Signaling through CD70 Regulates B Activation and IgG Production Ramon Arens, Martijn A. Nolte, Kiki Tesselaar, Bianca Heemskerk, Kris A. Reedquist, René A. W. van Lier and This information is current as Marinus H. J. van Oers of September 24, 2021. J Immunol 2004; 173:3901-3908; ; doi: 10.4049/jimmunol.173.6.3901 http://www.jimmunol.org/content/173/6/3901 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Signaling through CD70 Regulates B Cell Activation and IgG Production

Ramon Arens,1*† Martijn A. Nolte,1*† Kiki Tesselaar,2† Bianca Heemskerk,3† Kris A. Reedquist,† Rene´A. W. van Lier,† and Marinus H. J. van Oers4*

CD70, the cellular ligand of the TNF family member CD27, is expressed transiently on activated T and B cells and constitutively on a subset of B cell chronic lymphocytic leukemia and large B cell . In the present study, we used B cells constitutively expressing CD70 to study the functional consequences of signaling through CD70. In vitro, CD70 ligation with anti-CD70 mAbs strongly supported proliferation and cell cycle entry of B cells submitogenically stimulated with either anti-CD40 mAb, LPS, or IL-4. In this process, the cell surface receptors CD25, CD44, CD69, CD95, and GL7 were up-regulated, whereas the expression of CD21, CD62L, surface IgM (sIgM), and sIgD was decreased. Addition of CD70 mAb to low dose LPS-stimulated CD70-positive B cells strongly diminished IgG secretion and enhanced production of IgM. Signaling through CD70 on B cells was Downloaded from dependent on the initiation of both PI3K and MEK pathways. In vivo exposure to either CD70 mAb or the CD70 counterreceptor CD27 down-regulated CD62L and sIgM on CD70-positive B cells. CD70 signaling during -dependent immune responses also decreased IgG-specific Ab titers. Together, the in vitro and in vivo data demonstrate that CD70 has potent reverse signaling properties in B cells, initiating a signaling cascade that regulates expansion and differentiation. The Journal of Immunology, 2004, 173: 3901–3908. http://www.jimmunol.org/ fter encountering Ags, B cells undergo a series of acti- LIGHT (12) have been shown to possess ca- vation and maturation events, eventually leading to the pacity. Thus, bidirectional signaling might be a general phenom- A formation of Ab-secreting plasma cells and memory enon in interactions of TNF-TNFR family , thereby abro- cells. In T cell-dependent B cell responses, initial B cell activation gating the discrimination between receptor and ligand. is linked to cell contact-mediated interactions and to stimulation by The TNFR family member CD27 is expressed in both mice and . In delivering contact-dependent signals, members of the humans on the majority of T cells and and on subsets TNF-TNFR family guide survival, proliferation, and the formation of NK cells, and hemopoietic stem cells (13–15). In humans, ex- of memory cells and plasma cells (1). pression of CD27 was found on Ag-experienced B cells (16). Its

Fine-tuning of the signals from the TNF and TNFR family ligand CD70 is transiently expressed on the surface of activated B, by guest on September 24, 2021 members is largely achieved by tight regulation of their expres- T, and dendritic cells (17, 18). Functional studies have shown that sion. Most of the effects of TNFR-TNF interactions have been CD27 ligation promotes TCR-driven T cell expansion and effector attributed to intracellular signaling events evoked by members of cell formation (19–22). In addition to these costimulatory effects the TNFR family that can couple to two principal classes of cy- on T cells, several in vitro studies have indicated that CD27 liga- toplasmic adaptor proteins: TNF receptor-associated factors and tion on B cells augments differentiation of B cells into Ab-secret- death domain-containing molecules (2). However, functional ex- ing plasma cells (23–25). periments have also provided evidence for signaling through TNF In contrast to the tightly regulated physiological expression of family members (also called reverse signaling). In particular, TNF CD70, constitutive expression of human CD70 is found on several (3), CD153 (CD30L) (4), CD154 (CD40L) (5, 6), CD197 (CD95L) malignancies, including leukemias