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Human TCR Transgenic Bet v 1-Specific Th1 Cells Suppress the Effector Function of Bet v 1-Specific Th2 Cells

Alina Neunkirchner, Victoria M. Leb-Reichl, Klaus G. Schmetterer, Sonja Mutschlechner, Hans J. Kueng, Daniela Haiderer, Karina Schuch, Michael Wallner, Beatrice Jahn-Schmid, Barbara Bohle and Winfried F. Pickl
J Immunol October 15, 2011, 187 (8) 4077-4087; DOI: https://doi.org/10.4049/jimmunol.1003220
Alina Neunkirchner
*Christian Doppler Laboratory for Immunomodulation, Vienna A-1090, Austria;
†Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Victoria M. Leb-Reichl
*Christian Doppler Laboratory for Immunomodulation, Vienna A-1090, Austria;
†Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Klaus G. Schmetterer
†Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Sonja Mutschlechner
*Christian Doppler Laboratory for Immunomodulation, Vienna A-1090, Austria;
‡Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Hans J. Kueng
†Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Daniela Haiderer
†Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Karina Schuch
*Christian Doppler Laboratory for Immunomodulation, Vienna A-1090, Austria;
†Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Michael Wallner
§Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Salzburg A-5020, Austria; and
¶Department of Molecular Biology, University of Salzburg, Salzburg A-5020, Austria
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Beatrice Jahn-Schmid
‡Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Barbara Bohle
*Christian Doppler Laboratory for Immunomodulation, Vienna A-1090, Austria;
‡Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Winfried F. Pickl
*Christian Doppler Laboratory for Immunomodulation, Vienna A-1090, Austria;
†Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria;
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Abstract

Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1142–153 plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αβ TCR specific for the immunodominant epitope Bet v 1142–153. cDNAs encoding TCR α- and β-chains were amplified from a Bet v 1142–153-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1142–153 peptide or coexpressing invariant chain::Bet v 1142–153 fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1142–153-presenting but not Bet v 14–15-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.

Birch pollen is one of the main causes of pollinosis in the Northern Hemisphere from spring to early summer (1). More than 95% of birch pollen-allergic individuals mount an IgE Ab response against Bet v 1, the major birch pollen allergen (2). T cells from these patients preferentially recognize the immunodominant epitope Bet v 1142–153. This region is located at the highly conserved C terminus of Bet v 1 and shares considerable homology with related food and pollen allergens (3). Recent studies have demonstrated that various Bet v 1 peptides bind with high affinity to different HLA-DR molecules (4). The structures targeted by both the humoral and cellular immune responses to Bet v 1 are sufficiently characterized at the molecular level to allow the development of knowledge-based therapies for treatment of birch pollen atopic disorders (3, 5).

Individuals allergic to birch pollen frequently develop type I hypersensitivity reactions against certain foods; for example, apple, celery, carrot, or hazelnut (6, 7). Ingestion of these foods causes the oral allergy syndrome, such as an itching, burning, or tingling sensation of the lips, mouth, or pharynx (8, 9). The molecular basis for these symptoms is Bet v 1-specific IgE Abs, which cross-react with tertiary structural determinants on homologous proteins within these foods (10, 11). Moreover, Bet v 1-specific T cells are also efficiently activated by Bet v 1-homologous food allergens (3). But, in marked contrast to IgE-mediated effects, T cell cross-reactivity is not abolished by cooking or by digestion of birch pollen-related food allergens (12) because these processes do not destroy T cell epitopes (13). Thus, ingestion of pollen-related food allergens can lead to aggravation of T cell-mediated immunopathology, such as exacerbation of eczema in patients with atopic dermatitis (14).

In the past, experimental tools have been devised that allow exploration of T cell responses in more detail. For instance, the Ag specificity of T cells can be conveniently transferred to other T cells by αβ TCR gene transfer (15–17). How might allergy research benefit from recombinant TCRs recognizing major allergens? The answers are severalfold: first, recombinant allergen-specific TCRs should allow the generation of large numbers of specific T cells in a short period of time independent of the allergic status of the donor and the allergy season; second, the availability of well-defined allergen-specific T cells favors the creation of biological model systems in which all three components of the allergen-specific synapse (HLA molecule, allergen peptide, and TCR) are well defined; and third, the recombinant TCR technology should allow engineering of allergen-specific T cells with immunomodulatory capacity (e.g., for adoptive transfer to modulate an existing Th2-dominated allergen-specific response).

Initial steps toward the realization of these objectives have been made possible by the transfer of a human allergen (Art v 1)-specific TCR into human T lymphocytes (18). The resulting TCR transgenic (TCRtg) T cells strictly responded according to the two-signal model described by Bretscher and Cohn (18, 19). In the absence of costimuli, TCRtg T cells receiving solely allergen-specific signal 1 developed a state of unresponsiveness (20). These studies have afforded a rational strategy for anergizing allergen-specific CD4+ T lymphocytes.

In the current study, we characterized a Bet v 1-specific TCR that recognizes the immunodominant epitope Bet v 1142–153 (3). For this purpose, we isolated cDNA encoding the α- and β-chains of the TCR of a Bet v 1-specific T cell clone (TCC), transferred the TCR chains into Jurkat T cells and peripheral blood (PB) T cells of nonallergic individuals, and studied T cell function upon coculture with APCs presenting allergen plus various combinations of costimulators. Furthermore, we analyzed whether the transferred TCR retained its original fine specificity (i.e., the capacity to cross-react with Bet v 1-related food and pollen allergens). Finally, by combining recently developed APC technologies, we asked whether strictly Th1-polarized TCRtg Bet v 1-specific PB T cells could be conveniently generated and whether such T cells would be able to modulate the effector function of Th2-differentiated cells.

Materials and Methods

Cell lines and primary cells

293 cells and Jurkat TCC 41-19 expressing an IL-2 enhancer/promoter driving luciferase were cultured as described (21). Bet v 1-specific TCCs and immortalized B cells were established from PBMCs of birch pollen-allergic individuals with typical clinical history and positive skin-prick tests to birch pollen extract as described (22). TCR gene usage was determined by RT-PCR with TRAV and TRBV primers (Clontech, Heidelberg, Germany) as described (23). PBMCs from allergic and nonallergic individuals were obtained upon informed consent in accordance with institutional ethics guidelines.

