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Desensitising allergen immunotherapy has been practised for many decades. Although time consuming, this form of therapy is antigen-specific and disease-modifying, in contrast to palliative pharmacotherapy. However, the use of allergen extracts containing native allergen molecules frequently results in allergic adverse reactions to treatment. Several strategies to reduce the allergenicity of therapeutic preparations, while maintaining their therapeutic benefit, are being developed. Peptide immunotherapy is one such approach. Short synthetic peptides, comprising T cell epitopes of major allergens, were unable to crosslink allergen-specific IgE molecules on basophils in vitro. Treatment of allergic volunteers with allergen peptides resulted in reduced skin, lung and nasal sensitivity to allergen challenge and improved their subjective ability to tolerate allergen exposure. Peptides reduced pro-inflammatory cytokine secretion from peripheral blood cells, whilst increasing the immunosuppressive cytokine IL-10. Furthermore, peptide therapy was associated with the induction of a population of CD4+ T cells with a suppressive functional phenotype. Thus, peptide therapy may be suitable for the antigen-specific treatment of allergic diseases.

Allergic and autoimmune diseases are forms of immune hypersensitivity that increasingly cause chronic ill health. Most current therapies treat symptoms rather than addressing underlying immunological mechanisms. The ability to modify antigen-specific pathogenic responses by therapeutic vaccination offers the prospect of targeted therapy resulting in long-term clinical improvement without nonspecific immune suppression. Examples of specific immune modulation can be found in nature and in established forms of immune desensitization. Understanding and exploiting common mechanisms such as the ability to induce antigen-specific regulatory cells should allow the development of effective therapeutic strategies for both forms of immunopathology. Targeting pathogenic T cells using vaccines consisting of synthetic peptides representing T cell epitopes is one such strategy that is currently being evaluated with encouraging results. Future challenges in the development of therapeutic vaccines include selection of appropriate antigens and peptides, optimization of peptide dose and route of administration and identifying strategies to induce bystander suppression.

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.

Allergic diseases such as asthma, rhinitis, and eczema are increasing in prevalence and affect up to 15% of populations in Westernized countries. The description of Tregs as T cells that prevent development of autoimmune disease led to considerable interest in whether these Tregs were also normally involved in prevention of sensitization to allergens and whether it might be possible to manipulate Tregs for the therapy of allergic disease. Current data suggest that Th2 responses to allergens are normally suppressed by both CD4+CD25+ Tregs and IL-10 Tregs. Furthermore, suppression by these subsets is decreased in allergic individuals. In animal models, Tregs could be induced by high- or low-dose inhaled antigen, and prior induction of such Tregs prevented subsequent development of allergen sensitization and airway inflammation in inhaled challenge models. For many years, allergen-injection immunotherapy has been used for the therapy of allergic disease, and this treatment may induce IL-10 Tregs, leading to both suppression of Th2 responses and a switch from IgE to IgG4 antibody production. Improvements in allergen immunotherapy, such as peptide therapy, and greater understanding of the biology of Tregs hold great promise for the treatment and prevention of allergic disease.

In the atopic allergic individual, challenge with allergen elicits manifestations of both the humoral (IgE-mediated or early-phase reaction) and the cell-mediated immune response (late-phase reaction). Detailed study of late-phase cell-mediated events is confounded by the effects of the earlier IgE-mediated response which results in mast cell and basophil activation. Thus the relative contributions to allergic inflammation of individual cell types, such as T cells and eosinophils, are difficult to define. In this review we describe experiments, largely from our own group, in which we have attempted to dissociate early and late allergic reactions. To this end, cell-mediated responses were induced in the absence of preceding IgE-mediated events, by the delivery of synthetic peptides representing T cell epitopes of the allergen. Activation of T cells resulted in airway narrowing and an increase in airway reactivity to non-specific stimuli. Furthermore, we describe the induction of antigen-specific hyporesponsiveness or "tolerance" following intradermal, but not mucosal, peptide delivery. The induction of peptide-induced hyporesponsiveness could be temporally dissociated from the initial T cell activation resulting in bronchoconstriction and likely occurred through a different mechanism. Analysis of in vitro allergen responses of peripheral blood cells revealed that hyporesponsiveness was associated with reductions in both Th1 and Th2 cytokines, together with a concomitant increase in the regulatory cytokine IL-10. We conclude that activation of T cells in vivo may result in manifestations of chronic allergic inflammation including bronchoconstriction and hyperreactivity. Additionally, when administered systemically at low dose, peptides may induce long-lasting hyporesponsiveness in the T cell compartment, through a mechanism that is associated with induction of IL-10.

Desensitising therapy for allergic diseases has changed little over almost a century of practice. Administration of increasing doses of extracts of allergen source material has been shown to be reproducibly effective when patients are carefully selected and appropriate concentrations of allergen employed. However, specific immunotherapy is limited by the interaction of specific IgE with allergen, leading to a relatively high frequency of adverse events including anaphylaxis and death. Several strategies have been developed to tackle this issue. Most of these rely on reducing the allergenicity of the treatment, whilst maintaining the immunogenicity. The use of short, synthetic peptide sequences corresponding to T-cell epitopes from the allergen has been shown to modify surrogate markers of allergy including cutaneous responses to allergen challenge and ex vivo parameters of T-cell activation. This review discusses recent advances in our understanding of the mechanisms and potential efficacy of this form of therapy.

