Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice.

Antigen-specific CD4+ T cells play an important role in the allergic immune response to house dust mite (HDM) allergens in humans. The group 1 allergen of Dermatophagoides spp. is a major target antigen in both B and T cell recognition of HDM. In vitro studies have shown that the presentation of peptides to human T cells under appropriate conditions may lead to a state of specific nonresponsiveness. Therefore, to determine if peptides are able to modulate the function of allergen-reactive T cells in vivo, we have used a murine model of T cell recognition of the HDM allergen Der p 1. The results demonstrate that inhalation of low concentrations of peptide containing the major T cell epitope of Der p 1 (residues 111-139), induces tolerance in naive C57BL/6J mice such that they become profoundly unresponsive to an immunogenic challenge with the intact allergen. When restimulated in vitro with antigen, lymph node T cells isolated from tolerant mice secrete very low levels of interleukin 2, proliferative poorly, and are unable to provide cognate help to stimulate specific antibody production. Furthermore, intranasal peptide therapy was able to inhibit an ongoing immune response to the allergen in mice and this has potential implications in the development of allergen-based immunotherapy.

Peptide modulation of allergen-specific immune responses.

In vitro peptide stimulation of allergen-reactive T-helper type 1 and type 0 cells, in the absence of costimulatory signals, induces anergy that is accompanied by the modulation of cell surface phenotype and changes in cytokine production. In experimental animal models, the administration of allergen-derived peptides may result in the downregulation of cytokine and antibody production, which is preceded by transient activation of CD4+ T cells, without the induction of effector immunity. Preliminary results of clinical trials using allergen-derived peptides for desensitization are becoming available and should provide some insight into the efficacy of peptide therapy in man.

The T cell surface protein, CD28.

The cluster of differentiation (CD) antigen CD28 is a 44-kDa, disulphide-bonded, homodimeric glycoprotein, which is constitutively expressed on the surface of all murine T cells and the majority of human T cells. Ligation of CD28 by its counter receptor, B7, expressed on the surface of antigen presenting cells, has been shown to induce signals that, in synergy with those derived from engagement of the T cell receptor by an antigen bound to a major histocompatibility complex, enhance proliferation and cytokine production. Manipulation of this interaction can have dramatic effects on the outcome of T cell activation. Blocking CD28/B7 interactions may be useful in preventing unwanted activation in allergy and autoimmune diseases, whereas enhancing this interaction can promote tumour rejection. Thus, CD28 and its signalling pathways may prove to be useful targets in the development of new therapeutic treatments.

T-cell responses to orally administered antigens. Study of the kinetics of lymphokine production after single and multiple feeding.

Mice fed a protein antigen develop a phenomenon called oral tolerance which is defined classically by the inability to respond to a parenteral challenge with the same antigen. In a recent report we showed that antigen-reactive T cells are not depleted following the development of oral tolerance to the soluble antigen ovalbumin (OVA). Instead mice remain highly sensitized so OVA-reactive T cells can be detected in the mesenteric lymph nodes (MLN), Peyer's patches and spleen. In the present study we show that OVA-specific T cells become sensitized in the MLN within 24 hr of feeding and that lymphokine responses peak 48-96 hr after feeding. T cells produced large amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) but no interleukin-2 (IL-2) following activation in vitro. Responsiveness as measured by GM-CSF declined by days 8-11 while the ability to stimulate IFN-gamma secretion was more persistent. It was found in experiments with repeated feeding, 1 week apart, that the T-cell responsiveness was restimulated after each feed and that the magnitude and duration of the IFN-gamma or GM-CSF responses were almost identical to primary, even after 10 feeds.

Regulation of T cell function in mucosal tolerance.