Recombinant allergens

Recombinant allergens (Api g 1.0101; Bet v 1.0101; Dau c 1.0103; Cor a 1.0103; Mal d 1.0108) were purchased from Biomay AG (Vienna, Austria) or produced and purified to homogeneity [Car b 1.0109 (EU283857); Cas s 1.0101 (FJ390843.1); Fag s 1.0101 (FJ390846.1); Que a 1.0301 (EU283863); accession numbers in parentheses were submitted to GenBank] as described elsewhere (24).

Cloning and characterization of αβ TCRs

mRNA from the Bet v 1142–153-specific HLA-DRB1*07:01–restricted, Th2-like TCC SD334 (3) was amplified with TRAV6.01-forward 5′-CGCGGGAAGCTTGCCACCATGGAGTCATTCCTGGGAGGTGT-3′ and TRAC-reverse 5′-CCCGCGGCGGCCGCTTTAGCTGGACCACAGCCGC-3′ or TRBV20.01-forward 5′-CGCGGGAAGCTTGCCACCATGCTGCTGCTTCTGCTGCTT-3′ and TRBC-reverse 5′-CCCGCGGCGGCCGCTTTAGAAATCCTTTCTCTTGACCATG-3′, digested with NcoI (bold) or HindIII (italics) and NotI (underlined), and gel purified. DNA fragments were ligated into the retroviral expression vector pMMP412 (R.C. Mulligan, Children’s Hospital, Boston, MA) and sequenced (VBC Genomics, Vienna, Austria). [Note: HLA nomenclature in this article is given according to the 2010 update and using colons (:) in the allele names to act as delimiters of the separate fields according to Marsh et al. (25).]

Cloning of HLA-DR constructs

mRNA from autologous EBV-immortalized B cells was amplified with HLA-DRB1*07:01-forward 5′-CGCGGGAAGCTTGCCACCATGGTGTGTCTGAAGCTCCC-3′ and HLA-DRB1*07:01-reverse 5′-CCCGCGGCGGCCGCTTTAGCTCAGGAATCCTGTTGGCT-3′, digested with HindIII (bold) and NotI (underlined). Gel-purified DNA fragments were ligated into pEAK12 expression vector (EdgeBio, Gaithersburg, MD) and sequenced.

Generation of Bet v 1-containing invariant chain constructs

pcDNA1.1/Amp containing an invariant chain (Ii) with an SfuI–Eco47III cassette instead of CLIP was kindly provided by J. van Bergen (Department of Immunohematology and Blood Transfusion, Leiden, The Netherlands) (26). The following double-stranded oligonucleotides were inserted into SfuI/Eco47III digested pcDNA1.1/Amp-Ii: Bet v 14–15-upper 5′-CGAACTACGAGACCGAGACCACCAGCGTGATCCCCGC-3′, Bet v 14–15-lower 5′-GCGGGGATCACGCTGGTGGTCTCGGTCTCGTAGTT-3′; Bet v 1112–123-upper 5′-CGTCCATCCTGAAGATCTCCAACAAGTACCACACCAA-3′, Bet v 1112–123-lower 5′-TTGGTGTGGTACTTGTTGGAGATCTTCAGGATGGA-3′; Bet v 1142–153-upper 5′-CGACCCTGCTGCGCGCCGTGGAGTCCTACCTGCTGGC-3′, Bet v 1142–153-lower 5′-GCCAGCAGGTAGGACTCCACGGCGCGCAGCAGGGT-3′. Ii containing CLIP or HA309–317 was constructed as described (26). Proper DNA insertion was confirmed by sequence analysis (VBC Genomics).

Transduction of αβ TCRs into Jurkat and PB T cells

Bet v 1142–153-specific TCR constructs in pMMP412.TRAV6 and pMMP412.TRBV20 or Art v 125–36-specific TCR constructs pMMP412.TRAV17 and pMMP412.TRBV18 were transduced into Jurkat T cells, bulk or sorted naive CD4+CD45RA+ PB T cells (Miltenyi, Bergisch Gladbach, Germany; purity of sorted T cells >98%) as described previously (18). In some experiments, a multicistronic strategy was used to express the Bet v 1-specific TCR, linking TRAV6 and TRBV20 via a P2A sequence in an IRES-GFP–containing version of pMMP. TCRtg Jurkat clones were obtained by limiting dilution. (The nucleotide sequences for the Bet v 1142–153-specific TCR α and β chains have been deposited in the GenBank database under accession numbers GQ179994 and GQ179995, respectively; http://www.ncbi.nlm.nih.gov/genbank/.)

Immunofluorescence analyses and intracellular cytokine determination

mAbs CD3–FITC (UCHT1), pan TCRαβ–PE (BMA031), CD4–FITC (VIT4), CD8–PE (VIT8b), and control Ig FITC and PE were obtained from An der Grub (Kaumberg, Austria). TCRβ-chain–specific mAbs TRBV12–FITC (56C5) and TRBV20–PE (MPB2D5) were from Immunotech (Marseille, France), and CD45RA (MEM-56) was from Invitrogen (Carlsbad, CA). HLA class II–FITC (1/47) and Ii–FITC (5-329) were kindly provided by O. Majdic (Institute of Immunology, Medical University of Vienna, Vienna, Austria). Staining and flow cytometry was performed as described (18). Intracellular cytokine production was analyzed after pretreatment for 6 h with GolgiStop (1:1500; Becton Dickinson, Palo Alto, CA) using Fix and Perm (An der Grub) and using Abs specific for IFN-γ (25723-PerCP; R&D Systems, Minneapolis, MN), IL-2 (MQ1-17H12-allophycocyanin; eBioscience, San Diego, CA), IL-4 (MP4–25D2-PE; Invitrogen, Camarillo, CA), IL-5 (JES1-39D10-PE; BioLegend, San Diego, CA), IL-10 (JES3-9D7; eBioscience), and IL-13 (JES10-5A2-allophycocyanin; BioLegend).

