CD27/CD70 Interaction Augments IgE Secretion by Promoting the Differentiation of Memory B Cells into Plasma Cells

This information is current as Haruo Nagumo, Kazunaga Agematsu, Koji Shinozaki, Sho of October 2, 2021. Hokibara, Susumu Ito, Masaya Takamoto, Toshio Nikaido, Kozo Yasui, Yoshio Uehara, Akihiro Yachie and Atsushi Komiyama J Immunol 1998; 161:6496-6502; ; http://www.jimmunol.org/content/161/12/6496 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD27/CD70 Interaction Augments IgE Secretion by Promoting the Differentiation of Memory B Cells into Plasma Cells

Haruo Nagumo,* Kazunaga Agematsu,1* Koji Shinozaki,* Sho Hokibara,* Susumu Ito,† Masaya Takamoto,‡ Toshio Nikaido,§ Kozo Yasui,* Yoshio Uehara,¶ Akihiro Yachie,ʈ and Atsushi Komiyama*

The induction of IgE switching in B cells requires several signals given by and cell contact-delivered signals. Here, we investigated the role of CD27/CD70 interaction in B cell IgE synthesis. The addition of CD27 ligand (CD70) transfectants to B cell cultures increased the IgE synthesis synergistically in the presence of IL-4 plus anti-CD40 mAb (anti-CD40). The effect of CD70 ,transfectants was dose dependent and was completely blocked by anti-CD70 mAb. CD27؉ B cells had the ability to produce IgE which was increased by contact with CD70 transfectants, whereas CD27؊ B cells did not produce IgE. CD27/CD70 interaction enhanced B cell proliferation in the presence of IL-4 or IL-4 plus anti-CD40. The augmentation of B cell proliferation by CD70 Downloaded from transfectants was apparent in CD27؉ B cells, but was mild in CD27؊ B cells. The helper activity for IgE synthesis by the CD27/CD70 interaction did not contribute to the enhancement of germline ⑀ transcripts. Flow cytometric and morphological analyses demonstrated that the addition of CD70 transfectants to B cell cultures remarkably promoted differentiation into plasma cells in the presence of IL-4 and CD40 signaling. Finally, CD27 cross-linking resulted in the up-regulation of positive regulatory domain I-binding factor-1. Taken together, our findings indicate that signaling via CD27 on B cells induces IgE synthesis, in cooperation with IL-4 and CD40 signaling, by promoting the generation of plasma cells through up-regulation of positive regu- http://www.jimmunol.org/ latory domain I-binding factor-1. The Journal of Immunology, 1998, 161: 6496–6502.