and lymphomas (26–28), car- (7), CD137 (4-1BBL) (8), CD134 (OX40L) (9), TNF-related ac- cinomas (29, 30), and brain tumors (31). Previously, we showed tivation-induced (TRANCE)5 (10), TRAIL (11), and that in vitro cross-linking of CD70 on CD70-expressing malignant B cells augmented proliferation, raising the possibility that CD70 functions as an activation receptor on malignant B cells (28). To *Department of Hematology and †Laboratory for Experimental Immunology, Aca- demic Medical Center, University of Amsterdam, Amsterdam, The Netherlands directly examine the consequences of CD70 signaling on B cell Received for publication January 7, 2004. Accepted for publication July 16, 2004. responses in vitro and in vivo, we used B cells constitutively ex- pressing CD70. Our results demonstrate that CD70 ligation results 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 in B cell proliferation and differentiation, especially in conjunction with 18 U.S.C. Section 1734 solely to indicate this fact. with TLR4, CD40, and IL-4R occupation. 1 R.A. and M.A.N. contributed equally to this work. 2 Current address: Department of Pediatrics, Leiden University Medical Center, Lei- Materials and Methods den, The Netherlands. Animals 3 Current address: Department of Clinical Viro-Immunology, Sanquin Research at CLB, Amsterdam, The Netherlands. C57BL/6, CD70 transgenic (Tg), CD27Ϫ/Ϫ (21), CD27Ϫ/Ϫϫ CD70 Tg ␥Ϫ/Ϫ ␥Ϫ/Ϫϫ 4 Address correspondence and reprint requests to Dr. Marinus H. J. van Oers, Aca- mice (22), IFN- (32), and IFN- CD70 Tg (22) on a B6 back- demic Medical Center, Department of Hematology, F4-224, University of Amster- ground were bred and maintained in the facilities of The Netherlands Can- dam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail address: cer Institute (Amsterdam, The Netherlands). B cell-specific CD70 Tg mice [email protected] were generated by expression of the murine CD70 under control of 5 Abbreviations used in this paper: TRANCE, TNF-related activation-induced cyto- the human CD19 promoter, as previously described (22). The analysis of kine; HPE, high performance ELISA; KLH, keyhole limpet hemocyanin; PKB, pro- the frequency and absolute numbers of mature follicular, marginal zone, tein kinase B; sIg, soluble Ig; Tg, transgenic; TNP, trinitrophenol; WT, wild type. and transitional type 1 and type 2 B cells resulted in similar findings among

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 3902 CD70 SIGNALING IN B CELLS

wild-type (WT), C27Ϫ/Ϫ, and CD27Ϫ/Ϫϫ CD70 Tg mice (data not shown). Determination of Ig titers by ELISA All mice used were 6–12 wk old. All animal experiments were performed according to institutional and national guidelines. Maxisorb 96-well plates (Nunc, Roskilde, Denmark) were coated with 1 ␮g/ml TNP-BSA in 0.1 M sodium carbonate buffer (pH 9.7) for 16 h at Immunization and adoptive transfers 4°C. After blocking for 1 h with 2% milk in PBS at room temperature, sera were added at an initial dilution of 1/100 with high performance ELISA ␮ Mice were immunized by i.p. injection with 100 g of trinitrophenol (HPE) buffer (CLB, Amsterdam, The Netherlands) and 1/3 sequential di- (TNP)-keyhole limpet hemocyanin (KLH) emulsified in alum (Biosearch lutions, and incubated for3hatroom temperature. Plates were subse- Technologies, Novato, CA). Sera were collected at days 0, 7, and 14. quently washed six times and incubated with 0.1 ␮g/ml biotinylated rat Anti-TNP-specific Ig levels were determined by ELISA, as described anti-mouse Ig (Southern Biotechnology Associates) of the indicated iso- below. In vivo CD70 cross-linking was accomplished by i.p. injection with type in HPE buffer for1hatroom temperature. After washing, plates were 0.5 mg of anti-mouse CD70 mAb (clone 6D8) (18). For adoptive transfer, incubated with streptavidin-conjugated HRP streptavidin for 45 min, ϫ 6 purified splenic B cells (20 10 ) were resuspended in PBS and washed, and developed with tetramethylbenzidine substrate. The reaction injected i.v. was stopped with2MH2SO4, and OD was measured at 450 nm and endpoint titers were expressed as log . B cell purification 3 For measurement of Ig concentrations in 7-day-old cultures, plates were