Generation of artificial APCs

293 cells (3 × 106) were transiently cotransfected with CD80::GPI, CD54::GPI, cathepsin S, HLA-DRA*01:01, and either HLA-DRB1*07:01 or HLA-DRB1*01:01 in combinations as indicated. Alternatively, artificial Ag-presenting cells (aAPCs) were transfected in addition with Ii–allergen fusion constructs Ii::Bet v 14–15, Ii::Bet v 1112–123, Ii::Bet v 1142–153, or Ii::Art v 125–34 and used 72 h after transfection. For polarization experiments, aAPCs were variably transfected with single-chain IL-12::GPI. IL-12::GPI had been constructed from pORF–hIL-12 (Invivogen, San Diego, CA) with primers IL-12 forward 5′-GCGAAGGAGGGCCACCATGGGTC-3′ and IL-12 reverse 5′-CCCGCGGCTAGCGGAGCCGCCGCCGCCGCTGCCGCCGCCGCCGCTGCCGCCGCTGGAGGAAGCATTCAGATAGCTCATC-3′ followed by digestion with NcoI (bold) and NheI (underlined) and ligation together with a HindIII–NcoI linker into pEAK12_CD80::GPI (21).

Generation of monocyte-derived dendritic cells

Monocyte-derived dendritic cells (MDDCs) were differentiated as described previously (27) and incubated at 2 × 105/well in 96-well flat-bottom culture plates with allergens (range, 0.01 × 10−6 to 9.4 × 10−6 M) in a total volume of 100 μl at 37°C overnight. Subsequently, TCRtg Jurkat T cells (1 × 105/well) were added and cocultured (total volume 200 μl) for 6 h.

Jurkat IL-2 promoter activity assays

aAPCs expressing the earlier described Ii constructs or aAPCs preincubated with peptides (3 × 10−5 M) Bet v 14–15, Bet v 1112–123, Bet v 1142–153, Art v 125–36, or medium alone (control) for 3 h were generated. Subsequently, Bet v 1142–153-specific or Art v 125–36-specific TCRtg Jurkat cells (1 × 105) were added to 5 × 104 of the above-described aAPCs and cocultured for 6 h. PMA (10−7 M) plus PHA (5 μg/ml) served as positive and medium alone as negative control. Luciferase activity was assayed as described (21). Stimulation of Jurkat T cells with MDDCs followed an analogous protocol.

T cell proliferation and Th1 polarization

TCRtg T cells (5 × 104; starting population bulk PB T cells or naive CD4+CD45RA+ T cells) were incubated with 5 × 104 irradiated (60 Gy) aAPCs expressing recombinant immunomodulatory molecules in 96-well flat-bottom plates for 3 d. After 24 to 72 h, cell culture supernatants were harvested and concentrations of IL-2, IL-4, IL-5, IL-10, IL-13, and IFN-γ determined by multiplex analysis (20). Duplicate plates were pulsed after 72 h with methyl-[3H]thymidine (1 μCi/well) for 18 h and processed as described (20). Medium alone or anti-CD3/CD28 microbeads (5 × 104) served as controls. Proliferation assays with allergen-specific TCC and Bet v 1-related food and pollen allergens were performed as described (3). Th1-polarized cells were generated by coculture with irradiated (60 Gy) allergen-specific aAPCs coexpressing IL-12::GPI for 24 h, harvested, washed extensively, and cytometrically sorted for expression of GFP cotransduced with the recombinant TCR.

Inhibition of effector function of Th2-differentiated T cells

For the generation of TCRtg T cells, CD4+CD45RA+ PB T cells of allergic and nonallergic individuals were isolated, preactivated for 2 d with CD3/CD28-coated microbeads (ratio 1:2 of beads/cells), followed by transduction with the allergen-specific TCR as described. For Th1 differentiation, T cells were supplemented with IL-2 (100 U/ml; Peprotech) throughout the 9-d culture period. Final Th1 polarization was achieved by coculture of these T cells with irradiated (60 Gy) allergen-specific aAPCs coexpressing IL-12::GPI for 24 h. Th2 differentiation was achieved by supplementing T cells with IL-2 (10 ng/ml; Peprotech), IL-4 (50 ng/ml; Peprotech), anti–IFN-γ mAb (10 μg/ml; R&D Systems, Minneapolis, MN), and anti–IL-12 mAb (3 μg/ml; Ustekinumab; Jansen-Cilag, Titusville, NJ) for the 10-d culture period. After harvesting and extensive washing, Th2-differentiated cells (0.5 × 105/well) and Th1-polarized cells (0.5 × 105 to 1.5 × 105/well) were either cultured alone or in combination with allergen-specific or allergen-nonspecific aAPCs coexpressing CD80 (5 × 104/well, irradiated) in 96-well flat-bottom culture dishes in triplicate for up to 72 h. At various times, supernatants were harvested and subjected to multiplex cytokine analyses. After 4 d, cellular proliferation was monitored by pulsing with methyl-[3H]thymidine (1 μCi/well) for the last 18 h of cultivation.

Statistical analyses

For multiple group comparisons, a linear mixed-model ANOVA was performed using SPSS software (IBM SPSS, Chicago, IL). For comparison of more than three groups, a Bonferroni correction was applied. Comparison between two groups was performed using the Student t test. Statistically significant values are denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

Results

Molecular cloning of the αβ TCR of a Bet v 1142–153-specific, HLA-DRB1*07:01–restricted TCC

A Bet v 1142-153-specific CD4+ TCC (SD334) was isolated from PB of a birch pollen-allergic individual (HLA-DRB1*07, *15; DRB4*01; DRB5*01; DQB1*02, *06). This clone specifically reacted with the epitope RAVESY located within the immunodominant peptide Bet v 1142–153 in an HLA-DRB1*07:01–restricted manner (3). cDNA cloning and sequencing revealed that the functional α-chain gene of SD334 is formed by the rearrangement of TRAV6*01 to a TRAJ21*01 segment. The β-chain resulted from a TRBV20-1*01–TRBD2*02–TRBJ2-7*01–TRBC2 rearrangement (Fig. 1). In the α-chain, two non-templated codons resulting from N-diversification are present, whereas the C-terminal TRAV6*01 codon is deleted. In the β-chain CDR3 region, the C-terminal codon of TRBV20-1 has been deleted with P- and N-diversification giving rise to three non-germline-encoded codons at the 3′-end of TRBD2*02.