he IgE immune response is an important event in vivo addition, it has been demonstrated that the CD27/CD70 interaction because it may lead to the development of allergic dis- is involved in the differentiation of B cells into plasma cells (17, 18). T eases such as atopy and asthma (1). The induction of IgE In the present study we clarified the role of CD27/CD70 inter- switching in B cells requires at least two signals: a class switching action in B cell IgE synthesis by investigating the effects of CD27 factor, most commonly IL-4, and the engagement of CD40. The signaling on B cell proliferation, germline ⑀ transcripts, differen- combination of IL-4 and anti-CD40 mAb can stimulate IgE pro- tiation into plasma cells, and the expression of transcription factors duction in vitro (2–4), whereas several other regulatory molecules involved in B cell differentiation such as positive regulatory do- by guest on October 2, 2021 may be involved in the regulation of B cell IgE secretion (5–7). main I-binding factors 1 (PRDI-BF1)2 and B cell-specific activator The enhancement of IgE synthesis by CD4ϩ T cell clones in the protein (BSAP). presence of IL-4 plus CD40 signaling suggest a different B cell pathway for IgE production (4). Life et al. demonstrated that T cell Materials and Methods clones from an X-linked hyper-IgM syndrome patient induced IgE Abs and reagents synthesis in vitro despite the expression of nonfunctional CD154, Anti-CD27 mAb (8H5; IgG1), which does not block the ligation of CD27/ indicating the involvement of T cell surface molecules other than CD70 (16, 19), was provided by Dr. T. Morimoto (Dana-Farber Cancer CD154 in the induction of IgE synthesis (8). Institute, Boston, MA). FITC-conjugated anti-CD20 mAb and PE-conju- CD27, characterized in detail by van Lier et al. (9, 10), is a type gated anti-CD20 mAb were purchased from Dako Japan (Tokyo, Japan). FITC-conjugated anti-CD38 mAb (T16; IgG1), anti-CD40 mAb (MAB89; I expressed on T and B cells and in B cell malignan- IgG1), anti-CD70 mAb (HNE51; IgG1), and anti-CD62L mAb (TQ1; cies (11). Recently, we have demonstrated that the interaction be- IgG1) were purchased from Immunotech (Marseille, France). PE-conju- tween CD27, which belongs to the nerve receptor/ gated anti-mouse Ig mAb was obtained from Southern Biotechnology As- TNF receptor family (12), and CD27 ligand (CD70), which sociates (Birmingham, AL). Conjugation of biotin to anti-CD27 mAb belongs to the TNF family (13) and is expressed not only on ac- (8H5) was performed by the standard technique, using N-hydroxysuccin- ϩ imido-biotin (Sigma, St. Louis, MO) in our laboratory. G418 was pur- tivated B but also on T cells, especially activated CD4 CD45RO chased from Life Technologies (Grand Island, NY), and human IL-4 was T cells (14), can enhance Ig production by B cells (15, 16). In obtained from Genzyme (Cambridge, MA). The L cell line expressing murine Fc␥II receptor (CD32L) was obtained from American Type Culture Collection (Manassas, VA). The CD154-CD8 fusion protein (cell culture † ‡ § Departments of *Pediatrics, Blood Transfusion, Parasitology, and Obstetrics and supernatant, soluble CD154) was provided by Dr. A. Aruffo (Bristol- Gynecology, Shinshu University School of Medicine, Matsumoto, Japan; ¶Nagano ʈ Myers/Squibb Pharmaceutical Research Institute, Seattle, WA). Children’s Hospital, Toyoshina, Japan; and Department of Laboratory Sciences, School of Health Sciences, Faculty of Medicine, Kanazawa University, Kanazawa, Cell preparation Japan Received for publication April 3, 1998. Accepted for publication August 26, 1998. Adult human PBMNCs were isolated from healthy volunteers by Ficoll- Hypaque (Pharmacia, Piscataway, NJ) density gradient centrifugation and 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. 2 Abbreviations used in this paper: PRDI-BF1, positive regulatory domain I-binding 1 Address correspondence and reprint requests to Dr. Kazunaga Agematsu, Depart- factor-1; BSAP, B cell-specific activator protein; PE, phycoerythrin; EϪ, erythrocyte ment of Pediatrics, Shinshu University School of Medicine, Asahi 3–1-1, Matsumoto rosette-negative; SAC, Staphylococcus aureus Cowan strain; Blimp-1, B - 390-0802, Japan. E-mail address: [email protected] induced maturation protein-1.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 6497 were separated with 5% sheep erythrocytes into erythrocyte rosette-posi- fication (Biotex, Houston, TX). First-strand cDNA copies were synthe- tive and -negative (EϪ) populations (20). After depleting monocytes with sized by using murine Moloney leukemia virus reverse transcriptase (Life silica (IBL, Fujioka, Japan) or by adherence to the plastic surface, EϪ cells Technologies, Grand Island, NY) with oligo(dT) (Life Technologies) as a were further purified into B cells by positive selection with anti-CD19 primer in a total volume of 20 ␮l, and then PCR was performed. The mAb-coated immunomagnetic beads (Dynal, Oslo, Norway), and then anti- following oligonucleotide primers were used for PCR: for C⑀ exon, sense CD19 mAb was removed by the use of Detach a Bead (Dynal). Ninety- primer, 5Ј-CATGCGGTCCACGACCAAGAC-3Ј; antisense primer, 5Ј- seven percent of the B cell population was reactive with anti-CD20 mAbs. CCACTGCACAGCTGGATGGAG-3Ј; for BSAP, 5Ј-CAACCAAC Proliferation and activation were not observed from the B cells thus ob- CAGTCCCAGCTTC-3Ј and 5Ј-TCACAATGGGGTAGGACTGCG-3Ј; tained. CD20ϩ CD27Ϫ or CD20ϩ CD27ϩ B cells were isolated from the and for PRDI-BF1, 5Ј-AGCTGACAATGATGAATCTCA-3Ј and 5Ј-CT monocyte-depleted EϪ cells by sorting with a FACStarPlus (Becton Dick- TGGGGTAGTGAGCGTTGTA-3Ј. Two microliters of cDNA were am- inson, Mountain View, CA) under sterile conditions. Both populations ob- plified in PCR using each primer and Taq DNA polymerase (Life Tech- tained were Ͼ98% pure. nologies). The amplified products were analyzed on a 1.2% agarose gel ␤ containing ethidium bromide and visualized by UV illumination. 