Single-cell suspensions of splenocytes were obtained by mincing through coated with 1 ␮g/ml unlabeled rat anti-mouse Ig of the indicated isotype, cell strainers, and erythrocytes were lysed with ammonium chloride solu- and collected supernatants were 1/10 diluted in HPE buffer. Subsequent tion. B cells were positively enriched by using CD19 MACS microbeads steps were performed, as described above. Ig concentrations were cal- and the MACS system, according to the manufacturer’s guidelines (Milte- culated from linear standard curves generated with affinity-purified nyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated cells mouse Ig (Southern Biotechnology Associates). was verified by flow cytometric analysis (Ͼ95% B220ϩ). Immunoblotting Downloaded from Determination of B cell characteristics in vitro B cells (1 ϫ 107) were stimulated with anti-CD70 mAb (25 ␮g/ml; clone Cell culture and stimulation. Purified B cells were cultured in IMDM 6D8) for 5 and 15 min, and subsequently lysed in ice-cold lysis buffer (50 medium (Invitrogen Life Technologies, Gaithersburg, MD) supplemented mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 137.5 mM NaVl, 1% glycerol, 1 Ϫ5 with 10% heat-inactivated FCS, L-glutamine, gentamicin, and 5 ϫ 10 M mM sodium orthovanadate, and 0.5 mM EDTA, pH 8.0). After centrifu- 2-ME. B cells were seeded at 1 or 5 ϫ 105 cells/well in a final volume of gation, cell lysates were resolved by 9 or 10.5% SDS-PAGE and electro- 200 ␮l in 96-well flat-bottom plates and incubated for the indicated time blotted to polyvinylidene difluoride membrane. Blots were blocked in 2% periods at 37°C in a humidified atmosphere containing 5% CO . Cells were milk and incubated with primary Abs. Anti- kinase B (PKB), anti-

2 http://www.jimmunol.org/ stimulated with various concentrations of either LPS (Sigma-Aldrich, St. phospho-PKB, anti-ERK1/2, and anti-phospho-ERK1/2 Abs were pur- Louis, MO), anti-CD40 mAb (clone HM-40.3; BD Pharmingen, San Di- chased from Technology (Beverly, MA). After incubation Ј ego, CA), goat anti-mouse IgM F(ab )2 (Jackson ImmunoResearch Labo- with HRP-conjugated goat anti-rabbit, bound Igs were detected using ECL ratories, West Grove, PA), or IL-4 (50 U/ml) in presence or absence of (Amersham Biosciences), according to manufacturer’s instructions. anti-mouse CD70 mAb (clone 6D8) (18). For stimulation with CD27, B cells were cocultured 1:1 with T cells derived from WT or CD27Ϫ/Ϫ mice. Results T cells were purified, as described previously (21), and either stimulated Enhanced B cell proliferation after CD70 ligation with CD3 mAb (clone 145-2C11) or left unstimulated. B cell proliferation assays. Cultures were pulsed with 0.2 ␮Ci of [3H]thy- To study the effects of CD70 signaling in B cell responses, we used midine during the last 16 h of 3 days, and incorporation was determined by B cells from mice that constitutively express murine CD70 on their using an automatic cell harvester and liquid scintillation counter. Data are surface and concomitantly lack CD27 (22), thereby preventing sig- by guest on September 24, 2021 Ϯ displayed as mean SD of triplicate cultures. Each experiment was per- naling via the latter molecule (Fig. 1A). Cross-linking of CD70 formed three times, yielding similar results, and the results of one repre- Ϫ/Ϫϫ sentative experiment are shown. with anti-CD70 mAbs enhanced proliferation of these CD27 Cell cycle analysis. Cell cycle progression was analyzed by flow cytom- CD70 Tg B cells, as evidenced by thymidine incorporation (Fig. etry using CFSE. B cells (1 ϫ 107) were washed three times with PBS, and 1A). In contrast, addition of anti-CD70 mAbs had no effect on pro- subsequently CFSE was added to a final concentration of 5 ␮M in PBS. liferation of B cells obtained from WT or CD27Ϫ/Ϫ mice (Fig. 1B). After 10 min at 37°C, labeling was stopped by adding 10% FCS-containing Upon stimulation through cell surface receptors such as TLR4, IMDM and cells were washed twice. CFSE-labeled cells were cultured, as described above, with 0.25 ␮g/ml LPS or 1 ␮g/ml anti-CD40 in the pres- CD40, BCR, and IL-4R resting B cells become activated and pro- ence of anti-CD70 mAb or control mAb for 2, 3, or 4 days. liferate. To