In individuals with atopy and asthma, allergen-derived T cell peptides injected intradermally induce isolated late asthmatic reactions (LARs) followed by bronchial hyporesponsiveness to peptide, inhibition of the allergen-induced cutaneous late-phase reaction, and altered T cell function in vitro. Laboratory animal data indicate that "activation" and "tolerance" also occur if peptides are inhaled. In this study, we show that inhalation of Fel d 1-derived peptides induced isolated LAR in individuals with asthma sensitive to cat allergen comparable with that previously demonstrated using intradermal injection. LARs were accompanied by eosinophilia and nonsignificant elevations of total cysteinyl leukotrienes in the sputum. Unlike the intradermal route, repeated inhalation of peptides was not associated with abrogation of the LAR and produced a sputum eosinophilia comparable with the first exposure. In addition, there was no inhibition of the cutaneous late-phase reaction to whole cat dander. Thus, isolated LAR induced by inhaled, allergen-derived peptides represent a novel model of provoked asthma and are not associated with the induction of hyporesponsiveness ("tolerance") in the skin or lung.

The only disease-modifying treatment available for IgE-mediated disease is specific immunotherapy, but the retention of B cell epitopes in whole allergen preparations confers a risk of IgE-mediated systemic reactions to their administration. Compelling evidence for the central role of T cells in allergic disease suggests that IgE-binding epitopes could be removed from such therapy, improving safety without affecting efficacy. Short, allergen-derived peptides lack the conformational determinants required for IgE crosslinking and are, therefore, an attractive therapeutic possibility. However, human leukocyte antigen (HLA) polymorphism means that T cell peptide epitopes present a huge diversity, which makes the design of peptide-based vaccines problematic. Over the past 10 years, advances in our understanding of epitope selection and major histocompatibility complex (MHC)-peptide-T cell receptor interactions have taken this therapy forward to early clinical trials with human volunteers.

There is considerable evidence to support a role for T cells in asthma, particularly the involvement of T(H)2 cells both in atopic allergic asthma and in nonatopic and occupational asthma. There might also be a minor contribution from T(C)2 CD8+ T cells. Several T(H)2 cytokines have the potential to modulate airway inflammation, particularly IL-13, which induces airway hyperresponsiveness independently of IgE and eosinophilia in animal models. The identification of transcription factors controlling T(H)1 and T(H)2 development further support the T(H)2 hypothesis because GATA3 is overexpressed and T-bet is underexpressed in the asthmatic airway. Specific T cell directed immunotherapy might allow induction, modulation, or both of T-cell responses, and elucidation of the mechanisms of regulatory T cells might allow further optimization of immunotherapy. Recent advances in our understanding of dendritic cell function in directing T-cell responses might uncover further therapeutic targets. The efficacy of cyclosporin A and anti-CD4 treatment in patients with chronic severe asthma argues for continued T-cell involvement, but whether remodeling contributes to pathology inaccessible to anti-inflammatory treatment or T-cell immunotherapy will be an important future question.

The efficacy of conventional allergen-specific immunotherapy (SIT) for allergic conditions and venom hypersensitivity is well documented. However it's use is limited due allergic side effects including anaphylaxis and the difficulty of standardising proteins in complex allergenic mixtures. The aim of new therapeutic strategies is to circumvent these limitations and approaches include allergen non-specific therapy, such as anti-IgE and anti-cytokine therapy and other allergen specific techniques including the peptide based vaccines (PBV), modified allergens (allergoids) and DNA vaccines. PBV are small linear peptide fragments containing T cell epitopes which are designed to reduce the ability to cross link antigen-specific IgE. Studies in animal models have confirmed proof of principle demonstrating the induction of hyporesponsiveness using high doses of peptides. However, the principle limitation to clinical use of PBV is the polymorphism of HLA class II molecules. There are ongoing clinical studies using peptide-based vaccines for cat, bee and grass allergies--looking at both immunological mechanisms and clinical outcome measures. The mechanisms underlying the efficacy of PBV appear to be similar to those described for classical immunological tolerance. Thus, the peptides may induce anergy due to absence of co-stimulation, activation-induced cell death, a switch from a Th2 to a Th1 cytokine profile, the induction of regulatory T cells or combinations of these mechanisms. Successful immunotherapy, in bee sensitive individuals, is associated with the elaboration of IL-10. Clonal deletion is unlikely as an overall mechanism as there is evidence that the subsequent in vitro response to associated, non-injected, peptides can be suppressed. Mechanistic studies continue to provide insight into the mode of action of whole allergen and peptide-based immunotherapy. Clinical studies designed on the basis of these observations hold the promise of safer vaccines with improved efficacy. Whether this strategy can be used for allergy to complex allergen mixtures such as dust mites will need further evaluation.

