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The Journal of Immunology, 2008, 180, 8168 -8175
Copyright © 2008 by The American Association of Immunologists, Inc.
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Tolerization of a Type I Allergic Immune Response through Transplantation of Genetically Modified Hematopoietic Stem Cells1

Ulrike Baranyi*, Birgit Linhart{dagger}, Nina Pilat*, Martina Gattringer*, Jessamyn Bagley{ddagger}, Ferdinand Muehlbacher*, John Iacomini{ddagger}, Rudolf Valenta2,{dagger} and Thomas Wekerle2,3,*

* Division of Transplantation, Department of Surgery, and {dagger} Division of Immunopathology, Department of Pathophysiology, Center of Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria; and {ddagger} Transplantation Research Center, Renal Division, Brigham and Women’s Hospital and Children’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The pathophysiological hallmark of type I allergy is the Th2 cell-driven production of IgE against otherwise harmless Ags (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 that 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 used 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 the 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, 10, 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 toward self (12). A major mechanism of self-tolerance is mediated by subpopulations of hematopoietic cells expressing self-Ags (13, 14). This principle has been emulated in organ transplantation by introducing donor hematopoietic stem cells (HSC)4 into the recipient in a way to create a chimeric state in which recipient and donor bone marrow (BM) coexist, thereby inducing tolerance toward donor (allo)-Ags (15, 16). Alternatively, disease-associated Ag(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 Ag(s), leading to so-called molecular chimerism (17). Where successful, regimens relying on hematopoietic chimerism are characterized by a state of Ag-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 Ags) (18, 19), a xenogeneic response (introducing the enzyme {alpha}-1,3-galactosyltransferase) (20), and selected autoimmune responses (21, 22). However, other studies have failed to achieve tolerance in particular autoimmune disease models (23) and have 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Female BALB/c mice were purchased from Charles River Laboratories. All mice were housed under specific pathogen-free conditions and were used between 6 and 12 wk 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.

Retroviral constructs and production of retroviruses

To generate membrane-anchored Phl p 5, full-length Phl p 5 (accession number X74735) was fused to a signal sequence and a transmembrane domain (both pDisplay, Invitrogen) by overlapping PCR technique (24). The original signal sequence of Phl p 5 was replaced by the murine Ig 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'; transmembrane domain: 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 NcoI site; the stop codon was inserted with the BamHI site. For virus production plasmids, pMMP-Phl p 5-TM, pMD.G, encoding for vesicular stomatitis virus (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 (33,620 x g for 2 h). Mock viruses were produced in the same manner using empty pMMP vector.

Retroviral transduction of BM cells

BALB/c donors were injected i.p. with 5-fluorouracil (150 mg/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 of 3–5.

BM transplantation (BMT)

One day before BMT, recipients received 8 Gy total body irradiation and a depleting dose of anti-CD8 (2.43; 0.5 mg/mouse) and anti-CD4 (GK1.5; 0.5 mg/mouse) mAbs. On the day of reconstitution mice were transplanted with 2–4 x 106 transduced BM cells i.v. After BMT mice received anti-CD40L mAb (MR1; 0.5 mg/mouse). Anti-CD4, anti-CD8, and anti-CD40L were used, as they were shown to enhance engraftment of transduced BM (28). All mAbs used in vivo were purchased from BioExpress.

Recombinant allergens and immunization of mice

Purified recombinant (r) timothy grass pollen and birch pollen allergens (rPhl p 5, rBet v 1) were obtained from BIOMAY. All groups of mice were immunized s.c. with 5 µg rPhl p 5 and 5 µg rBet v 1 adsorbed to aluminum hydroxide (Alu-Gel-S, Serva) as described previously (29).

Secondary BMT

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 x 107 BM cells harvested from one chimera.

Flow cytometric analysis

Nonspecific Fc{gamma} receptor binding was blocked with mAb against mouse Fc{gamma}II/III receptor (CD16/CD32). Phl p 5 polyclonal antiserum against full-length rPhl p 5 was purified from rabbit serum (Charles River Laboratories) by a protein G column (Pierce) according to the manufacturers’ instructions. Polyclonal anti-Phl p 5 IgG was biotinylated and developed with PE streptavidin. To detect Phl p 5+-expressing cells among various leukocyte lineages, white blood cells were stained with FITC-conjugated Abs against CD4, CD8, B220, Mac-1, and isotype controls (all Abs from BD Pharmingen) 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– 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– 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) was used for acquisition, and EXPO32 ADC software (Applied Cytometry Systems) was used for analysis of flow cytometric data.