Administering antigens through mucosal surfaces leads to the induction of antigen-specific T cell unresponsiveness. This property of the mucosal immune system is now beginning to be exploited in the design of immunotherapeutic strategies aimed at targeting disease-inducing T cell populations. The induction of high dose mucosal tolerance leads to the induction of T cell anergy. Recent studies have suggested that the induction of anergy in vivo may not necessarily be due to a lack of costimulation by APC. Instead, recognition of mucosal antigen leads to transient T cell activation which eventually gives rise to a population of regulatory T cells whose function is to modulate, rather than promote antigen-specific immune responses. These regulatory T cells mediate linked suppression in vivo thus enabling T cell responses directed to a multideterminant protein to be effectively controlled. The manner in which T cell responses to mucosally delivered antigens are regulated are examined herein.

Peptide-mediated regulation of the allergic immune response.

A major key to successful immunotherapy may depend on altering the qualitative nature of the immune response in allergic patients. In this review we examine how immune responses to environmental allergens are regulated, and the mechanisms used by the immune system to prevent allergic sensitization. We also discuss future prospects of using allergen-derived peptides in immunotherapy and the possibility of 'reprogramming' the immune responses by immunizing under conditions that promote Th1 responses instead of Th2 responses.

Linked suppression in peripheral T cell tolerance to the house dust mite derived allergen Der p 1.

BACKGROUND: Peripheral tolerance is required to maintain balance within the immune system. A feature of peripheral tolerance is linked suppression, in which tolerance induced to a single T cell epitope inhibits the response to all epitopes in the same protein. It is suggested that this phenomenon is mediated by regulatory T cells through either the activity of immunopressive cytokines or direct cell contact. In previous experiments we failed to detect inhibitory cytokines when T cells from mice rendered tolerant by intranasal delivery of the immunodominant peptide of Der p 1 (p 1, 110-131) were restimulated with peptide in vitro. Therefore, the aim of this study was to determine if cognate interactions between T cells mediated by Notch/Delta signaling induce and maintain peripheral T cell tolerance. METHODS: Using in situ hybridization and viral mediated gene transfer, the expression and function of Delta1 were investigated in a murine model of T cell tolerance to Der p 1 in vivo. RESULTS: Delta1 expression is increased on peripheral T cells during the induction of tolerance with high-dose peptide delivered intranasally and when tolerant animals are rechallenged under immunogenic conditions. Peptide p 1, 110-131-specific CD4+ T cells transfected with Delta1 inhibited the response of antigen-primed T cells and induced linked suppression. CONCLUSIONS: High-dose peptide delivered intranasally induces transient expression of Delta 1 on inhibitory CD4+ T cells. Ligation of the Notch1 receptor on neighbouring T cells by Delta1+ regulatory T cells inhibits clonal expansion of the former and mediates linked suppression.

Peptide-mediated immunoregulation.

Quantitative and qualitative characteristics of the signals received by a T cell determine whether receptor ligation results in cell activation, cell death, or the induction of antigen-specific non-responsiveness. Environmental factors such as the nature of costimulation, antigen-presenting cell type, peptide structure and cytokine levels also influence the differentiation of CD4+ helper T cells into functionally distinct subsets, which now appear pivotal in many immune-mediated disorders, including autoimmunity and allergy. Selective manipulation of the immune response, such as the functional inactivation or deviation of the cytokine secretion patterns of specific T cells, may be an effective strategy for immunotherapy.

Susceptibility of Branhamella catarrhalis to sulphamethoxazole and trimethoprim.

Fifty strains of Branhamella catarrhalis were examined for susceptibility to sulphamethoxazole, trimethoprim and a combination of the two by determinating minimum inhibitory concentrations (MICs) and fractional inhibitory concentrations (FICs). All strains were susceptible to sulphamethoxazole and resistant to trimethoprim. On the basis of the MIC results it was predicted that greater synergy between sulphamethoxazole and trimethoprim would be observed with approximately equal proportions of each component. The lowest FIC values were obtained with a ratio of 1:1 and the greatest synergy was observed at this ratio with 39 strains (78%). Only seven strains were most synergistically inhibited at the ratio of 20:1 (sulphamethoxazole: trimethoprim) although this ratio was still synergic for most strains. Overall the 1:20 ratio was not synergic.