Allergic conditions contribute significantly to the burden of chronic disease in the industrialized world. Current treatments offer varying degrees of palliation. The sole proven disease-modifying strategy, specific or whole-allergen immunotherapy, is limited because of the associated risk of systemic adverse effects, such as anaphylaxis. Short, linear allergen-derived peptides, corresponding to T cell epitopes, offer the possibility of a safer approach as they are capable of inducing allergen-specific hyporesponsiveness without cross-linking mast cell-bound IgE. This review evaluates the scientific basis of peptide immunotherapy and clinical experience in allergy up to the present time.

Background: In general, T cells from normal, nonatopic individuals respond to aeroallergens with synthesis and release of IFN-gamma. In contrast, release of T(H)2-type cytokines by activated lymphocytes is a feature of allergic rhinitis and atopic asthma., Objective: The purpose of this study was to determine differences in T-cell recognition of epitopes within allergenic sequences, in terms of proliferation and cytokine production, in subjects with atopic asthma compared with subjects with allergic rhinitis and normal controls., Methods: Proliferative responses and IL-5/IFN-gamma release patterns from PBMCs from cat-allergic asthmatic, cat-allergic rhinitic, and non-cat-allergic asthmatic subjects and nonatopic normal controls were determined in primary cultures. Cells were challenged with 7 overlapping peptides spanning chain 1 of the major cat allergen, Fel d 1., Results: The 4 groups did not differ with respect to the ability to mount proliferative responses to Fel d 1 peptides. In all groups, the IFN-gamma responses were predominantly to the amino terminus peptides. Cat-allergic and non-cat-allergic asthmatic subjects (and not cat-allergic rhinitic subjects and normal controls) made IL-5 responses to most of the Fel d 1 peptides, the result being a mixed (T(H)0) cytokine response at the N-terminus and a restricted (T(H)2) response at the C-terminus., Conclusion: Proliferative and IL-5/IFN-gamma responses of T cells from asthmatic and atopic rhinitic subjects and normal controls to allergen peptides can be dissociated. Furthermore, differing cytokine responses to peptides derived from a single antigen suggest that certain domains of the molecule might preferentially induce IL-5 rather than IFN-gamma and as a result could be more important in disease pathogenesis.

Background: Isolated late asthmatic reactions can be provoked by intradermal challenge of allergen-derived T-cell peptide epitopes., Objective: The purpose of this study was to determine whether the isolated LAR is associated with the local accumulation of inflammatory cells, the expression of T(H)2 cytokines, and the production of pharmacologic mediators., Methods: A randomized, placebo-controlled, crossover study design was used. The investigation involved bronchial and skin biopsies and bronchoalveolar lavage (BAL) fluids from 8 cat-allergic subjects who developed significant late asthmatic reactions 6 hours after intradermal injection of Fel d 1 chain 1-derived peptides (FC1Ps)., Results: Immunostaining of bronchial biopsy specimens showed no changes in the numbers of eosinophils, neutrophils, basophils, mast cells, CD3(+), CD4(+) or CD8(+) T cells, CD25(+) cells or macrophages, or cells mRNA(+) for IL-4, IL-5, or IL-13 when the FC1P day was compared with the diluent control day. There were also no significant differences in eosinophil numbers, either in BAL fluids or in peripheral blood after FC1P challenge. Furthermore, there were no significant alterations in the concentrations of histamine, histamine-releasing factors, or eicosanoids (LTC(4)/D(4)/E(4), PGD(2), PGE(2), TXB(2), PGF(2alpha)) in BAL fluids. FC1Ps induced a significant (P <.05) elevation in CD8(+) cells in the skin and an unexpected decrease in IL-5 in BAL fluids (P =.043)., Conclusion: Part of the asthma process might involve T cell-dependent airway narrowing with no requirement for IgE, mast cells, or infiltrating inflammatory cells.

Allergen-derived peptides can induce T cell tolerance in naive and Ag-primed mice. This is preceded by transient T cell activation. In humans, intradermal administration of short allergen-derived T cell peptide epitopes provokes IgE-independent isolated late asthmatic reactions (LARs) in sensitized subjects. In this study, we determine whether, as in mouse models, such peptides produce hyporesponsiveness to rechallenge with peptides, or whole allergen, either clinically or in terms of in vitro T cell responses. We found that a second injection of cat allergen (Fel d 1)-derived T cell peptides was associated with a marked reduction, or absence, of the LAR, and that up to 40 wk was required for return to baseline values. The cutaneous late-phase reaction to whole cat dander was also inhibited, even in subjects who did not experience an initial LAR. These observations were associated with a significant decrease in peptide- and whole allergen-induced proliferation of PBMCs and the production of IL-4, IL-13, and IFN-gamma in cultures. Thus, allergen-derived peptides induce tolerance to subsequent peptide injection in the target organ (the lung), reduce late-phase cutaneous responsiveness to whole allergen, and alter in vitro T cell reactivity.

The mouse is now the animal of choice for laboratory-based medical research. Although its contribution to advancing understanding of our inner workings is indisputable, we should acknowledge that mice and humans are tangibly different. This article highlights, and attempts some rationale for, discrepancies between the two species' immune systems.

