Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy

The mechanisms by which signaling by the innate immune system controls susceptibility to allergy are poorly understood. In this report, we show that intragastric administration of a food allergen with a mucosal adjuvant induces allergen-specific IgE, elevated plasma histamine levels, and anaphylactic symptoms in three different strains of mice lacking a functional receptor for bacterial LPS (Toll-like receptor 4 (TLR4)), but not in MHC-matched or congenic controls. Susceptibility to allergy correlates with a Th2-biased cytokine response in both the mucosal (mesenteric lymph node and Peyer's patch) and systemic (spleen) tissues of TLR4-mutant or -deficient mice. TLR4-mutant mice are not inherently impaired in their ability to regulate Th1 cytokine production because they respond to stimulation via TLR9. Coadministration of CpG oligodeoxynucleotides during sensitization of TLR4-mutant mice with allergen plus CT abrogates anaphylactic symptoms and Ag-specific IgE, and results in a Th1-polarized cytokine response. When the composition of the bacterial flora is reduced and altered by antibiotic administration (beginning at 2 wk of age), TLR4 wild-type mice become as susceptible to the induction of allergy as their TLR4-mutant counterparts. Both allergen-specific IgE and Th2 cytokine responses are reduced in antibiotic-treated mice in which the flora has been allowed to repopulate. Taken together, our results suggest that TLR4-dependent signals provided by the intestinal commensal flora inhibit the development of allergic responses to food Ags.     

Blockade of CD28-B7, but not CD40-CD154, prevents costimulation of allogeneic porcine and xenogeneic human anti-porcine T cell responses

Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 microg/ml) was considerably more inhibitory than anti-CD154 mAb (100 microg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-gamma by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-gamma and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.     

Tolerization of a type I allergic immune response through transplantation of genetically modified hematopoietic stem cells

Allergy represents a hypersensitivity disease that affects >25% of the population in industrialized countries. The underlying type I allergic immune reaction occurs in predisposed atopic individuals in response to otherwise harmless Ags (i.e., allergens) and is characterized by the production of allergen-specific IgE, an allergen-specific T cell response, and the release of biologically active mediators such as histamine from mast cells and basophils. Regimens permanently tolerizing an allergic immune response still need to be developed. We therefore retrovirally transduced murine hematopoietic stem cells to express the major grass pollen allergen Phl p 5 on their cell membrane. Transplantation of these genetically modified hematopoietic stem cells led to durable multilineage molecular chimerism and permanent immunological tolerance toward the introduced allergen at the B cell, T cell, and effector cell levels. Notably, Phl p 5-specific serum IgE and IgG remained undetectable, and T cell nonresponsiveness persisted throughout follow-up (40 wk). Besides, mediator release was specifically absent in in vitro and in vivo assays. B cell, T cell, and effector cell responses to an unrelated control allergen (Bet v 1) were unperturbed, demonstrating specificity of this tolerance protocol. We thus describe a novel cell-based strategy for the prevention of allergy.     

Immunosuppression in Early Postnatal Days Induces Persistent and Allergen-Specific Immune Tolerance to Asthma in Adult Mice

Bronchial asthma is a chronic airway inflammatory condition with high morbidity, and effective treatments for asthma are limited. Allergen-specific immunotherapy can only induce peripheral immune tolerance and is not sustainable. Exploring new therapeutic strategies is of great clinical importance. Recombinant adenovirus (rAdV) was used as a vector to make cells expressing cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4Ig) a soluble CTLA4 immunoglobulin fusion protein. Dendritic cells (DCs) were modified using the rAdVs together with allergens. Then these modified DCs were transplanted to mice before allergen sensitization. The persistence and specificity of immune tolerance were evaluated in mice challenged with asthma allergens at 3 and 7 months. DCs modified by CTLA4Ig showed increased IL-10 secretion, decreased IL-12 secretion, and T cell stimulation in vitro. Mice treated with these DCs in the early neonatal period developed tolerance against the allergens that were used to induce asthma in the adult stage. Asthma symptoms, lung damage, airway reactivity, and inflammatory response all improved. Humoral immunity indices showed that this therapeutic strategy strongly suppressed mice immune responses and was maintained for as long as 7 months. Furthermore, allergen cross-sensitization and challenge experiments demonstrated that this immune tolerance was allergen-specific. Treatment with CTLA4Ig modified DCs in the early neonatal period, inducing persistent and allergen-specific immune tolerance to asthma in adult mice. Our results suggest that it may be possible to develop a vaccine for asthma.     

