides pteronyssinus group 1-specific human CD4+ T cells from atopic donors into Th cell effectors showing a prevalent Th1, instead of Th2, cytokine profile. This latter effect was completely blocked by the neutralization of IL-12 and IFN (alpha and gamma) in bulk culture, suggesting that the Th1-inducing activity of phosphorothioate ODNs was mediated by their ability to stimulate the production of these cytokines by monocytes, dendritic, and NK cells. Cytosine methylation abolished the Th1-inducing activity of ODNs; however, CpG dinucleotide-containing ODNs exhibited the Th1-shifting effect independently of the presence or the absence of CpG motifs (5'-pur-pur-CpG-pyr-pyr-3'). Moreover, the inversion of CpG to GpC resulted only in a partial reduction of this activity, suggesting that the motif responsible for the Th1-skewing effect in humans is at least partially different from that previously defined in mice. These results support the concept that the injection of allergens mixed to, or conjugated with, appropriate ODNs may provide a novel allergen-specific immunotherapeutic regimen for the treatment of allergic disorders.

Highly Th2-Skewed Cytokine Profile of β-Lactam-Specific T Cells from Nonatopic Subjects with Adverse Drug Reactions

Abstract A positive lymphocyte transformation test to β-lactams (β-L) was found in 12 of 29 subjects with adverse drug reaction (ADR) to β-L, irrespective of either the type of clinical manifestation or the presence of specific serum IgE. Short-term T cell lines specific for penicillin G, amoxicillin, and ampicillin could be generated only from subjects with ADR (eight with positive and one with negative lymphocyte transformation test), while streptokinase and Dermatophagoides pteronyssinus group 1 (Der p 1)-specific T cells were obtained from all these subjects, from 7 atopic Der p-sensitive donors without history of ADR and 17 healthy nonatopic donors. Streptokinase-specific T cells from all subjects showed intracellular expression of IFN-γ with poor or no IL-4, whereas Der p 1-specific T cells exhibited IFN-γ but low or no IL-4 expression in nonatopics, and remarkable IL-4 expression in atopic donors. By contrast, all penicillin G-, ampicillin-, and amoxicillin-specific short-term T cell lines showed high intracellular expression of IL-4, IL-5, and IL-13, but poor or no expression of IFN-γ, thus exhibiting a clear-cut Th2 profile. Accordingly, most penicillin G-specific T cell clones derived from two subjects with ADR released high concentrations of IL-4 alone or IL-4 and IFN-γ. These data suggest that cytokines produced by Th2 cells play an important role in all β-L-induced ADR, even when late clinical manifestations occur and an IgE-mediated mechanism is apparently indemonstrable.

IgE and IgG 4 antibodies from patients with mite allergy recognize different epitopes of Dermatophagoides pteronyssinusgroup II antigen (Der p 2)

BACKGROUND: Der p 2, composed of 129 amino acids, is one of the major allergens of Dermatophagoides pteronyssinus . Several groups reported epitope mapping of IgE antibody but not of IgG or IgG subclass antibodies in Der p 2. OBJECTIVE: The purpose of this study was to compare the epitopes recognized by Der p 2–specific IgE antibodies with those recognized by IgG 4 antibodies. METHODS: Recombinant fusion proteins containing a full-length peptide or overlapping five fragments of Der p 2 were prepared and analyzed for their reactivity with IgE and IgG subclass antibodies in sera from patients with mite allergy by means of Western blot procedure. RESULTS: IgE antibodies in 12 of 13 sera tested bound to full-length Der p 2 (1-129). In 11 of these 12 sera, IgE antibodies bound to one or more of fragments 41-80, 64-105, or 81-129 but not to those of 1-40 or 20-63. Of the 11 sera in which the IgE antibodies to the fragments were detected, only four contained detectable levels of IgG 4 antibodies reactive to the fragments. The binding spectra of the IgG 4 antibodies against the fragments were rather wide and differed from those of IgE antibodies. CONCLUSION: Results revealed that there are differences in epitope recognition between IgE and IgG 4 antibodies; IgE antibodies recognize restricted parts of Der p 2 antigen compared with IgG 4. (J A LLERGY C LIN I MMUNOL 1996;97:638-45.)

Peptide induced anergy of human allergen-specific T cells.

