Mechanisms of Sublingual Immunotherapy Guy Scadding ab ;Stephen Durham a a

This article was downloaded by: [Milani, Massimo] On: 20 April 2010 Access details: Access Details: [subscription number 921439129] Publisher Informa Healthcare Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Asthma Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713597262 Mechanisms of Sublingual Immunotherapy Guy Scadding ab ;Stephen Durham a a Allergy and Clinical Immunology, Imperial College, London, United Kingdom b Allergy, Royal Brompton Hospital, London, United Kingdom To cite this Article Scadding, Guy anddurham, Stephen(2009) 'Mechanisms of Sublingual Immunotherapy', Journal of Asthma, 46: 4, 322 334 To link to this Article: DOI: 10.1080/02770900902785729 URL: http://dx.doi.org/10.1080/02770900902785729 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

Journal of Asthma, 46:322 334, 2009 Copyright C 2009 Informa Healthcare USA, Inc. ISSN: 0277-0903 print / 1532-4303 online DOI: 10.1080/02770900902785729 REVIEW ARTICLE Mechanisms of Sublingual Immunotherapy Guy Scadding, M.A., M.B.B.S. 1,2 and Stephen Durham 1, 1 Imperial College, Allergy and Clinical Immunology, Sir Alexander Fleming Building, London, SW7 2AZ United Kingdom 2 Royal Brompton Hospital, Allergy, Fulham Road, London, SW7 United Kingdom Allergen-specific sublingual immunotherapy is now recognized to be an efficacious and well-tolerated treatment for allergic rhinitis. Emerging treatment strategies are also aimed at the primary treatment of allergic asthma, particularly allergy to house dust mites. Knowledge of the exact mechanisms of action of sublingual immunotherapy is at a basic level, although there appear to be similarities to the immunological changes seen in subcutaneous immunotherapy. An improved understanding should allow the development of more effective treatment programs and widen the potential use of this form of immunotherapy. This review discusses the possible mechanism of action of sublingual immunotherapy, including data from animal and clinical studies, while comparing this with the current understanding of subcutaneous immunotherapy. Keywords immunotherapy, sublingual, mechanism, allergic rhinitis, asthma Introduction Subcutaneous Immunotherapy is effective in the treatment of selected patients with immunoglobulin-e (IgE)-mediated allergies who fail to respond to avoidance measures and usual pharmacotherapy. Immunotherapy involves the repeated administration of allergen extracts to reduce symptoms and improve quality of life. In view of concerns regarding safety and convenience, recent attention has focused on sublingual immunotherapy as an alternative route of administration. In this article, we present a brief clinical overview of the role of immunotherapy followed by a review of the immunological mechanisms of subcutaneous immunotherapy. There follows a review, largely based on murine studies, of possible events within the oral mucosa and local lymph nodes that may contribute to the mechanism of sublingual immunotherapy. Finally, current knowledge concerning the effects of sublingual immunotherapy on the human immune system is evaluated. Clinical Overview Allergen immunotherapy is indicated in patients with IgE-mediated respiratory allergies and for the management of life-threatening anaphylaxis due to insect stings. Immunotherapy is particularly effective in patients with seasonal allergic rhinitis with/without associated seasonal asthma. Traditionally administered by the subcutaneous route, this treatment is less effective in chronic perennial asthma with multiple allergies, where efficacy is less and side effects, including the remote risk of anaphylaxis, are increased. Anti-allergic drugs and immunomodulatory agents such as anti-ige only treat symptoms with relapse following their discontinuation. In contrast, immunotherapy may induce long-term remission (1, 2), reduce the onset of new sensitisations (3), and prevent disease progression from rhinitis to asthma (4, 5). Corresponding author: Professor Stephen Durham, Imperial College, Allergy and Clinical Immunology, Sir Alexander Fleming Building, London, SW7 2AZ United Kingdom; E-mail: s.durham@imperial.ac.uk 322 In view of the need for specialist diagnosis and administration, the inconvenience of repeated physician attendances and the increased risks of side effects, recent attention has focused on the use of adjuvants or a combination of subcutaneous immunotherapy with anti-ige to reduce side effects and improve ease of administration. Importantly, the sublingual route has emerged as an effective, safer alternative compared with the subcutaneous route. Although the indication for sublingual immunotherapy should be a specialist decision, daily self-administration in the patients home is feasible. The efficacy of the sublingual route (6) as for the subcutaneous route (7) has been confirmed in Cochrane systematic reviews and meta-analyses. The dose dependency has been established with two independent definitive dose-response studies (8, 9), confirming the need for high-dose therapy, with daily administration of grass allergen (in these studies in tablet form) from at least 8 weeks before the pollen season. Increased efficacy compared with placebo was observed with more prolonged therapy with up to 4 to 6 months of pre-seasonal followed by seasonal treatment. An important question is how effective is the sublingual route? At present there are few head-to-head comparisons with either subcutaneous immunotherapy or usual pharmacotherapy with anti-allergic drugs. Although data for the sublingual route is encouraging, more data are needed to confirm whether long-term benefit after discontinuation and suppression of new sensitisations result, as observed for the subcutaneous route. A recent study in patients with seasonal allergic rhinitis confirmed sustained clinical benefit during 2 years continuous treatment with a sublingual rapiddissolving grass allergen tablet (10) (Figure 1). This study is now into its 4th year and should yield important information on potential long-term benefits. In the light of these studies that confirm the efficacy of sublingual immunotherapy there is considerable interest in attempting to understand the underlying mechanisms. This knowledge might inform the identification of biomarkers of successful treatment and allow manipulation to improve