ELISA

To measure Ag-specific Abs in the sera of immunized mice, ELISAs were performed as described previously (31). Plates were coated with rPhl p 5 (5 µg/ml), sera were diluted 1/20 for IgE, 1/100 for IgM, IgA, IgG2a, and IgG3, respectively, and 1/500 for IgG1, and bound Abs were detected with monoclonal rat anti-mouse IgM, IgG1, IgE, IgA, IgG2a, and IgG3 Abs (BD Pharmingen) diluted 1/1000 and a HRP-coupled goat anti-rat antiserum (Amersham Biosciences) diluted 1/2000. The substrate for HRP was ABTS (60 mM/L citric acid, 77 mM/L Na2HPO4 x 2H2O, 1.7 mM/L ABTS (Sigma-Aldrich), 3 mM/L H2O2).

Lymphocyte proliferation assay

Spleens were removed under aseptic conditions (weeks 29/40) and homogenized. Suspended splenocytes were plated into 96-well round-bottom plates at a concentration of 2 x 105 cells/well in triplicates and stimulated with Con A (0.5 µg/well, Sigma-Aldrich), rPhl p 5 (2 µg/well), and rBet v 1 (2 µg/well). On day 5 cultures were pulsed with 0.5 µCi/well [3H]thymidine (Amersham Biosciences) and harvested ~16 h 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, 35).

Rat basophil leukemia (RBL) cell degranulation assay

RBL-2H3 cell subline (33) was cultured as described previously (34) in RPMI 1640 medium (Biochrom) containing 10% FCS. Cells (4 x 104) were plated in 96-well tissue culture plates (Greiner Bio-One), loaded with 1/50 diluted mouse sera, and incubated for 2 h 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, 0.5 mM MgCl2, 1.8 mM CaCl2, 0.4 mM NaH2PO4, 5.6 mM D-glucose, 12 mM NaHCO3, 10 mM HEPES, and 0.1% (w/v) BSA (pH 7.2)). Preloaded cells were stimulated with rPhl p 5 or rBet v 1 (0.03 µg/well) for 30 min at 37°C. The supernatants were analyzed for β-hexosaminidase activity by incubation with the substrate 80 µM 4-methylumbelliferyl-N-acetyl-β-D-glucosamide (Sigma-Aldrich) in citrate buffer (0.1 M (pH 4.5)) for 1 h at 37°C. The reaction was stopped by addition of 100 µl glycine buffer (0.2 M glycine, 0.2 M NaCl (pH 10.)7) and the fluorescence was measured at {lambda}ex: 360/{lambda}em: 465 nm using a fluorescence microplate reader (PerkinElmer Wallac). Results are reported as percentage of total β-hexosaminidase released after addition of 1% Triton X-100. Determinations were done in triplicates and are displayed as mean values + SD.

Cutaneous type I hypersensitivity reaction

Thirty weeks after BMT, mice were injected i.v. with 100 µl of 0.5% Evans blue (Sigma-Aldrich). Subsequently, 30 µl of rPhl p 5 and rBet v 1 (0.5 µg/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 (20 µg/ml, Sigma-Aldrich) was injected intradermally whereas PBS was injected as a negative control. Twenty minutes after injection, mice were sacrificed and the blue color intensity of a positive skin reaction due to vascular permeability was compared with the individual positive and negative control on the inverted skin.

Statistical analysis

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. Values of p < 0.05 were considered statistically significant. Error bars indicate SDs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Membrane-anchored expression of an allergen on murine BM after retroviral transduction

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 cotransfection of plasmids carrying viral structural proteins and VSV-G protein envelope and pMMP-Phl p 5-TM into 293 T cells resulted in VSV-Phl p 5-TM pseudotyped recombinant retroviruses (27). BALB/c donors were treated with 5-fluorouracil, and BM was isolated 7 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).