2m Preparation of transfectants sense primer 5Ј-GCTATGTGTCTGGGTTTCAT-3Ј and antisense primer 5Ј-ATCTTCAAACCTCCATGATG-3Ј were used as controls (24). The preparation of CD70 transfectants or mock transfectants has been de- scribed previously (16). Briefly, total RNA was isolated by the single-step ϩ Quantitative RT-PCR of germline C⑀ method (21) from the CD70 human B cell line (Daudi cells). After RT- PCR, the amplified cDNA was digested with EcoRI and XbaI and ligated In the germline C⑀ transcripts, the amounts of mRNA were estimated by with the mammalian expression vector pcDLSR␣296 (22). The resulting coamplification of parvo virus DNA for a competitor. PCR amplification of plasmid or the vector alone was transfected into the murine pre-B cell line, parvo virus DNA, which includes germline C⑀ primers in both sides, was 300-19 (23) by electroporation. The transfectants were selected by growth conducted with flanking primers. The product was 505 bp in length and in a culture medium containing a neomycin analogue (G418, Life Tech- was separated by PAGE and purified. Each template contained the same nologies) at 1 mg/ml. amounts of cDNA from RNA extracted from highly purified B cells, and Downloaded from one of fivefold dilutions of germline C⑀ transcript competitor from 3.2 ϫ Flow cytometric analysis 10Ϫ1 to 2.6 ϫ 10Ϫ3 attomoles. The equivalent point was determined by PAGE. To ensure that the amounts of germline C⑀ cDNA in the samples Activated purified B cells were stained with anti-CD38-FITC and anti- ␤ CD20-PE. Two-color analysis of B cell surface molecules was performed were the same, the amounts of the -actin cDNA were evaluated as an by FACScan (Becton Dickinson). Ab-coated cells were gated on living internal mRNA control. cells by cell size and granularity, and dead cells were removed by pro- pidium iodide staining and then enumerated by flow cytometric analysis. Results http://www.jimmunol.org/ Augmentation of B cell IgE production by CD27/CD70 Fixation of transfectant cells interaction The CD70/300-19 or mock/300-19 transfectants were incubated with 1% paraformaldehyde in PBS for 5 min. After washing with PBS three times, Since triggering via CD27 by CD70 transfectants, which greatly the cells were cultured in RPMI 1640 and 10% FCS for 30 min and then express CD70 on the surface (Fig. 1A), induces IgA, IgM, and IgG used for the analysis. synthesis in B cell activation systems with SAC plus IL-2 (15) and IL-10 (25), we investigated whether CD27/CD70 interaction par- Ig assay by ELISA ticipates in B cell IgE synthesis. To clarify the direct effect of Highly purified peripheral blood B cells were cultured with 50 ng/ml of CD27 signaling on IgE synthesis, we highly purified B cells from ␮ by guest on October 2, 2021 IL-4 and 1 g/ml of anti-CD40 in the presence of various concentrations adult peripheral blood. As reported previously (26), anti-CD40 or of fixed CD70/300-19 or mock/300-19 with or without 1 ␮g/ml of anti- CD70 mAb for 12–14 days at 37°C in a humidified atmosphere with 5% IL-4 alone did not induce IgE synthesis by B cells, whereas in the ϫ 5 ␮ CO2. The final cell density was 5–10 10 /ml in a volume of 200 l/well. presence of anti-CD40 plus IL-4, B cells produced substantial The cultured supernatants were harvested, and the supernatants and the amounts of IgE. Most significantly, CD70 transfectants greatly standard human IgE (Chemicon, Temecula, CA) were added to 96-well flat augmented IgE production in the presence of IL-4 plus anti-CD40, ELISA plates (Nunc, Roskilde, Denmark). The plates were coated with which was about fourfold compared with that after the addition of human IgE mAbs (CIA-E-7.12 and CIA-E-4.15, provided by Dr. A. Saxon, Division of Clinical Immunology/Allergy, University of California School mock transfectants (Fig. 1B). The augmentation of IgE synthesis of Medicine, Los Angeles, CA). After an overnight culture at 4°C, the by CD70 transfectants was dose dependent, and the enhancement supernatants were discarded, and the wells were washed with 0.05% of IgE production by CD70 transfectants was completely blocked Tween 20 in PBS. Alkaline phosphatase-labeled goat anti-human IgE by the initial addition of anti-CD70 mAb (Fig. 1B), but not by the (Sigma, St. Louis, MO) at a dilution of 1/5000 was added. After2hof incubation at room temperature, color detection was performed by control mAb (data not shown). On the other hand, the addition of 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer containing IL-4 or anti-CD40 to CD70 transfectants did not induce detectable p-Nitrophenyl phosphate (pNPP) (Sigma). Calibration was performed with levels of IgE secretion (Fig. 1B). When we stimulated B cells by PBS at standard zero levels. The limit of sensitivity for IgE was 5 ng/ml. cross-linking with anti-CD40 plus CD32 transfectants and by the B cell proliferation assay addition of soluble CD154 in the presence of IL-4, IgE production was 32 Ϯ 7 and 21 Ϯ 5 ng/ml, respectively. These data demon- Highly purified unseparated B cells and CD20ϩ CD27ϩ and CD20ϩ Ϫ strate that triggering via CD27 by its ligand, CD70, synergistically CD27 B cells were cultured with fixed mock/300-19 or CD70/300-19 (20% of the B cells added) in the presence of 50 ng/ml IL-4 or IL-4 plus induces B cell IgE synthesis in the presence of IL-4 plus anti- 1 ␮g/ml anti-CD40 at a final cell density of 2.5–5 ϫ 105/ml in a volume CD40, indicating that the CD27/CD70 interaction is involved in B of 200 ␮l/well in 96-well round-bottom plates (Nunc) for 3 days at 37°C cell IgE synthesis by direct cell to cell contact. in a humidified atmosphere with 5% CO2. Then the cultures were pulsed 3 with 0.5 ␮Ci of [ H]thymidine. After 18 h of incubation, the cells were Production of IgE by CD27ϩ B cells, but not by CD27Ϫ B cells, harvested with an automatic cell harvester (Packard, Meriden, CT), and in the presence of IL-4 plus anti-CD40 [3H]thymidine incorporation was measured on a liquid scintillation ana- lyzer (Packard). Having demonstrated that peripheral blood B cells can be sepa- ϩ Ϫ RT-PCR of germline ⑀, BSAP, and PRDI-BF1 rated by CD27 expression, and that CD27 B cells, but not CD27 B cells, produce large amounts of IgA, IgM, and IgG (27), we ϩ ϩ ϩ Ϫ Highly purified unseparated B cells and CD20 CD27 or CD20 CD27 conducted additional experiments to determine whether B cell sub- B cells (1 ϫ 106 cells) were stimulated with 50 ng/ml IL-4 or IL-4 plus 1 ␮ populations separated by CD27 have different functions with re- g/ml anti-CD40 in the presence of mock transfectants or CD70 transfec- ϩ ϩ tants for 16 h or 4 days. Total RNA was extracted by the acid-guanidine gard to the IgE synthesis. We purified CD20 CD27 B cells and ϩ Ϫ thiocyanate-phenol-chloroform method using a RNAzol rapid RNA puri- CD20 CD27 B cells by flow cytometry; the purities of the two 6498 AUGMENTATION OF IgE SYNTHESIS BY CD27/CD70