MECHANISMS OF SUBLINGUAL IMMUNOTHERAPY 323 Figure 1. Daily average scoring of symptoms and medication use in placebo and Grazax (grass pollen tablets; 75,000 standardized units once a day) treated individuals during the grass pollen seasons of 2005 and 2006. (Reprinted from Dahl et al 2008 (10)with permission.) efficacy and simplify treatment regimens. Sublingual immunotherapy provides a unique opportunity to study the evolution of human antigen-specific tolerance. Recent interest has focused on the role of the oral dendritic cell and its interaction with T-cells locally, possibly leading to the induction of regulatory T-cells. Such interactions are likely to orchestrate changes in both local and systemic responses to specific allergen. Mechanisms of Subcutaneous Immunotherapy In patients with allergic rhinitis, successful subcutaneous immunotherapy may involve blunting of the seasonal increase in serum pollen allergen-specific IgE concentrations (11) in addition to substantial increases in allergen-specific IgG4 (11 13). The increase in the ratio of specific IgG4 to specific IgE may be crucial. Recent studies have confirmed elevations in allergen-specific IgA after immunotherapy (13, 14). However, such changes in immunoreactive serum antibody concentrations appear to relate more to the dose of allergen administered rather than necessarily correlate with clinical improvement (15). Rather than measure simply antibody concentrations, it is possible to measure the functional activity of these antibodies. Antigen-specific IgG4 may simply have a role in mopping up allergen by competing with IgE. This may itself be protective as IgG4 is non-inflammatory since it is unable to fix complement and does not form immune complexes. B- cells are able to take up allergen bound to IgE via the surface receptor CD23. They subsequently process this allergen and present epitopes to T-cells. This results in effective T-cell activation at low concentrations of allergen. This process had been modeled in vitro using Ebstein-Barr virus transformed B-cells incubated with allergen-ige complexes (11, 16). Complexes bound to CD23 on the surface of B-cells can be quantified by flow cytometry. Serum obtained from subjects with hay fever after successful immunotherapy has been shown to inhibit allergen-ige binding to B-cells (11), with the effect mediated by IgG4. This system has provided an in vitro assay of the efficacy of blocking antibodies induced by immunotherapy. Effector cells including mast cells and basophils possess IgG receptors that contain intracellular immunoreceptor tyrosine-based inhibition motifs (ITIMs) capable of down regulating the response of these cells to allergen (17, 18). Allergen cross-linking of IgE and IgG on the surface of these cells can subsequently inhibit the activation signals transduced via the high-affinity IgE receptor FcεR1 and prevent degranulation (19, 20). Post-immunotherapy serum fractions of IgA are also capable of inducing release of the regulatory cytokine IL-10 from blood monocytes in vitro (14). Subcutaneous immunotherapy has been shown to decrease the numbers of effector cells at mucosal sites, both during seasonal allergen exposure and after allergen challenge (21, 22), as well as reducing effector cell reactivity in vitro (12). Regulatory T-cells (Tregs) are important in normal health and may possibly be deficient in allergic individuals (23). Allergic disease may result from a relative imbalance between the effects of regulatory cells and Th2 cells. Regulatory T-cells can be divided into naturally occurring thymus derived CD4+ CD25+ cells, which are positive for the

324 transcription factor Foxp3, and adaptive regulatory cells, either Tr1 IL-10 secreting cells or Th3 TGF-β secreting cells (24). Many studies of SCIT have looked at the possible induction of regulatory T-cells. Successful immunotherapy in patients with grass pollen (25) and with mite (13) allergy has been shown to be accompanied by increased production of IL-10 in allergenstimulated peripheral T cell cultures. In one study this was accompanied by suppression of allergen-induced T cell proliferation, possibly mediated by CD4+ CD25+ T-cells and inhibited by strategies that blocked IL-10 or TGF-β (13). Additionally, subcutaneous immunotherapy has been associated with alterations in peripheral T cell responses, with immune deviation in favor of Th1 responses (26). However, changes in T cell responses to allergen have not been universally observed in cells derived from peripheral blood (27, 28). Local changes in the nasal mucosa have been measured after immunotherapy. These include skewing of cytokine profiles in favor of Th1 responses (22, 29) and the local induction of regulatory T cells with increases in IL-10 (11) and TGF-β (14). Foxp3 is a transcription factor that is closely linked with regulatory T cells, although transient Foxp3 expression may also be detected during T cell activation. By G. SCADDING AND S. DURHAM use of triple immunofluorescence microscopy, increases in CD3+CD25+Foxp3+ phenotypic Tregs have been demonstrated in the nasal mucosa after successful grass pollen immunotherapy, with further increases after immunotherapy was detected during natural allergen exposure during the pollen season (30). Regulatory T-cells may directly inhibit Th2 cell activation, proliferation, and cytokine secretion. IL-10 can inhibit signalling via the T-cell co-receptor CD28 leading to prevention of activation (anergy) (31). In addition, IL10 is able to induce B-cell heavy chain class switching in favor of IgG4 production with inhibition of switching to IgE (32). TGF-β, which is also elevated locally after immunotherapy, is the major switch factor responsible for induction of IgA2 production by B-cells. The proposed mechanisms of subcutaneous immunotherapy are summarized in Figure 2. Immunotherapy and the Oral Mucosa Modest elevations in allergen specific IgG, particularly IgG4, are detectable in serum obtained after sublingual immunotherapy with different allergens. It is likely that the allergen-immune system interactions underpinning these changes occur in the oral mucosa and within regional lymph Figure 2. (A) Postulated mechanisms of subcutaneous immunotherapy. Repeated high-dose allergen administered with adjuvant acts via antigen presenting cells (APC) leading to enhancement of Th1 and regulatory T cell (Tr) responses. These T cells inhibit Th2 responses and encourage B-cell heavy chain switching, leading to enhanced IgG and IgA production. Specific IgG, particularly IgG4, may inhibit further natural allergen presentation. IgA may encourage further IL-10 release from monocytes and other cells. (B) Postulated mechanisms of sublingual immunotherapy given current evidence. Repeated high-dose allergen acts via antigen presenting cells within the sublingual mucosa. These cells then stimulate the development of regulatory or Th1 cells in preference to Th2 cells. Regulatory T cells may enhance B cell IgG4 production.