test whether CD70-derived signals act synergistically ϩ Inhibition of signal transduction pathways. The following signaling in- with these B cell stimuli, CD70 B cells were cultured with bac- hibitors were used: SB203580 at 10 ␮M (Calbiochem, San Diego, CA), terial LPS, anti-CD40 mAb, anti-IgM Ab, or IL-4 either in the LY294002 at 10 ␮M (Sigma-Aldrich), rapamycin at 2 ng/ml (Sigma- presence or absence of anti-CD70 mAb. Increased [3H]thymidine ␮ Aldrich; kindly provided by L. Evers), protein phosphatase 1 at 10 M incorporation was observed in response to suboptimal concentra- (SanverTech, Boechout, Belgium; kind gift from H. Versteeg), pertussis toxin at 100 ng/ml (Sigma-Aldrich; kindly provided by J. Wormmeester), tions of LPS, a stimulus that activates B cells independently of PD98059 at 50 ␮M (BIOMOL, Plymouth Meeting, PA), and U0126 at 10 BCR signaling via TLR4 (Fig. 1C). In addition, CD70 ligation also ␮M (BIOMOL). All stock solutions were made in DMSO, except for rapa- synergized with anti-CD40 mAb or IL-4, whereas only marginal mycin and pertussis toxin, which were dissolved in ethanol and RPMI additive effects of anti-CD70 mAb on [3H]thymidine incorporation 1640, respectively. Purified B cells were pretreated with the inhibitors for 1hat37°C before activation, and the inhibitors were present in culture were found with (sub)optimal anti-IgM Ab stimulation. Thus, during the stimulation period. CD70 cross-linking on B cells, either alone or in combination with TLR4-, CD40-, and IL-4R-derived signals enhanced B cell Flow cytometry proliferation. Cells (1–4 ϫ 105) were incubated with Abs for 30 min at 4°C in staining buffer (PBS containing 0.5% BSA and 0.05% azide). All stainings included CD70 ligation on B cells promotes cell cycle entry preincubation with the anti-Fc receptor (CD16/CD32) Ab 2.4G2 (BD The increased [3H]thymidine incorporation observed after CD70 Pharmingen) to reduce Fc receptor-mediated binding. Propidium iodide (2 ␮g/ml) was added to samples before acquisition to exclude apoptotic or ligation might reflect enhanced cell survival, increased cell cycle necrotic cells. Data acquisition was performed with FACSCalibur, and progression, or both. To distinguish between these possibilities, we analysis was conducted using CellQuestPro software (BD Biosciences, San first examined cell viability by flow cytometric detection using Jose, CA). mAbs used were: FITC-, PE-, or allophycocyanin-labeled anti- propidium iodide staining. However, no obvious increase in sur- B220, CD21/CD35, CD23, CD25, CD44, CD54, CD62L, CD69, CD86, ϩ CD95, CD138, and GL-7 (BD Pharmingen). FITC-labeled anti-IgM and vival of CD70 B cells was found after CD70 ligation (data not PE-labeled anti-IgD were purchased from Southern Biotechnology Asso- shown). Cell cycle progression was assessed at various time points ciates (Birmingham, AL). after stimulation by monitoring CFSE dilution. We observed a The Journal of Immunology 3903 Downloaded from http://www.jimmunol.org/

FIGURE 1. Enhanced B cell proliferation after CD70 ligation. A, Purified splenic B cells were stained for B220 and anti-CD70 mAbs. Spleens of WT, CD27Ϫ/Ϫ, and CD27Ϫ/Ϫϫ CD70 Tg mice were isolated, and B cells were purified using MACS magnetic separation system. B, Increased CD70-induced B cell proliferation. Purified splenic B cells from WT, CD27Ϫ/Ϫ, and CD27Ϫ/Ϫϫ CD70 Tg mice were cultured in triplicates in 96-well microplates in presence of 5 ␮g/ml anti-CD70 mAb (f) or control Ab (Ⅺ). Proliferation was measured by [3H]thymidine incorporation during the last 16 h of a 3-day culture. The data are displayed as the mean cpm values Ϯ SE of one representative experiment of three. C, CD70-induced B cell proliferation is enhanced by CD40, TLR4, and IL-4R signaling. Purified splenic B cells from CD27Ϫ/Ϫϫ CD70 Tg mice were stimulated with the indicated concentrations of (␮g/ml)