A common feature of allergic disorders is the presence of allergen-specific antibodies of the IgE isotype which bind to high affinity receptors on mast cells and basophils. Upon interaction of the mast cell-bound IgE antibody with allergen, soluble mediators, like histamine and leukotriens, are released which cause allergic reactions in the various target organs. In house dust mite-induced allergy IgE antibodies that react with a variety of allergens are detected; the predominant IgE response however is directed at the Der-matophagoides pteronyssinus group I (Derp I) and group II (Derp II) allergens(l).KeywordsAntigen Present CellCell CloneCell EpitopeCD40L ExpressionHouse Dust Mite AllergenThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Inhibition of specific IgE response in vivo by allergen-gene transfer.

DNA immunization has been an attractive approach in altering the host immune response to antigen. To examine the utility of DNA immunization in allergic response, we examined the in vivo efficacy of an 'allergen-gene immunization' approach in the modulation of allergen-specific IgE responses in mice. Our results showed first that I.m. injection of a gene construct (pCMVD) containing an important house dust mite allergen gene (Dermatophagoides pteronyssinus group 5 allergen; Der p 5) results in the induction of Der p 5-specific IgG antibodies, but not IgE antibody. We next examined the effect of transduced allergen gene on the expression of specific IgE response in mice after i.p. challenge with recombinant Der p 5 (rDer p 5). Both vector (mock) control- and pCMVD-treated mice were i.p. sensitized with rDer p 5 at 3 weeks after injection of gene construct. Results showed that there is a 90% reduction in the level of specific IgE in pCMVD-treated mice when compared with mock-treated mice. Furthermore, the suppression of specific IgE response can be adoptively transferred with CD8+ T cells from pCMVD-treated mice and such inhibition is in an antigen-specific manner, since the level of specific IgE to an irrelevant allergen, Der p 1, remained unchanged in comparison to that of the mock-treated group. In addition, Der p 5-specific CD8+ T cells could produce high levels of IFN-gamma which probably inhibit allergen-specific IgE responses. Taken together, our results suggest that allergen-gene transfer is effective in the modulation of allergen-specific IgE responses and may provide a novel therapeutic approach.

Polyinosinic acid: polycytidylic acid promotes T helper type 1-specific immune responses by stimulating macrophage production of interferon-alpha and interleukin-12.

The effects exerted on the development in vitro of Dermatophagoides pteronyssinus group I (Der p I)-specific T cell lines and T cell clones by addition of polyinosinic acid: polycytidylic acid (poly I:C) in lymphocyte bulk culture were examined. Der p I-specific T cell lines generated in presence of poly I:C exhibited reduced ability to produce interleukin (IL)-4 and IL-5 and developed into Der p I-specific CD4+ T cell clones showing a T helper (Th) type 0 or Th1, instead of Th0/Th2 cytokine profile. This effect was prevented by addition to lymphocyte bulk cultures of a mixture of antibodies specific for interferon (IFN)-alpha and IL-12, whereas the addition of anti-IFN-alpha or anti-IL-12 antibody alone was uneffective. Poly I:C also showed the ability to stimulate the production of noticeable amounts of both IFN-alpha and IL-12 by human monocytes. Taken together, these data suggest that poly I:C is a Th1-inducing agent whose activity is mediated by its ability to stimulate the production of IFN-alpha and IL-12 by monocytes.

Interleukin-1 favours the in vitro development of type 2 T helper (Th2) human T-cell clones

The effects exerted by interleukin-1 (IL1) on the growth and differentiation of human Th1 and Th2 cells were examined. Neither IL1 nor the IL1 receptor antagonist (IL1ra) had detectable activity toward the antigen- or anti-CD3 antibody-induced proliferative response of already established type 1 T helper (Th1) or type 2 T helper (Th2) clones. Moreover, neither exogenous IL1 addiction to, nor neutralization of, endogenously produced IL1 in bulk cultures before cloning changed the Th1-like cytokine profile of PPD-specific T-cell lines. Likewise, IL1 addition in bulk culture before cloning did not significantly affect the Th2-like cytokine profile of Der.p.I-specific T-cell lines (Dermatophagoides pteronyssinus group I). However, Der.p.I-specific T-cell lines, derived in the presence of anti-IL1 Ab, IL1ra or the M-20 IL1 inhibitor, exhibited the reduced ability to produce IL4 and an increased ability to produce interferon gamma (IFN gamma). More importantly, Der.p.I-specific T-cell lines derived in the presence of IL1ra developed into Der.p.I-specific CD4+ T-cell clones showing a Th0/Th1-like, instead of a Th0/Th2-like, cytokine profile. These data suggest that IL1 is not required for the growth of already established human Th1 or Th2 CD4+ T-cell clones and has no regulatory effects on the in vitro development of Th1-like cells, but it plays a critical role in the development of Th2-like cells.