MECHANISMS OF SUBLINGUAL IMMUNOTHERAPY 325 Figure 3. Proposed pathway of allergen and mechanisms of sublingual immunotherapy within the oral mucosa and local lymph nodes. (A) Allergen is taken up by oral Langerhans cells (olc) within the sublingual epithelium, possibly mediated by the high-affinity IgE receptor, FcεRI. (B) Following allergen uptake olcs migrate across the submucosa to draining lymphatic vessels. (C) Within the regional lymph nodes (submaxillary, cervical, internal jugular), olcs present allergen to T cells. olc may produce IL-10 and TGF-β and up regulate IDO (indoleamine 2,3-dioxygenase) during interaction with T cells. This may lead to the development of regulatory T cells and Th1 cells and inhibition of Th2 cells. (D) Regulatory T cells subsequently encourage B cell immunoglobulin class switching to IgG4 and IgA, likely via the secretion of IL-10 and TGF-β, respectively. Regulatory T cells may inhibit Th2 cells via secretion of these cytokines or by direct cell-cell contact. Regulatory T cells may subsequently migrate to allergen-exposed effector mucosae. nodes. The speculated local and regional mechanisms of sublingual immunotherapy are summarized in Figure 3. In this model, allergen is taken up by dendritic cells within the sublingual epithelium. These cells then migrate to regional lymph nodes where they present allergen to T cells. This interaction induces allergen-specificth1 or Treg cells, rather than Th2 cells. These cells then orchestrate redirection of the specific and innate immune system response to allergen via B cell immunoglobulin class-switching and inhibition of Th2-mediated inflammation. Studies on the pharmacodynamics of allergen trafficking within/from the oral mucosa using radio-labeled allergen suggest that some allergen remains within the oral mucosa for several hours after application and that there is little systemic absorption direct from the mouth (33, 34). This is despite the highly vascular nature of the sublingual mucosa, which enables use of this region as a means of drug delivery (glyceryl trinitrate, for example). Muco-adhesive formulations, designed to maximize duration of contact with the oral mucosa, have shown increased efficacy in an animal model (35), although no such studies have been performed to date in humans. Allergen uptake by antigen-presenting cells is presumed to be the initial step (Figure 3). The prime candidate for this is the oral Langerhans cell, an analogue of the skin Langerhans cell. These cells constitutively express the highaffinity IgE receptor, FcεR1, with levels in atopics correlating with systemic IgE levels (36). FcεR1 receptor-mediated allergen uptake may be crucial during sublingual immunotherapy. Conversely, allergen uptake in vitro by cord-blood derived Langerhans cells has been demonstrated to occur by macropinocytosis using fluorescently labeled allergens (37). The local environment within the mouth is considered to have a degree of immune privilege. Inflammatory responses are rare despite microbial colonization and exposure to foreign materials in the form of foodstuffs. A high degree of tolerance is likely the result of several factors, crucially the properties of both local antigen presenting cells and T cells, but with possible contributions from epithelial/structural cells, secreted IgA, and non-pathogenic resident micro-organisms themselves. As well as the presence of the Langerhans cell network, the oral mucosa has previously been considered to have a relative paucity of effector cells compared with skin or other mucosal sites. This may mean that allergens are

326 G. SCADDING AND S. DURHAM encountered in a hypo-inflammatory environment during sublingual immunotherapy. However, mast cells have been identified throughout the oral mucosa, with levels comparable to the skin (38). Local side effects may occur within the mouth during treatment, with symptoms similar to those occurring in pollen-sensitized individuals with oral allergy syndrome. These effects are presumed to be caused by local mast cell degranulation. Other cell types may play a role in the natural tolerogenic capacity of the mouth. Epithelial cells, monocytes, and other cells may produce IL-10, activin A or TGF-β, as has been demonstrated at other mucosal sites (11, 39). The abundant secreted IgA may have an antiinflammatory effect. Dendritic cells have been shown to have tolerance inducing properties in both animal and human studies. In mice, prophylactic administration of OVA to the nasal cavity before sensitization resulted in a reduction of the in vitro activation of OVA-specific T-cells (40). Pulmonary dendritic cells isolated from these mice transiently expressed IL-10 and could stimulate the production of high amounts of IL- 10 from T-cells in vitro. Adoptive transfer of these dendritic cells could induce antigen-specific hypo-responsiveness in recipient mice on subsequent challenge. Murine studies from other groups have also demonstrated a pro-tolerogenic, IL-10 driven role of certain dendritic cells (41). Similar properties have been observed with oral Langerhans cells. Van Wilsem et al. (42) demonstrated migration of oral Langerhans cells to regional lymph nodes after antigen application to the oral mucosa in mice. Furthermore, this oral antigen application was found to reduce the magnitude of delayed-type hypersensitivity reactions on subsequent allergen challenge. However, transfer of dendritic cells isolated from regional lymph nodes of orally treated animals could not prevent sensitization and subsequent delayed-type hypersensitivity reactions in naïve recipient mice. Human oral Langerhans cells constitutively express FcεR1, plus high amounts of MHC class I and II, as well as co-stimulatory and co-inhibitory molecules (36), making them highly suitable for allergen presentation. Cross-linking of FcεR1 on monocytes results in the production of IL-10 (43) and induction of indoleamine 2,3-dioxygenase (44). The latter is believed to lead to reduced tryptophan levels with impaired T-cell stimulatory capacity as a consequence. Oral Langerhans cells derived from human mucosa can produce IL-10 in vitro. Moreover, ligation of toll-like receptor 4 (the LPS receptor and part of the innate immune system s bacterial