Critical role of preconceptional immunization for protective and nonpathological specific immunity in murine neonates

Expression of Th2 immunity against environmental Ags is the hallmark of the allergic phenotype and contrasts with the Th1-like pattern, which is stably expressed in healthy adults throughout life. Epidemiological studies indicate that the prenatal environment plays an important and decisive role in the development of allergy later in life. Since the underlying mechanisms were unclear, an animal model was developed to study the impact of maternal allergy on the development of an allergic immune response in early life. An allergic Th2 response was induced in pregnant mice by sensitization and aerosol allergen exposure. Both, IgG1 and IgG2a, but not IgE, Abs cross the placental barrier. Free allergen also crosses the placental area and was detected in serum and amniotic fluids of neonatal F(1) mice. These F(1) mice demonstrated a suppressed Th1 response, as reflected by lowered frequencies and reduced levels of IFN-gamma production. Development of an IgE response against the same allergen was completely prevented early in life. This effect was mediated by diaplacental transfer of allergen-specific IgG1 Abs. In contrast, allergic sensitization against a different allergen early in life was accelerated in these mice. This effect was mediated by maternal CD4 and OVA-specific Th2 cells induced by allergic sensitization during pregnancy. These data indicate a critical role for maternal T and B cell response in shaping pre- and postnatal maturation of specific immunity to allergens.     

In vivo CD86 blockade inhibits CD4+ T cell activation, whereas CD80 blockade potentiates CD8+ T cell activation and CTL effector function

To address whether a functional dichotomy exists between CD80 and CD86 in naive T cell activation in vivo, we administered anti-CD80 or CD86 blocking mAb alone or in combination to mice with parent-into-F(1) graft-vs-host disease (GVHD). In this model, the injection of naive parental T cells into unirradiated F(1) mice results in either a Th1 cytokine-driven, cell-mediated immune response (acute GVHD) or a Th2 cytokine-driven, Ab-mediated response (chronic GVHD) in the same F(1) recipient. Combined CD80/CD86 blockade beginning at the time of donor cell transfer mimicked previous results seen with CTLA4Ig and completely abrogated either acute or chronic GVHD by preventing the activation and maturation of donor CD4(+) T cells as measured by a block in acquisition of memory marker phenotype and cytokine production. Similar results were seen with selective CD86 blockade; however, the degree of CD4 inhibition was always less than that seen with combined CD80/CD86 blockade. A more striking effect was seen with selective CD80 blockade in that chronic GVHD was converted to acute GVHD. This effect was associated with the induction of Th1 cytokine production, donor CD8(+) T cell activation, and development of antihost CTL. The similarity of this effect to that reported for selective CTLA4 blockade suggests that CD80 is a critical ligand for CTLA4 in mediating the down-regulation of Th1 responses and CD8(+) T cell activation. In contrast, CD86 is critical for the activation of naive CD4(+) T cells in either a Th1 or a Th2 cytokine-mediated response.     

Prevention of house dust mite induced allergic airways disease in mice through immune tolerance

Allergic airways disease is a consequence of a Th2 response to an allergen leading to a series of manifestations such as production of allergen-specific IgE, inflammatory infiltrates in the airways, and airway hyper-reactivity (AHR). Several strategies have been reported for tolerance induction to allergens leading to protection from allergic airways disease. We now show that CD4 blockade at the time of house dust mite sensitization induces antigen-specific tolerance in mice. Tolerance induction is robust enough to be effective in pre-sensitized animals, even in those where AHR was pre-established. Tolerant mice are protected from airways eosinophilia, Th2 lung infiltration, and AHR. Furthermore, anti-CD4 treated mice remain immune competent to mount immune responses, including Th2, to unrelated antigens. Our findings, therefore, describe a strategy for tolerance induction potentially applicable to other immunogenic proteins besides allergens.     

Allograft rejection by primed/memory CD8+ T cells is CD154 blockade resistant: therapeutic implications for sensitized transplant recipients

We have shown that CD8(+) CTLs are the key mediators of accelerated rejection, and that CD8(+) T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8(+) T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8(+) T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8(+) T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8(+) T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8(+) T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8(+) T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance.     

CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment

Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.     

Treatment of allograft recipients with donor-specific transfusion and anti-CD154 antibody leads to deletion of alloreactive CD8+ T cells and prolonged graft survival in a CTLA4-dependent manner

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanism of allograft survival, we determined the fate of tracer populations of alloreactive transgenic CD8+ T cells in a normal microenvironment. We observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state, in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive transgenic CD8+ T cells. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by costimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade of costimulation.     

