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* Department of Hematology and
Laboratory for Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fine-tuning of the signals from the TNF and TNFR family members is largely achieved by tight regulation of their expression. Most of the effects of TNFR-TNF interactions have been attributed to intracellular signaling events evoked by members of the TNFR family that can couple to two principal classes of cytoplasmic adaptor proteins: TNF receptor-associated factors and death domain-containing molecules (2). However, functional experiments have also provided evidence for signaling through TNF family members (also called reverse signaling). In particular, TNF (3), CD153 (CD30L) (4), CD154 (CD40L) (5, 6), CD197 (CD95L) (7), CD137 (4-1BBL) (8), CD134 (OX40L) (9), TNF-related activation-induced cytokine (TRANCE)5 (10), TRAIL (11), and LIGHT (12) have been shown to possess signal transduction capacity. Thus, bidirectional signaling might be a general phenomenon in interactions of TNF-TNFR family proteins, thereby abrogating the discrimination between receptor and ligand.
The TNFR family member CD27 is expressed in both mice and humans on the majority of T cells and thymocytes and on subsets of NK cells, and hemopoietic stem cells (13, 14, 15). In humans, expression of CD27 was found on Ag-experienced B cells (16). Its ligand CD70 is transiently expressed on the surface of activated B, T, and dendritic cells (17, 18). Functional studies have shown that CD27 ligation promotes TCR-driven T cell expansion and effector cell formation (19, 20, 21, 22). In addition to these costimulatory effects on T cells, several in vitro studies have indicated that CD27 ligation on B cells augments differentiation of B cells into Ab-secreting plasma cells (23, 24, 25).
In contrast to the tightly regulated physiological expression of CD70, constitutive expression of human CD70 is found on several malignancies, including leukemias and lymphomas (26, 27, 28), carcinomas (29, 30), and brain tumors (31). Previously, we showed 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 directly examine the consequences of CD70 signaling on B cell responses in vitro and in vivo, we used B cells constitutively expressing CD70. Our results demonstrate that CD70 ligation results in B cell proliferation and differentiation, especially in conjunction with TLR4, CD40, and IL-4R occupation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6, CD70 transgenic (Tg), CD27–/– (21), CD27–/–x CD70 Tg mice (22), IFN-
–/– (32), and IFN-–/–x CD70 Tg (22) on a B6 background were bred and maintained in the facilities of The Netherlands Cancer Institute (Amsterdam, The Netherlands). B cell-specific CD70 Tg mice were generated by expression of the murine CD70 gene under control of the human CD19 promoter, as previously described (22). The analysis of the frequency and absolute numbers of mature follicular, marginal zone, and transitional type 1 and type 2 B cells resulted in similar findings among wild-type (WT), C27–/–, and CD27–/–x CD70 Tg mice (data not shown). All mice used were 6–12 wk old. All animal experiments were performed according to institutional and national guidelines.
Immunization and adoptive transfers
Mice were immunized by i.p. injection with 100 µg of trinitrophenol (TNP)-keyhole limpet hemocyanin (KLH) emulsified in alum (Biosearch Technologies, Novato, CA). Sera were collected at days 0, 7, and 14. Anti-TNP-specific Ig levels were determined by ELISA, as described below. In vivo CD70 cross-linking was accomplished by i.p. injection with 0.5 mg of anti-mouse CD70 mAb (clone 6D8) (18). For adoptive transfer, purified splenic B cells (20 x 106) were resuspended in PBS and injected i.v.
B cell purification
Single-cell suspensions of splenocytes were obtained by mincing through cell strainers, and erythrocytes were lysed with ammonium chloride solution. B cells were positively enriched by using CD19 MACS microbeads and the MACS system, according to the manufacturer’s guidelines (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated cells was verified by flow cytometric analysis (>95% B220+).
Determination of B cell characteristics in vitro
Cell culture and stimulation. Purified B cells were cultured in IMDM medium (Invitrogen Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS, L-glutamine, gentamicin, and 5 x 10–5 M 2-ME. B cells were seeded at 1 or 5 x 105 cells/well in a final volume of 200 µl in 96-well flat-bottom plates and incubated for the indicated time periods at 37°C in a humidified atmosphere containing 5% CO2. Cells were stimulated with various concentrations of either LPS (Sigma-Aldrich, St. Louis, MO), anti-CD40 mAb (clone HM-40.3; BD Pharmingen, San Diego, CA), goat anti-mouse IgM F(ab')2 (Jackson ImmunoResearch Laboratories, West Grove, PA), or IL-4 (50 U/ml) in presence or absence of anti-mouse CD70 mAb (clone 6D8) (18). For stimulation with CD27, B cells were cocultured 1:1 with T cells derived from WT or CD27–/– mice. T cells were purified, as described previously (21), and either stimulated with CD3 mAb (clone 145-2C11) or left unstimulated.