pattern recognition mechanism) enhances IL-10 production by these cells (45). This is accompanied by decreased T-cell proliferation in co-culture experiments (mixed lymphocyte reactions) and the induction of T-cells with a possible regulatory phenotype. One hypothesis is that these innate receptors enhance the tendency toward tolerance to antigens presented in the microbe-rich oral environment. It is not yet known whether such mechanisms may operate to induce tolerance via the oral mucosa during sublingual immunotherapy in humans. The tolerogenic properties of both murine and human dendritic cells may be enhanced by adjuvants in vitro (46). In a mouse model of sublingual immunotherapy it was shown that a combination of vitamin D3 and dexamethasone administered sublingually with allergen suppressed airway hyperresponsiveness to a significantly greater extent than allergen alone. This was accompanied by an increased percentage of CD4+CD25+FOXP3+ cells subsequently isolated from the spleens of these animals (46). After allergen uptake, oral Langerhans cells probably undergo maturation, including alteration in chemokine receptors, allowing migration to the draining regional lymph nodes. Involvement of these nodes was demonstrated in a mouse model of sublingual immunotherapy for allergic rhinitis (47). Mice were sensitized to Timothy grass by intraperitoneal injection. Allergic rhinitis symptoms were inducible by nasal allergen challenge. Sublingual treatment with high-dose grass pollen extract before nasal challenge significantly reduced these symptoms and objective measures of inflammation. There was an associated significant decrease in proliferative response to grass pollen allergen in vitro in cells derived from the cervical lymph nodes. This was associated with a reduction of allergen-stimulated in vitro production not only of IL-4 and IL-5 but also of IL- 10 and IFN-γ. Conversely, allergen-stimulated proliferation and cytokine secretion was not significantly altered versus placebo in cells extracted from the spleens of these animals. The authors conclude that inhibition of proliferation was probably mediated by allergen-specific anergy rather than the induction of regulatory cells. Although of interest, sensitization in this murine model involved the intraperitoneal route, and the relevance of these findings to clinical tolerance after sublingual immunotherapy in humans remains to be tested. Antigen-bearing cells are presumed to encounter T cells within regional lymph nodes. Oral Langerhans cells may interact with naïve T-cells, resulting in the generation of allergen-specific regulatory T-cells. Alternatively, interaction with allergen-specific memory Th2 cells may result in down regulation of function or even redirection to a regulatory or Th1 phenotype. It remains to be determined whether lymph nodes draining the oral mucosa have additional properties that aid the development of immune tolerance beyond those of nodes in other areas of the body. Alternatively, interaction between Langerhans cells, and T-cells may occur locally within T-cell rich areas in the oral mucosa as well as within the draining regional glands (38). Assuming a redirection of T-cell function toward a regulatory or Th1 phenotype, further downstream events, as in subcutaneous immunotherapy, may include B-cell classswitch to IgG4 and IgA production rather than IgE, down regulation of mucosal effector cells, and decreased hyperresponsiveness at mucosal sites. Regulatory T-cells likely interact with B-cells within the lymph node; however, other functions would require migration to mucosal sites as in subcutaneous immunotherapy (11, 30). It has yet to be determined whether regulatory T-cells migrate to the oral mucosa during sublingual immunotherapy. Immunological Effects of Sublingual Immunotherapy The majority of clinical studies to date have assessed the role of sublingual immunotherapy in subjects with sensitivity to aeroallergens. The dose of allergen administered and duration of therapy have varied considerably. There are also

MECHANISMS OF SUBLINGUAL IMMUNOTHERAPY 327 differences in criteria for patient selection in these studies and a wide variability in the different laboratory techniques used to measure putative biomarkers. All of these factors may either confound or compromise the interpretation/comparison of clinical and immunological outcomes. Effects on Specific Antibody Levels Two recent, large-scale, double-blind placebo-controlled studies demonstrate dose-dependent specific antibody changes during grass pollen sublingual immunotherapy (8, 9). Durham et al. (8) enrolled 855 individuals with seasonal allergic rhinitis from Europe and Canada. Placebo or one of three different doses of grass allergen (Timothy grass) tablet; 2500, 25,000 and 75,000 SQ-T (corresponding to 0.5, 5, and 15 µg of the major allergen Phleum p5) were administered daily. Administration began from approximately 8 weeks before the start of the grass pollen season and was continued for a total of 18 weeks. Efficacy was determined by diary recordings of daily symptom scores and use of rescue medication. Measured immunological parameters included grass pollen specific IgE and IgG, plus IgX (the capacity of non-ige serum components to inhibit binding of IgE with grass pollen allergen). A dose-response relationship was observed in clinical measures of efficacy, with patients taking 75,000 SQ-T having significant improvements versus placebo. These changes were paralleled by dose-dependent increases in specific IgG and IgE (see Figure 4), as well as increases in IgX. Specific IgG levels were raised at 8 weeks in the highest dose group and continued to increase, along with levels in the intermediate dose group, up to 18 weeks/post-treatment. Conversely, levels of specific IgE peaked at 8 weeks and remained elevated post-treatment, but without further seasonal rise. In contrast, placebo-treated patients showed no early changes in specific IgG but significant seasonal increase in specific IgE. This early increase in systemic IgE, Th2-priming, before blunting of increases in seasonal IgE is also observed following subcutaneous immunotherapy. Whether this event is necessary for efficacy or a bystander phenomenon is unknown, but this transient event does not appear to be associated with exacerbation of disease. Didier et al. (9) performed a similar international placebocontrolled trial in 628 adults with seasonal allergic rhinitis. A sublingual tablet consisting of equal amounts of allergen extracts from five different grasses was used, as opposed to Timothy grass alone. Participants were given placebo, 100, 300, or 500 IR tablets. 