FIGURE 1.
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FIGURE 1.

Nucleotide and amino acid sequence of the HLA-DRB1*07:01–restricted TCR of the Bet v 1142–153-specific TCC SD334. A, TCR α-chain sequence. B, TCR β-chain sequence. Codon numbering and nomenclature according to the ImMunoGeneTics web resource for T cell receptors (44). The 5′-ends of C-region sequences are shown. CDRs (boxed), P-nucleotides (gray), and amino acids from P-diversification (magenta) and from N-diversification (turquoise) are indicated.

Retroviral transfer and expression of the Bet v 1142–153-specific TCR

cDNAs encoding the Bet v 1142–153-specific TCR α- and β-chains were introduced by retroviral transduction into Jurkat T cells harboring a human IL-2 promoter/enhancer controlling luciferase expression (21). The tgTRBV20+ TCR was expressed on >99.8% of the cells of Bet v 1 TCRtg Jurkat clone no. 21 obtained by limiting dilution from bulk cultures of transduced cells (Fig. 2). Both the parental line and clone no. 21 expressed CD3 and TCR αβ, low levels of CD4, and no CD8. Similar results were obtained with other clones (data not shown).

FIGURE 2.
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FIGURE 2.

Bet v 1142–153-specific TCRtg Jurkat T cells. Expression pattern of TCR/CD3, Bet v 1142–153-specific transgenic TCR, and coreceptor molecules on wild-type and TCRtg single-cell clone no. 21 are shown. Expression of endogenous TRBV12 and transgenic TRBV20 is shown in the right panel. Numbers indicate percentage values within quadrants. Data are representative of two independently performed experiments.

The Bet v 1142–153-specific TCR is functionally intact in Jurkat T cells

To assess whether Bet v 1-specific TCRtg Jurkat T cells retain the antigenic specificity of the original TCC, Jurkat clone no. 21 was coincubated with aAPCs expressing pools of defined HLA class II molecules in medium alone or after pulsing with saturating concentrations of Bet v 14–15, Bet v 1112–123, Bet v 1142–153 peptides or with Art v 125–36 peptide, the immunodominant epitope of the major mugwort pollen allergen Art v 1, the latter serving as a negative control. Fig. 3A illustrates the specific recognition by Jurkat clone no. 21 of Bet v 1142–153-peptide presenting by HLA-DRB1*07:01+ aAPCs, resulting in a significant increase in IL-2 promoter activity (p < 0.001). Coculture of Jurkat clone no. 21 with Bet v 1- or Art v 1-pulsed aAPCs expressing a collection of non-DR7 HLA class II molecules consistently revealed negative results. Experiments with aAPCs expressing single HLA-DRB1 specificities confirmed the HLA-DRB1*07:01 restriction of Jurkat clone no. 21 (data not shown). Half-maximal stimulation (ED50) was achieved with 0.30 ± 0.07 μM Bet v 1142–153 peptide (data not shown). Wild-type Jurkat T cells did not react with Bet v 1142–153 but otherwise responded well to the superantigen staphylococcal enterotoxin E (data not shown).

FIGURE 3.
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FIGURE 3.

Functional activity of the transgenic Bet v 1142–153-specific TCR. A, IL-2 promoter activity of Bet v 1 TCRtg Jurkat T cells upon coculture with aAPCs transfected with the indicated pools of HLA class II cDNAs and pulsed with the indicated peptides or medium (no peptide). PMA/PHA (white bar) served as positive control (n = 3). Recognition of aAPCs expressing Ii-borne allergenic peptides. B and C, aAPCs expressing the indicated molecules and Ii::fusion proteins or vector control were coincubated with Bet v 1142–153-specific (n = 6) (B) or Art v 1-specific (n = 6) (C) TCRtg Jurkat T cells. Medium or PMA/PHA in the absence of APCs served as controls. Mean values + SEM of triplicates are shown. ***p < 0.001. AU, arbitrary units; CatS, cathepsin S.

aAPCs harboring an Ii::Bet v 1142–153 fusion construct along with expression of HLA-DRA*01:01, HLA-DRB1*07:01, CD80::GPI, CD54::GPI, and cathepsin S also significantly stimulated (p < 0.001) Bet v 1142–153-specific TCRtg Jurkat T cells comparable with the positive control PMA/PHA (Fig. 3B). In contrast, clone no. 21 was not activated by aAPCs transfected with Ii::Bet v 14–15, Ii::Bet v 1112–123, or control vector. Furthermore, Art v 125–36 had no stimulatory effect on Bet v 1142–153-specific TCRtg T cells in the context of HLA-DR1, which otherwise significantly (p < 0.001) stimulated Art v 1-specific TCRtg Jurkat T cells (Fig. 3C). Generally, T cell activation was moderate in the absence (stimulation index: 3.2 ± 0.6 fold background) but became pronounced in the presence (stimulation index: 24.2 ± 3.4 fold background) of costimulation, for example, by CD80 (Supplemental Fig. 1).

Bet v 1142–153-specific TCRtg T cells cross-react with Bet v 1-related food and pollen allergens

In a further step aimed at evaluating the fine specificity of the recombinant TCR, TCRtg Jurkat T cells were incubated with HLA-DR7+ MDDCs pulsed with Bet v 1, Bet v 1-related food (Dau c 1, carrot; Api g 1, celery; Mal d 1, apple) or pollen (Cor a 1, hazelnut; Cab a 1, horn beam; Cas s 1, chestnut; Fag s 1, beech; and Que a 1, oak) allergens. Fig. 4A and 4B show that, apart from Bet v 1 (ED50 0.22 ± 0.01 μM), the food-derived Api g 1 (ED50 0.78 ± 0.09 μM) and the pollen-derived Cor a 1 (ED50 0.16 ± 0.05 μM) and Car b 1 (ED50 0.38 ± 0.08 μM) were recognized by TCRtg T cells. In contrast, Dau c 1 was only poorly stimulatory, and Mal d 1 remained unrecognized. Among the pollen allergens, Cas s 1 was poorly recognized, with responses seen only at the highest concentrations, whereas Que a 1 and Fag s 1 remained unrecognized by the TCRtg T cells. Art v 1 from mugwort used as negative control did not activate TCRtg Jurkat T cells (data not shown). The original TCC SD334 from which the TCR was derived displayed an almost identical reaction pattern to the tested food and pollen allergens when incubated with 0.3 μM allergens (Fig. 4C, 4D). Neither reactivity with Bet v 1 nor cross-reactivity with related allergens was observed when HLA-DR7− MDDCs were used as aAPCs (data not shown).