Ј ␮ f Ⅺ by guest on September 24, 2021 LPS, anti-CD40 mAb, polyclonal F(ab )2 goat anti-mouse IgM, or IL-4 (ng/ml) in presence of 5 g/ml anti-CD70 mAb ( ) or control mAb ( ). Proliferation was measured as in B. The data are displayed as the mean cpm values Ϯ SE of one representative experiment of three. strongly increased entry into the cell cycle of CD70-triggered B not with addition of anti-IgM Ab (data not shown). Thus, CD70 cells in combination with LPS or anti-CD40 mAb stimulation ligation on B cells promotes cell cycle entry. when compared with LPS or CD40 stimulation alone (Fig. 2). Remarkably, a fraction of the activated B cells without CD70 li- CD70 ligation mediates expression of B cell activation and gation had undergone more cell divisions than that of CD70-trig- differentiation molecules gered B cells. Similar results were found with IL-4 addition, but Activation of B cells changes the expression of various molecules, thereby reflecting the altered activation and maturation state. We examined the effects of CD70 ligation on the expression of cell surface B cell markers by flow cytometry. At day 3 after stimula- tion, expression of CD25, CD44, CD69, CD86, CD95, and GL7 was increased on B cells triggered with anti-CD70 mAb and in combination with either LPS or anti-CD40 mAb, whereas expres- sion of soluble IgM (sIgM), sIgD, and CD62L was down-regulated (Fig. 3A). Comparable effects on the expression of B cell surface markers were found in presence of IL-4, whereas stimulation in combination with anti-IgM Ab only had marginal effects (data not shown). The anti-CD70 mAb had no effect on CD70-negative WT and CD27Ϫ/Ϫ B cells (data not shown). Increase of CD54 and CD23 was only found after CD70 ligation in combination with FIGURE 2. CD70 ligation promotes cell cycle entry. For B cell division LPS, whereas CD21/CD35 was found to be down-regulated in Ϫ/Ϫϫ measurement, purified B cells of CD27 CD70 Tg were labeled with combination with anti-CD40 mAb (Fig. 3A). In addition, spleen- the fluorescence dye CFSE, and incubated with 0.25 ␮g/ml LPS or 1 ␮g/ml derived B cells showed comparable expression patterns of B cell anti-CD40 in presence or absence of 5 ␮g/ml anti-CD70 mAb. Loss of CFSE fluorescence, indicating cell division progression, was measured by surface molecules after CD70 signaling as B cells derived from flow cytometry on days 2, 3, and 4. Dead cells were excluded by propidium peripheral lymph nodes (data not shown). iodide staining. Solid lines indicate CD70-activated cells, and shaded his- The effect of CD70 ligation on the B cell activation was dose tograms indicate controls. Data are representative of three independent dependent; higher concentrations of anti-CD70 mAb increased the experiments. expression of CD69 as well as the expression of CD95 (Fig. 3B). 3904 CD70 SIGNALING IN B CELLS

FIGURE 3. CD70 ligation medi- ates expression of B cell activation and maturation molecules. A, Effects of CD70 signaling on B cell surface markers. Purified splenic B cells from CD27Ϫ/Ϫϫ CD70 Tg mice were cul- tured in medium alone or stimulated with 0.25 ␮g/ml LPS or 1 ␮g/ml anti- CD40 mAb in presence of either 5 ␮g/ml anti-CD70 mAb (solid line his- tograms) or control mAb (shaded his- tograms). After 1 day of culture in medium alone or 3 days after LPS or CD40 stimulation, B cells were ana- lyzed by flow cytometry for surface ex- pression of CD25, CD44, CD69, CD86, CD95 (Fas/APO-1), GL-7, CD62L, CD21/35, CD23, CD54, sIgM, and

IgD. B, Dose-dependent effects of Downloaded from CD70-induced up-regulation of CD69 and CD95 by anti-CD70 mAb. Cells were stimulated with 1 ␮g/ml LPS and increasing amounts of anti-CD70 mAbs. Data represent the mean fluores- cence intensity (MFI, f) and percent- ϩ ϩ age of CD69 and CD95 samples http://www.jimmunol.org/ (Ⅺ). C, Stimulation of CD70ϩ B cells by CD27. CD27Ϫ/Ϫϫ CD70 Tg B cells were cultured with CD3-stimulated WT T cells (CD27ϩ/ϩ) in presence or ab- sence of blocking anti-CD27 mAb or with CD3-stimulated CD27 knockout (CD27Ϫ/Ϫ) T cells. The histogram rep- resents the mean B cell surface expres- sion of CD69 and CD95 after 3 days of by guest on September 24, 2021 culture with WT T cells relative to WT T cells cultured in presence of anti- CD27 mAb or cultured in presence of CD27Ϫ/Ϫ T cells. Similar effects were observed with unstimulated T cells. All in vitro activation assays are represen- tative of at least three independent ex- periments. Only live cells were ana- lyzed, as dead cells were excluded by propidium iodide staining.