300 IR is equivalent to 25 µg of the group 5 major allergens. Efficacy, as determined by symptom scores and use of rescue medication, was significantly greater than placebo over the entire season in both 300 and 500 IR groups, but with no differences between these groups. Moreover, significant improvements were seen from the very start of the season. In parallel, levels of grass pollen specific IgG4 were increased relative to placebo in a dose-dependent manner. Increases were in the region of three times those seen at baseline broadly similar to those demonstrated in the study by Durham et al. Specific IgE levels increased only in the treatment groups, by a factor of about 2, slightly less than the equivalent observations in the Durham study. These trials show a clear relationship between dose, efficacy, and specific antibody levels. The absence of a baseline year before starting intervention means it is not possible to make within-patient comparisons between efficacy and specific antibody levels. The ratio of specific IgG to IgE in these studies was not separately addressed. The magnitude of induction of IgG/IgG4 is several times lower than that observed during subcutaneous immunotherapy. Durham et al. used a modification of a previously developed method (48) to measure the interference of postimmunotherapy sera containing IgG on allergen-ige binding. Future large-scale studies should further evaluate the clinical relevance of functional assays of IgG antibodies after immunotherapy. Figure 4. Immunologic changes after treatment with placebo or grass pollen tablets; 2,500, 25,000, or 75,000 standardized units. The amounts of specific IgG (A) and IgE (B) to Phleum pratense allergen are given in RU and ku/l, respectively. App, Approximately. (Reprinted from Durham et al. (8) with permission from Elsevier.)

328 G. SCADDING AND S. DURHAM The long-term efficacy of sublingual immunotherapy is currently being assessed. Having established the optimal dose and need for a longer pre-seasonal duration of immunotherapy (8), the same group, as part of a long-term 5-year study in progress, have reported the effects of 2 years of continuous treatment with grass pollen tablets (GRAZAX) (10). Symptom and medication scores were on average 30% and 38% better than placebo in the first year and 36% and 46% better in the second year of study. Immunological parameters were also reported. After an early increase in specific IgE at 2 months a gradual decline back to near baseline was seen by 22 months. The usual seasonal increases in IgE were again absent, in contrast to placebo-treated patients. Specific IgG4 levels increased, on average, to 10 times pre-treatment levels at 10 months and 23 times at 22 months. This was paralleled by increases in allergen-ige blocking antibodies. These results imply a progressive treatment effect over more than one season, as observed with subcutaneous immunotherapy. They also suggest an increase in the specific IgG4 to IgE ratio during prolonged treatment. Of note, an equivalent 2 years of subcutaneous immunotherapy for grass pollen increases specific IgG4 100-fold (11), as opposed to the 23-fold observed here. However, as stated earlier, such changes in IgG have not consistently correlated with clinical outcomes during subcutaneous immunotherapy. Although there are no adequately powered head-to-head comparisons between subcutaneous and sublingual immunotherapy (for separate meta-analyses see references 7 and 49), these data imply that the correlation between clinical efficacy and magnitude of change in immunological parameters such as IgG4 may not be in proportion and that additional local mechanisms may be involved for the sublingual route. Several other studies provide evidence that sublingual immunotherapy for seasonal allergic rhinitis raises specific IgG4 levels, mostly for grass pollen allergy (50 52), but also other seasonal aeroallergens (53) (Table 1). High-dose regimens were used for all of these above studies with cumulative monthly doses up to several hundred times higher than equivalent monthly subcutaneous doses. Some of these studies are less robust than those described in detail above, including lacking placebo control subjects (50, 52) or blinding (52). Others demonstrated significant increases in specific IgG4 between treatment and placebo groups in the absence of demonstrable efficacy in primary clinical outcomes (51, 53). This raises the issue of causality versus bystander effects of specific IgG4. Equally, it is possible that IgG4 changes are necessary but not sufficient for efficacy. Alternatively, a failure to reach clinical significance may be due to insufficient power as a consequence of fewer participants. Sublingual immunotherapy for house dust mite allergy in asthmatic children has also been assessed. A doubleblind randomized placebo- controlled trial using a dose approximately 200 times higher than the equivalent for subcutaneous immunotherapy produced significantly increased specific IgE and IgG4 in the treatment group (54). These differences were not statistically significant until 2 years into the study. Moreover, there were few statistical differences in clinical outcomes between treatment and placebo groups other than rhinitis score. Lue et al (55) did see significant increases in specific IgG4 after 6 months treatment for children with mite-allergic asthma. This was accompanied by raised total, but not mite-specific IgE, a feature not noted elsewhere. Other studies of sublingual immunotherapy for mite allergy found no significant changes in specific IgG4 (58 61), although again this may have related to the lower doses used. Rolinck- Werninghaus et al. (57) found no differences between placebo and active groups in specific IgG4 after treatment of grass pollen allergic children. However, of note is the relatively low dose