Inhibition of human T‐cell responses by allergen peptides

Of the many significant advances in the field of immunology that have occurred in the last 30 years arguably the most important, at least from a T-cell biologists point of view, has been the definition of major histocompatibility complex (MHC) restriction1 and the crystal structure of human leucocyte antigen (HLA) molecules.2–4 These demonstrations have enabled detailed study of the way in which T cells recognize antigen and also how this process differs from that in B cells. With a few notable exceptions, production of specific immunoglobulin by B cells is T-cell dependent. Furthermore, recruitment of non-lymphoid effector cells into areas of inflammation is a process largely marshalled by the T lymphocyte. It follows therefore that inhibition of T-cell function may have beneficial outcomes in situations in which detrimental immune responses occur and give rise to pathology. Suppression of T-cell function has been achieved with the use of pharmacological agents, such as glucocorticosteroids and cyclosporin, and more recently with biological agents, such as monoclonal antibodies directed against CD3 and CD4. Whilst achieving the desired aim of immunosuppression, these approaches suffer from serious weaknesses, the most obvious of which is a lack of specificity for the antigen(s) which drive the inflammatory process. The same may be said to be true of other therapeutic approaches currently being developed which aim to neutralize the effects of individual mediators in the inflammatory cascade. Here, a notable success has been the use of monoclonal antibodies to tumour necrosis factor (TNF) to treat patients with rheumatoid arthritis.5 However, the long-term consequences of removing individual mediators remain to be seen. Of concern is the recent report of subjects receiving anti-TNF therapy developing a lupus-like syndrome thought to be a direct result of the therapy itself.6 The challenge of introducing specificity into strategies aimed at modulating T-cell responses is one that has been met with considerable success in animal models by the use of peptide epitopes from the antigen in question. Peptide-induced tolerance has been demonstrated in models of experimental autoimmune encephalomyelitis (EAE),7,8 collagen-induced arthritis9 and, more recently, in models of allergic disease. Briner and colleagues sensitized mice to the cat allergen Fel d I and subsequently demonstrated the ability of allergen-derived peptides to inhibit T-cell cytokine and antibody production.10 Hoyne and colleagues demonstrated the ability of peptides from the house dust mite allergen Der p II to down-regulate T-cell and antibody responses to challenge with intact protein and also to prevent sensitization by prior administration.11,12 Bauer administered a dominant T-cell epitope of Bet v I to CBA/J mice either prior to or following sensitization with the whole allergen. No change in antibody isotype was observed but T-cell proliferative responses were inhibited by both treatments.13 More recently, mice sensitized to bee venom allergen phospholipase A2 (PLA2), were treated by intranasal administration of a mixture of three long peptides spanning the entire molecule. A marked reduction in specific immunoglobulin E (IgE) was observed coupled with a decreased interleukin-4 (IL-4) to interferon-γ (IFN-γ) ratio. Pre-administration of peptide resulted in a failure to develop IgE sensitization to subsequent immunization with the whole molecule.14 In addition to animal models, the ability of high-dose peptides to modulate human T-cell responses has also been demonstrated. In vitro studies with human T cells were performed by Lamb and colleagues, who demonstrated the induction of antigen-specific non-responsiveness following the incubation of T-cell clones with supraoptimal doses of specific ligand in the absence of professional antigen-presenting cells.15 Collectively, these studies provided a logical basis for the translation of a peptide-based therapeutic approach in man. Allergic diseases have, in some quarters, been regarded as the ‘poor relation’ of immunology; a group of diseases which are not particularly severe and can be managed adequately with conventional pharmacotherapy. The reality is that allergic sensitization is increasing in prevalence at an alarming rate. Furthermore, sensitization to previously innocuous food stuffs, such as peanuts and tree nuts, have increased dramatically and represent a significant threat to life. There is therefore a significant unmet need in relation to disease-modifying therapies (as opposed to palliation) in atopic disorders. In fact, disease-modifying therapy is available in the form of conventional whole allergen immunotherapy often referred to as SIT (specific immunotherapy). First described by Freeman and Noon working at St Mary's Hospital in London, SIT involves administration, initially by injection but more recently by sublingual or swallow, of increasing doses of whole allergen extract. This approach is widely used but due to a number of drawbacks, is unlikely ever to be adopted on a large scale. The primary problems associated with this form of therapy are the risk of systemic IgE-mediated reactions resulting from cross-linking by intact allergen, the relatively crude and unstandardized extracts employed and also the length of time that effective desensitization takes. For example, subcutaneous