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Allergy represents a hypersensitivity disease which affects more than 25% of the population in industrialized countries. The underlying type I allergic immune reaction occurs in predisposed atopic individuals in response to otherwise harmless antigens (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 (HSC) to express the major grass pollen allergen Phl p 5 on their cell membrane. Transplantation of these genetically modified HSC led to durable multi-lineage molecular chimerism and permanent immunological tolerance towards 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 non-responsiveness persisted throughout follow-up (40 weeks). 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.
The pathophysiological hallmark of type I allergy is the Th2 cell-driven production of IgE against otherwise harmless antigens (i.e. allergens) in predisposed atopic individuals. 1,2 IgE-mediated allergy manifests itself clinically either locally (e.g. as hay fever, allergic asthma, or food allergy) or systemically, as is the case in anaphylaxis. Although infrequent, anaphylaxis is an acute life-threatening condition (e.g. induced by food allergens or insect venoms). 3 Allergy is mainly being treated symptomatically by various drugs which are associated with considerable side-effects and cost.
Allergen-specific immunotherapy, whereby increasing doses of the sensitizing allergen are repeatedly administered in the form of crude extracts, is employed in selected patients and is currently the only allergen-specific treatment of allergy. 4 However, allergen-specific immunotherapy is associated with limited effectiveness and substantial risks, as exemplified by anaphylactic reactions or therapy-induced sensitization to additional allergens. 5 Today the molecular structure of most common allergens has been revealed and advanced experimental allergen-specific strategies have been developed. 6 They include the use of allergen-derived T cell epitope-containing peptides 7, genetically engineered allergens 5 and DNA-based forms of treatment. 8 Several experimental approaches for tolerance induction in allergy have also been explored but are characterized by limited robustness and relatively short-lived effects. 9-11 So far no robust allergen-specific tolerance approach has been reported that permanently prevents allergy.
It is one of the main features of the immune system to be tolerant towards self. 12 A major mechanism of self-tolerance is mediated by subpopulations of hematopoietic cells expressing self antigens. 13;14 This principle has been emulated in organ transplantation by introducing donor HSC into the recipient in a way to create a chimeric state in which recipient and donor bone marrow (BM) co-exist, thereby inducing tolerance towards donor (allo)antigens. 15;16 Alternatively, disease-associated antigen(s) can be introduced into an individual by transplanting autologous (i.e. in the experimental rodent setting syngeneic) HSC after they have been genetically modified in vitro to express the relevant antigen(s), leading to so-called molecular chimerism. 17 Where successful, regimens relying on hematopoietic chimerism are characterized by a state of antigen-specific tolerance that is particularly robust and long-lasting.
Molecular chimerism models have been used experimentally to tolerize an allogeneic immune response (using single MHC antigens), 18,19 a xenogeneic response (introducing the enzyme alpha (1,3) galactosyltransferase) 20 and selected autoimmune responses. 21,22 However, other studies failed to achieve tolerance in particular autoimmune disease models 23 and even enhanced the susceptibility for disease development. No studies attempting tolerization of the distinct allergic immune response through molecular chimerism have been reported so far.
We wanted to investigate whether the immune response of IgE-mediated allergy can be tolerized by transplantation of syngeneic HSC expressing an allergen.
Female BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany). All mice were housed under specific pathogen-free conditions and were used between 6 and 12 weeks of age. All experiments were approved by the local review board of the Medical University of Vienna, and were performed in accordance with national and international guidelines of laboratory animal care.