of allergen used, coupled with only modest improvement in clinical outcomes. Overall, there is substantial data linking the induction of specific IgG with successful sublingual immunotherapy, but firm evidence for a causative role is still lacking. The affinity for allergen and ability to prevent allergen-ige binding is likely to be more important than absolute numbers of antibodies induced. During high-dose sublingual immunotherapy with pollen allergens, specific IgE levels appear to be initially increased but plateau relatively quickly without further subsequent seasonal increases (51, 10). The immunological effects of treatment with perennial allergens such as house dust mites are less clear. Effects on Effector-Cells in the Allergic Mucosa Passalacqua et al. (62) conducted a placebo-controlled trial of modified house dust mite allergen, applied sublingually, to treat adult patients with perennial rhino conjunctivitis. The dose used was about 20 times that given during subcutaneous immunotherapy. Conjunctival inflammation was assessed both before and after allergen-specific conjunctival challenge. After one year the active treatment group had significantly fewer neutrophils and eosinophils in conjunctival fluid after allergen challenge than placebo-treated patients. Intercellular adhesion molecule 1 (ICAM-1) expression was also decreased in conjunctival cell scrapings. These findings were accompanied by significantly lower rhinoconjunctivitis symptom scores after 9 months. The actively treated group also had significantly lower serum concentrations of eosinophil cationic protein (ECP) at 12 and 24 months of treatment. The same group also studied the effect of sublingual immunotherapy on allergen-specific nasal provocation tests (63). Patients with Parietaria species-induced seasonal rhino-conjunctivitis were treated with allergen extract or placebo. Fewer eosinophils and neutrophils were present in nasal secretions after nasal allergen challenge in the treatment group compared with placebo after one season of treatment. A lower level of expression of ICAM-1 on nasal epithelial cells was also observed after immunotherapy. These studies suggest that, as with subcutaneous immunotherapy, sublingual immunotherapy may have effects on allergen-mediated inflammation at mucosal sites. This might explain why treatments with doses that are inadequate to elicit systemic antibody changes have still been reported to have significant clinical benefit. Conversely, Bahceciler et al. (60) did not find any difference in eosinophil numbers within nasal smear samples between baseline and 6 or 12 months of a trial of sublingual immunotherapy for dust mite allergic children. These measurements were obtained in the absence of a nasal allergen challenge and therefore may be less sensitive. Decreases in serum ECP (65, 62) and blood

Table 1. Summary of studies, as discussed in the text, with published data on the effects of sublingual immunotherapy on allergen-specific antibody levels. References in brackets. DBRPCT, double-blinded, randomised, placebo-controlled trial. SCIT, subcutaneous immunotherapy. HDM, house dust mite. Clinical efficacy Specific IgE Specific IgG4 Comments Course length Approx. dose/month Participants recruited Allergen Study DBRPCT, IgX ; blocking antibody activity also increased Dose-dependent increases versus placebo (IgG rather than IgG4) Dose-dependent increases versus placebo 18 weeks Yes; at top dose versus placebo Durham 2006 (8) 855 Timothy grass Up to 420 µg major allergen DBRPCT Dose-dependent increases versus placebo Dose-dependent increases verses placebo 22 weeks Yes; at top 2 doses versus placebo Didier 2007 (9) 628 5 grass extract Up to 1,120 µg major allergens Increased versus placebo Increased versus placebo DBRPCT, Decreased late phase skin response in active group 12 to 18 months Primary outcomes not significantly different versus placebo Lima 2002 (51) 56 Timothy grass 900 µg major allergen No placebo/ control group. Pediatric study. Increased versus baseline at 6 months but not at 3 months. Increased versus baseline at 6 months but not at 3 months 6 months Symptom/ medication scores significantly lower in high versus low dose group Marcucci 2005 (50) 71 3 grass extract Up to 700 µg major allergens No placebo, increase in IgG4 1 /4 of that seen after 8 weeks grass pollen SCIT Increased versus baseline in both groups 17 19 weeks n/a No change in high-dose group Up to 672 µg major allergen Rossi 2007 (52) 45 Timothy grass/ 5 grasses Increased versus placebo Increased versus placebo Adult and pediatric study. 6.5 months Significant in top dose group only Andre 2003 (53) 110 Ragweed pollen Up to 7,200 µg Major allergen No change Increased versus placebo Pediatric study. Raised total IgE in treatment group. 6 months Only night-time symptom score significantly better than placebo Lue 2006 (55) 20 HDM 280 µgderp1 500 µg Derf1 No change DBRPCT 16 weeks n/a Dose-dependent increases versus placebo Aberer 2007 (56) 40 Timothy grass Up to 420 µg major allergen Asthmatic adults and children, DBRPCT Increased versus placebo at 25 months Increased versus placebo at 25 months 24 months Significant improvement in rhinitis only Bousquet 1999 (54) 85 HDM 180 µg Der p1, 300 µg Derf1 No change Combined adult and paediatric study Decreased versus placebo at 24 months 24 months Significant difference in rhinitis score versus placebo at 2 years Tonnel 2004 (59) 32 HDM 50 µg Der p1, 61 µgderf1 No change No change Asthmatic children recruited. DBRPCT. 24 months Decreased asthma and medication scores at 2 years Pajno 2000 (58) 24 HDM 9.6 µg Der p1, 4.8 µg Derp2 Decreased versus baseline No change No placebo or control group 6 or 12 months Improved measures of lung function versus baseline Bahceciler 2005 (60) 31 HDM 93 µg Der p1, 163 µgderf1 No change No change DBRPCT Pediatric study 2 years Significant improvement in medication scores only versus placebo 29 5 grass extract 6 µg major allergen mix Rolinck- Werninghaus 2005 (57) No change at 8 weeks No change at 8 weeks Retrospective study of immune parameters in children with good response to treatment >1 year Patient-reported improvement at 1 year versus baseline 9.6 µg Der p1/f1, 4.8 µg Phl p5, 52.8 µgbetv1 Dehlink 2006 (61) 13 HDM, grass, tree pollen 329

