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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.

The potential for development of mycobacteria as T helper type 1 (Th1) vaccines capable of induction of Th1 responses to recombinant antigens was explored in a model system based on an immunodominant peptide from house dust mite. Different recombinant mycobacterial preparations were compared for their ability to induce a Th1 response to the peptidea. It was found that mycobacterial viability was not a prerequisite for Th1 immunogenicity. A dominant interferon-gamma (IFN-gamma) response to peptide was observed in splenocytes from C57BL/6J mice immunized with live or heat-killed preparations of recombinant Mycobacterium vaccae or with live attenuated bacillus Calmette-Guèrin (BCG) vaccine expressing the antigen. Interleukin-5 (IL-5), a marker of a Th2 response, was detected only in mice receiving live M. vaccae. A similar pattern was observed in BALB/b mice, although the magnitude of the IFN-gamma response was much lower. Control and immunized mice were subsequently exposed to allergen using a Th2-inducing challenge protocol. A significant shift from a Th2 to a Th1 response was observed in immunized mice, as judged by cytokine expression by splenocytes and by subclass of circulating antibody. The effect was seen in three inbred mouse strains differing in their innate bias towards Th1 or Th2 responses. It was dependent on the presence of specific antigen in the mycobacterial preparation and, under the immunization conditions tested, was more pronounced with dead M. vaccae than with live BCG as carrier vaccine. The results demonstrate the potency of killed mycobacteria as Th1 adjuvants and suggest a potential application for recombinant mycobacteria in antigen-specific immune modulation.

Recognition of antigen by T cells in the periphery may lead either to the generation of productive immunity or the induction of tolerance. These two functional outcomes are a consequence of distinct pathways of T-cell differentiation. T cells are selected to become regulatory cells and their function is to maintain homeostasis with the immune system. In this review we discuss the cell-fate decisions that T cells might make allowing them to promote immunity or induce tolerance in the context of the role that Notch signalling may play in this process.

Exposure to optimal peptide antigen concentrations induces human CD4+ T-cell clones to proliferate and secrete various cytokines. Higher (> 10-fold optimal) antigen concentrations cause long-term proliferative unresponsiveness, which can be reversed by exogenous interleukin-2 (IL-2). We call this condition 'tolerance'. We used intracellular cytokine staining and flow cytometric analysis to investigate the kinetics of interferon-gamma, tumour necrosis factor-alpha, IL-4 and IL-5 production during the initial phase of tolerance induction. Single cell analysis of interferon-gamma and IL-4 or IL-5 coexpression showed functional heterogeneity of cloned human CD4+ T cells. Superstimulation with phorbol 12-myristate 13-acetate and ionomycin (PI) revealed enhanced responsiveness shortly after tolerizing treatment, followed by reduced responsiveness. Both tolerized and activated T cells had similarly reduced cytokine responses when further stimulated with antigen during the following 48 hr, with limited enhancement following additional stimulation with PI. We conclude that cytokine induction is normally followed by a refractory phase, but that the expression of cytokines is enhanced in the initial phase of tolerance induction.

The different biological activities of T-cell-derived cytokines and their level of production influences the qualitative nature of immune responses and, in certain forms of T-cell tolerance, the lack of antigen responsiveness is associated with the production of transforming growth factor-beta (TGF-beta) and interleukin-4 (IL-4). In this study we have investigated the effects of T-cell receptor (TCR) ligation with peptide analogues and the native peptide, in the presence and absence of costimulation, on cytokine production by human T-helper type 0 (Th0) cells reactive with influenza virus haemagglutinin (HA) peptide (HA306-318) and restricted by HLA-DRB1*0101. We observed that resting Th0 cells constitutively produced TGF-beta, but when stimulated with peptide and antigen-presenting cells (APC) under conditions that induce clonal expansion, TGF-beta secretion was abrogated. Furthermore, exposure of the T cells to the wild-type HA peptide under conditions that induce T-cell anergy resulted in the secretion of TGF-beta, and subsequent antigenic rechallenge was unable to override this signal and down-regulate TGF-beta production. Stimulation with altered TCR ligands that failed to induce proliferation also resulted in marked production of TGF-beta, although in many instances the levels were less than those observed in the total absence of antigen, suggesting that partial signalling has occurred. Although in general, there was a direct positive correlation between proliferation and the production of IL-2, IL-4 and interferon-gamma (IFN-gamma) following stimulation with certain analogues, the production of selected cytokines was dissociated.