To generate membrane-anchored Phl p 5, full length Phl p 5 (accession number ×74735) was fused to a signal sequence and a transmembrane domain (TMD) (both pDisplay, Invitrogen, Carlsbad, CA) by overlapping PCR technique. 24 The original signal sequence of Phl p 5 was replaced by the murine immunoglobulin signal sequence pDisplay. Primer sequences are used as follows: leader peptide: 5′-GGCGCCATGGAGACAGACACACTCCTG-3′, 5′-GTAACCGAGATCGGCGTCACCAGTGGA-3′, Phl p 5: 5′-ACTGGTGACGCCGATCTCGGTTAC-3′, 5′-GCC CAC AGC GAC TTT GTA GCC ACC-3′, TMD: 5′-TACAAAGTCGCTGTGGGC-3′, 5′-GGCGGATCCTAACGTGGCTTCTTCTG-3′. PCR product was cloned into the retroviral vector pMMP NcoI and BamHI sites resulting in pMMP-Phl p 5-TM. The start codon was inserted with the Nco I site, the stop codon was inserted with the BamHI site. For virus production plasmids pMMP-Phl p 5-TM, pMD.G, encoding for VSV-G protein and pMLV, encoding for viral proteins gag and pol, were cotransfected using the calcium phosphate method 25 into 293 T cells resulting in VSV-Phl p 5-TM viruses. Viral supernatants were concentrated by ultracentrifugation (33620 g for 2h). Mock viruses were produced in the same manner using empty pMMP vector.
BALB/c donors were injected intraperitoneally with 5-fluorouracil (150mg/KG) 7 days before BM isolation. 26 Mice were sacrificed and BM was harvested from tibiae, femurs, humeri and pelvis. BM cells were cultured and transduced with VSV-Phl p 5 or mock transduced as described by Bagley et al 27 with a multiplicity of infection (MOI) of 3-5.
One day before BMT, recipients received 8 Gy total body irradiation (TBI) and a depleting dose of anti-CD8 (2.43; 0.5mg/mouse) and anti-CD4 (GK1.5; 0.5mg/mouse) monoclonal antibodies (mAb). On the day of reconstitution mice were transplanted with 2-4×106 transduced BM cells intravenously. After BMT mice received anti-CD40L mAb (MR1; 0.5mg/mouse). Anti-CD4, anti-CD8 and anti-CD40L were used as they were shown to enhance engraftment of transduced BM. 28 All mAb used in vivo were purchased from Bioexpress (New Lebanon, NH).
Purified recombinant (r) timothy grass pollen and birch pollen allergens (rPhl p 5, rBet v 1) were obtained from Biomay (Vienna, Austria). All groups of mice were immunized subcutaneously with 5 μg rPhl p 5 and 5μg rBet v 1 adsorbed to aluminumhydroxid (Alu-Gel-S, Serva, Ingelheim, Germany) as described previously. 29
Forty weeks after BMT, BM cells were harvested from primary recipients and transplanted into secondary BALB/c mice preconditioned like primary recipients (described above). Each secondary recipient received 3×107 BM cells harvested from one chimera.
Non-specific Fcγ receptor binding was blocked with mAb against mouse FcγII/III receptor (CD16/CD32). Phl p 5 polyclonal antiserum against full length rPhl p 5 was purified from rabbit serum (Charles River) by a protein G column (Pierce, Rockford, USA) according to the manufacturer's instructions. Polyclonal anti-Phl p 5 IgG was biotinylated and developed with phycoerythrin streptavidin. To detect Phl p 5+ expressing cells among various leukocyte lineages white blood cells were stained with FITC-conjugated antibodies against CD4, CD8, B220, Mac-1 and isotype controls (all antibodies from Pharmingen, San Diego, CA) and analyzed by flow cytometry. Propidium iodide staining was used to exclude dead cells. Two-color flow cytometric analysis was used to determine the percentage of Phl p 5-expressing cells of particular lineages. The percentage of Phl p 5+ cells (i.e. molecular chimerism) was calculated by subtracting control staining from quadrants containing Phl p 5+ and Phl p 5 negative cells expressing a particular lineage marker, and by dividing the net percentage of Phl p 5+ cells by the total net percentage of Phl p 5+ plus Phl p 5 negative cells of that lineage as described. 30 Mice were considered chimeric if they showed at least 1% Phl p 5 + cells within the myeloid lineage and at least one lymphoid lineage. An Epics XL-MCL flow cytometer (Beckman Coulter, IL Alliance, Vienna, Austria) was used for acquisition and EXPO32 ADC Software, Applied Cytometry Systems (West Sheffield, UK), was used for analysis of flow cytometric data.