FIGURE 2. Production of IgE in CD27ϩ B cells but not in CD27Ϫ B cells. CD20ϩ CD27ϩ B cells and CD20ϩ CD27Ϫ B cells were separated from adult peripheral blood EϪ cells by means of flow cytometry (purity was Ͼ97%, respectively). The highly purified CD27ϩ and CD27Ϫ B cells were cultured with IL-4 plus anti-CD40 in the presence of mock transfec-

tants or CD70 transfectants with or without anti-CD70 mAb at a final cell Downloaded from density of 0.5 ϫ 105/well for 12–14 days. IgE content was measured by ELISA. The data were representative of three independent experiments. Results are expressed as the mean Ϯ SD of triplicate determinations.

hancing effect. In addition, the augmentation of B cell proliferation http://www.jimmunol.org/ FIGURE 1. Expression of CD70 by mock and CD70 transfectants and by CD70 transfectants was completely blocked by the initial ad- enhancement of B cell IgE synthesis by CD27/CD70 interaction. A, Mock dition of anti-CD70 mAb (Table I). B cell proliferation was greatly and CD70 transfectants were stained with mouse anti-human CD70 mAb (HNE51) followed by (goat) anti-mouse Ig-PE. Expression of CD70 on increased by IL-4 plus anti-CD40 plus CD32 transfectants Ϯ ϭ mock and CD70 transfectants is shown with a log scale (solid line). Iso- (18,952 2,802 cpm; n 3) or IL-4 plus soluble CD154 type-matched PE-labeled mAb was used as the negative control (dotted (15,916 Ϯ 2,156 cpm; n ϭ 3) compared with IL-4 plus anti-CD40 line). B, Highly purified B cells were cultured with medium, IL-4, anti- stimulation (2,409 Ϯ 1,067 cpm; n ϭ 7). As the baseline levels of CD40, or IL-4 plus anti-CD40 in the presence of mock transfectants or B cell proliferation were high in these systems with IL-4 plus CD70 transfectants with or without anti-CD70 mAb at a final cell density anti-CD40 plus CD32 transfectants or IL-4 plus soluble CD154 by guest on October 2, 2021 of 1 ϫ 105/well in 96-well round-bottom plates for 12–14 days. IgE content only, the enhancing effects of CD70 transfectants on B cell pro- was measured by ELISA. Similar results were obtained in five independent liferation were not prominent: the values with CD70 transfectants Ϯ experiments. Results are expressed as the mean SD of triplicate deter- were 21,304 Ϯ 4,012 cpm (n ϭ 3) in the former and 19,185 Ϯ minations, and error bars are sometimes smaller than the plot symbol. 3,231 cpm (n ϭ 3) in the latter system. This enhancement of B cell proliferation by CD70 transfectants was obtained in CD27ϩ B cells, but was very mild in CD27Ϫ B cells (Table II). Thus, these cell populations were Ͼ98%. CD27ϩ B cells produced IgE in the presence of IL-4 plus anti-CD40, but not in the presence of IL-4 or anti-CD40 alone. The production of IgE by CD27ϩ B cells was strongly enhanced by the addition of CD70 transfectants in the Table I. B cell proliferation by CD70 transfectants in the presence of IL-4 or IL-4 plus anti-CD40 mAb presence of IL-4 plus anti-CD40, whereas mock transfectants did not have such an enhancing effect. The enhancement by the CD70 3 a transfectants was specifically blocked by the initial addition of [ H]Thymidine Incorporation anti-CD70 mAb (Fig. 2). On the other hand, IgE secretion from Expt. 1 Expt. 2 CD27Ϫ B cells was below measurable levels in the presence of Medium 239 Ϯ 52b 322 Ϯ 73 IL-4 plus anti-CD40, even with the addition of CD70 transfectants c Ϯ Ϯ ϩ IL-4 356 80 723 175 (Fig. 2). These data demonstrate that CD27 B cells, but not ϩ mock-trans.d 387 Ϯ 78 572 Ϯ 101 CD27Ϫ B cells, have ability to produce IgE and that signaling via ϩ CD70-trans.d 1227 Ϯ 315 1389 Ϯ 216 the CD27 molecule participates in the IgE synthesis. ϩ CD70-trans. ϩ HNE51e 365 Ϯ 81 654 Ϯ 155 ϩ CD70-trans. ϩ TQ1e 1305 Ϯ 322 1443 Ϯ 319 ϩ c Ϯ Ϯ B cell proliferation by CD27/CD70 ligation in the presence of IL-4 anti-CD40 1161 216 2949 465 ϩ mock-trans. 1396 Ϯ 329 2820 Ϯ 498 IL-4 or IL-4 plus anti-CD40 ϩCD70-trans. 2193 Ϯ 476 8174 Ϯ 1,279 We next investigated whether CD27 molecule on B cells contrib- ϩCD70-trans. ϩ HNE51 1185 Ϯ 237 3066 Ϯ 793 ϩ ϩ Ϯ Ϯ uted to their proliferation in the presence of IL-4 or IL-4 plus CD70-trans. TQ1 2218 531 7792 1378 anti-CD40. IL-4 alone induced low levels of proliferation of un- a Proliferation assay was performed as described in Materials and Methods using ϫ 4 separated B cells. The B cell proliferation was enhanced by the 5 10 /well of highly purified peripheral blood B cells. b Results are expressed as the mean Ϯ SD of triplicates. addition of anti-CD40 to IL-4 compared with that with IL-4 alone. c The final concentration of IL-4 and anti-CD40 mAb was 50 ng/ml and 1 ␮g/ml, Furthermore, the proliferation of B cells was augmented by the respectively. d The transfectants were present as 20% of the B cell numbers added. addition of CD70 transfectants in the presence of IL-4 or IL-4 plus e Both Abs were added at a concentration of 1 ␮g/ml at the initiation of cell anti-CD40, whereas mock transfectants did not have such an en- culture. The Journal of Immunology 6499