CD4+ T-cells are likely to be involved as a source of pro-inflammatory cytokines in asthma. This study assessed the effects of an infusion of keliximab (IDEC CE9.1), an anti-CD4+ monoclonal antibody, on peripheral blood CD4+ T-cells in corticosteroid-dependent asthmatics. Three cohorts of patients (termed C0.5: n=6, C1.5: n=5, and C3.0: n=5) received a single infusion of 0.5, 1.5 or 3.0 mg x kg(-1), respectively, with a fourth receiving placebo (Cpl: n=6), and were followed-up for 4 weeks. By flow cytometry in peripheral blood, pre- and postinfusion assessment was made of: a) CD4 and CD8 counts and mean fluorescence; b) CD25, human leukocyte antigen-DR (HLA-DR), CD45RO and CD45RA expression on CD4+ T-cells; and c) interferon (IFN)-gamma, interleukin (IL)-4 and IL-5 expression in CD4+ T-cells. Keliximab's in vitro effects on allergen-specific peripheral blood mononuclear cells (PBMC) proliferation in atopic asthmatics were also evaluated. There was a significant increase in lung function (peak expiratory flow rate) in the C3.0 group. Following infusion in C0.5, C1.5 and C3.0 but not Cpl: 1) the CD4, but not CD8 count was significantly decreased; 2) there was total loss of Leu3a staining; 3) there were significant reductions in the mean fluorescence of OKT4 binding; and 4) there were significant reductions in the numbers of CD25, HLA-DR, CD45RO and CD45RA/CD4+ cells. There were no changes in CD4+ cell expression of IFN-gamma, IL-4 or IL-5. Keliximab caused a significant reduction in T-cell proliferation as compared to a control monoclonal antibody. Keliximab, as an anti-CD4 monoclonal antibody, leads to a transient reduction in the number of CD4+ T-cells and modulation of CD4+ receptor expression in severe asthmatics. The effects of keliximab may be mediated through a decrease in CD4+ surface expression and T-lymphocyte numbers, in addition to a reduction in allergen-induced proliferation.

Short peptide sequences corresponding to T cell epitopes have been identified in the major cat allergen Fel d 1. In order to directly activate allergen-specific T cells in cat-allergic asthmatic individuals, peptides were administered by intradermal injection. Subsequently, a proportion of subjects experienced a delayed reduction of airway calibre manifested as a decrease in FEV(1). Changes in lung function occurred approximately 3 h after peptide injection, peaked at 6 h and resembled an isolated late asthmatic reaction (LAR). Using molecular tissue typing techniques, it was determined that many of the individuals experiencing isolated LAR expressed particular HLA-DR molecules. These molecules were shown in subsequent experiments to bind individual peptides within the preparation and thus to activate T cells in a major histocompatibility complex (MHC)-restricted fashion. The precise mechanisms whereby MHC-restricted activation of allergen-specific T cells gives rise to bronchoconstriction are currently under investigation., (Copyright 2001 S. Karger AG, Basel)

Conventional immunotherapy using whole allergen extracts has been shown to be an effective, disease-modifying treatment in carefully selected patients with allergic conjunctivo-rhinitis, asthma and bee and wasp venom hypersensitivity. However, this form of therapy is associated with the risk of systemic anaphylaxis, which, when severe, can be life threatening. A potentially significant reduction in the incidence of IgE-mediated events during immunotherapy may be achieved by the use of short peptides corresponding to T cell epitopes which, by virtue of their size, are incapable of cross-linking allergen-specific IgE bound to the surface of mast cells and basophils. Initial clinical studies have demonstrated degrees of efficacy which have, in some cases, been associated with adverse events occurring immediately or several hours after peptide administration. Preliminary data from studies employing shorter peptides (20 amino acids or less) suggest that improved efficacy may be achieved by using peptides of defined major histocompatibility complex-binding specificity administered in an incremental dose fashion comparable to conventional immunotherapy. This review will discuss the concept of peptide immunotherapy and the implications of recent studies., (Copyright 2000 S. Karger AG, Basel.)

Background: Blood eosinophils from hypereosinophilic donors were previously reported to possess the functional high-affinity IgE receptor (Fc epsilon RI), so providing a potential mechanism to account for eosinophil degranulation in atopic allergic disease. Furthermore, tissue eosinophils from allergic tissue reactions were shown to be mRNA(+) for the alpha, beta, and gamma subunits of Fc epsilon RI and gave positive immunostaining with an anti-Fc epsilon RI-alpha antibody. Recent studies, however, revealed negative surface staining on peripheral blood eosinophils, but intracellular Fc epsilon RI-alpha protein was identified by Western blot analysis., Objective: Our purpose was to examine on peripheral blood eosinophils from atopic subjects (1) surface expression and mRNA for Fc epsilon RI-alpha, (2) up-regulation of Fc epsilon RI-alpha by allergy-associated tissue factors, and (3) Fc epsilon RI-alpha-dependent release of eosinophil peroxidase (EPO)., Methods: We measured (1) Fc epsilon RI mRNA expression by in situ hybridization, (2) Fc epsilon RI-alpha by flow cytometry and immunocytochemistry (with use of nonpermeabilized and permeabilized cells), and (3) Fc epsilon RI-alpha-dependent release of EPO., Results: Eosinophils from atopic donors had negligible surface expression of Fc epsilon RI-alpha, which was not enhanced by culture with IgE, IL-3, IL-4, IL-5, GM-CSF, or fibronectin or coculture with fibroblasts. Permeabilization, however, revealed appreciable intracellular staining for Fc epsilon RI-alpha. The majority of eosinophils were mRNA(+) for the alpha, beta, and gamma subunits of Fc epsilon RI. Small but significant (P =.03) increases in alpha chain mRNA expression were observed after coculture of eosinophils with fibroblasts but not with IgE, IL-4, or fibronectin. Cross-linking of Fc epsilon RI on the surface of eosinophils from atopic donors did not lead to detectable EPO release., Conclusion: Human blood eosinophils express negligible, nonfunctional membrane Fc epsilon RI-alpha but have intracellular Fc epsilon RI-alpha protein and mRNA expression for the alpha, beta, and gamma subunits.