Cellular reactivity to Der p 2, a major allergen of the house dust mite (HDM) Dermatophagoides pteronyssinus, was studied in a group of 41 symptomatic HDM sensitive patients, using fresh peripheral blood mononuclear cells (PBMC) and assays of proliferation. Sixty per cent of the patients responded to Der p2, with reactivities being greater in patients with asthma as one of their clinical manifestations and also in those who had skin-test reactivity to a number of allergens. HLA-DR and -DQ serotyping was undertaken in 39 of the patients and the magnitude of T-cell proliferative responses to Der p 2 were found to be positively associated with DQ7 and negatively associated with DQ2. T-cell determinants within the Der p 2 molecule were identified by assays using a series of overlapping peptides (15- to 19-mers) spanning the entire protein. Fifty-nine per cent of the 41 HDM-sensitive patients responded to one or more of the peptides. All of the peptides were antigenic for at least one of the individuals, indicating the heterogeneity of the human repertoire reactive with Der p 2. There was a substantial variability in the number and location of epitopes recognized by T cells from the different allergic patients, the mean number per patient being 2.3 +/- 1.3 (SD). The most frequently recognized peptide was that spanning residues 111-129, being stimulatory in 66.7%, the other peptides were each recognized by between 8 to 25% of individuals. There was no correlation between the epitope recognized and the presence of particular HLA-DQ antigens.

Cytokines can influence the selection of functional subsets (Th1 or Th2) of CD4+ T cells. However, quantitative changes in affinity of peptide/major histocompatibility complex (MHC) class II/T-cell receptor (TCR) interactions may alter antigen density and modulate T-cell effector function. The possibility exists to use peptide analogues to induce a partial signal to dissociate production of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) by T-helper type-0 (Th0) cells and, consequently, to regulate T-cell function. Based on binding assays and resolution of the crystalline structure of an influenza virus haemagglutinin peptide (HA 306-318) bound to the human MHC class II molecule DRB1*0101, we synthesized HA peptide analogues with amino acid substitutions predicted to modify either MHC class II/peptide density or TCR/peptide interactions. When we examined their antigenicity using cloned human Th0 cells, the analogues, in general, elicited a gradation in potency reflected by a reduction in both proliferation and cytokine production (IL-2, IL-4 and IFN-gamma). Although the analogue HA-R309 diminished IL-2 production, none of the analogues tested could selectively induce only IL-4 or IFN-gamma. Since, in general, the effector functions of the Th0 cells examined here were resistant to selective manipulation by the peptide analogues, this suggests that for some clones of chronically activated T cells modulation of selected functions may be difficult to achieve.

The induction of IgE antibodies to aeroallergens depends upon antigen-specific CD4+ helper T cells of an 'interleukin-4 (IL-4)-dominant' phenotype. Nuts also drive IgE-mediated hypersensitivity and are the most dangerous of the orally encountered allergens. We have studied the polyclonal T-cell responses of atopic and non-atopic individuals to extracts of peanut, brazilnut and hazelnut. Strong proliferative responses were observed in all patients but specific IgE was only present in the nut-allergic patients suggesting a similar pathogenic mechanism to aeroallergen-mediated hypersensitivity. To investigate this hypothesis a panel of peanut-reactive T-cell clones was raised from a peanut- and brazilnut-allergic individual without hazelnut allergy. The antigen specificity, major histocompatibility complex (MHC) class II restriction and cytokine profiles of the T-cell clones were determined. With the exception of one T-cell clone, which proliferated in response to both peanut and hazelnut extract, the peanut T-cell clones were not cross-reactive with hazelnut or brazilnut. The T-cell clones recognized antigen in association with HLA-DR and HLA-DP but not HLA-DQ class II molecules. The peanut-specific clones produced high levels of IL-4 and low levels of interferon-gamma (IFN-gamma), exhibiting the 'TH2-like' profile which dominates the aeroallergen response. In contrast, the T-cell clone that was cross-reactive on both peanut and hazelnut allergen had a Th0-like phenotype, consistent with the lack of specific serum IgE to hazelnut. These results support the importance of functionally distinct T-cell populations that recognize oral allergens. The relative production of IL-4 and IFN-gamma of the cloned T cells in the peanut-allergic patients plays a role in determining whether or not IgE antibody responses are induced with the associated potential to develop anaphylactic reactions.

