Coupling of a Major Allergen to the Surface of Immune Cells for Use in Prophylactic Cell Therapy for the Prevention of IgE-Mediated Allergy.

Up to a third of the world's population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.

Resistance of parvalbumin to gastrointestinal digestion is required for profound and long-lasting prophylactic oral tolerance.

BACKGROUND: Early introduction of food allergens into children's diet is considered as a strategy for the prevention of food allergy. The major fish allergen parvalbumin exhibits high stability against gastrointestinal digestion. We investigated whether resistance of carp parvalbumin to digestion affects oral tolerance induction. METHODS: Natural Cyp c 1, nCyp c 1, and a gastrointestinal digestion-sensitive recombinant Cyp c 1 mutant, mCyp c 1, were analyzed for their ability to induce oral tolerance in a murine model. Both antigens were compared by gel filtration, circular dichroism measurement, in vitro digestion, and splenocyte proliferation assays using synthetic Cyp c 1-derived peptides. BALB/c mice were fed once with high doses of nCyp c 1 or mCyp c 1, before sensitization to nCyp c 1. Immunological tolerance was studied by measuring Cyp c 1-specific antibodies and cellular responses by ELISA, basophil activation, splenocyte proliferations, and intragastric allergen challenge. RESULTS: Wild-type and mCyp c 1 showed the same physicochemical properties and shared the same major T-cell epitope. However, mCyp c 1 was more sensitive to enzymatic digestion in vitro than nCyp c 1. A single high-dose oral administration of nCyp c 1 but not of mCyp c 1 induced long-term oral tolerance, characterized by lack of parvalbumin-specific antibody and cellular responses. Moreover, mCyp c 1-fed mice, but not nCyp c 1-fed mice developed allergic symptoms upon challenge with nCyp c 1. CONCLUSION: Sensitivity to digestion in the gastrointestinal tract influences the capacity of an allergen to induce prophylactic oral tolerance.

Belatacept/CTLA4Ig: an update and critical appraisal of preclinical and clinical results.

INTRODUCTION: The B7/CD28/CTLA4 signaling cascade is the most thoroughly studied costimulatory pathway and blockade with CTLA4Ig (abatacept) or its derivative belatacept has emerged as a valuable option for pharmacologic immune modulation. Several clinical studies have ultimately led to the approval of belatacept for immunosuppression in kidney transplant recipients. Areas covered: This review will discuss the immunological background of costimulation blockade and recent preclinical data and clinical results of CTLA4Ig/belatacept. Expert commentary: The development of belatacept is a major advance in clinical transplantation. However, in spite of promising results in preclinical and clinical trials, clinical use remains limited at present, in part due to increased rates of acute rejection. Recent efforts showing encouraging progress in refining such protocols might be a step toward harnessing the full potential of costimulation blockade-based immunosuppression.

A B Cell Epitope Peptide Derived from the Major Grass Pollen Allergen Phl p 1 Boosts Allergen-Specific Secondary Antibody Responses without Allergen-Specific T Cell Help.

More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope-derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope-derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines.

Cell Therapy for Prophylactic Tolerance in Immunoglobulin E-mediated Allergy.

BACKGROUND: Therapeutic strategies for the prophylaxis of IgE-mediated allergy remain an unmet medical need. Cell therapy is an emerging approach with high potential for preventing and treating immunological diseases. We aimed to develop a cell-based therapy inducing permanent allergen-specific immunological tolerance for preventing IgE-mediated allergy. METHODS: Wild-type mice were treated with allergen-expressing bone marrow cells under a short course of tolerogenic immunosuppression (mTOR inhibition and costimulation blockade). Bone marrow was retrieved from a novel transgenic mouse ubiquitously expressing the major grass pollen allergen Phl p 5 as a membrane-anchored protein (BALB/c-Tg[Phlp5-GFP], here mPhl p 5). After transplantation recipients were IgE-sensitized at multiple time points with Phl p 5 and control allergen. RESULTS: Mice treated with mPhl p 5 bone marrow did not develop Phl p 5-specific IgE (or other isotypes) despite repeated administration of the allergen, while mounting and maintaining a strong humoral response towards the control allergen. Notably, Phl p 5-specific T cell responses and allergic airway inflammation were also completely prevented. Interestingly allergen-specific B cell tolerance was maintained independent of Treg functions indicating deletional tolerance as underlying mechanism. CONCLUSION: This proof-of-concept study demonstrates that allergen-specific immunological tolerance preventing occurrence of allergy can be established through a cell-based therapy employing allergen-expressing leukocytes.

Belatacept: from rational design to clinical application.

Gradually improved immunosuppression has contributed significantly to the progress achieved in transplantation medicine so far. Nevertheless, current drug regimens are associated with late graft loss--in particular as a result of immunologic damage or drug toxicity--and substantial morbidity. Recently, the costimulation blocker belatacept (marketed under the name Nulojix®) has been approved for immunosuppression in renal transplantation. Belatacept (a mutated version of CTLA4Ig) is a fusion protein rationally designed to block CD28, a critical activating receptor on T cells, by binding and saturating its ligands B7-1 and B7-2. In phase II and III trials, belatacept was compared with cyclosporine (in combination with basiliximab, MMF, and steroids). Advantages observed with belatacept include superior graft function, preservation of renal structure and improved cardiovascular risk profile. Concerns associated with belatacept are a higher frequency of cellular rejection episodes and more post-transplant lymphoproliferative disorder (PTLD) cases especially in EBV seronegative patients, who should be excluded from belatacept-based regimens. Thus, after almost three decades of calcineurin inhibitors as mainstay of immunosuppression, belatacept offers a potential alternative. In this article, we will provide an overview of belatacept's preclinical development and will discuss the available evidence from clinical trials.

Expression of a major plant allergen as membrane-anchored and secreted protein in human cells with preserved T cell and B cell epitopes.

BACKGROUND: Expression of allergens in human cells is a prerequisite for the development of antigen-specific cell therapy in IgE-mediated allergy. We developed a strategy how the clinically relevant major grass pollen allergen Phl p 5 can be efficiently secreted or expressed on the surface of human cells with preserved allergenic activity. METHODS: The cDNA of Phl p 5 was fused to a leader peptide with or without a transmembrane domain and both constructs were ligated into a mammalian expression vector. Transfection of these plasmids into human cells resulted in a membrane-anchored or secreted version of Phl p 5, respectively, as determined by ELISA or flow cytometric analysis. RESULTS: Both the secreted and membrane-anchored Phl p 5 proteins bound IgE from allergic patients in an immunoblot assay and induced specific histamine release and CD203c upregulation in basophils of grass pollen-allergic patients. Proliferation of peripheral blood mononuclear cells from Phl p 5-allergic individuals was induced upon stimulation with both variants of Phl p 5 expressed in human cells similar to recombinant Phl p 5. CONCLUSIONS: Secreted and membrane-anchored Phl p 5 expressed in human cells preserved B cell as well as T cell epitopes and may be used to develop and test various cell-based strategies for allergen-specific immunomodulation and to delineate the tolerance mechanisms involved therein.

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.