To measure antigen-specific antibodies in the sera of immunized mice ELISAs were performed as described previously. 31 Plates were coated with rPhl p 5 (5μg/ml), sera were diluted 1:20 for IgE, 1:100 for IgM, IgA, IgG2a and IgG3 respectively and 1:500 for IgG1 and bound antibodies were detected with monoclonal rat anti-mouse IgM, IgG1, IgE, IgA, IgG2a and IgG3 antibodies (Pharmingen, San Diego, CA) diluted 1:1000 and a HRP-coupled goat anti-rat antiserum (Amersham, Biosciences, UK) diluted 1:2000. The substrate for HRP was ABTS (60mM/l citric acid, 77mM/l Na2HPO4 × 2H2O, 1.7mM/l ABTS [Sigma, St. Lois, MO.], 3mM/l H2O2).
Spleens were removed under aseptic conditions (week 29/40) and homogenized. Suspended splenocytes were plated into 96-well round-bottom plates at a concentration of 2×105 cells / well in triplicates and stimulated with concanavalin A (Con A; 0.5μg/well, Sigma), rPhl p 5 (2μg/well) and rBet v 1 (2μg/well). On day 5 cultures were pulsed with 0.5μCi/well [3H]thymidine (Amersham, Biosciences, UK) and harvested approximately 16 hours thereafter. The proliferative response was measured by scintillation counting. The stimulation index (SI) was calculated as the ratio of the mean proliferation after allergen stimulation and medium control values. 32
RBL-2H3 cell subline 33 was cultured as described previously 34, in RPMI 1640 medium (Biochrome AG, Berlin, Germany) containing 10 % fetal calf serum. 4×104 cells were plated in 96 well tissue culture plates (Greiner, Bio-One, Stuttgart, Germany), loaded with 1:50 diluted mouse sera and incubated for 2 hours at 37°C and 5 % CO2. Supernatants were removed and the cell layer was washed with 2x Tyrode's buffer (137 mM NaCl, 2.7 mM KCL, 0.5mM MgCl2, 1.8mM CaCl2, 0.4mM NaH2PO4, 5.6mM D-glucose, 12mM NaHCO3, 10mM HEPES and 0.1 % w/v BSA, pH 7.2). Preloaded cells were stimulated with rPhl p 5 or rBet v 1 (0.03μg per well) for 30 min. at 37°C. The supernatants were analyzed for ß-hexosaminidase activity by incubation with the substrate 80μM 4-methylumbelliferyl-N-acetyl-ß-D-glucosamide (Sigma-Aldrich, Vienna, Austria) in citrate buffer (0.1M, pH4.5) for 1 hour at 37°C. The reaction was stopped by addition of 100μl glycine buffer (0.2M glycine, 0.2M NaCl, pH 10.7) and the fluorescence was measured at λex: 360/λem: 465nm using a fluorescence microplate reader (Wallac, Perkin Elmer, Vienna, Austria). Results are reported as percentage of total ß-hexosaminidase released after addition of 1 % Triton X-100. Determinations were done in triplicates and are displayed as mean value + SD.
Thirty weeks after BMT mice were injected intravenously with 100μl of 0.5 % Evans blue (Sigma, St. Louis, MO). Subsequently, 30μl of rPhl p 5 and rBet v 1 (0.5μg/ml each, diluted in PBS) were injected intradermally into the shaved abdominal skin as described previously. 35 As positive control, the mast cell-degranulating compound 48/80 (20μg/ml, Sigma) was injected intradermally whereas PBS was injected as a negative control. Twenty minutes after injection, mice were sacrificed and the blue colour intensity of a positive skin reaction due to vascular permeability was compared with the individual positive and negative control on the inverted skin.