The resistance and susceptibility of T cells to human immunodeficiency virus (HIV)-gp120 induced anergy was examined. Antigen-dependent proliferation of polyclonal T cells was markedly inhibited by gp120, whereas from the analysis of monoclonal populations, T cells resistant to the effects of gp120 could be identified. Similarly, exposure of monoclonal T cells to gp120 in the absence of accessory cells, also demonstrated that some T cells could resist the induction of anergy. Loss of antigen recognition was associated with phenotypic modulation of CD3 and CD28, which was not observed in T cells resistant to functional inactivation by gp120. Modulation of CD4 was not related to induction of anergy in the monoclonal T cells examined in this study. Inhibition of T-cell responses by anti-CD4 antibodies was compared to that by gp120. Anti-CD4 antibodies, which cross-compete with gp120 for binding to CD4, inhibited the response to antigen of monoclonal T cells. In contrast, no tolerogenic signals were delivered by pretreating T cells with the anti-CD4 antibodies in the absence of accessory cells, indicating that inhibition was due to abrogation of the interaction of CD4 with major histocompatibility complex (MHC) class II molecules expressed on accessory cells. Although the free CD4-binding region peptide of gp120 could inhibit polyclonal T-cell responses, only the carrier-bound peptide was able to modulate cloned T cells, suggesting a conformational requirement for functional inactivation through engagement of CD4. The results reported here using clonal CD4+ T-cell populations demonstrate that effects of gp120 on antigen-dependent proliferation are not uniform, and that therapeutic intervention might be directed at T-cell populations identified as susceptible to HIV-gp120 induced anergy.

We have established previously that human thyroid epithelial cells (TEC) from patients with autoimmune thyroiditis are able to synthesize cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6). This paper examines TEC in sections from autoimmune thyroiditis for the in vivo production of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using the combined techniques of in situ hybridization and immunohistochemistry. Thyroid tissue from patients with Graves' disease, Hashimoto's disease and non-toxic goitre was examined and both mRNA and the protein of TNF-alpha were detected in TEC on frozen sections. Representative figures of only Graves' samples are illustrated in this paper. In contrast, using the same methods, IFN-gamma was detected only in the infiltrating cells and not in TEC of thyroid tissue from the patients.

The Nb2 rat lymphoma cell line has the unique property that its growth is dependent on lactogenic pituitary hormones. Cell surface staining with monoclonal antibodies showed expression of class I MHC alloantigens of the RT1u haplotype, but no expression of class II MHC antigens. Staining for differentiation markers was strongly positive with antibodies OX52, W3/13 and OX44. Partial and weaker staining was obtained with CD2, P4/16 and the transferrin receptor. Nb2 cells were negative with CD5, OX40 and CD4, whilst CD8 stained only a minor fraction (1%) and certain variant clones of the cell line. This general pattern of staining is consistent with the phenotype of a small subpopulation of immature T cells. Nb2 cells proliferated in response to recombinant human IL-2, although they did not stain with antibodies against the IL-2 receptor. Enhancement of the stimulation by IL-2 in the presence of a submitogenic concentration of hGH indicated a synergism between these two hormones, and responses were suppressed by a similar dose of cyclosporin A (ID50=2 microg/ml). Although IL-2 could not be identified in culture supernatants, the presence of mRNA for IL-2, IL-2R and IL-4 was demonstrated by dot blot analysis. Finally, evidence that the Nb2 lymphoma is of T-cell lineage was given by Northern blot detection of mRNA for the alpha and beta chains of the T-cell receptor.

Human lymphoproliferative responses to a rabbit anti-idiotypic antibody (anti-Id TB71) and the corresponding mycobacterial protein antigen [38,000 molecular weight (MW)] have been investigated in a number of donors. It was found that responsiveness to anti-Id TB71 correlated with responder and non-responder (four subjects each) status to the 38,000 MW antigen. Furthermore, the induction of T-cell proliferation by both the 38,000 MW antigen and the anti-Id TB71 was dependent on accessory cells. When taken together with the concordance between the 38,000 MW antigen and anti-Id responsiveness, this implies that the 38,000 MW antigen and anti-Id TB71 stimulate related, or at least partially overlapping, repertoires of T cells. This was confirmed by the finding that cloned T cells reactive with the 38,000 MW antigen also proliferated in response to the anti-Id TB71. These observations are readily explained if the anti-idiotypic antibody contains an internal image of, and can therefore mimic, the antigen.