FIGURE 4.
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FIGURE 4.

The Bet v 1142–153-specific TCR cross-reacts with Bet v 1-related food and pollen allergens. A and B, HLA-DR7+ MDDCs preincubated overnight with Bet v 1 and Bet v 1-related (A) food or (B) pollen allergens were cocultured the next day with TCRtg Jurkat cells for 6 h before luciferase activity was determined. Allergens: Bet v 1 (birch), Api g 1 (celery), Dau c 1 (carrot), Mal d 1 (apple), Cor a 1 (hazelnut), Car b 1 (hornbeam), Cas s 1 (chestnut), Fag s 1 (beech), Que a 1 (oak). C and D, Proliferation of the original TCC from which the TCR was derived in response to (C) food and (D) pollen allergens. Data show mean values + SD of triplicate cultures representative of two independently performed experiments. AU, arbitrary units; conc., concentration.

The Bet v 1142–153-specific TCR is functionally active in human PB T cells

Next, we tested whether it was possible to generate human Bet v 1-specific T lymphocytes from nonallergic individuals. PBLs from five donors (HLA-DR7+ or HLA-DR7−) were transduced with the Bet v 1142–153-specific TCR, resulting in 63 ± 2% compared with 7 ± 2% CD3+TRBV20+ T cells in TCR versus control transduced T cells, respectively (Fig. 5A). TCRtg T cells from all four donors significantly proliferated in an HLA-DR7–restricted manner upon coincubation with aAPCs coexpressing Ii::Bet v 1142–153 but not with aAPCs coexpressing Ii::Bet v 14–15 or Ii::Bet v 1112–123 (Fig. 5B). Moreover, Ii::Bet v 1142–153-induced proliferation was strictly costimulation dependent (Fig. 5C).

FIGURE 5.
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FIGURE 5.

TCRtg PB T cells are specific for Bet v 1142–153. A, Expression levels of TRBV20 and CD3 on control and TCRtg PB T cells. Data are representative for five independent experiments using five different donors. Numbers indicate percentage values within indicated quadrants. B, Proliferation of TCRtg PB T cells cocultured with aAPCs coexpressing the indicated molecules. Control, control vector. Anti-CD3/CD28 microbeads and medium (no peptide) were used as controls. Data show mean proliferation + SD of triplicate cultures corrected for proliferation of GFPtg T cells (9.8 kcpm; range, 8.1–12.5 kcpm). C, Costimulation requirement of TCRtg PB T cells. Data show relative proliferation rates of TCRtg PB T cells cocultured with aAPCs expressing Ii::Bet v 1142–153 in the context of HLA-DR7 in the presence or absence of CD80::GPI. Data are representative for four different donors and seven experiments performed. **p < 0.01, ***p < 0.001. CatS, cathepsin S.

Th1 polarization of TCRtg PB T lymphocytes by aAPCs coexpressing HLA/allergen plus membrane-bound IL-12

We next asked whether aAPCs could polarize de novo-generated, TCRtg allergen-specific T cells from nonallergic and allergic donors toward the Th1 phenotype. For that purpose, we created aAPCs expressing HLA/allergen in the absence or presence of the costimulatory molecule CD80 and/or membrane-bound IL-12 (28). Allergen-specific aAPCs coexpressing CD80 induced significant levels (>40 pg/ml) of IFN-γ, IL-2, and IL-13, whereas no IL-4 (data not shown) and only minute amounts of IL-10 were detectable (Fig. 6 and Supplemental Table I). Significantly, coexpression of membrane-bound IL-12 (p40::p35::GPI fusion protein) (28) in the absence of CD80 strongly increased IFN-γ secretion levels during primary allergen-specific stimulation (p < 0.05), which was even more pronounced in the additional presence of CD80 (p < 0.05). When compared with CD80 costimulated T cells, IL-12–costimulated allergen-specific T cells (in the absence of CD80) produced significantly less IL-2 and IL-13 (<15 pg/ml; p < 0.05 and p < 0.001, respectively). In the absence of specific Ag, the levels of CD80-induced or IL-12–induced cytokines were consistently low similar to cytokine levels obtained upon stimulation of T cells with signal 1 alone (Fig. 6 and Supplemental Table I). In contrast, the combination of CD80 and IL-12 led to Ag-independent IFN-γ production. However, the additional presence of an Ag-specific signal 1 increased IFN-γ levels by 23.0 ± 10.4 fold (p < 0.01).

FIGURE 6.
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FIGURE 6.

Predominant IFN-γ secretion by Bet v 1142–153–TCRtg PB T cells stimulated with aAPCs expressing Bet v 1142–153 in the context of HLA-DR7 plus membrane-bound IL-12. TCRtg allergen-specific T cells were generated from allergic and nonallergic donors (n = 6) and stimulated with irradiated (60 Gy) aAPCs equipped with or without (mock) the indicated costimulatory molecules in the presence (Ii::Bet v 1142–153) or absence (mock) of allergen. Data show mean cytokine levels (ng/ml) of supernatants (24 h) of triplicates + SEM. *p < 0.05, ***p < 0.001. CatS, cathepsin S; ns, not significant.

Similar results were obtained with an Art v 1-specific TCR introduced into T cells of an HLA-DR1+ individual (Supplemental Table II). Comparable Th1-biased polarization was observed, irrespective of whether bulk PB or naive CD4+CD45RA+ PB T cells were used as the starting population (data not shown).