The reported p-values are results of Wilcoxon-Mann-Whitney U test and exact significances. SPSS statistical software system 14.0 was used for calculations. P-values ≤ 0.05 were considered statistically significant. Error bars indicate standard deviations (SD).
We generated a membrane-anchored fusion protein of full-length Phl p 5 (phleum pratense 5, timothy grass), 36 one of the most relevant respiratory allergens. The fusion gene was cloned into retroviral backbone pMMP 37 (Fig. 1A). Transient co-transfection of plasmids carrying viral structural proteins and Vesicular Stomatitis Virus G protein (VSV-G) envelope and pMMP-Phl p 5-TM into 293T cells resulted in VSV-Phl p 5-TM pseudotyped recombinant retroviruses. 27 BALB/c donors were treated with 5-fluorouracil, BM was isolated seven days later, cultured ex vivo and transduced with VSV-Phl p 5-TM retrovirus. Following transduction 35% and 55% of BM cells, respectively (in two independent experiments), expressed Phl p 5 on their membrane (Fig. 1B).
Transduced BM cells were transplanted into pre-conditioned BALB/c recipients (Fig. 2A). The percentage of cells expressing Phl p 5 among various leukocyte lineages (i.e. molecular chimerism) was determined in blood by flow cytometry at multiple time points post-BM transplantation (BMT). All mice transplanted with Phl p 5-transduced BM (n= 10) developed high levels of chimerism in all tested lineages (e.g. 11% Phl p 5+ B cells and 22% Phl p 5+ T cells, 25 weeks post-BMT). Multi-lineage chimerism persisted throughout follow-up (> 39 weeks) (Fig. 2B). Comparable levels of chimerism in recipients of Phl p 5-transduced BM were also found in spleen and BM at the time of sacrifice (data not shown). Recipients of mock-transduced BM did not show any detectable Phl p 5-expression on leukocytes (data not shown). Persistent multi-lineage chimerism beyond 39 weeks in recipients of Phl p 5-transduced BM is indicative of successful transduction and engraftment of HSC. 38 To directly test whether HSC had been successfully transduced with Phl p 5-integrating recombinant retroviruses we harvested BM cells from Phl p 5 chimeras 40 weeks post-BMT and transplanted them into myeloablatively irradiated secondary BALB/c recipients (n= 3). Multi-lineage chimerism was again detectable and persisted for the length of follow up (15 weeks after secondary BMT, Fig. 2C), demonstrating that HSC had indeed been transduced to express Phl p 5 and had successfully engrafted and survived in the primary recipients.
To assess whether tolerance was induced, we employed an established model in which Phl p 5-immunized BALB/c mice develop characteristics of clinical type I allergy such as production of allergen-specific IgE, other allergen-specific isotypes and IgE-mediated effector cell degranulation. 29;39 Following this protocol we sensitized BMT recipients through repeated immunization with the recombinant allergens Phl p 5 and an unrelated control allergen, the major birch pollen allergen Bet v 1 (Fig. 2A). No Phl p 5-specific IgE was detectable in sera of any of the Phl p 5 chimeric mice throughout follow-up as determined by ELISA (Fig. 3A). In contrast, Phl p 5 chimeric mice developed high levels of Bet v 1-specific IgE post-immunization (Fig. 3A). Recipients of mock-transduced BM (n= 3) and non-BMT immunized mice (i.e. naïve mice treated with the same immunization regimen but not receiving BMT, n= 10) developed high levels of IgE in response to both allergens. Likewise, Phl p 5-chimeras developed no Phl p 5-specific IgG1 whereas recipients of mock-transduced BM and non-BMT immunized mice showed high levels of Phl p 5-specific IgG1 upon repeated immunizations (Fig. 3B). Bet v 1-specific IgG1 levels were comparably high in all groups of mice. Similar results were obtained for allergen-specific IgA, IgG2a and IgG3 (Fig. 3C-E). Phl p 5-specific low affinity IgM could be detected in sera of Phl p 5 chimeras (Fig. 3F) reminiscent of natural autoantibodies directed against self-antigen. 40 Thus transplantation of Phl p 5-transduced BM led to specific tolerance towards the allergen at the B cell level, preventing the production of allergen-specific IgE and other high affinity isotypes.