Oral administration of ovalbumin after sensitization attenuates symptoms in a mouse model of food allergic enteropathy

Oral immunotherapy (OIT) is a promising treatment of food allergy. To administer an appropriate oral dose of an allergenic component as OIT to individuals sensitized with a food allergen may prevent inducing food allergic inflammation in them. So we attempted to establish a mouse model to evaluate efficacy for oral administration of food allergen after sensitization. In BALB/c mice sensitized by injecting ovalbumin (OVA) with alum twice, OVA was administered before inducing inflammation by feeding the mice with egg white (EW) diet. Severe inflammatory responses, such as enteropathy, weight loss, IL-4 production, and increase of IgE antibody levels, were suppressed by administration with 4 mg of OVA 7 times before feeding EW diet. OVA administration alone induced a slight Th2 response, but no symptoms. The current study demonstrated that severe food allergic enteropathy could be prevented by pre-administration with appropriate dose of OVA to sensitized mice.     

High-affinity IgE receptors on dendritic cells exacerbate Th2-dependent inflammation

The IgE-mediated and Th2-dependent late-phase reaction remains a mechanistically enigmatic and daunting element of human allergic inflammation. In this study, we uncover the FcεRI on dendritic cells (DCs) as a key in vivo component of this form of allergy. Because rodent, unlike human, DCs lack FcεRI, this mechanism could be revealed only by using a new transgenic mouse model with human-like FcεRI expression on DCs. In the presence of IgE and allergen, FcεRI(+) DCs instructed naive T cells to differentiate into Th2 cells in vitro and boosted allergen-specific Th2 responses and Th2-dependent eosinophilia at the site of allergen exposure in vivo. Thus, FcεRI on DCs drives the cascade of pathogenic reactions linking the initial allergen capture by IgE with subsequent Th2-dominated T cell responses and the development of late-phase allergic tissue inflammation.     

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7.

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.     

Antigen-specific T cell tolerance down-regulates mast cell responses in vivo

Fel d I is the major cat allergen that induces asthma and allergic rhinitis in humans. To investigate the mechanism of allergic responses to this allergen, a mouse model was developed. Mice sensitized to chain 1 of Fel d I exhibited T cell responses, B cell responses, and mast cell responses when challenged with the protein. Subcutaneous injections of peptides containing the dominant T cell epitopes of the allergen induced T cell tolerance in presensitized mice. When challenged with the allergen intratracheally, these tolerized mice produced a decreased amount of histamine in vivo. The decrease in histamine release was not solely dependent on the reduction of allergen-specific IgE. These data show that mast cell activity in mice with an ongoing sensitivity to allergen can be regulated through peptide-induced T cell tolerance.     

The CD28/CTLA-4-B7 signaling pathway is involved in both allergic sensitization and tolerance induction to orally administered peanut proteins

Dendritic cells are believed to play an essential role in regulating the balance between immunogenic and tolerogenic responses to mucosal Ags by controlling T cell differentiation and activation via costimulatory and coinhibitory signals. The CD28/CTLA-4-CD80/CD86 signaling pathway appears to be one of the most important regulators of T cell responses but its exact role in responses to orally administered proteins remains to be elucidated. In the present study, the involvement of the CD28/CTLA-4-CD80/CD86 costimulatory pathway in the induction of allergic sensitization and oral tolerance to peanut proteins was investigated. In both an established C3H/HeOuJ mouse model of peanut hypersensitivity and an oral tolerance model to peanut, CD28/CTLA-4-CD80/CD86 interactions were blocked using the fusion protein CTLA-4Ig. To examine the relative contribution of CD80- and CD86-mediated costimulation in these models, anti-CD80 and anti-CD86 blocking Abs were used. In the hypersensitivity model, CTLA-4Ig treatment prevented the development of peanut extract-induced cytokine responses, peanut extract-specific IgG1, IgG2a, and IgE production and peanut extract-induced challenge responses. Blocking of CD80 reduced, whereas anti-CD86 treatment completely inhibited, the induction of peanut extract-specific IgE. Normal tolerance induction to peanut extract was found following CTLA-4Ig, anti-CD86, or anti-CD80 plus anti-CD86 treatment, whereas blockade of CD80 impaired the induction of oral tolerance. We show that CD28/CTLA-4-CD80/CD86 signaling is essential for the development of allergic responses to peanut and that CD86 interaction is most important in inducing peanut extract-specific IgE responses. Additionally, our data suggest that CD80 but not CD86 interaction with CTLA-4 is crucial for the induction of low dose tolerance to peanut.     