Next, we assessed cytokine expression levels in allergic and nonallergic individuals (n = 5) also on the cellular level by flow cytometry. Fig. 7 shows a clear-cut increase in the percentage of IFN-γ–positive cells in allergic and nonallergic donors upon costimulation with IL-12 compared with that of T cells receiving signal 1 alone (mean ± SD: 5.3 ± 1.6 fold and 4.6 ± 1.6 fold, respectively). Of note, the IL-12–induced percentage of IFN-γ–producing cells was even higher than upon CD80 costimulation (mean ± SD: 1.9 ± 1.3 fold and 2.4 ± 0.3 fold, respectively). In contrast, the percentages of IL-2– and IL-13–positive cells remained low compared with CD80-costimulated cells (mean ± SD of IL-2: 0.4 ± 0.1% and 0.9 ± 0.6% versus 5.7 ± 2.1% and 6.5 ± 3.1%, respectively; mean ± SD of IL-13: 0.9 ± 0.6% and 1.3 ± 0.5% versus 3.6 ± 0.9% and 5.2 ± 2.4%, respectively). Similar intracellular cytokine pattern was obtained in an HLA-DR1–positive individual and applying the Art v 1-specific TCR (Supplemental Fig. 2).

FIGURE 7.
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FIGURE 7.

Intracellular cytokine levels of allergen-specific TCRtg T cells upon stimulation with different forms of allergen-specific or nonspecific APCs. TCRtg allergen-specific T cells were generated from allergic (n = 2) and nonallergic (n = 3) donors and stimulated with the indicated irradiated (60 Gy) aAPCs in the presence or absence of allergen (1st signal: Ii::Bet v 1142–153) and equipped or nonequipped with costimulatory molecules (2nd signal). After 24 h, cocultures were incubated with GolgiStop (Becton Dickinson) for 6 h, followed by intracellular cytokine staining using Fix and Perm (An der Grub) and analysis by flow cytometry. Data show dual parameter dot plots of one representative allergic and nonallergic individual. Markers were set according to the results obtained with nonbinding control Abs. Numbers indicate percentage positive cells in respective quadrants.

Allergen-specific Th1-polarized TCRtg T cells inhibit the effector function of allergen-specific Th2-differentiated T cells

To investigate whether IL-12–induced Th1 cells, differentiated in the absence of CD80 costimulation, could inhibit allergen-specific Th2 cells, we introduced the TCR into naive CD4+CD45RA+ T cells of allergic and nonallergic donors undergoing Th1 (as described earlier) or Th2 polarization. Fig. 8 shows that both cell types when cultured individually secreted typical signature cytokines upon restimulation with Bet v 1142–153-specific aAPCs coexpressing CD80. However, upon coculture of Th2-polarized cells of allergic (Fig. 8A) or nonallergic (Fig. 8B) individuals with syngeneic Th1-polarized cells at a 1:2 ratio (i.e., 0.5 × 105 Th2 cells to 1 × 105 Th1 cells), Th2-differentiated T cells significantly reduced production of their signature cytokines IL-5 and IL-13 (p < 0.01, respectively) and also significantly downregulated IL-2 and IL-10 production (p < 0.01 and p < 0.05, respectively) (Fig. 8A, 8B). Subset-specific cytokine secretion was strongly dependent on allergen-specific activation by aAPCs coexpressing CD80, as only minute quantities of cytokines (e.g., IFN-γ <10 pg/ml) were secreted in the absence of allergen-specific aAPCs (data not shown) or in the presence of aAPCs expressing the noncognate Bet v 14–15 peptide and CD80 (Fig. 8, white bars). Furthermore, allergen specificity was also confirmed by determination of intracellular cytokine levels (Fig. 9 and Supplemental Fig. 3).

FIGURE 8.
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FIGURE 8.

Allergen-specific TCRtg Th1-polarized T cells suppress the effector function of allergen-specific Th2-differentiated T cells. A and B, Naive CD4+CD45RA+ T cells from (A) allergic and (B) nonallergic individuals were transduced with the Bet v 1142–153-specific TCR and cultured under Th2-differentiating conditions in the presence of IL-2, IL-4, anti–IFN-γ, and anti–IL-12. Th1-polarized T cells were generated by coculture of Bet v 1142–153-specific TCRtg T cells with APC Bet v 1142–153 peptide in the context of HLA-DR7 and membrane-bound IL-12. Data show mean + SD of secreted cytokine levels of individual Th cell types or Th1/Th2 cocultures upon incubation with allergen-specific (Ii::Bet v 1142–153, black bars) or allergen-nonspecific (Ii::Bet v 14–15, white bars) aAPCs coexpressing CD80 for 48 h (IFN-γ, IL-2) or 72 h (IL-5, IL-10, IL-13). Data are representative of four individuals and seven experiments performed. *p < 0.05, **p < 0.01, ***p < 0.001. CatS, cathepsin S.

FIGURE 9.
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FIGURE 9.

Intracellular cytokine levels of polarized allergen-specific T cells upon individual culture and coculture. A and B, Th1 (GFP+) and Th2 (GFP−) differentiated T cells from an allergic individual cocultured with (A) allergen-specific (Ii::Bet v 1142–153) or (B) allergen-nonspecific (Ii::Bet v 14–15) aAPCs coexpressing CD80 were analyzed for intracellular cytokine levels individually or upon Th1/Th2 coculture. After 48 h, cultures were incubated with GolgiStop (Becton Dickinson) for 6 h, followed by intracellular cytokine staining using Fix and Perm (An der Grub) and the indicated directly conjugated cytokine-specific mAbs and analyzed by flow cytometry. Data show dual parameter dot plots of one representative experiment of several performed (n = 5). Markers were set according to negative controls; numbers indicate percentage positive cells within the GFP− and GFP+ cell fractions.