ϩ Ј Ϫ/Ϫϫ Stimulation of CD70 B cells with F(ab )2 fragments of anti- Therefore, CD27 CD70 Tg B cells were treated with specific CD70 mAb resulted in similar findings as with whole Ab, thereby inhibitors of signal transduction pathways and subsequently cul- excluding Fc receptor-mediated effects (data not shown). To in- tured in presence of anti-CD70 mAb or control mAb. Cell surface vestigate whether CD70 ligation could also be accomplished by its expression levels of CD25 and CD69 were determined by flow Ϫ/Ϫϫ natural counterreceptor CD27, we cultured CD27 CD70 Tg cytometry after 24 h as readout. CD70-mediated up-regulation of ϩ/ϩ B cells with WT (CD27 ) T cells in presence or absence of CD25 and CD69 was blocked by preincubation with the PI3K inhib- Ϫ/Ϫ blocking CD27 mAb or with CD27 knockout (CD27 ) T cells. itor LY294002 and the MAPK/ERK kinase (MEK) inhibitors The expression of CD69 and CD95 was increased on the CD70ϩ PD98059 and U0126, whereas the p38 MAPK inhibitor SB203580, B cells cultured with CD27ϩ/ϩ T cells as compared with cultures Ϫ Ϫ the mammalian target of rapamycin kinase inhibitor rapamycin, the containing blocking CD27 mAb or CD27 / T cells (Fig. 3C), Src tyrosine kinase inhibitor PPI, and the Gi␣-protein inhibitor per- indicating that functional CD70 ligation can occur after interaction with CD27. In summary, stimulation through CD70 on B cells not tussis toxin had no effect (Fig. 4A). To demonstrate that CD70 sig- only promotes cell cycle entry, but also induces activation and naling couples to PI3K and MEK pathways, we determined whether differentiation of these cells. their downstream targets, PKB (also known as Akt) and ERK, respectively, were activated. Cross-linking of CD70 increased CD70 signaling couples to PI3K and MEK pathways phosphorylation of PKB and ERK1/2 without altering PKB and To elucidate the molecular basis of CD70 signaling, signal trans- ERK1/2 protein levels (Fig. 4B). Finally, while LY294002 inhib- duction pathways activated by CD70 triggering were analyzed. ited the CD70-mediated phosphorylation of PKB, but not ERK1/2, The Journal of Immunology 3905 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. CD70 signaling couples to PI3K and MEK pathways. A, CD70-induced enhancement of CD69 up-regulation is blocked by the PI3K inhibitor LY294002 and the MEK inhibitors U0126 and PD98059. Purified CD27Ϫ/Ϫϫ CD70 Tg B cells were pretreated with 10 ␮M SB203580, 10 ␮M LY294002, 2 ng/ml rapamycin, 100 ng/ml pertussis toxin, 10 ␮M protein phosphatase 1, 50 ␮M PD98059, and 10 ␮M U0126 for 1 h and then cultured with 5 ␮g/ml anti-CD70 mAb or control mAb for 24 h. The cells were then collected and stained for cell surface expression of CD25 and CD69. B, Engagement of CD70 on CD27Ϫ/Ϫϫ CD70 Tg B cells enhances activity of the PI3K target PKB and the MEK target ERK. Immunoblot analysis of total cellular lysates for ERK and PKB phosphorylation (pERK and pPKB) and expression (ERK and PKB). Before stimulation, purified B cells were pretreated for 30 min with 10 ␮M LY294001 (the PI3K inhibitor) or 10 ␮M U0126 (the MEK inhibitor). Subsequently, cells were stimulated with anti-CD70 mAb for 5 and 15 min or left unstimulated (0 min). Data are representative of three independent experiments.

U0126 inhibited phosphorylation of ERK1/2, but not PKB (Fig. day 4 after transfer, CD27Ϫ/Ϫϫ CD70 Tg (Ly-5.2ϩ) B cells in 4B). This indicates that CD70-mediated induction of these path- recipients displayed increased CD44 expression and decreased ways apparently works in parallel rather than in series. Taken to- CD62L and sIgM expression as compared with non-CD70 Tg B gether, CD70 engagement on B cells couples to both PI3K and cells, indicating that CD27 contact is capable of modifying the MEK pathways, resulting in phosphorylation of their targets PKB phenotype of CD70-expressing B cells (Fig. 5B). In addition, flu- Ϫ Ϫ and ERK1/2. orescence-labeled CD27 / ϫ CD70 Tg B cells adoptively trans- ferred into WT (CD27ϩ/ϩ) recipient mice displayed the charac- Effects of CD70 ligation in vivo teristic CD44ϩCD62LϪIgMlow phenotype compared with Ϫ/Ϫ To test whether the CD70-mediated changes on B cell surface CD27 recipient mice. In summary, these data indicate that li- molecules can also occur in vivo, we analyzed the effect of CD70- gation of CD70 on B cells either by CD70 mAb and its cellular specific mAb on B cells in CD27Ϫ/Ϫϫ CD70 Tg mice. Ligation of ligand CD27 results in substantial B cell activation. CD70 in vivo with anti-CD70 mAb increased CD44 expression and decreased sIgM and CD62L expression, consistent with the Inhibition of formation and IgG secretion after data obtained in vitro (compare Figs. 3a and 5a). CD70 ligation To investigate whether CD70 ligation could be stimulated by its The hallmark of terminal differentiation of B cells is the formation counterreceptor CD27 in vivo, we adoptively transferred CD27Ϫ/Ϫ of plasma cells and their secretion of Igs. B cell cultures stimulated or CD27Ϫ/Ϫϫ CD70 Tg B cells into Ly-5.1 (CD27ϩ/ϩ) mice. At with LPS and CD70 showed a decrease of B cells positive for the 3906 CD70 SIGNALING IN B CELLS Downloaded from http://www.jimmunol.org/

FIGURE 5. CD70 signaling in vivo. A, Induction of CD70 signaling in B cells by CD70 mAb in vivo. CD27Ϫ/Ϫ and CD27Ϫ/Ϫϫ CD70 Tg mice were injected with 0.5 mg anti-CD70 mAb (solid line) or control mAb (shaded histogram). At day 4, splenic B cells were isolated and stained with Abs to the indicated cell surface markers. B, Induction of CD70 signaling by CD27 in vivo. B cells from CD27Ϫ/Ϫ or CD27Ϫ/Ϫϫ CD70 Tg mice (Ly-5.2) were adoptively transferred independently into Ly-5.1 recipient Ϫ/Ϫ Ϫ/Ϫϫ mice. Gated Ly-5.2 CD27 (shaded histogram) and Ly-5.2 CD27 by guest on September 24, 2021 CD70 Tg (solid line) B cells are shown. A representative result from two independent adoptive transfer experiments is shown.