Together with B-lymphocytes, T-lymphocytes (or T-cells) comprise the antigen (Ag)-specific arm of the immune response. The Ag receptors of both cell types are generated by recombination of multiple gene segments (1), giving rise to a large repertoire of receptors of differing specificity. When a lymphocyte encounters Ag, cellular activation occurs via aggregation of cell surface Ag receptors (2). The consequences of activation are numerous, but, in the case of the T-cell, include clonal expansion, via proliferation, and also secretion of soluble mediators and growth factors, such as interleukin-2 (for a review of the role of the T-lymphocyte in allergic disease, see ref. 3).

Intradermal administration of short overlapping peptides derived from chain 1 of the cat allergen Fel d 1 (FC1P) that did not cross-link IgE, elicited isolated late asthmatic reactions with no visible early or late cutaneous response in 9/40 cat-allergic asthmatics. Four of the nine were human histocompatibility leukocyte antigen DR13-positive, as compared with only 1/31 nonreactors. The other five reactors expressed either DR1 or DR4. To confirm major histocompatibility complex restriction, fibroblast cell lines transfected with HLA-DR molecules were used to present FC1Ps to cat allergen-specific T cell lines derived from subjects before peptide injection. FC1P3 (peptide 28-44 of Fel d 1 chain 1) was recognized in the context of DR13 alleles (DRB1*1301, 1302) and induced specific T cell proliferation and IL-5 production. T cells from a DR1(+) responder proliferated and produced IL-5 in the presence of FC1P3 and DR1 (DRB1*0101) fibroblast cell lines, whereas T cells from a DR4(+) subject recognized FC1P2 (peptide 22-37) when presented by DRB1*0405. We conclude that short, allergen-derived peptides can directly initiate a major histocompatibility complex-restricted, T cell-dependent late asthmatic reaction, without the requirement for an early IgE/mast cell-dependent response, in sensitized asthmatic subjects.

Atopic allergic asthma is characterized by activation of Th2-type T cells in the bronchial mucosa. Previous reports have suggested an important role for costimulation through the CD28/CTLA4-CD80/CD86 pathway in allergen activation of T cells in animal models of inhaled allergen challenge. However, human allergen-specific lines and clones were reported to be costimulation independent. We therefore examined CD80 and CD86 dependence of allergen-induced T cell proliferation and cytokine production in peripheral blood and bronchoalveolar lavage from atopic asthmatic subjects and controls. Both allergen-induced proliferation and IL-5 production from PBMC were inhibited by CTLA4-Ig fusion protein and anti-CD86, but not anti-CD80 mAbs. When allergen-specific CD4+ T cell lines from peripheral blood were examined, proliferation and cytokine production were found to be independent of CD80 or CD86 costimulation. However, when cells obtained directly from the airways were examined, allergen-induced proliferation of bronchoalveolar lavage T cells from atopic asthmatic subjects was inhibited by anti-CD86 but not anti-CD80. In addition, bronchoalveolar lavage-adherent cells from asthmatic, but not control subjects showed APC activity to autologous T cells. This was also inhibited by anti-CD86 but not anti-CD80. Thus allergen-induced T cell activation and IL-5 production in the airway in asthmatic subjects is susceptible to blockade by agents interfering with costimulation via CD86, and this may hold therapeutic potential in asthma.

