SEQUENTIAL EVENTS IN ALLERGENSPECIFIC IMMUNOTHERAPY AND THEIR
UNDERLYING MECHANISMS Very early desensitization effect
The underlying immunological mechanisms of allergen- specific immunotherapy (SIT) are continuously being elucidated (Table I; Fig 1). Very early effects are related to mast cell and basophil desensitization. Intermediate effects are related to changes in allergen-specific T cells, and late affects are related to B cells and IgE as well as mast cells, basophils, and eosinophils. Although definite decrease in IgE antibody levels and IgE-mediated skin sensitivity normally requires years of SIT, most patients are protected against bee stings already at an early stage of venom-SIT. An important observation starting from the first injection is an early decrease in mast cell and basophil activity for degranulation and systemic anaphylaxis (Fig 1). Although it seems similar to rapid desensitization for hypersensitivity reactions to drugs, the mechanism of this desensitization effect is yet unknown. Acute oral desensitization to penicillinVin mice demonstrated that antigen specificmast cell desensitization is one of themain underlying mechanisms for oral desensitization.6 It has been shown that mediators of anaphylaxis (histamine and leukotrienes)
are released during SITand sting challenges without inducing a systemic anaphylactic response.7 Their piecemeal release below the threshold of systemic anaphylaxis may decrease the granule content of mediators and also may affect the threshold of activation of mast cells and basophils, because decreased mediator release in these cells is a well demonstrated feature in in vitro analysis a short time after the start of allergen-SIT.7-9 Although there are fluctuations and a risk for developing systemic anaphylaxis during the course of allergen-SIT, the suppression of mast cells and basophils continues to be affected by changes in other immune parameters such as the generation of allergen-specific T-regulatory (Treg) cells and decreased specific IgE.
Generation of Treg cells and peripheral
The induction of a tolerant state in peripheral T cells represents an essential step in allergen-SIT (Table I; Fig 1). Peripheral T-cell tolerance is characterized may by generation of allergen-specific Treg cells, suppressed proliferative and cytokine responses against the major allergen (Fig 2).10-12 Subsets of Treg cells with distinct phenotypes and mechanisms of action include the naturally occurring thymic selected CD41CD251 Treg cells and the inducible type 1 Treg (Tr1) cells.13 In allergen-SIT, peripheral T-cell tolerance is initiated by autocrine action of IL-10 and TGF-b, which is increasingly produced by the antigen-specific T cells.11,14,15 The suppression by these cells could be partially blocked by the use of neutralizing antibodies against secreted or membranebound IL-10 and TGF-b.12 However, these cells do express CD4 and CD25, raising the question whether these are inducible Tr1 cells, which have upregulated CD25, or naturally occurring CD41CD251 Treg cells that produce suppressive cytokines.13 In addition, it has been shown that CD41CD251 Treg cells from atopic donors have a reduced capability to suppress the proliferation of CD41CD252 T cells.16 Therefore, it has been suggested that upregulation of CD41CD251 Treg cells plays a role in allergen-SIT. TGF-b plays a dual role in allergic disease: it suppresses allergen-specific T cells and plays a role in remodeling of the tissues. It remains to be elucidated in allergic inflammation whether the remodeling and the suppressive roles of TGF b show an imbalance that aggravates the disease instead of controlling the immune response.17
Studies on the mechanisms by which immune responses to nonpathogenic environmental antigens lead to either allergy or nonharmful immunity have demonstrated that Treg cells are dominant in healthy individuals. 