Tissue localization and frequency of antigen-specific effector CD4 T cells determines the development of allergic airway inflammation

Previous activation of effector Th2 cells is central to the development of allergic inflammatory responses. We have observed that priming of allergen-specific Th2 cells in C57BL/6 or B10.A mice with allergen delivered via the i.p. or s.c. routes results in very different outcomes following subsequent airway exposure to the same allergen. Systemic allergen immunization (via the i.p. route) resulted in the formation of a lung-resident population of allergen-specific T cells, and mice developed severe allergic airway inflammation in response to inhaled allergen. The localization of cells to the lung did not require the presence of antigen at this site, but reflected a large pool of circulating activated allergen-specific T cells. In contrast, localized immunization (via the s.c. route) resulted in a small T-cell response restricted to the draining lymph node, and mice were not responsive to inhaled allergen. These data indicate that prior sensitization to an allergen alone was not sufficient for the induction of allergic inflammation; rather, responsiveness was largely determined by precursor frequency and tissue localization of the allergen-specific effector Th2 cells.     

IL-13 overexpression predisposes to anaphylaxis following antigen sensitization

Anaphylaxis represents an extreme form of allergic reaction. This acute-phase component of allergy and asthma is triggered by allergen-induced degranulation of mast cells following the cross-linking of cell surface-bound, allergen-specific IgE, resulting in the liberation of inflammatory mediators and the development of bronchoconstriction. We used IL-13 transgenic mice to investigate the role of this Th2 cell-derived cytokine in the onset of allergic disease. Strikingly, IL-13-transgenic mice were highly predisposed to fatal anaphylaxis following Ag sensitization. This response correlated with substantially elevated levels of circulating Ag-specific IgE, mast cell degranulation, and histamine release. Furthermore, allergen exposure also induced phenotypic changes typical of asthma, including pulmonary fibrosis, goblet cell hyperplasia, elevated Th2 cytokines, eosinophilia, and airways occluded by mucus and Charcot-Leyden crystals. Expression of IL-4 was not required for the induction of IgE-mediated responses. These data represent the first characterization of a functional role for IL-13-induced IgE in the generation of immediate hypersensitivity reactions and highlight the importance of IL-13 in the development of the symptoms of atopy. The systemic regulation of this response makes these mice an important resource for studying atopic responses.     

Control of memory CD4 T cell recall by the CD28/B7 costimulatory pathway

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62L(high)) and effector memory (T(EM); CD62L(low)) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-gamma production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-gamma production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly T(EM) in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on T(EM) responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of T(EM) cells in autoimmunity and chronic disease.     

A hypoallergenic hybrid molecule with increased immunogenicity consisting of derivatives of the major grass pollen allergens, Phl p 2 and Phl p 6

Allergen-specific immunotherapy is currently based on the administration of allergen extracts containing natural allergens. However, its broad application is limited by the poor quality of these extracts. Based on recombinant allergens, well-defined allergy vaccines for allergen-specific immunotherapy can be produced. Furthermore, they can be modified to reduce their allergenic activity and to avoid IgE-mediated side effects. Here, we demonstrate that the immunogenicity of two grass pollen-derived hypoallergenic allergen derivatives could be increased by engineering them as a single hybrid molecule. We used a hypoallergenic Phl p 2 mosaic, generated by fragmentation of the Phl p 2 sequence and reassembly of the resulting peptides in an altered order, and a truncated Phl p 6 allergen, to produce a hybrid protein. The hybrid retained the reduction of IgE reactivity and allergenic activity of its components as shown by ELISA and basophil activation assays. Immunization with the hybrid molecule demonstrated the increased immunogenicity of this molecule, leading to higher levels of allergen-specific IgG antibodies compared to the single components. These antibodies could inhibit patients' IgE binding to the wild-type allergens. Thus, the described strategy allows the development of safer and more efficacious vaccines for the treatment of grass pollen allergy.     

Toll-like receptor 4 is required for optimal development of Th2 immune responses: role of dendritic cells

LPS potently induces dendritic cell maturation and the production of proinflammatory cytokines, such as IL-12, by activation of Toll-like receptor 4 (TLR4). Since IL-12 is important for the generation and maintenance of Th1 responses and may also inhibit Th2 cell generation from naive CD4 T cell precursors, it has been inferred that TLR4 signaling would have similar effects via the induction of IL-12 secretion. Surprisingly, we found that TLR4-defective mice subjected to sensitization and pulmonary challenge with a protein allergen had reductions in airway inflammation with eosinophils, allergen-specific IgE levels, and Th2 cytokine production, compared with wild-type mice. These reduced responses were attributable, at least in part, to decreased dendritic cell function: Dendritic cells from TLR4-defective mice expressed lower levels of CD86, a costimulatory molecule important for Th2 responses. They also induced less Th2 cytokine production by antigenically naive CD4 T cells in vitro and mediated diminished CD4 T cell Ag-specific pulmonary inflammation in vivo. These results indicate that TLR4 is required for optimal Th2 responses to Ags from nonpathogenic sources and suggest a role for TLR4 ligands, such as LPS derived from commensal bacteria or endogenously derived ligands, in maturation of the innate immune system before pathogen exposure.