Figure 1
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  		FIGURE 1. Efficient retroviral transduction of BM with membrane-bound allergen. A, Schematic representation of the MMP-Phl p 5-TM retroviral construct. Phl p 5 was fused to a signal peptide (S) and a transmembrane domain (TM). LTR represents long terminal repeats; SD and SA, splicing donor and splicing acceptor. The drawing is not to scale. B, Histogram depicting flow-cytometric analysis of Phl p 5 expression on the surface of VSV-Phl p 5-transduced BM immediately after transduction (solid line). Dashed line represents mock-transduced BM. One of two similar experiments is shown.

 
Long-term molecular chimerism after transplantation of allergen-transduced HSC

Transduced BM cells were transplanted into preconditioned 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 after 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 wk post-BMT). Multilineage chimerism persisted throughout follow-up (>39 wk) (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 multilineage chimerism beyond 39 wk 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 wk post-BMT and transplanted them into myeloablatively irradiated secondary BALB/c recipients (n = 3). Multilineage chimerism was again detectable and persisted for the length of follow-up (15 wk 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.


Figure 2
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  		FIGURE 2. Transplantation of syngeneic HSC retrovirally transduced to express Phl p 5 leads to high levels of permanent multilineage molecular chimerism. A, Schematic drawing of the experimental in vivo protocol for the transplantation of Phl p 5-transduced BM. Preconditioned BALB/c mice received 2–4 x 106 transduced BM cells. Mice were repeatedly immunized s.c. with rPhl p 5 and Bet v 1 at weeks 6, 9, 12, and 22. At the end of follow-up, BM of chimeras was isolated and transplanted into secondary preconditioned recipients. B, Percentages of Phl p 5+ cells among various leukocyte lineages were determined in blood by two-color flow cytometry in recipients of Phl p 5-transduced BM (n = 3) at multiple time points and are presented as means. Results from one of two independent similar experiments are shown. C, In secondary recipients the molecular chimerism within the Mac-1+ and B220+ populations was analyzed in peripheral blood 15 wk after secondary BMT (right panels). A representative plot of one secondary recipient is shown. Left panels show white blood cells of a naive BALB/c mouse as control.

 
Specific absence of Phl p 5-specific humoral responses in recipients of Phl p 5-transduced BM

To assess whether tolerance was induced, we used 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 postimmunization (Fig. 3A). Recipients of mock-transduced BM (n = 3) and non-BMT immunized mice (i.e., naive 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-Ag (40). Thus, transplantation of Phl p 5-transduced BM led to specific tolerance toward the allergen at the B cell level, preventing the production of allergen-specific IgE and other high-affinity isotypes.


Figure 3
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  		FIGURE 3. Recipients of Phl p 5-transduced BM are specifically tolerant toward Phl p 5 at the B cell level. Allergen-specific Ab levels in sera of recipients of Phl p 5-transduced BM (n = 10), mock-transduced BM (n = 3), and non-BMT immunized mice (n = 10) were analyzed by ELISA at the indicated time points. Mean Ab levels (+SD) are shown for each group. Dotted horizontal lines represent the baseline preimmune (pre imm) allergen-specific Ab level (collected 6 wk after BMT). A, Phl p 5-specific and Bet v 1-specific IgE levels; B, Phl p 5-specific and Bet v 1-specific IgG1 level; C–F, Phl p and Bet v 1-specific Ab levels IgA, IgG2a, IgG3, and IgM at the indicated time points. Values of p for recipients of Phl p 5-transduced BM vs recipients of mock-transduced BM are shown. Results are from pooled data of two independent experiments.

 
Specific T cell unresponsiveness in recipients of Phl p 5-transduced BM

In the course of an IgE-mediated allergic immune reaction, APCs 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 wk and 29 wk after BMT, respectively (Fig. 4). Proliferation in response to Phl p 5 was reduced by 90% in recipients of Phl p 5-transduced BM cells compared with 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.


Figure 4
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  		FIGURE 4. Recipients of Phl p 5-transduced BM are specifically tolerant toward the Phl p 5 at the T cell level. Results from in vitro allergen-specific proliferation assays performed at the time of sacrifice (weeks 29/40). Splenocytes of naive age-matched mice (n = 5), recipients of Phl p 5-transduced BM (n = 4), and non-BMT immunized mice (n = 7) were stimulated with rPhl p 5 (A) and rBet v 1 (B). Columns represent the mean stimulation indices (SI + SD) from two independent experiments.