18,19 If a detectable immune response is mounted, Tr1 cells specific for common environmental allergens consistently represent the dominant subset in healthy individuals. They use multiple suppressive mechanisms, IL-10 and TGF-b as secreted cytokines, and cytotoxic T-lymphocyte antigen 4 and programmed death 1 as surface molecules. Healthy and allergic individuals exhibit all 3—TH1, TH2, and Tr1 allergen-specific subsets—in different proportions.18 Accordingly, a change in the dominant subset and the balance between TH2 and Treg cells may lead to either allergy development or recovery. Another study on healthy immune response to allergens demonstrated that CD41CD251 Treg cells have been associated with the spontaneous remission of cow’s milk allergy. Children who outgrew their allergy (tolerant children) had higher frequencies of circulating CD41CD251 T cells and decreased in vitro proliferative responses to bovine b-lactoglobulin in PBMCs compared with children who maintained clinically active allergy.20 Several studies have been reported in other diseases in the same line. The in vitro proliferative response to nickel of human CD41 T cells from healthy, nonallergic individuals was strongly augmented when CD41CD251 Treg cells were depleted.21 Furthermore, a human in vivo study on immunotherapy of rheumatoid arthritis also showed a marked increase in the number of FoxP31CD41CD251 Treg cells in peripheral blood.22 CD251 Treg cells are characterized by the expression of the transcriptional regulator Foxp3 (FOXP3 in human beings), which appears to be the master switch gene for Treg cell development and function. The spontaneous development of allergic airway inflammation, hyper-IgE, and eosinophilia in addition to various autoimmune diseases in Foxp3 mutant mice provides compelling evidence for its importance in allergic inflammation. 23 Human beings with immune dysregulation polyendocrinopathy enteropathy X-linked syndrome are similarly affected and mostly develop hyper-IgE and eczema because of mutations in the FOXP3 gene.23 Supporting these findings, a dysregulation of disease-causing effector T cells is observed in atopic dermatitis lesions in association with an impaired CD41CD251FoxP31 T-cell infiltration in the dermis.24 The role of Treg cells is not limited to suppression of TH2 cells. Peripheral tolerance uses multiple mechanisms to suppress allergic inflammation. Apparently, Treg cells contribute to the control of allergen-specific immune responses in 5 major ways: suppression of antigen-presenting cells that support the generation of effector TH2 andTH1 cells; suppression of TH2 and TH1 cells; suppression of allergen-specific IgE and induction of IgG4 and/or IgA; suppression of mast cells, basophils, and eosinophils; and interaction with resident tissue cells and remodeling.13
Modulation of allergen-specific IgE and IgG subtype responses during allergen-SIT
Specific IgE in serum and on the surface of mast cells and basophils bound to FceRI in patients with allergy is a hallmark of atopic disease. Although peripheral T-cell tolerance is rapidly induced during SIT, there is no evidence for B-cell tolerance in the early course.10 Natural exposure to a relevant allergen is often associated with an increase in the IgE synthesis. Similarly, SIT frequently induces a transient increase in serum specific IgE, followed by a gradual decrease over a period of months or years of treatment (Fig 1).25,26 In pollen-sensitive patients, desensitization prevents elevation of the serum specific IgE during the pollen season.27 However, the changes in IgE levels cannot explain the diminished responsiveness to specific allergen as a result of SIT, because the decrease in serum IgE is relatively late and does not correlate with clinical improvement after SIT. Antibody responses induced during allergen-SIT are functionally heterogeneous, which may account for the conflicting data in relation to the protective effects of IgG.12,25,26 Subclasses of IgG antibodies, especially IgG4, are thought to capture the allergen before reaching theeffector cell-bound IgE, and thus to prevent the activation of mast cells and basophils. However, the relationshipbetween the efficacy of SIT and the induction of allergen-specific IgG subgroups remains a controversial issue, with serum concentrations of allergen-specific IgG correlating with clinical improvement in some studies but not in others.28,29 Allergen-specific IgG may be directed against the same epitopes as IgE, resulting in direct competition for allergen binding and a blocking effect. By contrast, induction of IgG specific for other epitopes may result in a failure of the IgG response to compete with IgE, even when IgG is present in molar excess. The concept of blocking antibodies has recently been revaluated. Analysis of the IgG subtypes induced by SIT has shown specific increases in IgG1 and particularly IgG4, with levels increasing 10-fold to 100 fold.30,31 There is accumulating evidence that SIT also influences the blocking activity on IgE-mediated responses by IgG4, and cellular assays are commonly used to investigate these changes. Recently, a novel assay that detects