In the course of an IgE-mediated allergic immune reaction, antigen-presenting cells induce activation and proliferation of allergen-specific T cells. 1;2 We therefore determined T cell responsiveness in in vitro proliferation assays by stimulating splenocytes isolated from recipients of Phl p 5-transduced BM with Phl p 5 and Bet v 1. The lymphocytes were isolated at the end of follow up from chimeric mice of two independent experiments 40 weeks and 29 weeks after BMT, respectively (Fig. 4A and B). Proliferation in response to Phl p 5 was reduced by 90% in recipients of Phl p 5-transduced BM cells compared to proliferation of splenocytes from non-BMT immunized mice (stimulation indices of 21 vs. 219, P= 0.006) (Fig. 4A). In contrast, the proliferation response to Bet v 1 was high both in Phl p 5 chimeras and non-BMT immunized controls (Fig. 4B). Thus recipients of Phl p 5-transduced BM showed allergen-specific T cell tolerance.
Cross-linking of IgE on tissue mast cells and basophils by allergens results in local release of inflammatory mediators (including histamine) which cause many symptoms of the acute phase of an allergic reaction. 41 We therefore analyzed effector cell function in vitro and in vivo. In a rat basophil leukemia (RBL) degranulation assay sera from Phl p 5 chimeras and control groups were loaded onto RBL-cells, and mediator release (ß-hexosaminidase as surrogate marker for histamine 33 was measured after challenge with allergen. In recipients of Phl p 5-transduced BM no mediator release was detectable in response to Phl p 5 while release occurred upon challenge with Bet v 1 (Fig. 5A and B). Non-BMT immunized mice and recipients of mock-transduced BM cells showed mediator release in response to both Phl p 5 and Bet v 1. To investigate anaphylactic activity of skin mast cells in vivo we measured allergen-specific immediate-type hypersensitivity responses by intradermal allergen challenge and Evans blue staining. No positive skin reaction was detectable after intradermal challenge with Phl p 5 in 6 of 7 tested Phl p 5 chimeric mice, while a positive reaction was visible in all mice upon Bet v 1 challenge (Fig. 6B). Non-BMT immunized mice showed positive skin reactions to both allergens (Fig. 6A). In contrast naïve BALB/c mice did not show any mast cell skin reaction upon allergen challenge (Fig. 6C). The results from these in vitro and in vivo assays reveal that recipients of Phl p 5-transduced BM developed allergen-specific tolerance at the effector cell level.
The data presented herein provide proof of concept that tolerance towards an allergen can be induced through transplantation of genetically modified BM. This novel approach for tolerizing a type I allergic immune response has two unique characteristics: permanence and robustness. Tolerance persisted for the length of follow up (40 weeks). As our data point to the successful engraftment of HSC transduced with the allergen, it appears safe to assume that molecular chimerism would persist for the physiological lifespan of the recipient and would continue to maintain tolerance. With this approach all relevant levels of a type I allergic immune response, namely T cells, B cells and effector cells, were rendered specifically tolerant towards the immunogenic grass pollen allergen used in these experiments for BM transduction. Notably, allergen-specific IgE, IgG subtypes and IgA remained undetectable throughout follow-up. Besides, T cell responses and effector cell responses towards the allergen could not be detected. A comparably complete degree of tolerance has to the best of our knowledge not been reported with other experimental or clinical approaches that have been employed for allergy treatment or prevention.