B cell proliferation assays. Cultures were pulsed with 0.2 µCi of [3H]thymidine during the last 16 h of 3 days, and incorporation was determined by using an automatic cell harvester and liquid scintillation counter. Data are displayed as mean ± SD of triplicate cultures. Each experiment was performed three times, yielding similar results, and the results of one representative experiment are shown.
Cell cycle analysis. Cell cycle progression was analyzed by flow cytometry using CFSE. B cells (1 x 107) were washed three times with PBS, and subsequently CFSE was added to a final concentration of 5 µM in PBS. After 10 min at 37°C, labeling was stopped by adding 10% FCS-containing IMDM and cells were washed twice. CFSE-labeled cells were cultured, as described above, with 0.25 µg/ml LPS or 1 µg/ml anti-CD40 in the presence of anti-CD70 mAb or control mAb for 2, 3, or 4 days.
Inhibition of signal transduction pathways. The following signaling inhibitors were used: SB203580 at 10 µM (Calbiochem, San Diego, CA), LY294002 at 10 µM (Sigma-Aldrich), rapamycin at 2 ng/ml (Sigma-Aldrich; kindly provided by L. Evers), protein phosphatase 1 at 10 µM(SanverTech, Boechout, Belgium; kind gift from H. Versteeg), pertussis toxin at 100 ng/ml (Sigma-Aldrich; kindly provided by J. Wormmeester), PD98059 at 50 µM (BIOMOL, Plymouth Meeting, PA), and U0126 at 10 µM (BIOMOL). All stock solutions were made in DMSO, except for rapamycin and pertussis toxin, which were dissolved in ethanol and RPMI 1640, respectively. Purified B cells were pretreated with the inhibitors for 1 h at 37°C before activation, and the inhibitors were present in culture during the stimulation period.
Flow cytometry
Cells (1–4 x 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 preincubation with the anti-Fc receptor (CD16/CD32) Ab 2.4G2 (BD Pharmingen) to reduce Fc receptor-mediated binding. Propidium iodide (2 µg/ml) was added to samples before acquisition to exclude apoptotic or necrotic cells. Data acquisition was performed with FACSCalibur, and analysis was conducted using CellQuestPro software (BD Biosciences, San Jose, CA). mAbs used were: FITC-, PE-, or allophycocyanin-labeled anti-B220, CD21/CD35, CD23, CD25, CD44, CD54, CD62L, CD69, CD86, CD95, CD138, and GL-7 (BD Pharmingen). FITC-labeled anti-IgM and PE-labeled anti-IgD were purchased from Southern Biotechnology Associates (Birmingham, AL).
Determination of Ig titers by ELISA
Maxisorb 96-well plates (Nunc, Roskilde, Denmark) were coated with 1 µg/ml TNP-BSA in 0.1 M sodium carbonate buffer (pH 9.7) for 16 h at 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 (HPE) buffer (CLB, Amsterdam, The Netherlands) and 1/3 sequential dilutions, and incubated for 3 h at room temperature. Plates were subsequently washed six times and incubated with 0.1 µg/ml biotinylated rat anti-mouse Ig (Southern Biotechnology Associates) of the indicated isotype in HPE buffer for 1 h at room temperature. After washing, plates were incubated with streptavidin-conjugated HRP streptavidin for 45 min, washed, and developed with tetramethylbenzidine substrate. The reaction was stopped with 2 M H2SO4, and OD was measured at 450 nm and endpoint titers were expressed as log3.
For measurement of Ig concentrations in 7-day-old cultures, plates were coated with 1 µg/ml unlabeled rat anti-mouse Ig of the indicated isotype, and collected supernatants were 1/10 diluted in HPE buffer. Subsequent steps were performed, as described above. Ig concentrations were calculated from linear standard curves generated with affinity-purified mouse Ig (Southern Biotechnology Associates).
Immunoblotting
B cells (1 x 107) were stimulated with anti-CD70 mAb (25 µg/ml; clone 6D8) for 5 and 15 min, and subsequently lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 137.5 mM NaVl, 1% glycerol, 1 mM sodium orthovanadate, and 0.5 mM EDTA, pH 8.0). After centrifugation, cell lysates were resolved by 9 or 10.5% SDS-PAGE and electroblotted to polyvinylidene difluoride membrane. Blots were blocked in 2% milk and incubated with primary Abs. Anti-protein kinase B (PKB), anti-phospho-PKB, anti-ERK1/2, and anti-phospho-ERK1/2 Abs were purchased from Cell Signaling Technology (Beverly, MA). After incubation with HRP-conjugated goat anti-rabbit, bound Igs were detected using ECL (Amersham Biosciences), according to manufacturer’s instructions.
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Results |
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To study the effects of CD70 signaling in B cell responses, we used B cells from mice that constitutively express murine CD70 on their surface and concomitantly lack CD27 (22), thereby preventing signaling via the latter molecule (Fig. 1A). Cross-linking of CD70 with anti-CD70 mAbs enhanced proliferation of these CD27–/–x CD70 Tg B cells, as evidenced by thymidine incorporation (Fig. 1A). In contrast, addition of anti-CD70 mAbs had no effect on proliferation of B cells obtained from WT or CD27–/– mice (Fig. 1B).