Human T lymphocyte clones specific for the haemagglutinin (HA) molecule of A/Texas/1/77 were maintained in long-term culture with T cell-growth factor. The clones were analysed for their viral antigen specificity using serologically defined type A influenza subtypes and chemically synthesized peptides of the HA-1 molecule. With the exception of one T cell clone that recognized only the HA molecule used for immunization, the clones responded to determinants that showed partial or complete cross-reactivity amongst the strain A subtypes. The cross-reactive population of T cell clones were specific for peptides distinct from the antibody binding sites of HA. Furthermore, one peptide located at the carboxyl terminus of the HA-1 molecule appeared to be immunodominant, although the critical residues for T cell antigen recognition within that could not be identified.

Using cloned human T lymphocytes reactive with a 24 amino acid peptide (p20) of the carboxyl terminus of the HA-1 molecule of influenza haemagglutinin (HA), we have investigated the ability of solid-phase antigen to induce antigen-specific T-cell proliferation. The activation by nitrocellulose-bound virus and p20 was accessory-cell dependent and was not caused by immobilized antigen directly cross-linking the specific receptors. Furthermore, we report that separation of complex antigen mixtures such as influenza virus and HA by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) followed by transfer to a nitrocellulose membrane can be used to allow direct screening of individual polypeptides in T-cell proliferation assays. With this immunoblotting procedure the antigenic site recognized by HA-reactive T cells was confirmed to reside in the HA-1 molecule of influenza virus of only the appropriate subtype. The general application of this approach is discussed in the case of infections and autoimmune diseases in which the immune response is predominantly T-cell mediated and where antibody studies may fail to identify key antigenic determinants involved in the activation of T cells.

The exposure of human T-cell clones to supra-immunogenic concentrations of peptide antigen in the absence of accessory cells induces antigen-specific unresponsiveness. Using this model we have investigated the ability of cytokines to modulate the induction of, or reversal of, T-cell tolerance. Our findings demonstrate that interleukin-2 (IL-2), but not interferon-gamma (IFN-gamma) or interleukin-1 (IL-1), is able to inhibit the induction of T-cell unresponsiveness in a dose-dependent fashion. Moreover, IL-2 was able to reverse established antigen-dependent T-cell unresponsiveness. In order to determine if modulation of IL-2 receptors is able to induce or abrogate unresponsiveness, the T cells were treated with anti-Tac antibody alone or together with tolerizing concentrations of antigen. Anti-Tac antibody was neither able to induce nor inhibit the induction of tolerance. The application of this model in the manipulation of immune responses is discussed here.

In this report the antigen and restriction specificity of human T-cell clones induced with Dermatophagoides farinae (D. farinae) and isolated from an atopic individual with perennial rhinitis has been investigated. Of the six clones analysed, four were species specific and two showed cross-reactivity for the closely related Dermatophagoides pteronyssinus (D. pteronyssinus). Inhibition of antigen-dependent proliferation by murine monoclonal antibodies directed against HLA-D-region gene products revealed that all the clones were restricted by HLA-DR molecules. The restriction specificity was investigated further using a panel of histocompatible and allogeneic-presenting cells. Of the clones tested, one appeared to be DR5 restricted while the remainder showed complex patterns suggesting that DRw52 and DRw53 supertypic specificities may be the restriction elements presenting antigen.

Cloned human T lymphocytes and a mitogenic monoclonal antibody (UCHT1) that binds to the T3 antigen complex were used to study the role of inositol lipid hydrolysis in T-cell activation. Binding of the T3 molecular complex with anti-T3 antibody induced the generation of inositol trisphosphate after a lag of 1 min. While this is commensurate with the rise in cytosolic Ca2+ in these cells, examination of the inositol lipid revealed that phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate occurred before the generation of inositol trisphosphate. Thus, activation of an inositol lipid kinase appears to be one of the primary signals in cellular activation in human T lymphocytes.