Table II. CD27ϩ and CD27Ϫ B cell proliferation by CD70 transfectants in the presence of IL-4 or IL-4 plus anti-CD40 mAb

[3H] Thymidine Incorporationa

CD27ϩ B cells CD27Ϫ B cells

IL-4b ϩ mock-trans.c 537 Ϯ 142d 781 Ϯ 180 IL-4 ϩ CD70-trans.c 1511 Ϯ 325 1023 Ϯ 293 IL-4 ϩ anti-CD40b ϩ mock-trans. 1101 Ϯ 291 889 Ϯ 283 IL-4 ϩ anti-CD40 ϩ CD70-trans. 2054 Ϯ 446 1240 Ϯ 305

a Proliferation assay was performed as described in Materials and Methods using 3 ϫ 104/well of CD27ϩ or CD27Ϫ B cells. b The final concentration of IL-4 and anti-CD40 mAb was 50 ng/ml and 1 ␮g/ml, respectively. c The transfectants were present as 20% of the B cell numbers added. d Results are expressed at the mean Ϯ SD of triplicates. results suggest that the ligation of CD27 on B cells by CD70 trans- fectants enhances B cell proliferation in the presence of IL-4 or IL-4 plus anti-CD40. Downloaded from CD27 signaling in B cells does not enhance germline ⑀ transcripts Since B cell IgE production was strongly enhanced by the addition of CD70 transfectants in the presence of IL-4 plus anti-CD40, we investigated the expression of germline ⑀ transcripts, which is an early molecular markers of the isotype switch process, to examine http://www.jimmunol.org/ possible early switch events being induced by CD70 transfectants. FIGURE 3. CD27 signaling in B cells does not enhance germline ⑀ Germline ⑀ transcripts from highly purified B cells were not ob- transcript expression. The highly purified B cells were cultured with IL-4 tained by the addition of medium, CD70 transfectants, or anti- or IL-4 plus anti-CD40 for 16 h. After extraction of total RNA, quantitative CD40 alone (data not shown). Representative data by quantitative RT-PCR was performed as described in Materials and Methods. Each tem- RT-PCR showed that germline ⑀ transcripts were induced by IL-4. plate contained the same amount of cDNA from RNA extracted from 1 ϫ In our experiments, the transcript expression by B cells was not 106 highly purified B cells and one of fivefold dilutions of germline C⑀ enhanced by the addition of anti-CD40 to IL-4 (Fig. 3). We were transcript competitor from 3.2 ϫ 10Ϫ1 to 2.6 ϫ 10Ϫ3 attomoles. The ⑀ amounts of the germline C⑀ mRNA were quantitated by the amounts of the