allergen-IgE binding by using flow cytometry has been used to detect functional SIT-induced changes in IgG antibody activity. Results uggest that successful SIT is associated with an increase in IgG blocking activity that is not solely dependent on the quantity of IgG antibodies.32,33 It seems to be relevant to measure the blocking activity of allergen-specific IgG or IgG subsets, particularly IgG4 and also IgG1, instead of the crude levels in sera. In this context, the role of anti-IgE treatment in the induction phase of allergen-SIT on safety and efficacy has been questioned. Anti-IgE mAb pretreatment enhances the safety of SIT for allergic rhinitis and may be an effective strategy to permit more rapid and higher doses of allergen immunotherapy.34 Its function on long-term efficacy is still under investigation. The noninflammatory role for IgG4 may arise because the IgG4 hinge region has unique structural features that result in a lower affinity for certain Fcg receptors and the ability to separate and repair, leading to bispecific antibodies that are functionally monomeric.35 Furthermore, IgG4 does not fix complement and is capable of inhibiting immune-complex formation by other isotypes, giving this isotype anti-inflammatory characteristics. By using well defined recombinant allergen mixtures, all treated subjects developed very strong allergen-specific IgG4 and also increased IgG1 antibody responses. Some patients who were not initially sensitized to Phl p 5 developed strong specific IgG4, but not IgE antibody responses to that allergen.30 This demonstrates that extract-based antibody measurements may provide incorrect information, and studies on mechanisms of allergen-SIT should be performed with single allergens. Nevertheless, IgG4 antibodies can be viewed as a marker of introduced allergen dose, and they have the ability to modulate the immune response to allergen and thus the potential to influence the clinical response to allergen. In addition, the affinity of newly produced IgG4 and decreasing IgE to allergens has not been intensely studied and may have a very decisive role. Affinity maturation of specific antibodies in allergen immunotherapy and preseasonal versus postseasonal changes in their affinity remain to be elucidated.36 IL-10 that is induced and increasingly secreted by SIT appears to counterregulate antigen-specific IgE and IgG4 antibody synthesis.11 IL-10 is a potent suppressor of both total and allergen-specific IgE, and it simultaneously increases IgG4 production. Thus, IL-10 not only generates tolerance in T cells but also regulates specific isotype formation and skews the specific response from an IgEdominated to an IgG4-dominated phenotype (Fig 2).The healthy immune response to Der p 1 demonstrated increased specific IgA and IgG4, small amounts of IgG1, and almost undetectable IgE antibodies in serum.12 House dust mite–SIT did not significantly change specific IgE levels after 70 days of treatment; however, a significant increase in specific IgA, IgG1, and IgG4 was observed.12 The increase of specific IgA and IgG4 in serum coincides with increased TGF-b and IL-10, respectively. This may account for the role of IgA and TGF-b as well as IgG4 and IL-10 in peripheral mucosal immune responses to allergens in healthy individuals.11,37 Most probably the decrease in IgE/IgG4 ratio during allergen-SIT is a feature of skew from allergen-specific TH2 to Treg cell predominance. However, although Treg cell generation happens within days, a significant decrease in IgE/IgG4 ratio occurs after years. The reason for the long period between the change in T-cell subsets but not IgE/IgG4 levels is not easily explainable by the half-life of antibodies. In this context, the role of bone marrow-residing IgE-producing plasma cells with very long life spans remains to be investigated.38
Suppression of effector cells and inflammatory responses during allergen-SIT
Peripheral T-cell tolerance to allergens, which is characterized by functional inactivation of the cell to antigen encounter, can overcome both acute and chronic events in allergic reactions (Fig 2). Allergen-SIT efficiently modulates the thresholds for mast cell and basophil activation and decreases IgE-mediated histamine release.39 In addition, IL-10 was shown to reduce proinflammatory cytokine release from mast cells.40 Furthermore, IL-10 downregulates eosinophil function and activity and suppresses IL-5 production by human resting TH0 and TH2 cells.41 Moreover, although demonstrated in a model of myocarditis, IL-10 gene transfer significantly reduces mast cell density,local histamine concentration, and mast cell growth, and prevents mast cell degranulation.42