T-cell regulation of peripheral tolerance and immunity: the potential role for Notch signalling.

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.

Comparison of antigen presentation by lymph node cells from protein and peptide-primed mice.

Lymph node cells from mice primed with peptides from the allergens Der p I and Der p II (the group I and II allergens of Dermatophagoides pteronyssinus) were unable to recall responses to the protein antigen when cultured in vitro despite being able to mount large responses to the peptides. The T cells could however recall responses to the protein when spleen-adherent cells were added into culture. Treating the spleen accessory cells with the monoclonal antibody (mAb) 33D1 and complement largely abrogated the protein response of peptide-primed T cells which indicates that dendritic cells were mainly responsible for the antigen-presenting function. If mice were primed with two injections of peptide the lymph node cells obtained could respond to both protein and peptides in vitro without the need for exogenous accessory cells. Using either negative depletion with the J11D mAb or positive purification, it was found that the presentation of protein antigen to lymph node T cells primed with either protein or peptide was limited to antigen-specific B cells. Peptide antigens could however be presented by both B and non-B populations. In one case the peptide 105-129 from Der p II which contains a T-cell epitope could not be shown to induce T-cell responses in the lymph node unless presentation was mediated by spleen-adherent or B-specific cells. These results are important for peptide-based immunomodulation and in interpreting results obtained from lymph node cultures.

Inhibition of T-cell responses by feeding peptides containing major and cryptic epitopes: studies with the Der p I allergen.

H-2b mice respond to the 222 residue allergen Der p I by producing T cells sensitized to the dominant epitopes encompassed in peptides 21-49, 78-100, 110-131 and 197-212. Immunization with the synthetic peptides 120-143 and 144-169, however, revealed cryptic epitopes which could sensitize T cells for responses to the respective peptides and, providing splenic adherent cells were added to lymph node cultures, to the whole allergen. It is shown that feeding recombinant fusion peptides can markedly inhibit the ability of the whole antigen to immunize mice, as measured by the in vitro interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 release on stimulation with protein or peptides, although inhibition measured by IL-2 release was more marked. The inhibition extended to epitopes other than those in the fusion peptides used for feeding. Thus feeding peptide 101-154 inhibited responses to 110-131 and 78-100. Fusion peptides 1-14 and 188-222 did not inhibit responses, although 188-222 did contain an epitope. Inhibition was also obtained when mice were fed a fusion containing the cryptic epitope 144-169. The ability of peptides containing the cryptic epitopes to inhibit responses has significant implications for peptide-based immunotherapy.

Differences in epitopes recognized by T cells during oral tolerance and priming.

Feeding protein antigens to mice normally leads to the development of oral tolerance but under some circumstances, feeding can lead to immunity, for example, following pretreatment of mice with cyclophosphamide (CY). In both cases, however, it is possible to detect sensitized T cells in the spleen and mesenteric lymph nodes (MLN) by in vitro lymphokine release for granulocyte-macrophage-CSF (GM-CSF) and IFN-gamma. This study examines the recognition of the immunodominant T cell epitope on ovalbumin (OVA) following intragastric priming and tolerance. T cells from CY/OVA treated mice and cells from mice injected subcutaneously with OVA in CFA responded well to both OVA and the H2d restricted peptide epitope pOVA323-339 releasing GM-CSF. On the other hand MLN or spleen T cells from tolerized mice which responded to the protein in vitro did not recognize the immunodominant determinant. The cells responding from tolerized mice were restricted by the class II MHC so these results show there can be differential recognition of T cell epitopes between oral priming and tolerance.

T-cell lymphokine response to orally administered proteins during priming and unresponsiveness.