Background: IL-5-producing allergen-specific T cells are thought to play a prominent role in the pathogenesis of allergic inflammation. We hypothesized that T cell allergen-driven IL-5 synthesis is elevated in patients with atopic disease as compared with that in atopic patients free of disease and nonatopic control subjects., Objectives: The purpose of this study was to compare IL-5 and interferon-gamma (IFN-gamma) secretion and proliferation by peripheral blood T cells from sensitized atopic patients with asthma, rhinitis, and no symptoms and from nonatopic control subjects in response to the allergen Dermatophagoides pteronyssinus (Der p) and the control recall antigen Mycobacterium tuberculosis purified protein derivative (PPD)., Methods: To measure allergen-induced IL-5 production and proliferation, we developed a short-term culture technique that required a single antigenic stimulation of freshly isolated peripheral blood mononuclear cells (PBMC). With this technique, we measured Der p- and PPD-induced IL-5 production and proliferation in PBMC from atopic patients with asthma who were allergic to Der p, atopic patients with rhinitis, atopic patients with no symptoms, and a group of nonatopic normal control subjects. In four experiments, CD4+ or CD8+ T cells were depleted from PBMC to confirm that IL-5 synthesis was T cell dependent., Results: T cell IL-5 production, but not IFN-gamma production, in response to Der p was elevated in atopic patients with asthma and atopic patients with rhinitis compared with findings in atopic patients with no symptoms or nonatopic control subjects. IL-5 production was abrogated by depletion of CD4+, but not CD8+, T cells. In subjects with asthma, allergen-driven IL-5 production correlated with bronchial hyperreactivity. Allergen-induced proliferation was also higher in patients with asthma than in atopic subjects with no symptoms or nonatopic controls. T cell IL-5 and IFN-gamma production and proliferation in response to PPD were similar regardless of atopic status or disease., Conclusions: Elevated IL-5 production is a characteristic of allergen-specific peripheral blood CD4+ T cells from sensitized patients with atopic disease but not atopy per se.

We recently demonstrated bronchial mucosal expression of IL-4 and IL-5 at the mRNA and protein level in both atopic and nonatopic (intrinsic) asthma. In this report, using double immunohistochemistry (IHC) and in situ hybridization (ISH), we show that 70% of IL-4 and IL-5 mRNA+ signals co-localized to CD3+ T cells, the majority (>70%) of which were CD4+, although CD8+ cells also expressed IL-4 and IL-5 mRNA. The remaining IL-4 and IL-5 mRNA signals co-localized to mast cells and eosinophils. The cellular distribution of these mRNA species did not differ between atopic and nonatopic asthmatics. In contrast, double IHC showed that IL-4 and IL-5 immunoreactivity was predominantly associated with eosinophils and mast cells, with few IL-5 or IL-4 immunoreactive CD3+ T cells detectable. However, total IL-4- or IL-5-positive cells detected by IHC were <40% of the total mRNA+ cells, raising the possibility that insufficient cytokine protein accumulated within T cells to enable detection by IHC. We conclude that: 1) in atopic and nonatopic asthma CD8+ T cells, in addition to CD4+ T cells, mast cells and eosinophils express mRNA for IL-4 and IL-5; 2) whereas IL-4 and IL-5 mRNA expression was associated mainly with T cells, immunoreactivity for the corresponding protein products was detectable predominantly in eosinophils and mast cells; and 3) this discrepancy may be partly attributable to the relative insensitivity of double IHC technique that does not allow detection of cytokine protein in T cells where, unlike eosinophils and mast cells, there is no facility for storage and concentration in granules.

Four murine IgG1 monoclonal antibodies, each with specificity for a human tumour-associated antigen, have been tested for their in vivo immunogenicity using a rabbit model. Surprisingly, one of these antibodies, MR6, was significantly more immunogenic than the remaining three reagents. This enhanced MR6 immunogenicity was not restricted to the immunoglobulin molecule itself, but also applied to a hapten (fluorescein isothiocyanate, FITC) when conjugated to the monoclonal antibody. In addition, the secondary immune response to an independent antigen, human haemoglobin, was higher when the antigen was administered simultaneously with MR6 than when co-injected with an isotype-matched control monoclonal antibody. The presence of the target antigen, gp200-MR6, on both rabbit and human leucocytes and epithelium, and its known association with human IL-4 function, raises the possibility that antibody MR6 may not only target immunogens to antigen-presenting cells, but may also enhance the ability of these cells to present antigen to the immune system. Antibodies to the gp200-MR6 may therefore find important clinical application as in vivo adjuvants.

Mutations in the gene for CD40 ligand are responsible for the X-linked form of hyper IgM syndrome. However, no clinical or laboratory findings that reliably distinguish X-linked disease from other forms of hyper IgM syndrome have been reported, nor are there tests available that can be used to confidently provide carrier detection. To identify efficiently mutations in the gene for CD40 ligand, eight pairs of PCR primers that could be used to screen genomic DNA by single strand conformation polymorphism (SSCP) were designed. 11 different mutations were found in DNA from all 13 patients whose activated T cells failed to bind a recombinant CD40 construct. The exact nature of four of these mutations, a deletion and three splice defects, could not be determined by cDNA sequencing. In addition, SSCP analysis permitted rapid carrier detection in two families in whom the source of the mutation was most likely a male with gonadal chimerism who passed the disorder on to some but not all of his daughters. These studies document the utility of SSCP analysis for both mutation detection and carrier detection in X-linked hyper IgM syndrome.