Long-term SIT is associated with reduction of not only the immediate response to allergen provocation but also the late-phase reaction (LPR) in the nasal and bronchial mucosa or in the skin. The mechanism of LPR is different from the mast cell–mediated immediate reaction and involves the recruitment, activation, and persistence of eosinophils and activated T cells at the sites of allergen exposure.3 Successful SIT results not only in the increase of allergen concentration necessary to induce immediate or LPR in the target tissue but also in the decreased responses to nonspecific stimulation. Bronchial, nasal, and conjunctival hyperreactivity to nonspecific stimuli, which seems to reflect underlying mucosal inflammation, decreases after SIT and correlateswith clinical improvement.43 During birch pollen SIT, reduced plasma levels of eosinophil cationic protein, a marker of eosinophil activation, as well as chemotactic factors for eosinophils and neutrophils correlated with decreased bronchial hyperreactivity and clinical improvement.44 Inhibition by SIT of the seasonal increase in eosinophil priming has also been demonstrated. 45 In biopsies taken during grass pollen SIT, decreased eosinophil and mast cell infiltration in nasal and bronchial mucosa after SIT correlated with the anti-inflammatory effect.46
MECHANISMS OF SUBLINGUAL IMMUNOTHERAPY
Because of potentially severe, albeit infrequent, side effects associated with injection SIT, mucosal routes of administration are being investigated to conduct allergenic desensitization.47 Although its safety and efficacy have now been largely documented, much remains to be investigated on the immunologic mechanisms underlying sublingual immunotherapy (SLIT). A meta-analysis of the double-blind, placebo-controlled trials performed in the past decade has shown that SLIT is clinically efficacious although, at present, the treatment benefit is approximately half that achieved with subcutaneous SIT.48 The immunologic mechanisms of sublingual SIT seem to be similar to subcutaneous SIT, although the magnitude of the change in most parameters is modest or no change has been observed. It seems likely that the contact of the allergen with the oral mucosa is critical for the success of SLIT.49 It is postulated that most likely oral Langerhans cells are critically involved in this process.50 During SLIT, the allergen is captured within the oral mucosa by oral Langerhans cells, and subsequently these cells mature and migrate to proximal draining lymph nodes. Those local lymph nodes may favor the production of blocking IgG antibodies and the induction of T lymphocytes with suppressive function.51,52 Most studies using SLIT have reported increased levels of serum IgG4 with a relatively modest increase compared with injection immunotherapy. 53 There is no consistency in T-cell and IgE and effector cell responses, and a significant number of studies has failed to detect systemic immunologic changes.48,53 This may be related to the different doses of allergen administered in different studies, or to the development of more localized immunologic changes. In a recent study, PBMCs were stimulated with pollen allergen extract after 1 and 2 years of SLIT. The expression of IL-10 mRNA was increased in both high and low doses and showed a positive correlation with TGF-b expression. IL-5 was suppressed with a high dose, which negatively correlated with TGF-b.54 In a recently reported birch pollen extract SLIT, patients showed improved nasal provocation scores to birch pollen; however, apple-induced oral allergy syndrome caused by cross-reactivity was not significantly reduced. Bet v 1–specific T-cell tolerance to all epitopes and those cross-reactive with Mal d 1 from apple were shown. However, neither Mal d 1–specific IgE and IgG4 levels nor Mal d 1–induced T-cell proliferation changed significantly, probably because of noncross-reactive epitopes.55 These results may explain why pollen-associated food allergy is frequently not ameliorated by pollen immunotherapy. Although SLIT is increasingly being used, several points need further investigation, such as its efficacy in asthma, its mechanisms of action, the optimal dose and time to be administered, its combination with injection immunotherapy, age of onset for its safe use in young children, and its preventive role in the development of allergy.