unable to find differences in the expression of germline by B cells by guest on October 2, 2021 in CD70 transfectant-treated vs untreated B cells in the presence of competitive template that most nearly equaled the amplification of the cel- IL-4 or IL-4 plus anti-CD40 (Fig. 3). The addition of CD70 trans- lular cDNA (arrows). T, target cDNA products; C, competitive products. ␤ fectants also had no enhancing effect on transcription in the pres- The competitive PCR assay of the -actin in the same samples is shown, confirming that the amounts of cDNA used for the assay were the same. ence of IL-4 plus anti-CD40 plus CD32 transfectants (data not The fractions above represent the dilution series of the competitive C⑀ shown). This effect did not change with various culture times from ␤ ϫ Ϫ1 Ϫ DNA and the competitive -actin DNA (1 corresponds to 3.2 10 16–48 h (data not shown). Although CD27 B cells did not pro- attomoles). The data shown are representative of five separate experiments. duce IgE (Fig. 2), their germline ⑀ transcripts were obtained in the presence of IL-4 plus anti-CD40 (Fig. 4). Thus, it appears that the increased IgE secretion from peripheral blood B cells by CD27/ and CD70 interaction with anti-CD70 mAb (Fig. 5A). Morpholog- Ϫ high CD70 interaction does not occur in the process of germline ⑀ ical analysis showed that CD20 CD38 cells, which were in- transcripts. creased by CD27/CD70 interaction in the presence of IL-4 plus

Induction of plasma cells by CD27/CD70 interaction with IL-4 plus anti-CD40 Recently, we demonstrated that triggering via CD27 by CD70 on purified peripheral blood B cells yielded an increase in the number of plasma cells in the presence of IL-10 (17). The finding prompted us to ascertain whether CD27/CD70 interaction induced differen- tiation of B cells into plasma cells secreting IgE in the presence of IL-4 plus anti-CD40. Accordingly, we studied the effect of CD27 signaling on peripheral blood B cell differentiation stimulated with IL-4 plus anti-CD40 by flow cytometric and morphological anal- yses. Human peripheral blood B cells expressed high levels of ⑀ ϩ CD20 and CD40 and a low level of CD38, and plasma cells ex- FIGURE 4. Germline transcript expression by CD27 B cells and CD27Ϫ B cells. CD27ϩ and CD27Ϫ B cells were stimulated with IL-4 plus pressed CD38 strongly. Most interestingly, B cells stimulated with anti-CD40 for 16 h. After extraction of total RNA, quantitative RT-PCR CD70 transfectants in the presence of IL-4 plus anti-CD40 for was performed as described in Materials and Methods. Each template con- 12–14 days differentiated into plasma cells. In contrast, IL-4 plus tained the same amount of cDNA from RNA extracted from 1 ϫ 106 highly anti-CD40 without CD27 signaling induced a slight increase in the purified B cells and one of fivefold dilutions of germline C⑀ transcript number of plasma cells. The increase in the differentiation from B competitor from 3.2 ϫ 10Ϫ1 to 2.6 ϫ 10Ϫ3 attomoles. The data shown are cells to plasma cells disappeared by the blockage between CD27 representative of three separate experiments. 6500 AUGMENTATION OF IgE SYNTHESIS BY CD27/CD70

FIGURE 5. Induction of plasma cells by triggering via CD27 on B cells in the presence of IL-4 plus anti-CD40 mAb. A, Highly puri- fied B cells were cultured with IL-4 (50 ng/ml) plus anti-CD40 (1 ␮g/ml) in the presence of mock transfectants, CD70 transfectants, or CD70 transfectants plus anti-CD70 mAb (1 ␮g/ml) for 12–14 days. Then the cells were stained with anti-CD38-FITC and anti-CD20- PE. Ab-coated cells were gated on living cells by cell size and granularity, and dead cells were removed by propidium iodide (PI) stain- ing and then enumerated by flow cytometric analysis. Expressions of CD38 and CD20 on B cells are shown with a log scale. B, Highly pu- rified B cells were cultured in the presence of Downloaded from IL-4 plus anti-CD40 plus CD70 transfectants for 14 days. Then the cells were separated into CD38Ϫ CD20ϩ B cells (a) and CD38ϩ CD20Ϫ plasma cells (b) by sorting. The cells were cy- tospun and stained with May-Giemsa staining. http://www.jimmunol.org/

anti-CD40, were typical plasma cells with a basophilic cytoplasm BF1/Blimp-1, which has been shown to be involved in multiple with a pale Golgi zone and an eccentric nucleus. In contrast, steps of B cell differentiation leading to plasma cell development CD20ϩ CD38low cells revealed the morphology of B cells (Fig. (28) and the transcription factor BSAP. As shown in Fig. 6, PRDI- 5B). These results clearly demonstrated that, in agreement with the BF1 mRNA expression was up-regulated by the addition of CD70 IgE production measurement, CD27/CD70 interaction promotes transfectants compared with that with mock transfectants in the by guest on October 2, 2021 plasma cell differentiation in the presence of IL-4 plus anti-CD40. presence of IL-4 plus anti-CD40 after both 16 h and 4 days of culture. Up-regulation of BSAP mRNA expression was observed, Effect of CD27/CD70 interaction on the expression of although it was not significant, in CD70 transfectant-treated B cells PRDI-BF1 and BSAP in the presence of IL-4 plus anti-CD40. The enhancement of As a further assessment of the role of CD27 signaling on B cell PRDI-BF1 and BSAP mRNA levels by CD70 transfectants was differentiation into plasma cells, we finally investigated the effect completely blocked by the initial addition of anti-CD70 blocking of CD27/CD70 interaction on the mRNA expression of PRDI- mAb (data not shown). The murine pre-B cell lines, mock/300-19 and CD70/300-19, did not express mRNA for human PRDI-BF1, ␤ BSAP, or 2m (Fig. 6, lanes 1 and 2). These studies indicate that while CD27/CD70 interaction does not reduce the expression of BSAP, it promotes the generation of plasma cells at least in part by up-regulating PRDI-BF1 levels.