Allergen-specific immunotherapy, the only causative treatment of allergy currently available in the clinical setting, was suggested to lead to immunomodulation of T cell responses in large part through the induction of a Th1 shift and the generation of regulatory T cells. Additionally, the humoral response is affected by the induction of high levels of allergen-specific IgG (and other isotypes) which is then competing with allergen-specific IgE. 4 At the experimental level, dominant T cell epitope-containing polypeptides of three different allergens were administered intranasally in a murine mucosal tolerance approach. After subsequent sensitization with allergens, allergen-specific humoral and effector cell responses were merely reduced but not completely prevented. 10 Another recently published approach relied on the blockade of the ICOS-ICOS-ligand pathway which induced regulatory T cells and inhibited OVA-induced airway hyperreactivity, but OVA-specific IgE was still detectable. 9 A fusion protein consisting of an allergen and a truncated Fcγ1 portion which was designed for the purpose of immunomodulation was shown to inhibit allergen-induced basophil and mast cell degranulation by co-crosslinking of FcεRI and Fcγ receptors (follow-up ~6 weeks), but did not prevent antibody production. 11 Overall, the causative approaches described in the literature so far, as exemplified above, led to immunomodulation and reduction of an allergic reaction, but not to the complete, permanent absence of all relevant levels of an allergen-specific immune response. Molecular chimerism, in contrast, establishes such a state of complete tolerance towards an allergen. While detailed mechanistic studies are beyond the scope of this manuscript, we consider it likely that central tolerance plays a critical role in our model, as it does in all chimerism-based protocols 16. However, non-deletional mechanisms, in particular T regulatory cells, might also be of importance, as we have recently shown in an allogeneic mixed chimerism model 42;43.
Previously it had been shown that molecular chimerism can be used to tolerize allogeneic and xenogeneic immune responses, not only in rodents, but also in large animals. 20;27;44 This concept failed, however, in a specific autoimmune model for unclear reasons. 23 Thus, while molecular chimerism overall is a highly attractive tolerance approach, its effectiveness needs to be individually assessed for each particular immune response and regimens potentially need to be adapted accordingly. To the best of our knowledge, none of the reported molecular chimerism studies have investigated IgE responses and none have employed allergens.
Transplantation of retrovirally transduced BM has been able to correct life-threatening lymphoid and myeloid immunodeficiencies in the clinical setting 45;46, but was associated with serious side effects. 47 However, substantial advances in vector design are continuously being achieved so that safe vectors may some day become available. 47;48 Molecular chimerism relies on the transplantation of autologous BM modified to differ in a single antigen (or at most a small, limited number of antigens) thereby avoiding GVHD, one of the gravest risks associated with allogeneic BMT and cellular chimerism. Minimally toxic regimens for recipient conditioning have recently been developed for the experimental transplantation of allogeneic BM which could eventually also be employed for the molecular chimerism approach. 15,30,49,50 Besides, autologous and allogeneic BMT have become a therapeutic option in selected clinical cases of autoimmune disease and the range of indications for which BMT is a valid treatment is expected to increase substantially over the coming years. 51,52
According to advances made in the field of molecular allergen characterization a limited number (approx. 30) of major and clinically relevant allergens can be defined which cover the most relevant allergen sources 6. Using the recently described hybrid technology it should be possible to engineer a few hybrid molecules (5-6) which may be sufficient to tolerize populations against the most common allergen sources in certain areas 53.
The concept presented here provides a novel cell-based approach for tolerizing a type I allergic immune response through transplantation of genetically modified hematopoietic cells. As most relevant allergens have been cloned this approach may theoretically be used for the prevention of many common forms of allergy.
We thank Christian Lupinek for helpful assistance with statistical calculations.
1This work was supported by the Austrian Science Fund (FWF, F2310 to T.W. and FWF, F1815 to R.V.), and in part by the Christian Doppler Association and a research grant from Biomay.