330 G. SCADDING AND S. DURHAM eosinophil count (60) have also been demonstrated during the course of sublingual immunotherapy. There have been few studies of the effects of sublingual immunotherapy on the oral mucosa. Marcucci et al. (66) investigated sublingual tryptase and ECP levels in children undergoing grass pollen sublingual immunotherapy. Interestingly, they found a higher baseline ECP in sublingual secretions in atopics than non-atopics. Six months of sublingual immunotherapy significantly reduced the sublingual ECP level compared to baseline. However, there were no acute changes in either sublingual tryptase or ECP following application of treatment, effectively an allergen challenge to the oral mucosa, either at the start of treatment or during the course. This was even the case in individuals experiencing local side effects such as oral itching. The doses used for sublingual immunotherapy were low, a maximum of 0.5 µg per application. This may have been too low to produce measurable changes in sublingual secretions. This is in contrast to nasal challenges in which very low doses of allergen can induce symptoms and elevations of local tryptase in nasal lavage fluid (67). Oral mucosal biopsies were obtained from a single patient with prolonged local side effects to grass pollen sublingual treatment (68). Samples were taken before starting treatment and during maintenance treatment associated with intra-oral itching. Low numbers of mast cells and eosinophils were seen at both stages with no differences between the two. This led the authors to conclude that other cell types were responsible for the symptoms. In contrast to this, Allam et al. (38) have found significant numbers of mast cells in the oral mucosa. They took multiple biopsies from different regions within the oral mucosae of 10 cadavers. Levels of mast cells in the sublingual region were among the highest, comparable to those found in the skin. They also examined the distribution of oral Langerhans cells at each location. Of note, the ratio of mast cells to Langerhans cells in the sublingual region was among the highest of any area from which a biopsy was performed. In contrast, the mucosa of the oral vestibulum (the cavity between lips and teeth) contained fewer mast cells and greater Langerhans cells. The authors speculate that use of this latter region for allergen application may provide a more efficacious means of oral immunotherapy with fewer local side effects. The mechanism behind the local side effects has still to be confirmed but given the nature and time course of the reactions release of histamine and other mediators from mast cells seems the most plausible explanation. Only one study to date has investigated the effects of a course of sublingual immunotherapy on immune cells within the sublingual mucosa (51). Sublingual biopsy specimens were obtained after 18 months treatment with high-dose grass pollen extract (containing 20 µg Phl p5 daily) or placebo. No differences in absolute numbers of T-cells, CD1a+ dendritic cells, or macrophages were detectable. Similarly, no differences in IL-12 mrna levels were found. This may be because most of the crucial interactions take place at the level of the local lymph nodes rather than in the sublingual mucosa itself. Conversely, changes in cell function, including increased tolerogenicity, may take place in local epithelial cells, antigen presenting cells and T-cells, rather than changes in absolute cell numbers. Effects on T Cells and Cytokine Secretion Bohle et al. (69) studied in vitro proliferation of peripheral blood mononuclear cells (PBMCs) to birch allergen in nine patients undergoing birch pollen sublingual immunotherapy (4.5 µg major allergen per day). Proliferative response to Bet v 1 was significantly reduced at 4 and 52 weeks relative to baseline. Decreases in proliferative response to Mal d 1 (significant) and tetanus toxoid (trend only) were also seen in comparison to baseline at 4 weeks but not at 52 weeks. The role of possible regulatory T-cells was investigated by depletion of CD25+ cells. This was achieved by incubation of PBMCs with magnetic beads coated with anti-cd25 antibody followed by magnetic sorting. This produced a fraction containing less than 2% CD25+ cells as determined by flow cytometry. This resulted in increased in vitro proliferative responses to Bet v 1, Mal d 1 and tetanus toxoid at 4 weeks but not at 52 weeks. Similarly, adding anti-il-10 neutralising antibodies to the assay increased all proliferative responses at 4 weeks but not 52 weeks. The authors conclude that sublingual immunotherapy can induce regulatory T-cell activity, functioning in part via IL-10, with possible bystander suppression early on in the course of treatment. By 52 weeks suppression appeared to be allergen-specific and was associated with increased IFN-γ. A total of 24 patients with allergy to grass pollen (rhinitis and/or asthma) were treated with low-dose mixed grass pollen allergens for one year (70). The total cumulative allergen dose was approximately 80 µg major allergen. PBMC proliferative response to allergen was assessed in vitro at baseline, after a dose increase phase of approximately 3 months and after one year. Significant decreases in T cell proliferation to grass allergen were seen after 3 months and at one year, whereas no such changes were detectable to the control allergen, tetanus toxoid. Moreover, significant increases in allergen specific IgE and IgG4 were seen at one year of treatment. It is interesting that such low doses could achieve humeral and cellular systemic immune changes, especially considering that similar changes have not been consistently found during subcutaneous immunotherapy. This study was not placebo controlled and did not include data on clinical efficacy. Another study of sublingual treatment for house dust mite found a trend toward decreased in vitro PBMC proliferation in response to allergen without reaching significance (71). In contrast to these studies, several groups found no changes in allergen-specific PBMC proliferative responses in vitro during sublingual immunotherapy. Despite using a relatively high dose of house dust mite allergen, 51 µgevery second day, Ciprandi et al. (72) found no significant change in proliferative response to Dermatophagoides allergen. However, they did record decreased responses to C. albicans and Phytohaemagglutinin (PHA). Another study also found decreased proliferation to unrelated allergens (73). In a placebocontrolled study of grass pollen sublingual immunotherapy in children, no systemic immune changes, including PBMC allergen-specific proliferation assays, were observed (57). Doses were low but comparable to those used by Fanta et al. (70). Clinically, there was a positive effect on rescue medication use. Savolainen et al. (74) conducted a placebo-controlled trial with two doses of tree pollen extract compared with placebo