or intramuscular injections are given at least monthly for 3 or more years. It is, however, the ability to induce disease in allergic subjects in the clinical laboratory that separates this group of diseases from others driven by T cells. By careful selection of patients, it is possible to elicit exacerbations of disease in relative safety by challenge of the subject with the molecule to which they are allergic. Importantly, there is no evidence that such experimental challenges lead to any permanent worsening of the diseases and, moreover, challenge can be performed in a single day, allowing measurements of tissues (bronchial, nasal skin biopsies) and body fluids [e.g. blood, bronchoalveolar lavage fluid (BALF)] prior to despatching the patient with no ill effects. Such an approach is not possible in other areas. In autoimmunity for example, antigens remain incompletely defined (despite obvious HLA disease associations) and induction of disease exacerbation is unethical and impractical. Similar concerns are applicable to allograft transplantation. It follows then, that allergic disorders offer the only safe and ethical system in which to develop and evaluate novel forms of antigen-specific immunological therapy which, as described earlier, is the logical pinnacle of disease-modifying approaches. The model of allergen challenge has been successfully employed to study allergic and non-allergic asthma and to define the presence of increased numbers of CD4+ T cells and eosinophils in areas of allergic inflammation, both at baseline and following allergen challenge. Furthermore, the challenge model has enabled evaluation of therapeutic strategies directed against the T cell and has provided useful samples for the investigation of immunological mechanisms in pathogenesis.16,17 We recently took the allergen challenge model a step further by injecting subjects with allergic asthma with peptides derived from the allergen to which they were sensitive, in this case Fel d I, the major allergen of cats. The object of our experiment was to activate human antigen-specific T cells in vivo and attempt to induce symptoms of asthma, which would be both T-cell-dependent and IgE-independent since peptides were designed to be too short to cross-link adjacent IgE molecules on the surface of mast cells and basophils. In the study, peptides were administered intradermally to allergic asthmatic subjects, with moderate disease severity, resulting in the induction of isolated late asthmatic reactions in a proportion of individuals(18). This is unlike the response to whole allergen challenge. Following whole allergen challenge in the skin, or lung or elsewhere in an allergic subject, a bimodal response can be observed. The two phases are comprised of a type I hypersensitivity reaction (IgE-mast cell-mediated) peaking at 15 min (example: wheal and flare reaction) and a form of type IV hypersensitivity with an onset of 2–4 hr, peaking at 6–9 hr and requiring 24 hr or more to resolve. In the asthmatic, both early- and late-phase responses can lead to airway narrowing. In the peptide challenge model, each of the three injected peptides was capable of inducing peripheral blood mononuclear cell proliferation in a percentage of the subjects. The ability to induce isolated late asthmatic reactions did not correlate with peptide-induced proliferative responses since the latter may be dose-dependent and the dose administered in the study was the lowest dose demonstrated to induce late asthmatic reactions. T-cell responses to two of the three peptides were shown to be MHC-restricted and subjects experiencing late asthmatic reactions were shown to express HLA-DR molecules associated with peptide restriction. Thus, using human and murine fibroblasts transfected with individual HLA-DR molecules, we were able to determine that one of the three peptides could be presented to allergen-specific T-cell lines by DRB1*0405 and 0408, whereas another of the peptides was restricted by DRB1*0101 and both 1301 and 1302. The ability to design peptides which, theoretically, lack the three-dimensional structure required to interact with and cross-link specific IgE, leads to the notion that peptide immunotherapy may supersede conventional whole allergen immunotherapy. Peptide-based immunotherapy was recently evaluated in individuals with allergic disease induced by cats. Approximately 95% of subjects allergically sensitized to cats have IgE directed against the major cat allergen Fel d I. Epitope mapping studies by Mark and colleagues19 and Counsell and colleagues,20 defined the majority of T-cell proliferative responses to be induced by the N terminus of chain 1 (Fel d I is a heterodimer) of the molecule. Based upon these findings, two relatively large peptides, termed IPC-1 and IPC-2, were evaluated by a number of groups. Norman and colleagues21 administered four injections of peptide or placebo to 95 cat-sensitive patients in three dose groups (7·5 µg, 75 µg and 750 µg per injection). Clinical efficacy was observed only at the highest dose of peptide (4 × 750 µg). Both nasal and lung symptom scores improved in response to peptide therapy, although a significant placebo effect was observed. Treatment was associated with a significant incidence of adverse events, which occurred a few minutes to several hours after peptide injection. Further studies from the same group22 demonstrated reduced IL-4 production in