Feeding antigens induces an immunological unresponsiveness termed oral tolerance but under some conditions, for example following the administration of cyclophosphamide (CY), immunity can be induced. These observations have usually been made by studying antibody production and delayed hypersensitivity with little attention given to other measurements of cellular activation. We have therefore examined the lymphokines produced by T cells obtained after the induction of oral tolerance or intragastric priming. Cells isolated from the spleen and Peyer's patches (PP) of tolerized mice could secrete high levels of interferon-gamma (IFN-gamma) and moderate levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in response to antigen while interleukin-2 (IL-2), IL-3 and IL-4 could not be detected. Mesenteric lymph node (MLN) cells of tolerized mice did not respond to antigen unless spleen adherent cells were added to the cultures where IFN-gamma and GM-CSF were produced. Intragastric priming was achieved by feeding antigen to CY-treated mice. T cells from the spleen, MLN and PP of these mice could produce GM-CSF, IFN-gamma, some IL-3 but little or no IL-2 and IL-4. The ability of MLN cells to proliferate with antigen in vitro was low and corresponded to low IL-2 production. Thus T cells from fed mice secrete a defined pattern of lymphokines which differs in tolerizing and priming regimes.

Notch signalling in the regulation of peripheral immunity.

Notch signalling plays a critical role in embryogenesis, influencing the differentiation and growth of a variety of cell types across the species. In the mammalian immune system, Notch signalling operates at various levels; it controls the differentiation of haematopoietic stem cells and directs the early development of the T and B-cell lineages. It is also involved in the maturation of both CD4+ and CD8+ T cells in the thymus. The biological activities of this pathway extend beyond lymphocyte ontogeny; recent evidence has shown that it also contributes to the regulation of the peripheral immune system through its ability to influence cell survival and growth. In fulfilling this function, Notch signalling appears to act in conjunction with defined immunological signals such as cytokines, T-cell antigen receptor and co-stimulatory receptor-mediated signalling. In this review we discuss the potential of the Notch signalling pathway in the maintenance of homeostasis within the immune system affecting both peripheral tolerance and the negative feedback controlling productive immunity. The therapeutic manipulation of this pathway is likely to have broad application in a range of immunologically based diseases.

Regulation of house dust mite responses by intranasally administered peptide: transient activation of CD4+ T cells precedes the development of tolerance in vivo.

We have previously demonstrated that intranasal (i.n.) administration of an immunodominant peptide (p1-111-139) derived from the house dust mite (HDM) allergen Der p 1 inhibits antigen-specific CD4+ T cell responses in H-2b mice. Here we report that i.n. peptide induced a rapid but transient activation of MHC class II restricted CD4+ T cells that peaked 4 days after peptide treatment and was of similar magnitude to that induced by parenteral immunization with antigen in adjuvant. During the early phase of the response lymph node and splenic T cells secreted a range of lymphokines when re-stimulated in vitro with p1 111-139; however, by day 14 IL-2 and IFN-gamma secretion by T cells were down-regulated. Mice deficient in CD8+ T cells became tolerant by i.n. treatment with peptide, suggesting that CD8+ T cells are not involved in down-regulating the CD4+ T cell response. Rechallenging mice with a single dose of p1 111-139 21 days after the initial treatment elicited a further transient T cell response, which was subsequently down-regulated over time. Although the i.n. peptide induced a strong transient CD4+ T cell response, only low levels of peptide-specific antibodies were detected either after the initial or subsequent i.n. exposures to p1 111-139. Our findings address the mechanisms underlying peripheral T cell tolerance following i.n. administration of a high dose of immunogenic peptide and have implications for understanding the consequences of peptide immunothearapy.

Respiratory tract infections due to Branhamella catarrhalis: epidemiological data from Western Australia.

During a 3-year period Branhamella catarrhalis was isolated in significant numbers from 239 (1.3%) of 19,488 specimens of sputum sent for routine microbiological examination at a 700-bed general hospital. The majority of patients (83%) were over 60 years of age and 65% were male. There was a distinct seasonal variation in isolations with a peak incidence during the winter and early spring, a pattern not found with other pathogens. Susceptibility to amoxycillin decreased by approximately 50% over the 3 years, corresponding to an increased incidence of beta-lactamase-producing strains. There were minimal changes in susceptibility to other antimicrobial agents. Underlying pulmonary disease was the major factor predisposing to B. catarrhalis infection, and 71% of patients were smokers or ex-smokers.