 
IgE-mediated degranulation of basophils and mast cells is specifically abolished in recipients of Phl p 5-transduced BM

Cross-linking of IgE on tissue mast cells and basophils by allergens results in local release of inflammatory mediators (including histamine) that cause many symptoms of the acute phase of an allergic reaction (41). We therefore analyzed effector cell function in vitro and in vivo. In an RBL degranulation assay, sera from Phl p 5 chimeras and control groups were loaded onto RBL cells, and mediator release (β-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 whereas release occurred upon challenge with Bet v 1 (Fig. 5). 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, whereas 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, naive 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.


Figure 5
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  		FIGURE 5. Recipients of Phl p 5-transduced BM are specifically tolerant toward Phl p 5 at the effector cell level as determined in vitro. Results are from in vitro mediator release assays. RBL cells were loaded with sera collected at the indicated time points from Phl p 5 chimeras (n = 10), recipients of mock-transduced BM (n = 3), and non-BMT immunized mice (n = 10). Loaded cells were incubated with rPhl p 5 (A) or rBet v 1 (B). The mean percentages of allergen-specific β-hexosaminidase release (+SD) are shown. p values for recipients of Phl p 5-transduced vs recipients of mock-transduced BM are shown. Pre imm, preimmune.

 

Figure 6
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  		FIGURE 6. Recipients of Phl p 5-transduced BM are specifically tolerant toward the Phl p 5 at the effector cell level as determined by type I allergen-specific skin responses in vivo. Mice were injected i.v. with Evans blue dye and subsequently rPhl p 5 and rBet v 1 were injected intradermally into abdominal skin. Injection of the mast cell-degranulating compound 48/80 resulted in blue staining (positive control), whereas PBS resulted in no staining (negative control). The blue color intensity of the allergen-specific response was compared with the individual positive and negative controls. Reactions were assessed on the inverted abdominal skin. D, Scheme of intradermal injection. Representative skin sections of naive mice (n = 4) (C), non-BMT immunized mice (n = 5) (A), and Phl p 5 chimeras (n = 7) (B) are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The data presented herein provide "proof-of-concept" that tolerance toward 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 wk). 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 toward 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 toward 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 used 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 Fc{gamma}1 portion that was designed for the purpose of immunomodulation was shown to inhibit allergen-induced basophil and mast cell degranulation by co-crosslinking of Fc{epsilon}RI and Fc{gamma} receptors (follow-up ~6 wk), but it did not prevent Ab 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 toward an allergen. Although detailed mechanistic studies are beyond the scope of this paper, we consider it likely that central tolerance plays a critical role in our model, as it does in all chimerism-based protocols (16). However, nondeletional 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 has investigated IgE responses and none has used allergens.

Transplantation of retrovirally transduced BM has been able to correct life-threatening lymphoid and myeloid immunodeficiencies in the clinical setting (45, 46), but it 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 Ag (or at most a small, limited number of Ags), thereby avoiding graft-vs-host disease, 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 that could eventually also be used 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 during the coming years (51, 52).

According to advances made in the field of molecular allergen characterization, a limited number (~30) of major and clinically relevant allergens can be defined that 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) that may be sufficient to tolerize populations against the most common allergen sources in certain areas (53).

The concept presented herein 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.


   Acknowledgment
 
We thank Christian Lupinek for helpful assistance with statistical calculations.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
R.V. is consultant for Phadia, Uppsala, Sweden and Biomay, Vienna Austria; research support to T.W. and R.V. from Biomay; patent application No. 07450104.0 - Europe.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung, F2310 to T.W. and Fonds zur Förderung der Wissenschaftlichen Forschung, F1815 to R.V.) and in part by the Christian Doppler Association and a research grant from BIOMAY. Back

2 R.V. and T.W. are cosenior authors. Back

3 Address correspondence and reprint requests to Dr. Thomas Wekerle, Division of Transplantation, Department of Surgery, Vienna General Hospital, Waehringer Guertel 18, 1090 Vienna, Austria. E-mail address: Thomas.Wekerle{at}meduniwien.ac.at Back

4 Abbreviations used in this paper: HSC, hematopoietic stem cell; BM, bone marrow; BMT, BM transplantation; r, recombinant; RBL, rat basophil leukemia; SI, stimulation index; VSV, vesicular stomatitis virus. Back

Received for publication December 21, 2007. Accepted for publication April 1, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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