Recent studies show that most patients with X-linked hyper IgM syndrome have defects in the gene for CD40 ligand. We evaluated 17 unrelated males suspected of having X-linked hyper IgM syndrome. Activated T cells from 13 of the 17 patients failed to bind a soluble CD40 construct. In these patients, the sequence of CD40 ligand demonstrated mutations. By contrast, T cells from the remaining four patients exhibited normal binding to the CD40 construct. Sequencing of the cDNA for CD40 ligand from these patients did not show mutations. The possibility that hyper IgM syndrome in these four patients was due to abnormalities in the B cell response to CD40-mediated signals was examined. Peripheral blood lymphocytes were stimulated with anti-CD40 alone, IL4 alone or anti-CD40 plus IL4. In comparison with B cells from controls or patients with hyper IgM syndrome and mutant CD40 ligand, B cells from the patients with hyper IgM syndrome and normal CD40 ligand were defective in their ability to secrete IgE (P < 0.02) or express activation markers, CD25 and CD23 (P < 0.02) in response to stimulation with anti-CD40. The failure of these B cells to respond to CD40-mediated activation could not be attributed to a generalized deficiency in B cell activation because IL4 induced normal up-regulation of CD23 and CD25 expression. These findings indicate that hyper IgM syndrome may result from defects in expression of CD40 ligand by activated T cells or defects in CD40-mediated signal transduction in B cells.

mAb MR6 has previously been shown to block both IL-4-induced T cell proliferation and IL-4-dependent IgE production, suggesting a functional association between the antigen detected by MR6 (gp200-MR6) and the human IL-4 receptor. In this study the potential modulatory effects of mAb MR6 on IL-4 function have been further analysed in alloantigen-specific assays for cytotoxic and Th cell maturation, mature cytotoxic T cell killing, helper cell proliferation, and generation of IL-2- and IL-4-producing Th cells in a mixed lymphocyte reaction (MLR). Our data show that mAb MR6 has an inhibitory effect on both clonal expansion and maturation of cytotoxic T lymphocyte precursors within the alloreactive T cell population. mAb MR6 had no effect on the maturation of Th lymphocyte precursors (assayed by IL-2 production). However, this mAb had striking differential effects on cytokine production in MLR cultures, showing total ablation of IL-4 but no alteration of IL-2 levels in the supernatant medium. The absence of IL-4 from culture supernatants could be due to the fact that mAb MR6 is blocking cytokine production, that it is speeding up IL-4 internalization and utilization or that it inhibits the expansion of the T cell subset(s) that secretes IL-4. The data demonstrate that the action of mAb MR6 is focused on the IL-4-producing population and raise the possibility that gp200-MR6 may play an important role in this aspect of IL-4 function.

T lymphocytes recognise antigen in the form of short peptides complexed with the class I and II products of the Major Histocompatibility Complex (MHC). Cellular activation follows T cell recognition of peptide-MHC complexes at immunogenic cell surface concentrations together with the participation of the appropriate costimulatory signals. Interaction of TCRs and peptide-MHC complexes under inappropriate conditions may result in antigen-specific non-responsiveness, commonly referred to as anergy. Here we review some recent model systems which have been employed to study the phenomenon of anergy and the use of peptides to induce antigen-specific non-responsiveness both in vitro and in vivo.

TCR V alpha 3 and V alpha 5 transcripts in PBLs from healthy individuals of multiple age groups and from BMT recipients were analyzed. PCR, cloning, and sequencing studies revealed significant V-J junctional diversity among TCR transcripts from all tested blood samples, as provided both by N/P-region addition and exonuclease activity. However, results illustrated restrictions in TCR alpha diversity at several additional levels. First, V alpha 5 and V alpha 3 gene families, which were expected to be composed of multiple members, were dominated in each case by a single sequence at the transcript level. Second, restrictions existed in V-J pairing in that J alpha genes, which were encoded toward the 5' region of the locus, were rearranged frequently with V alpha 3 and rarely with V alpha 5. Conversely, J alpha genes encoded toward the 3' region of the locus preferentially rearranged with V alpha 5. Healthy individuals showed few differences with regard to V-J pairing patterns, while one of three BMT recipients demonstrated a skewed usage of 3' J alpha genes. In total, results demonstrated qualitative restrictions that may limit the working TCR repertoire in human peripheral tissues, both among BMT recipients and their healthy donors.

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-J gamma junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-J gamma joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to the V gamma 3-J gamma 1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. V gamma 3-J gamma 1 sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-J gamma joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical V gamma 3-J gamma 1 join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from V gamma 3-J gamma 1 joins.

Experiments were designed to test whether T cell progenitors are committed to particular T cell receptor (TcR) gene rearrangement and expression patterns (prior to rearrangement) or whether such patients are molded by the thymic microenvironment in which T cells develop. To this end, day 14 fetal thymocytes were removed from their normal environment and grown in organ culture (FTOC) for 5 to 12 days. RNA was extracted from organ-cultured cells, processed to cDNA, and TcR alpha sequences were amplified by the polymerase chain reaction for cloning and sequencing. By the examination of N-region additions and V-gene usage, and by the comparison of resultant patterns with those of early vs. adult stages of T cell differentiation in vivo, it was demonstrated that thymocytes in FTOC did not maintain early patterns of gene rearrangement. The thymocyte patterns were most dissimilar from those of normal, early ontogeny when interleukin-4 was added to FTOC. Taken together, results demonstrated the flexibility of T cell progenitors and that environment plays a critical role in the molding of TcR alpha rearrangement and expression patterns.