Natural killer cell stimulatory factor (interleukin 12 [IL-12]) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells.

The effects exerted on the in vitro development of antigen-specific T cell lines and T cell clones by addition or neutralization of interleukin 12 (IL-12) in lymphocyte bulk culture were examined. T cell lines specific for Dermatophagoides pteronyssinus group I (Der p I) derived in the presence of IL-12 exhibited reduced ability to produce IL-4 and increased ability to produce interferon gamma (IFN-gamma), and developed into Der p I-specific CD4+ T cell clones showing a T helper type 0 (Th0)- or Th1-, instead of Th2-, like cytokine profile. In contrast, purified protein derivative (PPD)-specific T cell lines derived in the presence of anti-IL-12 antibody exhibited an increased ability to produce IL-4 and developed into PPD-specific CD4+ T cell clones showing a Th0-, instead of Th1-, like profile. The influence of IL-12 on the cytokine secretion profile of Der p I-specific T cell lines was not prevented by addition to lymphocyte bulk cultures of anti-IFN-gamma antibody, but could be at least partially inhibited by the removal from bulk cultures of CD16+ cells. Thus, IL-12 and CD16+ cells appear to have inhibitory effects on the development of IL-4-producing cells and to play an inductive role in promoting Th1-like responses.

Cytokine production by allergen (Der pI)-specific CD4+ T cell clones derived from a patient with severe atopic disease

Twenty-four T cell clones (TCC) specific for purified Dermatophagoides pteronyssinus group I allergen (Der p I) were established from the peripheral blood of a patient with severe atopic disease and assessed for cytokine production in response to stimulation with both phorbol 12-myristate 13-acetate and anti-CD3 monoclonal antibody. All Der p I-specific TCC produced high amounts of interleukin (IL)-4 in association with IL-3, IL-5 and granulocyte monocyte-colony stimulating factor (GM-CSF), whereas they produced variable amounts of IL-2 and virtually no interferon-gamma. These data support the hypothesis that atopy is associated with preferential activation of type 2 T helper cells and suggest a deregulation in the function of the IL-3, IL-4, IL-5 and GM-CSF gene cluster in subjects with severe atopic disorders.

Reciprocal regulatory effects of IFN-gamma and IL-4 on the in vitro development of human Th1 and Th2 clones.

The effects exerted on the in vitro development of purified protein derivative (PPD)-specific or Dermatophagoides pteronyssinus group I (Der p I)-specific T cell lines (TCL) and T cell clones (TCC) by IL-4 or IFN-gamma addition or neutralization in human PBMC cultures were examined. PBMC from two normal individuals, which were stimulated with PPD and then cultured in IL-2 alone, developed into PPD-specific TCL and TCC able to produce IFN-gamma and IL-2 but not IL-4 and IL-5 (Th1-like). IFN-gamma or anti-IL-4 antibody addition in bulk cultures before cloning did not influence the PPD-specific TCL profile of cytokine production. In contrast, the addition of IL-4 resulted in the development of PPD-specific TCL and TCC able to produce not only IFN-gamma and IL-2 but also IL-4 and IL-5. PBMC from one atopic Der p I-sensitive patient, which were stimulated with Der p I and then cultured in IL-2 alone, developed into Der p I-specific TCL and TCC able to produce IL-5 and large amounts of IL-4 but no IFN-gamma (Th2-like). The addition in bulk cultures, before cloning, of either IFN-gamma or anti-IL-4 antibody markedly inhibited the development of Der p I-specific T cells into IL-4- and IL-5-producing TCL. Accordingly, the development into Der p I-specific Th2-like TCC was significantly reduced by the addition of IFN-gamma in bulk culture and was virtually suppressed by the presence of both IFN-gamma and anti-IL-4 antibody. These data suggest that the presence or the absence of IL-4 and IFN-gamma in bulk cultures of PBMC before cloning may have strong regulatory effects on the in vitro development of human CD4+ T cells into Th1 or Th2 clones.