UNDERLYING MECHANISMS OF NOVEL AND EMERGING SIT VACCINES
Intensive studies are being performed to improve efficacy and safety of allergen-SIT (Table II). A basic requirement for an allergen vaccine in achieving successful SIT without risk of anaphylaxis is to express T-cell epitopes, which induce T-cell tolerance and lack antibody-binding sites that mediate IgE cross-linking.56 Conformation dependence of B-cell epitopes and linearity of T-cell epitopes may induce a different regulation of allergen-specific T-cell cytokine toward a nonallergic phenotype. Native allergens use IgE-facilitated antigen presentation by dendritic cells and B cells, which activates T cells to produce TH2-type cytokines and B cells to produce further IgE in a secondary response. In contrast, B-cell epitope deleted allergens, which do not bind IgE, do not initiate effector cell degranulation. They use phagocytotic or pinocytic antigen uptake mechanisms in dendritic cells, macrophages, and B cells.57 T cells may be subsequently induced to generate a balanced TH0/TH1-type cytokine pattern in lower quantities as well as T-cell tolerance, which involves Treg cells. Accordingly, targeting T cells and bypassing IgE by modified allergens will enable the administration of higher doses of allergens, which is required to induce T-cell tolerance without the risk of anaphylaxis.57 In line with this concept, immunotherapy using peptid es (PIT) is an attractive approach for safe SIT. To date, clinical trials of PIT have been performed in 2 allergies. 58 Induction of T-cell tolerance and increased IL-10 production have been demonstrated both in cat Fel d 1 and bee venom Api m 1 PITs.58 A potential barrier to PIT of allergy is the apparent complexity of the allergen-specific T-cell response in terms of epitope use and dominant epitopes in human beings and stability of peptides. To overcome these problems, genetically engineered recombinant hybrid molecules that span the whole T-cell repertoire but do not bind IgE have been developed. In cysteine-containing proteins, folding is complicated by the formation of intramolecular and intermolecular disulphide bond formation, whereas any formed disulphide bond can fix the conformation and limits the freedom of further folding processes. With an increasing number of cysteines, the probability of a correct for nativelike folding rapidly decreases because of the increasing probability of incorrect disulfide-bound formation. A fusion protein consisting of the 2 major allergens of bee venom, Api m 1 and Api m 2, has been generated to investigate this concept. Destroyed conformational B-cell epitopes but intact T-cell epitopes of the 2 allergens characterize this protein. By providing decreased allergenicity with preserved T-cell tolerance– inducing capacity, the Api m [1/2] fusion protein represents a novel vaccine prototype for allergen-SIT.59
Another interesting approach was to cut the major allergens to fragments and fuse them in a different order without missing any T-cell epitopes in 1 reassembled mosaic allergen.60 In this study, 2 fragments of Api m 1, 3 fragments of Api m 2, and Api m 3 are reassembled in a different order with overlapping residues, so no T-cell epitopes are missed. Single injection of both vaccines, which only target T cells, demonstrated a preventive effect on IgE generation in mice. The advantage of these 2 approaches is that only 1 molecule has to be produced and purified instead of several recombinant allergens. T-cell epitopes are preserved, and B-cell epitopes can be deleted or preserved depending on the type of the fusion molecule. Another interesting approach was to use fragments and a trimer of major birch pollen allergen Bet v 1 to treat birch pollen allergy. A double-blind, placebocontrolled study has been completed in 3 centers, which demonstrated an increase in IgG1, IgG2, IgG4, and IgA and suppression of seasonal increases of IgE.61 In a different approach, the effectiveness of a mixture of 5 recombinant grass pollen allergens in reducing symptoms and a need for symptomatic medication in patients with grass pollen allergy were demonstrated. All treated subjects developed strong allergen-specific IgG1 and IgG4 antibody responses.30 Allergen-SIT vaccines are generally administered subcutaneously, intradermally, or sublingually, from where they must reach secondary lymphatic organs to induce an immune