Helper factors specifically stimulating cooperative antibody responses by normal mouse spleen cells to a dinitrophenylated protein antigen from Streptococcus mutans (DNP-SA) were produced in vitro from monkey peripheral blood leucocytes and mouse spleen cells. The factors were partially characterized by gel filtration on Sephadex G-75, isoelectric focusing, treatment with heat and degradative enzymes and binding to specific immunoadsorbents. Gel filtration of both the monkey and mouse factors showed coelution with human serum albumin, suggesting a molecular weight of approximately 70,000. The isoelectric points fell within the range of 4.9-5.2 for monkey and 6.4-6.7 for the mouse helper factors. The glycoprotein nature of both factors was suggested by their lability to heat and sensitivity to pronase and neuraminidase. The factors carried a small fragment of the stimulating antigen and showed specific binding to SA but not to keyhole limpet haemocyanin (KLH). Monkey factor bound to rabbit antisera directed against the Fc portion of monkey IgM, but not to the IgG or IgA isotypes. The mouse factor contained determinants coded for by the I-Ak but not I-Jk subregion of the MHC. Both factors were absorbed by an antiserum to helper factor raised in rabbits against a KLH-specific mouse helper factor as immunogen. A corresponding antiserum to suppressor factor failed to adsorb either factor. This emphasizes the specific identities of helper and suppressor factors and suggests an evolutionary relationship between those derived from monkey and mouse leucocytes.

Using cloned human helper T lymphocytes reactive with a 24 amino acid peptide (p20) of the carboxyl terminal of the HA-1 molecule of influenza haemagglutinin we have investigated the influence of antigen structure on the activation and in the induction of antigen specific unresponsiveness of T cells. For this analysis stereoisomers and structural isomers of p20 have been constructed. P20 in the form of a single loop created by a disulphide based between residue 306 and an additional cysteine at position 330 was able to activate the helper T cells in the presence of accessory cells but unable to induce tolerance. This result suggested that critical residues were prevented from direct interaction with the T-cell receptor and/or the MHC Class II determinants and required processing to expose them. The enantiomer (D-p20) and the inverted sequence (retro-L-p20) which have non complementary side chain topography as compared to the parent peptide neither activated nor tolerized the T cells. Furthermore the retro-D-p20 isomer which has the same side chain topography as L-p20 but with a reversal of amino and carboxyl acid groups also failed to stimulate or tolerize. Therefore T-cell antigen recognition is not determined by side chains alone. The results presented suggest that structure of extrinsic antigen influences T-cell antigen recognition.

In this report, T-cell and B-cell recognition of the house dust mite Dermatophagoides farinae (D. far.) is compared. Nitrocellulose immunoblots of polyacrylamide gel electrophoresis (SDS-PAGE)-fractionated D. far. were added to proliferation assays to map the antigen specificity of cloned human helper T cells and a long-term line induced with D. far. T-cell recognition was of a polypeptide of molecular weight 9000-13,000, that migrates with the serologically defined allergen Der fII (12,500 MW). Since the cloned T cells, unlike the polyclonal response, failed to respond to Dermatophagoides pteronyssinus (D. pter.), this suggests that they recognize a species-specific epitope. In contrast, analysis of the B-cell response using Western blotting demonstrated that, in addition to Der fII, antibodies reactive with the major allergens Der fI (26,000 MW) and Der fIII (29,000 MW) were present in the serum. Similar specificities were seen in the antibody response to D. pter., and while it has been reported that the B-cell response to D. far. and D. pter. are predominantly cross-reactive, our observations suggest that species-specific CD4-positive T cells are present in the overall cellular response to D. far.

T-cell differentiation is known to take place in the thymus, but the precise mechanisms involved remain unresolved. In order to analyse the role of the thymic microenvironment in thymocyte maturation and generation of the T-cell repertoire, we have raised monoclonal antibodies (mAb) to thymic stromal cells, and with these can recognize, in the non-lymphoid component of the thymus, several antigenically distinct compartments. One mAb, MR6, binds to both the cortical epithelium and medullary macrophages/dendritic (M phi/DC) cells in sections of the human thymus. Recently, the molecule detected by MR6 has also been detected at low levels on the surface of T lymphocytes. We now report that this molecule has a relative molecular mass of 145,000 (p145-MR6) and that this appears to be the same for both thymic lymphocytes and stromal cells. Functional studies show that mAb MR6 inhibits both the antigen-specific and the IL-2-induced proliferative response of MHC class II-restricted cloned helper T cells and peripheral blood mononuclear cells (PBMC). These results suggest that the molecule to which mAb MR6 binds could be responsible for the inhibition of T-cell proliferation to self-antigens, and hence may be involved in tolerance induction and MHC restriction.