FIGURE 6. CD70 ligation inhibits Ig secretion. A, Decreased expres- plasma cell differentiation marker CD138 (Syndecan-1) as com- sion of the plasma cell marker CD138 (Syndecan-1) after CD70 ligation. Ϫ/Ϫϫ pared with cultures stimulated with LPS alone (Fig. 6A). In all Purified CD27 CD70 Tg B cells stimulated with LPS or anti-CD40 CD40-stimulated B cell cultures, low frequencies of CD138-pos- mAb in presence (solid line) or absence (shaded histogram) of CD70 trig- gering with 5 ␮g/ml anti-CD70 mAb were analyzed at day 4 after stimu- itive cells were found (Fig. 6A). Engagement of CD70 in vitro on lation for CD138 expression. Data are representative of three independent LPS-stimulated B cells caused an increase in the cumulative con- experiments. B, Ig secretion after CD70 ligation in vitro. Enriched centration of IgM Abs after 7 days of culture, whereas IgG secre- CD27Ϫ/Ϫϫ CD70 Tg B cells were stimulated with 1 ␮g/ml LPS in pres- tion was inhibited (Fig. 6B). When B cells were stimulated with ence or absence of CD70 triggering with anti-CD70 mAb. The Ig concen- anti-CD40 mAb, CD70 ligation had no effect on IgM production, trations in the supernatant of the B cell cultures were measured by ELISA but inhibited IgG production (data not shown). at day 7 after stimulation. Data are displayed as OD (y-axis) vs 3-fold To test the effects of CD70 signaling on Ab responses in vivo, dilution steps (x-axis). The data shown are representative of three inde- we compared the T cell-dependent Ag response to TNP-KLH in pendent experiments. CÐE, T cell-dependent Ab response. C, Six-wk-old alum in CD27Ϫ/Ϫϫ CD70 Tg mice with or without anti-CD70 CD27Ϫ/Ϫϫ CD70 Tg mice injected with anti-CD70 mAb or control mAb; mAb treatment. Whereas the TNP-specific IgM levels were simi- D, WT vs CD70 Tg mice; and E, IFN-␥Ϫ/Ϫ vs IFN-␥Ϫ/Ϫϫ CD70 Tg mice lar, a considerable decrease in IgG levels was observed at day 7 were immunized with 100 ␮g of TNP-KLH emulsified in alum. Isotype- (Fig. 6C) and at day 14 (data not shown). To corroborate that specific anti-TNP titers were determined in sera by TNP-specific ELISA on CD27-CD70 interactions influence plasma cell differentiation in day 7 after immunization. Data are expressed as average values of five vivo, we examined T cell-dependent Ab response to alum-precip- mice per group. itated TNP-KLH in CD70 Tg mice that do express CD27. Despite lower B cell numbers, caused by excessive IFN-␥ secretion in these mice (21), the serum TNP-specific IgM Ab levels were in- Discussion creased as compared with WT mice, whereas lower NP-specific Effects of CD70 signaling on B cell terminal differentiation IgG titers were found (Fig. 6D). In CD70 Tg mice on an IFN-␥- In the present study, we have investigated the consequences of deficient background, which have normal B cell numbers (21), CD70 ligation in B cell activation and differentiation. By using B decreased TNP-specific IgG levels and similar IgM levels were cells from mice constitutively expressing CD70, but lacking found (Fig. 6E). Together, the in vitro and in vivo data suggest an CD27, we were able to specifically examine the role of CD70 inhibitory effect of CD70 ligation on the formation of IgG-secret- ligation without effects of CD27 signals. Addition of CD70 mAb ing plasma cells. to these B cells promoted entry into the cell cycle (Fig. 2) and The Journal of Immunology 3907 altered expression of cell surface markers, especially in combina- though it has been shown that BCR-induced phosphorylation of tion with TLR4-, CD40-, and IL-4R-derived signals (Figs. 3 and ERK is dependent on PI3K via the Ras pathway (40), we found 5). Moreover, CD70 signaling inhibited plasma cell formation and that CD70 triggering can activate MEK in a PI3K-independent IgG secretion (Figs. 6 and 7). manner. This implies either a common upstream signaling inter- Upon activation, B cells migrate toward the boundary of B and mediate or multiple independent signaling pathways initiated by T cell zones due to altered expression of receptors (33). CD70 signaling. Although we could show a clear effect of the At this particular site, B cells interact with activated, Ag-specific PI3K and MEK inhibitors on the induction of early activation Ags CD4ϩ T cells, receiving costimulatory signals. Because activated by CD70 triggering, this does not directly prove that both path- B cells express CD70 (17, 18) and activated T cells highly express ways are required for the long-term effects seen on cell