response. In a mouse study, an MHC class I–binding peptide from lymphocytic choriomeningitis virus enhanced immunogenicity by as much as 106 times compared with subcutaneous and intradermal vaccination. Intralymphatic administration induced CD8 T-cell responses with strong cytotoxic activity and IFN-g production that conferred long-term protection against viral infections and tumors.62 The efficacy of allergen-SIT vaccines administered directly into inguinal lymph nodes f human beings is currently being investigated. Allergen-SIT uses aluminium hydroxide as an adjuvant. These preparations have generally proven to be efficacious and have a good safety profile, but might be improved in efficacy. A new class of adjuvants—so-called immune response modifiers— act on antigen-presenting cells through the Toll-like receptors (TLRs), which recognize pathogen-associated olecular patterns on microorganisms. Depending on the type of TLR, different types of antigen-presenting cells can be targeted. TLR-triggering compounds that can control the overexpression of TH2 cytokines or skew the TH1:TH2 balance toward a TH1 profile have been effective in murine models of allergy.56 Oligodeoxynucleotides containing immunostimulatory
CpG motifs that trigger TLR9, linked to the allergen in ragweed allergy in human beings, have been
used. Amb a 1–immunostimulatory DNA sequence conjugate SIT led to a prolonged shift from TH2 immunity toward TH1 immunity and appeared to be safe.63 In a recent study, the same Amb a 1 CpG conjugate was shown to be effective for the treatment of allergic rhinitis in 2 consecutive seasons. Although an early increase in Amb a 1– specific IgE was observed during the injection phase, a seasonal increase in Amb a 1–specific IgE did not occur, and a reduction in the number of IL-4–positive basophils was reported.64 In another study, vaccination with a peptide antigen covalently coupled to highly repetitive viruslike particles induced high IgG antibody titers in human beings, suggesting the possibility of using allergens coupled to viruslike particles in allergen-SIT.65 As a different immunologic approach, the fusion of allergens with human Fcg has been reported to inhibit allergen-induced basophil and mast cell degranulation by cross-linking Fcg and FceRI receptors.66
There is increasing evidence to support IL-10 and/or TGF-b secreting Treg cells and immunosuppressive cytokines as key players in mediating successful allergen- SIT and a healthy immune response to allergens. In addition to allergy, these mechanisms may have implications in autoimmunity, graft-versus-host disease, tumor cell growth, parasite survival, chronic infections, and the development of AIDS. In sensitized individuals, peripheral T-cell tolerance represents the key mechanism in healthy immune responses to allergens. Peripheral tolerance to allergens induced by allergen-SIT involves control of the allergen-specific immune response in multiple phases including specific T-cell suppression, generation of noninflammatory antibody isotypes such as IgG4 and IgA, suppression of IgE and type 1 hypersensitivity responses, and suppression of LPRs and mast cells, eosinophils, and basophils. Taking the recent advances in knowledge of peripheral tolerance mechanisms into account, developments of safer approaches and more efficient methods of allergen-SIT await. New approaches to allergen-SIT, such as the use of ecombinant proteins, peptides, fragments, and hybrid allergens, are promising, but are only in an early stage of human clinical trials. There are several essential requirements underlying novel strategies for the development of safe and more efficient allergen-SIT vaccines: (1) the vaccine should be constituted of perfectly well standardized native proteins or recombinant allergens; (2) the vaccine should induce tolerance in allergen-specific effector T cells, suppress IgE production, and promote IgG4 or IgA isotype blocking antibody production; (3) the vaccine should not induce severe side effects and should be well tolerated; (4) the vaccine should be easily administered; (5) the treatment should achieve clinical success and long-term protection in a short time with few doses; and (6) early biological/immunologic markers to assess clinical success, before the onset or early in the course of the treatment, should be identified.
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