MECHANISMS OF SUBLINGUAL IMMUNOTHERAPY 331 in children with allergic rhino-conjunctivitis. Assays of peripheral blood mononuclear cells (PBMCs) demonstrated significantly greater IL-10 mrna levels in response to allergen in both treatment groups versus placebo, with IL-5 mrna also significantly lower in the high-dose group. In addition, treatment groups also had significantly higher IL- 18 (a Th1 cytokine) mrna. Moreover, IL-5 mrna levels correlated with worse symptom scores. No differences were found in expression of the Th2 transcription factor GATA-3 (75). Ciprandi et al. (76) studied a small cohort of patients with house dust mite induced allergic rhinitis. Three years of mite sublingual immunotherapy resulted in significantly better FEF 25 75 (forced expiratory flow between 25 and 75% of vital capacity) compared with allergic control subjects. They also reported significantly greater in vitro IL-10 production in the treated group. There was a significant positive correlation between FEF 25 75 and IL-10 production in the treated group. However, as in a previous study (72), IL-10 production was greater after stimulation with C. albicans than with D. farinae. Changes in the Th1-associated cytokines IFN-γ and IL-12 (64, 71) have not been consistently reproducible. Bohle et al. (69) measured cytokine mrna levels in unstimulated PBMCs and found a significant increase in IFN-γ at 52 weeks of treatment versus baseline. Fanta et al. (70) studied allergen-specific T cell clones from PBMCs of patients undergoing therapy for grass pollen allergy. Clones were classified as Th1, Th2, or Th0 depending on the ratio of IL- 4 to IFN-γ production. No differences in relative numbers of Th1, Th2, or Th0 clones were detectable after sublingual immunotherapy. Other groups also found no measurable effect on Th1, Th2, or T-reg cytokine profiles (57, 61). To some extent these contrasting results may be explained by different experimental techniques, allergen doses, and duration of treatment; lower treatment doses have generally failed to induce measurable changes in peripheral T-cell function. Table 2 summarizes the studies discussed above. The immunological effects of sublingual immunotherapy are likely mediated via allergen-specific T-cells, through induction of Tregs or Th1 cells; however, further substantive data on the role of T-cells are required, ideally to be included as part of large trials that also examine/confirm clinical efficacy. Subcutaneous immunotherapy for the treatment of grass pollen sensitized allergic rhinitics has been shown to induce IL-10 (11), TGF-β (14) and Tregs (30) in the nasal mucosa. These results were achieved through immunohistochemical staining and in situ hybridization of biopsies of nasal mucosa. Similar biopsy studies after sublingual immunotherapy may be informative concerning T cell and cytokine changes within the target tissue. Conclusions The sublingual route for allergen immunotherapy has emerged as an effective and safe alternative to the subcutaneous route in the treatment of IgE-mediated respiratory allergy. In particular, sublingual immunotherapy has been shown to reduce symptoms and requirements for rescue medication and improve the quality of life of seasonal allergic rhinitis patients. Outstanding issues include the need for more studies in children and head-to-head comparisons with the subcutaneous route and pharmacologic treatments. Equally important will be the assessment of possible long-term benefits, including long-term disease remission, suppression of new allergic sensitizations, and reduction of progression from rhinitis to asthma in children, as has been shown for the subcutaneous route. The sublingual route is generally perceived to be a distinct entity from classical oral tolerance induction. The currently accepted model proposes uptake of allergen by antigen presenting cells within the mucosa (perhaps oral Langerhans cells) and migration to regional lymph nodes, rather than absorption across the sublingual mucosa into the bloodstream or passage through the gut and involvement of the intestinal lymphoid system (77). This is supported to a greater extent in animal studies where dissection of regional lymph nodes is possible. Evidence in humans is limited to a few studies with radioactively labeled allergen, in vitro experiments on oral antigen presenting cells, and indirect evidence such as the observed success of sublingual-spit regimens. The immunological effects of sublingual immunotherapy and how these relate to clinical efficacy are similarly incompletely understood. Large-scale trials have confirmed the induction of allergen-specific IgG antibodies after several weeks of therapy, in common with subcutaneous immunotherapy. Moreover, this effect, as with clinical efficacy, appears to be dose dependent. In agreement with subcutaneous immunotherapy, there is no early suppression of allergen-specific IgE antibodies and a transient early increase in specific IgE antibodies is observed. Given the known redundancy of much of circulating IgE in terms of saturation of effector cell IgE receptors, clinical efficacy cannot simply be explained by decreased allergic antibodies, whereas functional assays of IgG antibodies generated after immunotherapy support their involvement as blocking antibodies. Current models of subcutaneous immunotherapy propose the induction of antigen-specific regulatory T cells, which then orchestrate the observed antibody and mucosal changes seen during treatment. 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