The observed downregulation of Th2-signature cytokine levels was also apparent when looking at the single-cell level of T cells from allergic individuals using intracellular cytokine staining. In fact, the percentage of IL-4–, IL-5–, and IL-13–positive cells was reduced from 3.3 to 0.7%, 1.0 to 0.3%, and 1.9 to 1.0%, respectively (Fig. 9A). These experiments also revealed that IL-2 consumption does not seem to be the major factor accounting for the observed decrease of IL-2 levels in cocultures, as also the percentage of IL-2–positive cells within the Th2 population was reduced upon coculture from 12.3% in Th2 cultures to 3.1% in Th1/Th2 cultures. Similar results were obtained with allergic and nonallergic donors (data not shown). Data concerning intracellular cytokine levels were confirmed by using an Art v 125–36-specific TCR and T cells from an HLA-DR1–positive individual (Supplemental Fig. 3). Cytokine expression was strictly Ag dependent, as no significant numbers of cytokine-positive cells were detectable when aAPCs used for coculture experiments presented the noncognate Bet v 14–15 peptide in the presence of CD80 (Fig. 9B). In summary, these results demonstrate the concept that the effector function of human Th2-differentiated T cells can be efficiently inhibited by allergen-specific TCRtg Th1-polarized T cells generated by priming with allergen-specific aAPCs coexpressing membrane-bound IL-12.

Discussion

In the current study, we have cloned, sequenced, and functionally characterized the TCR α- and β-chains of a CD4+ TCC (SD334) specific for the immunodominant T cell epitope of the major birch pollen allergen Bet v 1. Jurkat T cells as well as PB T cells of nonallergic individuals stably expressing this transgenic TCR after retroviral transduction specifically reacted with Bet v 1142–153 peptide-pulsed aAPCs, with aAPCs expressing Ii::Bet v 1142–153 fusion proteins, and with MDDC-processed Bet v 1 protein. Reactivity of TCRtg T cells depended on coexpression of costimulatory molecules such as CD80. The fine specificity of the TCC for Bet v 1-related food and pollen allergens was preserved in the TCRtg T cells. Coexpression of membrane-bound IL-12 on allergen-presenting aAPCs induced TCRtg T cells derived from allergic and nonallergic individuals to secrete high levels of IFN-γ, whereas IL-2 and IL-13 levels remained low, compatible with the induction of a Th1-polarized phenotype. Of note, analysis of cytokines at the single-cell level revealed similar results. Such Th1-polarized T cells, upon allergen-specific but not -nonspecific activation, significantly inhibited the effector function of Th2-differentiated allergen-specific T cells derived from naive CD4+CD45+ T cells. Similar results were obtained with an HLA-DR1–restricted TCR recognizing the mugwort major pollen allergen, Art v 125–34, confirming the general validity of our findings.

Birch pollen allergy and its associated oral allergy syndrome significantly reduce the quality of life of affected individuals throughout the year (8). Individuals with accompanying atopic dermatitis may additionally suffer cutaneous exacerbations (e.g., aggravation of atopic eczema) upon ingestion of Bet v 1-related food allergens (13). Significantly, Bet v 1-related food allergy cannot be palliated by conventional specific immunotherapy, leaving this aspect of birch pollen allergy currently intractable (29). To contribute to a better understanding of the Bet v 1-specific T cell response, in this study we devised a novel tool, a molecularly defined and transferable Bet v 1142–153-specific food and pollen cross-reactive TCR, which allows the creation of biological model systems in which the Bet v 1-specific T cell response can be studied in greater detail and which can be subjected to hypothesis-based modulation. In the Bet v 1-specific model system, all three components of the allergen-specific synapse (restriction element, immunodominant peptide, TCR) are well defined at the molecular level allowing the reproducible study and evaluation of new allergen variants/formulations for specific immunotherapy. The system also provides a solid basis for the establishment of “birch pollen-specific TCRtg mice” to study cross-reactivity and late-phase reactions in vivo in the near future. In principle, the recombinant TCR could also be used to evaluate the contribution of allergen-specific IgE in T cell-dominated in vivo model systems, for example, by adoptive transfer of recently developed mAbs with allergen specificity (30).

Upon transfer into heterologous T cells the Bet v 1142–153-specific TCR retained its typical cross-reactivity with the Bet v 1-related food allergen Api g 1 from celery and its weak but significant cross-reactivity with Dau c 1 from carrot. Similarly, cross-reactivity remained strong with the tree pollen allergens Cor a 1 from hazelnut and Car b 1 from hornbeam and weak with Cas s 1 from chestnut (3). Notably, the presence of the immunodominant epitope RAVESY, representing the amino acid residues 145–150 of Bet v 1, was not an absolute requirement for cross-reactivity with related food and pollen allergen. Only one of the cross-reactive allergens (i.e., Car b 1) has complete sequence identity at positions 145–150 with Bet v 1. Although Cor a 1 (hazelnut) and Api g 1 (celery) have one or three amino acid exchanges in the core binding region, respectively, they still functioned as highly cross-reactive allergens. These results together with the reliable identification of anchor residues and the availability of a high-throughput cellular test system should be useful for the design of therapeutically valuable immunomodulatory peptide ligands covering the immunodominant C-terminal epitope of Bet v 1 in the future.