cycle pro- CD27 (34), it might be that costimulation also occurs through gression and B cell differentiation. Along the same line, we cannot CD70. In addition, expression of both CD70 and CD27 has been exclude that other pathways are required for these effects. demonstrated on germinal center B cells (35, 36). The physiolog- ical outcome, in terms of the production of protective humoral Role of CD70 signaling in malignancies immune responses, of the bidirectional signaling via CD27-CD70 Besides the restricted expression of CD70 in normal tissues, strong interactions remains to be resolved. Although, on one hand, CD70 CD70 expression was found on Hodgkin’s , B cell ligation on B cells inhibits IgG secretion, triggering of CD27 on T chronic lymphocytic leukemia, and large B cell lymphomas (26– cells promotes their survival and will facilitate the generation of 28). In addition to these hematological malignancies, CD70 has Th cells. Because it has been demonstrated that CD70 transfectants also been reported to be present on various other malignancies promote B cell differentiation in responses to IL-4 and IL-10 in (29–31). The apparent deregulated expression of CD70 on malig- Downloaded from vitro (25), a third level of regulation of B cell differentiation by nant B cells suggests that CD70 might function as an agonistic these molecules can be envisaged. receptor for malignant B (28). A possible role for this A previous study showed that addition of CD27-transfected interaction in lymphomagenesis is suggested by the observations cells to T cell-dependent human B cell cultures resulted in marked that aggressive non-Hodgkin’s lymphomas abundantly express decreased IgG levels (23), which may now be explained by our CD70 and that blastoid transformation of mantle cell lymphoma observation that signaling through CD70 inhibits IgG production. coincides with the up-regulation of CD70 (41). In this respect, it is http://www.jimmunol.org/ Although CD70 ligation promoted cell cycle entry, in presence of of interest to note the paradox that certain malignancies with a LPS and to a lesser extent in presence of anti-CD40 mAb the poor prognosis (e.g., Hodgkin’s disease and anaplastic larger cell CD70-triggered B cells underwent less cell cycle progression com- lymphoma) are characterized by a high infiltration of T cells (42, pared with the fraction of rapidly dividing B cells cycling B cells 43). Potentially, these infiltrating T cells can provide the CD27 as without CD70 stimulation. This may explain the inhibitory effect ligand for CD70 on the tumor cell. Alternatively, some tumor cells of CD70 ligation on IgG production, given that IgG isotype express CD70 together with CD27, suggesting that this interaction switching is related to cell division number (37). In contrast to the may constitute an autocrine circuit regulating malignant cell effects of CD70 signaling, signaling through CD27 on B cells re- function. sults in enhanced plasma cell formation and increased IgG pro- Taken together, our results provide evidence that signals by guest on September 24, 2021 duction (23–25). Thus, CD70- and CD27-transduced signals in B through the TNF family member CD70 in B cells result in en- cells appear to have opposing effects in the fine-tuning of B cell hanced cell cycle entry while preventing differentiation into IgG- responses. secreting plasma cells. We also suggest that CD70 signaling might be important in CD70-expressing malignancies. Therefore, mod- Signal transduction pathways induced by CD70 signaling ulation of the CD70 signaling pathway might be able to control the Although signaling through TNF receptor family members by the extent of (malignant) B cell responses and potentially provide ther- TNF receptor-associated factors and death domain has been ex- apeutic benefits to treat certain immune system disorders. tensively studied (2), relatively little is known about the down- stream signaling pathways used by TNF family members. Al- Acknowledgments though the cytoplasmic domains of individual TNF family We thank Dr. J. Borst for critical reading of the manuscript. We thank the members are evolutionary conserved across species, comparison staff of the Animal Facility of The Netherlands Cancer Institute for excel- between different family members showed relatively little homol- lent animal care. ogy, and therefore provided no clues as to generic signaling mech- anisms used. 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