IPC-1/IPC-2-specific T-cell lines following therapy. A similar reduction in IL-4 production was reported by Pène and colleagues.23 Peripheral blood mononuclear cells were stimulated with cat dander extract before and after treatment with IPC-1/IPC-2. A significant reduction (compared to background levels) of IL-4 production was observed in the high-dose group (individual dose 750 µg; total dose 1500–4500 µg) but not in the lower dose groups. A reduction in allergen PD20[the provocative dose of inhaled allergen giving a 20% fall in forced expiratory volume in 1s (FEV1)] was also observed in response to both high (1500–4500 µg) and medium (individual dose 75 µg; total dose 150–450 µg) dose regimens when compared to baseline, but not when compared to placebo. The variability in dose between each group arose as a result of a staggered dose protocol in which a number of individuals received weekly injections for 2, 4, or 6 weeks. Simons and colleagues performed a double-blind, parallel group study administering weekly injections, for 4 weeks, of 250 µg of the same IPC-1 and IPC-2 preparation to 42 (21 active and 21 placebo) individuals with cat-induced rhinitis and/or asthma. The investigators observed late-onset symptoms of rhinitis, asthma and pruritis following treatment.24 No change in cytokine secretion patterns of peripheral blood mononuclear cells was observed before and after therapy. Furthermore, no change in early- and late-phase skin responses to whole allergen were recorded at several time-points (2, 6 and 24 weeks) after treatment. In a multicentre study of 133 patients, Maguire and colleagues reported an improvement in pulmonary function in subjects receiving eight injections of 750 µg IPC-1/IPC-2, who had diminished baseline 1-s forced expiratory volume (FEV1) but only at a single time-point (3 weeks) post-therapy25. Injections were given in two blocks of four, with two injections per week. A large number of adverse events were reported in this study, including some that required the use of adrenaline. In keeping with other studies evaluating IPC-1/IPC-2, late-onset adverse reactions diminished with successive doses of peptide, suggesting the induction of hyporesponsiveness. Thus, a relatively modest improvement in symptom scores combined with frequent adverse reactions resulted in cat and ragweed vaccines being withdrawn from clinical trials. At least some of the adverse events were likely to be due to cross-linking of IgE by the peptides which, by virtue of their relatively large size, may have been able to bridge adjacent IgE molecules on the surface of mast cells. A second, more common, manifestation was symptoms of breathlessness and wheeze with an onset several hours after peptide administration. Our own work has confirmed that these symptoms were probably T-cell dependent, IgE-independent, isolated late asthmatic reactions. Norman and co-workers reported a diminution in late adverse events with successive peptide administration compatible with the induction of hyporesponsiveness.21 In our own model, subjects who developed isolated late asthmatic reactions were rechallenged several weeks after their initial reaction. The second injection failed to elicit bronchospasm in the lung unless it was administered several months after the first. We have subsequently been able to demonstrate a ‘window’ of hyporesponsiveness which occurs between approximately 2 and 8 weeks after a single peptide injection (unpublished observations). From 8 weeks on, the ability to react to peptide challenge gradually returns but takes 4–6 months to reach baseline. It may be that a series of injections of increasing peptide dose may extend the refractory period and result in protection from rechallenge for a period of a year or more. Rechallenge demonstrated reduced responses to peptides in the lung. More relevant for therapy was the question of whether the induction of peptide-specific hyporesponsiveness would also result in reduced responses to challenge with intact allergen. Cat-allergic asthmatics were challenged cutaneously (intradermal administration) with whole cat dander allergen extract, before and after a single injection of 5 µg of each of 12 peptides in a mixture. The 12 peptides spanned the majority of the Fel d I molecule in order to enhance the chances of providing T-cell epitopes for the majority of the population. The results demonstrated that the magnitude of the cutaneous late-phase response to allergen could be markedly reduced even after a single administration of peptide. Furthermore, several subjects, who had received peptide but had not experienced a late asthmatic reaction, also displayed equivalent reductions in the size of the cutaneous late response to whole allergen, suggesting that late asthmatic reactions are not required to achieve T-cell hyporesponsiveness to whole allergen. No change was observed in the cutaneous early response to whole allergen after a single peptide challenge. However, in a subsequent study employing multiple peptide administrations, the magnitude of the IgE-mast cell-dependent early response was also reduced (unpublished data). Protection from rechallenge with whole allergen was also achieved by Muller and colleagues who treated five subjects allergic to bee venom, with a mixture of three peptides from the major venom allergen phospholipase A2.26 Each of the study subjects had experienced a previous moderately severe