Recent studies have demonstrated that the diversity of T-cell receptor alpha (Tcra) gene expression may be confined by a developmental program for gene rearrangement. To examine the effect of age on Tcra gene usage in peripheral tissues, a comparison of Tcr transcripts from newborn and adult mouse splenocytes was made. RNA was first isolated from the spleens of newborn (within five days from birth) and adult B10.BR mice. The polymerase chain reaction was then used to assess the presence of Tcra-V1, Tcra-V2, and Tcra-V3 gene sequences within the two RNA pools. The Tcra-V2 transcript was frequent in both newborn and adult populations and was therefore selected for sequencing analyses, by which V-gene family member and J gene usage could be delineated. Forty-one sequences were obtained, demonstrating Tcra-V2 gene family structure in the B10.BR mouse. Six family members were identified, of which four were new. Although there were differences in gene usage between newborn and adult animals, some junctional diversity added to the repertoire of both populations. A striking feature of V-J joining, as illustrated by this study, was the restriction of combinations based on the J gene location within the Tcra locus. The Tcra-V2 gene of dominant expression in the newborn (B10.BR.6) rearranged exclusively with the 30 most 5' Tcra-J genes. The Tcra-V2 gene of dominant expression at the adult stage (B10.BR.1) rearranged exclusively with the 21 most 3' Tcra-J genes in the locus. Thus, V-J combinatorial diversity was restricted in both newborn and adult mice, yielding a trend from 5'-3' Tcra-J gene usage with age. Inherent restrictions in V-J combinations should now be considered with regard to antigen responsiveness, particularly in the young animal. Qualitative restrictions in Tcr repertoire, compounding low T-cell numbers in peripheral tissues, may well contribute to functional voids and immunodeficiencies in early life.

This study was performed as a means to examine the effects of environment and stem cell origin on the molding of T cell receptor gene rearrangements during T cell development. Lethally irradiated adult mice were first reconstituted with (C57BL/6 x DBA/2)F1 stem cells, which derived either from a fetal liver or adult bone marrow source. The gene rearrangements in the irradiated thymi were then compared to those in normal fetal or adult thymi by hybridoma analysis. Results showed a general absence of gene rearrangements typical of the normal fetal thymus in both sets of chimeras. alpha and gamma gene rearrangements in chimeric mice matched normal adult patterns. However, chimeric thymocytes were unique in an unusually low frequency of rearrangements at the delta locus. The use of the bone marrow vs. fetal liver as stem cell sources in chimeric mice did not affect the patterns, indicating that environmental factors played a major role in the molding of gene rearrangement at multiple T cell receptor loci. Interestingly, the chromosomal sequence added an additional influence as homologous chromosomes showed allele-specific rearrangement patterns. We now question whether the unique patterns of rearrangement in irradiated adult thymi may substantially affect developing T cell populations in chimeric animals, particularly with regard to the gamma/delta T cell subset. Further analyses are warranted, both in experimental and clinical settings, regarding ultimate potentials for T cell diversity and function in transplantation recipients.

Normal T cell development depends upon an interaction between progenitor cells and their microenvironment. Previously raised monoclonal antibodies to subtypes of human thymic epithelium were used with gold-coupled reagents in immuno-electron microscopy to study the fine cellular distribution of the molecules to which these Mabs bind. Mab MR6, thought to recognize the human IL-4 receptor, shows strong surface labeling of cortical epithelial cells and thymic nurse cells, and very weak staining of thymocytes, medullary macrophages and interdigitating cells. Mab 1st 8.18 labels the surface of Hassall's corpuscles and associated medullary epithelial cells. The molecules detected by these two antibodies are therefore located in a position where they are available to interact with external cellular and soluble signals within the thymus. In contrast Mabs MR10 and 19 recognize Intracellular molecules within subcapsular, perivascular and some medullary epithelium. These molecules may represent soluble material awaiting secretion from the cell; alternatively, they may be internal structural proteins.

This paper attempts to quantitate immunohistological changes in the myasthenia gravis (MG) thymus and to correlate them with clinical and culture parameters in 40 untreated young onset patients covering a wide range of durations and serum anti-acetylcholine receptor (AChR) antibody titers. Total cellularities of both the thymic cortex and the medulla declined significantly with age. There was some hyperplasia of subcapsular and of medullary epithelial cells, often at the expense of cortex. A combined index of all hyperplastic changes correlated significantly with serum anti-AChR titre. Otherwise histological indices, e.g. of germinal centres (GC) were largely unrelated to any clinical parameters, especially duration of symptoms. Specific anti-AChR synthesis in culture (very closely related to serum titer) correlated better with the medullary lymph node-type T-cell areas; these were more widely prevalent and MG-specific. In contrast, basal and mitogen-stimulated total IgG productivity followed the GC indices more closely. We propose that the variability of GC is due to their dependence on extraneous immune complexes, and we discuss whether they or the T-cell areas are primary or secondary abnormalities. Finally, we conclude that autosensitization in MG with thymic hyperplasia and neoplasia probably arises through separate mechanisms.