Current approaches to the analysis of antigens involved in the cellular immune response to mycobacterial infection rely on the initial identification and isolation of molecular components using monoclonal antibodies. In order to overcome the constraints of this approach, we have utilized a procedure involving T-cell recognition of antigens fractionated by polyacrylamide gel electrophoresis (SDS-PAGE) and added to proliferation assays after blotting onto nitrocellulose membranes. Analysis of human T-cell responses to Mycobacterium tuberculosis and Mycobacterium bovis BCG by this procedure revealed distinctive patterns of reactivity to different molecular weight components indicative of the selective recognition of immunodominant and species-specific determinants. Human T-cell clones were subsequently derived, and SDS-PAGE immunoblotting was used to identify the antigen recognized by each clone. Three epitopes defined by individual T-cell clones were identified on separate polypeptides with molecular weights 16,000-18,000 (clone P53), 18,000-20,000 (clone P57) and 52,000-55,000 (clone P35). This study demonstrates the potential application of T-cell cloning in conjunction with SDS-PAGE immunoblotting for the dissection and analysis of the cellular immune response to pathogenic agents during human infection.

Here we report the presence of a low molecular weight (10,000 neutrophil chemotactic factor (NCF) in the supernatants of activated human T lymphocytes. Appreciable amounts of the 10,000 MW NCF were generated by peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 antibody, and comparable NCF was secreted by both long-term human T-helper (CD4+) cell lines reactive with house dust mite (Dermatophagoides farinae) and influenza A virus-immune T-cell clones. When the cloned T cells were stimulated with specific antigen in the presence of irradiated accessory cells (AC) or insolubilized anti-CD3 antibody the 10,000 MW NCF was readily identifiable in 24-hr culture supernatants. Cultures of AC and antigen alone produced negligible neutrophil chemotactic activity, as did control cultures using an irrelevant allergen (mixed grass pollen). These findings indicate that the 10,000 MW NCF may be T-lymphocyte derived and that formation and release are dependent upon stimulation via the antigen receptor.

Influenza virus immune human T-lymphocyte clones maintained in continuous culture in TCGF were analysed for helper activity and interleukin-2 (IL-2) production. The clones that functioned as helper cells in the production of specific antibody failed to release detectable amounts of IL-2. Conversely, the T cells that produced IL-2 were unable to provide either specific or non-specific helper function. These findings indicated the IL-2 is not an essential component for helper activity. However, phenotypic analysis revealed that both the functional subsets of T-cell clones expressed the helper phenotype in that they were T4+, T3+ and T11+. Nevertheless analysis with other antibodies revealed differences in that the IL-2 releasing clone showed greater staining with the anti-T-cell subset antibodies 9.3 and Leu 8, confirming that there is phenotype as well as functional heterogeneity within the helper inducer T-cell population.

An allergen-dependent in vitro model of immunoglobulin E (IgE) synthesis by human B cells is reported. Using this model, it is demonstrated that polyclonal T cells and CD4+ Dermatophagoides spp. (house dust mite)-specific T-cell clones derived from an atopic, house dust mite (HDM)-allergic individual are able to support IgE synthesis by autologous B cells. The helper activity was interleukin-4 (IL-4) dependent as only cloned T cells expressing detectable mRNA for IL-4 were able to induce IgE synthesis without the addition of exogenous IL-4. Peripheral and cloned T cells reactive with HDM could also be identified from a non-atopic individual but neither population was able to support IgE production even in the presence of exogenous IL-4.

The complex pleiotropic effects of the T-cell derived lymphokine interleukin-4 (IL-4) are becoming increasingly well documented; however, functional studies have been hampered by the lack of reagents directed against the receptor for this factor. In this report, we present data which suggest that the monoclonal antibody MR6 binds to the human interleukin-4 receptor (IL-4R). Addition of MR6 to cultures of T cells proliferating in response to IL-4 inhibited this response in a dose-dependent fashion, giving total inhibition at 10 micrograms/ml. Similarly, the IL-4-dependent production of specific antigen-induced IgE by B-cell populations was completely abrogated by MR6. Flow cytometric studies of the modulation of cell surface molecules after T-cell activation suggest that expression of the molecule detected by MR6 (p145-MR6) correlates inversely with that of the interleukin-2 receptor (IL-2R). These data, together with the previously determined molecular weight and tissue distribution of this molecule, strongly indicate that MR6 binds to the human IL-4R.