Discussion B cell IgE production is regulated by cytokines such as IL-4 (29– 31) and IL-13 (5, 6) and by direct contact between B and Th cells (2, 29, 32, 33), in which CD40/CD154 interaction is important (3). Gascan et al. reported that the addition of both anti-CD40 and the CD4ϩ T cell clones to purified B cells in the presence of IL-4 had FIGURE 6. Effect of CD27 cross-linking on PRDI-BF1 and BSAP ex- synergistic effects on IgE synthesis, suggesting that different B cell pression. Mock transfectants alone (lane 1), CD70 transfectants alone (lane activation pathways can result in IgE switching (4). Life et al. (8) 2), highly purified B cells with medium alone (lanes 3 and 6), mock trans- also suggested the involvement of T cell surface molecules, other fectants (lanes 4 and 7), or CD70 transfectants (lanes 5 and 8) were cul- than CD154, in the induction of IgE synthesis, because T cell tured in the presence of IL-4 plus anti-CD40 for 16 h (lanes 3–5) or 4 days clones with nonfunctional CD154 molecules obtained from (lanes 6–8). After extraction of total RNA, RT-PCR was performed as described in Materials and Methods. A 1-kb ladder was used as a m.w. X-linked hyper-IgM syndrome patients induced IgE synthesis. As previously reported, peripheral blood B cells can be separated by marker (M). The C lanes represent control of contamination-free reaction, ϩ ␤ ␤ CD27 expression, and CD27 B cells produce IgA, IgM, and IgG, in which all PCR reagents are present, but there is no cDNA. 2m( 2-MG) was used as a positive control. Data are representative of the results of three which are augmented with CD70 transfectants by promoting their experiments using different donors. differentiation into Ab-secreting cells in various B cell stimulation The Journal of Immunology 6501 systems (17, 18, 27). These findings prompted us to ascertain These findings suggested that CD27/CD70 interaction does not whether CD27/CD70 interaction participates in B cell IgE participate in the IL-4-induced germline ⑀ expression. However, synthesis. one possibility remains that CD27 signaling may induce IgE class It is known that IL-4 has a potent function in the production of switching following the increase in germline ⑀ transcripts with IgE by B cells (34, 35). However, IL-4 alone induces B cell germ- IL-4 or IL-4 plus CD40 signaling. To clarify this point, we per- line ⑀ transcripts but not IgE production. By the addition of CD40 formed quantitative RT-PCR using mature ⑀ primers. Unfortu- signaling to IL-4, the mature ⑀ transcripts can be induced, and IgE nately, as we could not detect mature ⑀ bands using several primer synthesis finally occurs (4, 3, 26). In our experiments, CD70 trans- pairs in the presence of IL-4 plus anti-CD40 plus CD32 transfec- fectants significantly augmented IgE production by highly purified tants, we failed to estimate the effects of CD27/CD70 interaction in B cells cultured with IL-4 plus anti-CD40. Since the blockage of the IgE class switching. CD27/CD70 interaction by mAb completely blocks the augmen- As reported previously, the CD27 signal resulted in the terminal tation of B cell IgE production by CD70 transfectants (Fig. 1), differentiation of peripheral blood memory B cells into plasma CD27 signaling evidently plays an important role in IgE synthesis. cells in B cell activation systems by IL-10 or IL-10 plus IL-2 (17) When we used highly purified CD27ϩ B cells, their IgE production and SAC or SAC plus IL-2 (18). In our experiments, B cells also was increased more than that of unseparated B cells in the presence differentiated into plasma cells by triggering via CD27 in the pres- of IL-4 plus anti-CD40, whereas CD27Ϫ B cells did not entirely ence of IL-4 plus anti-CD40 (Fig. 5), but not in the presence of produce IgE in the presence of IL-4 plus anti-CD40 (Fig. 2). These IL-4 alone (data not shown). Since CD154 is expressed on T cells findings support the view that CD27ϩ B cells are primed memory soon after the activation, and CD70 is expressed on T cells later B cells, and CD27Ϫ B cells are unprimed naive B cells as we previ- after the activation (17), CD40 triggering probably participates in Downloaded from ously demonstrated in the synthesis of IgA, IgM, and IgG (27). the early steps of the IgE switch recombination and then CD27/ Several reports indicated that CD70 transfectants induced B cell CD70 interaction may promote the differentiation of class- proliferation in the presence of SAC plus IL-2 (15, 16, 18). In our switched B cells into IgE-producing plasma cells (35, 39, 40). experiment, CD70 transfectants augmented B cell proliferation to CD40/CD154 interaction also induces the expansion of memory B about 3 times that found in the presence of IL-4 (Table I). In cells expressing CD27 on the surface. CD27 may be important in disagreement with our data, Jumper et al. reported that stimulation the induction of plasma cells when the expansion of CD27-positive via CD27 had no effect on B cells in the presence of IL-2 or IL-4, B cells has already occurred (17, 18). Thus, as we demonstrated http://www.jimmunol.org/ suggesting that the role of CD70 in regulating B cell growth and here and previously, CD27 is crucial in controlling the differenti- differentiation is questionable (36). The reasons for the discrep- ation of all isotype-committed memory B cells into plasma cells. ancy in B cell proliferation via CD27 between the reported data PRDI-BF1/Blimp-1, a recently described zinc finger transcrip- (36) and our present results are not clear, but several possibilities tion factor, is involved in various steps of B cell terminal differ- could be postulated. One possibility is that the degree of the CD70 entiation (28), indicating that its expression participates in the gen- expression of the transfectants may be different; CD70 expression eration of plasma cells. Since CD27 cross-linking on B cells of our transfectants is prominent compared with that of the re- enhanced the mRNA expression of PRDI-BF1/Blimp-1, even after