In allergic individuals, the net output of the cognate allergen/APC–T cell interaction is an imbalanced, Th2-dominated immune response favoring IgE production along with recruitment and arming of potent effector cells (31) such as eosinophils and mast cells. It is generally believed that, among other mechanisms, Th1 cells inhibit the de novo generation of Th2 cells by virtue of their secreted signature cytokines. Soluble IL-12 is a potent inducer of Th1 cells (32, 33); however, systemic administration of the cytokine has been shown to induce a plethora of adverse reactions (34, 35). To provide a more general solution for this problem and to restrict the action of cytokines to the site of Ag presentation, we have resorted to membrane attachment of cytokines to aAPCs (28). In this report, we sought to assess whether allergen-specific aAPCs expressing membrane-bound IL-12, in the absence of CD80 coexpression, would induce Th1-polarized Bet v 1-specific T cells. Our results clearly demonstrate that membrane-tethered IL-12p35::p40 fusion proteins in concert with HLA/allergen complexes elicited allergen-specific TCRtg T cells with a strict Th1 phenotype, exemplified by the sole secretion of large amounts of IFN-γ (Fig. 6 and Supplemental Table I). The increased IFN-γ levels were mirrored by elevated numbers of IFN-γ–producing cells as determined by intracellular cytokine measurements at the single-cell level (Fig. 7). Importantly, similar results were obtained irrespective of whether T cells were derived from allergic or nonallergic individuals. Of note, Th1 polarization was not a salient feature of the Bet v 1-specific TCR but was similarly observed with a mugwort, Art v 125–34-specific, TCR (Supplemental Table II and Supplemental Fig. 2). Moreover, we have demonstrated that such Th1-polarized T cells are capable of dominantly inhibiting the effector function of Th2-differentiated T cells with the same allergen specificity. Besides the significantly reduced Th2-signature cytokine levels in coculture supernatants, these experiments also revealed distinct inhibition of cytokine production at the single-cell level (Fig. 9). This indicates that cytokine consumption/deprivation (36) might not play a major role for the observed downregulation of cytokine levels in cocultures. Our findings with TCRtg T cells (i.e., inhibition of cytokine synthesis) are consistent with previous results obtained with Th2-differentiated human and murine TCCs, which were stimulated Ag-specifically in the presence of exogenously added IFN-γ (37, 38). In principle, redirection of Th cell polarization represents a potentially important strategy for treating Th2 cell-dominated diseases, such as type I allergies. However, future studies, which certainly are beyond the scope of this investigation, will have to address the question whether the Th2 cells under investigation have retained their flexibility and were thus diverted from their previous phenotype. Alternatively, suppression with subsequent deletion from cultures could explain the effects observed. The availability of well-characterized Bet v 1-specific TCRs along with adjustable aAPCs and noncellular Ag-presenting platforms (20) offers the possibility to generate allergen-specific, Th1-polarized T lymphocytes ex vivo on a large scale (e.g., for subsequent adoptive transfer into severely allergic individuals in the future).

Before initiation of such studies, however, it remains to be explored whether and how polarized T cells could be applied in vivo to restore the Th1/Th2 balance without harming the host. In fact, certain allergic diseases have a biphasic T cell response in which a systemic allergen-specific Th2 response is followed by a more Th1-dominated organ pathology, as for example in atopic dermatitis (39). Along those lines, also IL-17 has been described to have a dual nature in the pathogenesis of allergic diseases (40). While IL-17 is required for the establishment of asthma in sensitized mice, it suppresses asthma in the effector phase of the disease (41). Consequently, as of today our current study cannot exclude that adoptive transfer of allergen-specific Th1 cells might enhance disease-promoting mechanisms in the recipient. To clarify these important issues experimentally in the near future, humanized “allergy mice,” expressing the relevant Bet v 1-specific TCR and the human restriction elements described in this article, would offer an attractive model.

As an alternative to the above strategy, the engineering and application of allergen-specific regulatory T cells modified by direct gene transfer appears to represent an attractive option. In a pilot study, we have transduced PB T cells with multicistronic expression cassettes (42) harboring Bet v 1-specific TCR α- and β-chains along with regulatory genes, such as the FOXP3 gene, as a single translation product. This maneuver endows T cells with Ag specificity, a regulatory cell surface phenotype, and regulatory capabilities. The multicistronic approach reliably prevents inappropriate expression of single-gene products, avoiding the potential generation of undesired T cell specificities. Recently, we have demonstrated that Bet v 1-specific TCR/Foxp3 double-transgenic T cells suppress the effector function of Bet v 1-specific T cells in an activation-dependent manner (43).

In conclusion, the availability of well-characterized allergen-specific TCRs offers a broad range of possibilities for creating hypothesis-based, human-relevant in vitro and in vivo model systems to study the multifaceted aspects of allergic diseases. Biologically relevant pathways amenable to immunomodulation have been experimentally addressed in this report. The findings are of potential clinical importance and should also provide novel insights into the pathophysiology of allergic diseases and their possible cure in the future.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Jeroen van Bergen for providing the Ii construct, Dr. Otto Majdic for human HLA class II (1/47) and Ii (5-329) mAbs, and Margarethe Merio for perfect technical assistance with cytokine determinations. We are indebted to Dr. Brian Seed for discussion and invaluable comments.

Footnotes

  • This work was supported by grants from the Christian Doppler Research Association, the Austrian Science Foundation (SFB F1816-B13, SFB F1807-B13, and FWF 20011-B13), the Österreichische Forschungsförderungsgesellschaft (No. 812079), and Biomay AG, Austria.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    aAPC
    artificial Ag-presenting cell
    Ii
    invariant chain
    MDDC
    monocyte-derived dendritic cell
    PB
    peripheral blood
    TCC
    T cell clone
    TCRtg
    TCR transgenic.

  • Received September 28, 2010.
  • Accepted August 15, 2011.
  • Copyright © 2011 by The American Association of Immunologists, Inc.

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The Journal of Immunology: 187 (8)
The Journal of Immunology
Vol. 187, Issue 8
15 Oct 2011
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Human TCR Transgenic Bet v 1-Specific Th1 Cells Suppress the Effector Function of Bet v 1-Specific Th2 Cells
Alina Neunkirchner, Victoria M. Leb-Reichl, Klaus G. Schmetterer, Sonja Mutschlechner, Hans J. Kueng, Daniela Haiderer, Karina Schuch, Michael Wallner, Beatrice Jahn-Schmid, Barbara Bohle, Winfried F. Pickl
The Journal of Immunology October 15, 2011, 187 (8) 4077-4087; DOI: 10.4049/jimmunol.1003220

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Human TCR Transgenic Bet v 1-Specific Th1 Cells Suppress the Effector Function of Bet v 1-Specific Th2 Cells
Alina Neunkirchner, Victoria M. Leb-Reichl, Klaus G. Schmetterer, Sonja Mutschlechner, Hans J. Kueng, Daniela Haiderer, Karina Schuch, Michael Wallner, Beatrice Jahn-Schmid, Barbara Bohle, Winfried F. Pickl
The Journal of Immunology October 15, 2011, 187 (8) 4077-4087; DOI: 10.4049/jimmunol.1003220
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