Allergen-specific IgE production of committed B cells from allergic patients in vitro Peter Steinberger, MSca, Barbara Bohlea, Franco di Padova, MDb,

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Allergen-specific IgE production of committed B cells from allergic patients in vitro Peter Steinberger, MSca, Barbara Bohlea, Franco di Padova, MDb, Michael Wrann, PhDc, Ekke Liehl, PhDc, Otto Scheiner, PhDa, Dietrich Kraft, MDa, Rudolf Valenta, MDa Journal of Allergy and Clinical Immunology Volume 96, Issue 2, Pages 209-218 (August 1995) DOI: 10.1016/S0091-6749(95)70010-2 Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 1 Donor 1. Figs. 1 to 6 show comparison of specific and total IgE in culture supernatants (SN) of PBMCs from patients with allergy and from subjects without allergy. PBMCs from patients with allergy (donors 1 to 3) and from subjects without allergy (donors 4 and 5) were cultured without agonists (—), in the presence of rhu IL-4 (+IL-4) or with a combination of rhu IL-4 and anti-CD40 antibody (+IL-4 + αCD40). A neutralizing anti-IL-4 antibody was added as displayed at the bottom (−, without addition of anti-IL-4 antibody; or addition of 0.2, 6, and 50 μg/ml). IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens (A) and recombinant allergens (B and C) corresponds to determination of total IgE (D) in each lane. Group I and group V allergens migrate at 28 to 32 kd molecular weight, whereas profilin and group II and III allergens show molecular weight of 10 to 14 kd (A). Recombinant Phl p V was expressed in E. coli as a 140 kd β-galactosidase fusion protein (B), and purified recombinant birch profilin has a molecular weight of 14 kd (C). Allergens were in excess of IgE antibodies. Nitrocellulose strips were incubated with culture supernatants (SN) of unstimulated or stimulated donor PBMCs. IgE reactivity of corresponding serum (S) with blotted allergens is shown at right. IgE binding beneath Phl p V β-galactosidase fusion allergen results from partial degradation of the fusion protein. No IgE reactivity to natural or recombinant allergens could be detected with supernatants or sera from nonallergic donors (donors 4 and 5). Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 1 Donor 1. Figs. 1 to 6 show comparison of specific and total IgE in culture supernatants (SN) of PBMCs from patients with allergy and from subjects without allergy. PBMCs from patients with allergy (donors 1 to 3) and from subjects without allergy (donors 4 and 5) were cultured without agonists (—), in the presence of rhu IL-4 (+IL-4) or with a combination of rhu IL-4 and anti-CD40 antibody (+IL-4 + αCD40). A neutralizing anti-IL-4 antibody was added as displayed at the bottom (−, without addition of anti-IL-4 antibody; or addition of 0.2, 6, and 50 μg/ml). IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens (A) and recombinant allergens (B and C) corresponds to determination of total IgE (D) in each lane. Group I and group V allergens migrate at 28 to 32 kd molecular weight, whereas profilin and group II and III allergens show molecular weight of 10 to 14 kd (A). Recombinant Phl p V was expressed in E. coli as a 140 kd β-galactosidase fusion protein (B), and purified recombinant birch profilin has a molecular weight of 14 kd (C). Allergens were in excess of IgE antibodies. Nitrocellulose strips were incubated with culture supernatants (SN) of unstimulated or stimulated donor PBMCs. IgE reactivity of corresponding serum (S) with blotted allergens is shown at right. IgE binding beneath Phl p V β-galactosidase fusion allergen results from partial degradation of the fusion protein. No IgE reactivity to natural or recombinant allergens could be detected with supernatants or sera from nonallergic donors (donors 4 and 5). Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 1 Donor 1. Figs. 1 to 6 show comparison of specific and total IgE in culture supernatants (SN) of PBMCs from patients with allergy and from subjects without allergy. PBMCs from patients with allergy (donors 1 to 3) and from subjects without allergy (donors 4 and 5) were cultured without agonists (—), in the presence of rhu IL-4 (+IL-4) or with a combination of rhu IL-4 and anti-CD40 antibody (+IL-4 + αCD40). A neutralizing anti-IL-4 antibody was added as displayed at the bottom (−, without addition of anti-IL-4 antibody; or addition of 0.2, 6, and 50 μg/ml). IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens (A) and recombinant allergens (B and C) corresponds to determination of total IgE (D) in each lane. Group I and group V allergens migrate at 28 to 32 kd molecular weight, whereas profilin and group II and III allergens show molecular weight of 10 to 14 kd (A). Recombinant Phl p V was expressed in E. coli as a 140 kd β-galactosidase fusion protein (B), and purified recombinant birch profilin has a molecular weight of 14 kd (C). Allergens were in excess of IgE antibodies. Nitrocellulose strips were incubated with culture supernatants (SN) of unstimulated or stimulated donor PBMCs. IgE reactivity of corresponding serum (S) with blotted allergens is shown at right. IgE binding beneath Phl p V β-galactosidase fusion allergen results from partial degradation of the fusion protein. No IgE reactivity to natural or recombinant allergens could be detected with supernatants or sera from nonallergic donors (donors 4 and 5). Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 1 Donor 1. Figs. 1 to 6 show comparison of specific and total IgE in culture supernatants (SN) of PBMCs from patients with allergy and from subjects without allergy. PBMCs from patients with allergy (donors 1 to 3) and from subjects without allergy (donors 4 and 5) were cultured without agonists (—), in the presence of rhu IL-4 (+IL-4) or with a combination of rhu IL-4 and anti-CD40 antibody (+IL-4 + αCD40). A neutralizing anti-IL-4 antibody was added as displayed at the bottom (−, without addition of anti-IL-4 antibody; or addition of 0.2, 6, and 50 μg/ml). IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens (A) and recombinant allergens (B and C) corresponds to determination of total IgE (D) in each lane. Group I and group V allergens migrate at 28 to 32 kd molecular weight, whereas profilin and group II and III allergens show molecular weight of 10 to 14 kd (A). Recombinant Phl p V was expressed in E. coli as a 140 kd β-galactosidase fusion protein (B), and purified recombinant birch profilin has a molecular weight of 14 kd (C). Allergens were in excess of IgE antibodies. Nitrocellulose strips were incubated with culture supernatants (SN) of unstimulated or stimulated donor PBMCs. IgE reactivity of corresponding serum (S) with blotted allergens is shown at right. IgE binding beneath Phl p V β-galactosidase fusion allergen results from partial degradation of the fusion protein. No IgE reactivity to natural or recombinant allergens could be detected with supernatants or sera from nonallergic donors (donors 4 and 5). Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 2 Donor 2. PBMCs from patient with allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 2 Donor 2. PBMCs from patient with allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 2 Donor 2. PBMCs from patient with allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 2 Donor 2. PBMCs from patient with allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 3 Donor 3. PBMCs from patient with allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 3 Donor 3. PBMCs from patient with allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 3 Donor 3. PBMCs from patient with allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 3 Donor 3. PBMCs from patient with allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 4 Donor 4. PBMCs from subject without allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 4 Donor 4. PBMCs from subject without allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 4 Donor 4. PBMCs from subject without allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 4 Donor 4. PBMCs from subject without allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 5 Donor 5. PBMCs from subject without allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 5 Donor 5. PBMCs from subject without allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 5 Donor 5. PBMCs from subject without allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 5 Donor 5. PBMCs from subject without allergy. A, IgE reactivity of culture supernatants with nitrocellulose-blotted natural timothy grass pollen allergens. B and C, IgE reactivity of culture supernatants with recombinant allergens. D, Determination of total IgE. See legend to Fig. 1. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 6 Comparison of total and allergen-specific IgE synthesis in culture supernatants determined by radioimmunoassay. PBMCs from three patients (A to C) were cultured without and in presence of different concentrations of agonists. Total IgE in cultures was determined as in Figs. 1 to 5, whereas allergen-specific IgE synthesis was measured with nitrocellulose-dotted purified recombinant allergens (rBet v II, rPhl p V) followed by detection of bound IgE with 125I-labeled anti-human IgE (Pharmacia). Bound allergen-specific IgE was measured in counts per minute. Counts per minute with human serum albumin (negative control) representing background levels were subtracted from values obtained with recombinant allergens. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 6 Comparison of total and allergen-specific IgE synthesis in culture supernatants determined by radioimmunoassay. PBMCs from three patients (A to C) were cultured without and in presence of different concentrations of agonists. Total IgE in cultures was determined as in Figs. 1 to 5, whereas allergen-specific IgE synthesis was measured with nitrocellulose-dotted purified recombinant allergens (rBet v II, rPhl p V) followed by detection of bound IgE with 125I-labeled anti-human IgE (Pharmacia). Bound allergen-specific IgE was measured in counts per minute. Counts per minute with human serum albumin (negative control) representing background levels were subtracted from values obtained with recombinant allergens. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 6 Comparison of total and allergen-specific IgE synthesis in culture supernatants determined by radioimmunoassay. PBMCs from three patients (A to C) were cultured without and in presence of different concentrations of agonists. Total IgE in cultures was determined as in Figs. 1 to 5, whereas allergen-specific IgE synthesis was measured with nitrocellulose-dotted purified recombinant allergens (rBet v II, rPhl p V) followed by detection of bound IgE with 125I-labeled anti-human IgE (Pharmacia). Bound allergen-specific IgE was measured in counts per minute. Counts per minute with human serum albumin (negative control) representing background levels were subtracted from values obtained with recombinant allergens. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 7 Reactivity of supernatants from patients with allergy and from subjects without allergy with E. coli–phage proteins. The same supernatants from patients with allergy (donors 1 to 3) and from subjects without allergy (donors 4 and 5) and sera (right panel) as in Figs. 1 to 5 were probed with proteins from E. coli Y1089 infected with λ gt11 phage without insert. No specific IgE binding was observed with any sample. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 7 Reactivity of supernatants from patients with allergy and from subjects without allergy with E. coli–phage proteins. The same supernatants from patients with allergy (donors 1 to 3) and from subjects without allergy (donors 4 and 5) and sera (right panel) as in Figs. 1 to 5 were probed with proteins from E. coli Y1089 infected with λ gt11 phage without insert. No specific IgE binding was observed with any sample. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 7 Reactivity of supernatants from patients with allergy and from subjects without allergy with E. coli–phage proteins. The same supernatants from patients with allergy (donors 1 to 3) and from subjects without allergy (donors 4 and 5) and sera (right panel) as in Figs. 1 to 5 were probed with proteins from E. coli Y1089 infected with λ gt11 phage without insert. No specific IgE binding was observed with any sample. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 7 Reactivity of supernatants from patients with allergy and from subjects without allergy with E. coli–phage proteins. The same supernatants from patients with allergy (donors 1 to 3) and from subjects without allergy (donors 4 and 5) and sera (right panel) as in Figs. 1 to 5 were probed with proteins from E. coli Y1089 infected with λ gt11 phage without insert. No specific IgE binding was observed with any sample. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 8 DNA sequences of oligonucleotides. Oligonucleotides that were used for amplification of IgE-Fd and IgG-Fd cDNA are heavy-chain primers and corresponding variable-chain primers. Internal oligonucleotides specific for IgE and IgG were used to identify PCR products by hybridization. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 9 Amplification of IgG and IgE-Fd–cDNA from peripheral blood leukocytes derived from a patient with grass pollen allergy. RNA from fresh PBMCs was prepared, reversely transcribed, and PCR amplified with primers specific for IgG (lane 1) or IgE (lane 2) and equimolar mixture of two v region–specific primers. DNA was subjected to 1.5% agarose gel electrophoresis and ethidium bromide staining (left panel). Bands of approximately 650 base pairs were obtained, which, after transfer to nitrocellulose, could be hybridized with oligonucleotides specific for IgG (middle panel) and IgE (right panel), respectively. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions

FIG. 10 Influence of IL-4 and anti-CD40 treatment on amount of IgE-Fd cDNA amplified from blood leukocytes of patients with allergy. IgE-Fd cDNA was reversely transcribed and PCR-amplified from 3 × 106 peripheral blood leukocytes from a donor with allergy. Cells were isolated freshly by Ficoll density-gradient centrifugation (lane 1) and were then kept in culture for 8 days without stimulation (lane 2) or were stimulated by IL-4 (lane 3) or by combination of IL-4 and anti-CD40 antibodies (lane 4) for 8 days. Arrowhead indicates position of IgE-Fd cDNA at approximately 650 base pairs. Journal of Allergy and Clinical Immunology 1995 96, 209-218DOI: (10.1016/S0091-6749(95)70010-2) Copyright © 1995 Mosby, Inc. Terms and Conditions