ported cells (36). Alternatively, the concentrations of cytokines the overnight activation, the induction of plasma cells, which was by guest on October 2, 2021 used in the studies are probably different between the two studies. demonstrated by FACS, is probably due to the effect of the ex- The levels of B cell proliferation in our system using IL-4 plus pression of PRDI-BF1/Blimp-1 induced by CD27/CD70 interac- anti-CD40 were very low compared with the proliferation effects tion. We also investigated the transcription factor BSAP/Pax-5, previously reported (36). As cross-linking of anti-CD40 by CD32 which is involved in the regulation of B cell proliferation, because transfectants or soluble CD154 in the presence of IL-4 greatly it is expressed in all B cell developmental stages except for the promoted B cell proliferation, the effects of our stimulation sys- plasma cell stage (41). BSAP mRNA expression was unexpectedly tems with IL-4 plus anti-CD40 alone appear to be suboptimal. up-regulated by triggering via CD27 on B cells. A large majority Enhancement of B cell proliferation by CD70 transfectants were of the cultured cells in our system was composed of B cells, and remarkable in the presence of IL-4 plus anti-CD40, but were not plasma cells constituted a small population, even after 8 days of prominent in the presence of IL-4 plus anti-CD40 plus CD32 trans- culture. Thus, the remaining B cells may express BSAP. At least, fectants or IL-4 plus soluble CD154. Thus, our present suboptimal CD27/CD70 interaction on B cells did not directly reduce BSAP activation system is suitable for observing the enhancing effects of expression. CD27/CD70 interaction on B cell proliferation. On the other hand, In summary, CD70 transfectants significantly augmented IgE IgE synthesis was not as significantly increased in the presence of production from highly purified B cells, especially CD27ϩ B cells IL-4 plus anti-CD40 plus CD32 transfectants or IL-4 plus soluble cultured with IL-4 plus anti-CD40, by enhancing B cell prolifer- CD154 compared with that in the presence of IL-4 plus anti-CD40. ation and differentiation into plasma cells without any increase in The enhancement of IgE synthesis via CD27 signaling was most the expression of germline ⑀. Our findings presented in this paper significant in the presence of IL-4 plus anti-CD40. should be tested in the murine system including CD27Ϫ/Ϫ mutant B cell IgE synthesis is preceded by transcription of the germline mice (42), although the effects of CD27/CD70 interaction on B cell ⑀ gene, and the relationship between this expression and subse- Ig synthesis in the murine and human systems are somewhat dif- quent switching to C⑀ has been reported (37, 38). CD20ϩ CD27ϩ ferent (J. Borst, unpublished observations). Experiments to exam- and CD20ϩ CD27Ϫ B cells from healthy adults did not differen- ine these aspects are underway. tiate in their ability to produce ⑀ transcripts by exogenous IL-4 (Fig. 4). Therefore, IL-4-induced ⑀ transcripts occur similarly in Acknowledgments ϩ Ϫ ϩ ϩ CD20 CD27 naive B cell and CD20 CD27 B cell popula- We thank Drs. S. Nonoyama, T. Kobata, and C. Morimoto for support. tions. Jumper et al. (36) demonstrated that the addition of CD70 transfectants into EBV-transformed B cells did not augment the References germline ⑀ expression. Also, triggering via CD27 by CD70 in the 1. de Weck, A. L., and D. R. Stanworth. 1974. I. Anaphylactic hypersensitivity: present study exerted no enhancing effect on the expression of the introduction. In Progress in Immunology. L. Brent and J. Holborow, eds. North germline ⑀ in the presence of IL-4 or IL-4 plus anti-CD40 (Fig. 3). Holland, Amsterdam, p. 4. 6502 AUGMENTATION OF IgE SYNTHESIS BY CD27/CD70

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