The effect of drugs on the cell cycle of Giardia intestinalis.

Flow cytometric analysis of the binucleated protozoan parasite Giardia intestinalis gave DNA histograms with a broad G1 peak and a definable G2 + M peak with twice the DNA content of G1. Twenty-four hour treatment with metronidazole arrested cell cycle progression of susceptible trophozoites in the G2 + M phase, but had no effect, even at toxic doses, on the DNA histogram of a line selected for resistance to metronidazole. Furazolidone was inhibitory to both stocks, causing an arrest in the S and G2 + M phases. Inhibitors of the mammalian cell cycle were also tested. Hydroxyurea, which blocks mammalian cells in G1/S, and razoxane, which blocks in G2 + M, arrested trophozoites in the G2 + M phase whereas colchicine and gamma-irradiation had little or no effect on the cell cycle of G. intestinalis. These results suggest that the cell cycle of G. intestinalis may be controlled in a different manner from mammalian cells.

Characterization of the specificity and duration of T cell tolerance to intranasally administered peptides in mice: a role for intramolecular epitope suppression.

Mucosal administration of antigens in experimental animals leads to the induction of peripheral T cell tolerance. We have previously reported that in H-2b mice, intranasal (i.n.) or oral administration of a peptide containing the immunodominant T cell epitope will down-regulate the function of CD4+ T cells reactive with Der P 1, a major target antigen in both B and T cell responses to house dust mite. In the present study we have investigated the tolerogenicity of peptides containing both dominant and subdominant determinants when given i.n. to nalve mice. Induction of tolerance by the nasally administered immunodominant peptide leads to a diminution in all T cell-derived cytokines and modulation of delayed-type hypersensitivity responses, but IgE production did not seem to be affected, furthermore the induction of T cell tolerance was stable, lasting beyond 6 months. We have also examined the specificity of intramolecular epitope suppression which is a feature of mucosal tolerance induced by nasally administered peptides and demonstrate that regulatory CD4+ T cells may exert their suppressive effect by linked recognition of epitopes on the same or neighbouring antigen-presenting cells.

House dust mite allergy: from T-cell epitopes to immunotherapy.

CD4+ T-lymphocytes induce and regulate allergic inflammatory responses to common environmental aeroallergens derived from Dermatophagoides spp. (house dust mite, HDM), which cause clinical symptoms in approximately 10% of the population. Definition of the molecular structure of HDM proteins combined with the ability to isolate monoclonal populations of human CD4+ T-cells representative of the 'interleukin-4 (IL-4) dominant' functional phenotype, which support immunoglobulin E (IgE) synthesis, has allowed T-cell recognition of HDM to be examined in detail. The results of these investigations demonstrated extensive heterogeneity in both the antigen and HLA class II restriction specificity of the HDM reactive T-cell repertoire. Furthermore, long-lived clones of T-cells with oligoclonality in T-cell antigen receptor (TcR) usage, driven by chronic stimulation with HDM, have been identified in human peripheral blood. The presentation of specific peptides and superantigens under conditions that induce T-cell non-responsiveness has provided an in vitro model for analysing the mechanisms of CD4+ T-cell targeted immunotherapy. It appears that the mechanisms underlying T-cell anergy are accompanied by a transient downregulation of TcR and CD28 and mediated by a shift in the cytokine profile from that of the 'IL-4 dominant' to the 'interferon-gamma (IFN-gamma) dominant' functional phenotype of CD4+ T-cells. In parallel, using a murine model, it has been demonstrated that administration of an immunodominant peptide via the mucosal surfaces of the respiratory and alimentary tracts may tolerize an established response to intact HDM proteins. The potential application of these models in the development of novel approaches to immunotherapy is discussed.