systemic reaction following a wild bee sting. Peptides were administered at weekly intervals at increasing doses. The ‘induction’ phase consisted of seven injections (0·1, 1·0, 3·0, 6·0, 12·0, 25·0 and 50·0 µg). ‘Maintenance’ dose was three injections of 100 µg. A total dose of 397·1 µg was administered. Post peptide therapy, all five were able to tolerate challenge with 10 µg of purified PLA2. Furthermore, when subjects were challenged with a wild bee sting in the clinical laboratory, three experienced no reaction whilst the remaining two experienced mild localized reactions at the sting site. Analysing the in vitro T-cell responses to both peptides and whole allergen, the authors went on to demonstrate that the induction of clinical hyporesponsiveness was by reductions in T-cell proliferative responses and inflammatory studies an in allergen-specific in these subjects and which was by an in allergen-specific studies evaluating mechanisms in whole allergen immunotherapy have described a relative in T cytokine from T type 2 In an in the of IL-4 was with a change in the of allergen-specific IgE in of the Based upon the that allergic responses is the the success of conventional other approaches have been developed which, it is achieve the same but by For example, London, and are currently being evaluated in clinical trials. However, few any of the studies immune in immunotherapy have on such as and or cell such as the and colleagues provided evidence for a major for in hyporesponsiveness both in allergic individuals whole allergen or peptide immunotherapy and also in bee Our own studies with peptides derived from Fel d I that whilst are decreased after peptide therapy, production is markedly increased (unpublished observations). It then, that either two mechanisms in conventional immunotherapy to peptide therapy or whole venom allergen immunotherapy of that mechanisms are similar It is that the of to venom a response and that peptides, which not antigen and the of antigen-presenting lead to a of immune However, it is from the that have not been in studies of whole immunotherapy. such are or of mechanisms not be In the atopic subject, allergen challenge results in the and of cells that and on the to eosinophils that to the of challenge. IL-4 and as isotype for B cells resulting in IgE IgE mast cells for cross-linking and by allergen, to and allergen immunotherapy a change in the of allergen-specific IL-4 in of the the of IL-4 and cells in addition to isotype to with IgE for allergen, resulting in reduced mast cell immunotherapy to induce T cells cells and the of isotype to with IgE for allergen. Whilst induction of responses may to to allergic such a would be an to with the majority of autoimmune The then, of an such as the use of peptides, which to induce antigen-specific responses a upon which of therapeutic approaches may be the only autoimmune disease to have been with peptides is multiple many autoimmune multiple has HLA such as with In the major protein epitope and performed of in order to determine T-cell and and to peptide which may have therapeutic by virtue of their ability to induce reduced and peptide were evaluated in vivo in a model of experimental allergic encephalomyelitis of for at resulted in a peptide with for use in therapy. a peptide at several was developed and evaluated in clinical Both studies were prior to due to the high of adverse events, hypersensitivity reactions and disease of responses in vitro considerable T cells which were capable of both the and the peptide. However, of the T-cell responses a to be in multiple was also observed. Our own studies have that disease in the case of asthmatics the induction of a late asthmatic reaction, can occur following administration of doses of peptide as as 1 µg. In other studies with cat peptides allergic symptoms were also with of peptide and in both of were dose in of both and It is therefore to that in the two multiple studies described in which an peptide ligand was patients were administered doses of between 5 and of peptide on a weekly and monthly It is not therefore that exacerbations were observed. It is from the studies how such were However, it is that doses of peptide are required to induce in mice and in administered doses of 2 of peptide ligand to in Hoyne and colleagues employed multiple several to mice to house dust mite The studies of Muller and from our own laboratory that antigen-specific hyporesponsiveness in can be achieved with dramatically lower doses. in body into there may be a of approximately five of magnitude in the dose of peptide required to induce significant reductions in both in vivo and in vitro of between murine and human approaches to therapy offer the of disease on the of the immune only currently available approaches to the of they also to the of disease The incidence of allergic diseases including asthma is increasing particularly in developed to develop disease-modifying approaches to therapy give rise to and particularly in allergic may offer the of immune responses in a specific with a reduced risk of IgE-mediated adverse of evaluation of whole allergen immunotherapy have demonstrated that antigen-specific is although the risk associated with the use of whole allergen molecules is The use of short peptide with IgE cross-linking may give rise to and more immunotherapy for both allergic and autoimmune I to colleagues and for their to this work which was by the