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CD70 ligation on B cells promotes cell cycle entry
The increased [3H]thymidine incorporation observed after CD70 ligation might reflect enhanced cell survival, increased cell cycle progression, or both. To distinguish between these possibilities, we first examined cell viability by flow cytometric detection using propidium iodide staining. However, no obvious increase in survival of CD70+ B cells was found after CD70 ligation (data not shown). Cell cycle progression was assessed at various time points after stimulation by monitoring CFSE dilution. We observed a strongly increased entry into the cell cycle of CD70-triggered B cells in combination with LPS or anti-CD40 mAb stimulation when compared with LPS or CD40 stimulation alone (Fig. 2). Remarkably, a fraction of the activated B cells without CD70 ligation had undergone more cell divisions than that of CD70-triggered B cells. Similar results were found with IL-4 addition, but not with addition of anti-IgM Ab (data not shown). Thus, CD70 ligation on B cells promotes cell cycle entry.
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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 stimulation, 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 expression 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 CD27–/– B cells (data not shown). Increase of CD54 and CD23 was only found after CD70 ligation in combination with LPS, whereas CD21/CD35 was found to be down-regulated in combination with anti-CD40 mAb (Fig. 3A). In addition, spleen-derived B cells showed comparable expression patterns of B cell surface molecules after CD70 signaling as B cells derived from peripheral lymph nodes (data not shown).
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CD70 signaling couples to PI3K and MEK pathways
To elucidate the molecular basis of CD70 signaling, signal transduction pathways activated by CD70 triggering were analyzed. Therefore, CD27–/–x CD70 Tg B cells were treated with specific inhibitors of signal transduction pathways and subsequently cultured in presence of anti-CD70 mAb or control mAb. Cell surface expression levels of CD25 and CD69 were determined by flow cytometry after 24 h as readout. CD70-mediated up-regulation of CD25 and CD69 was blocked by preincubation with the PI3K inhibitor LY294002 and the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, whereas the p38 MAPK inhibitor SB203580, the mammalian target of rapamycin kinase inhibitor rapamycin, the Src tyrosine kinase inhibitor PPI, and the Gi
-protein inhibitor pertussis toxin had no effect (Fig. 4A). To demonstrate that CD70 signaling couples to PI3K and MEK pathways, we determined whether their downstream targets, PKB (also known as Akt) and ERK, respectively, were activated. Cross-linking of CD70 increased phosphorylation of PKB and ERK1/2 without altering PKB and ERK1/2 protein levels (Fig. 4B). Finally, while LY294002 inhibited the CD70-mediated phosphorylation of PKB, but not ERK1/2, U0126 inhibited phosphorylation of ERK1/2, but not PKB (Fig. 4B). This indicates that CD70-mediated induction of these pathways apparently works in parallel rather than in series. Taken together, CD70 engagement on B cells couples to both PI3K and MEK pathways, resulting in phosphorylation of their targets PKB and ERK1/2.
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To test whether the CD70-mediated changes on B cell surface molecules can also occur in vivo, we analyzed the effect of CD70-specific mAb on B cells in CD27–/–x CD70 Tg mice. Ligation of CD70 in vivo with anti-CD70 mAb increased CD44 expression and decreased sIgM and CD62L expression, consistent with the data obtained in vitro (compare Figs. 3a and 5a).
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Inhibition of plasma cell formation and IgG secretion after CD70 ligation
The hallmark of terminal differentiation of B cells is the formation of plasma cells and their secretion of Igs. B cell cultures stimulated with LPS and CD70 showed a decrease of B cells positive for the plasma cell differentiation marker CD138 (Syndecan-1) as compared with cultures stimulated with LPS alone (Fig. 6A). In all CD40-stimulated B cell cultures, low frequencies of CD138-positive cells were found (Fig. 6A). Engagement of CD70 in vitro on LPS-stimulated B cells caused an increase in the cumulative concentration of IgM Abs after 7 days of culture, whereas IgG secretion was inhibited (Fig. 6B). When B cells were stimulated with anti-CD40 mAb, CD70 ligation had no effect on IgM production, but inhibited IgG production (data not shown).
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secretion in these mice (21), the serum TNP-specific IgM Ab levels were increased as compared with WT mice, whereas lower NP-specific IgG titers were found (Fig. 6D). In CD70 Tg mice on an IFN--deficient background, which have normal B cell numbers (21), decreased TNP-specific IgG levels and similar IgM levels were found (Fig. 6E). Together, the in vitro and in vivo data suggest an inhibitory effect of CD70 ligation on the formation of IgG-secreting plasma cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have investigated the consequences of CD70 ligation in B cell activation and differentiation. By using B cells from mice constitutively expressing CD70, but lacking CD27, we were able to specifically examine the role of CD70 ligation without effects of CD27 signals. Addition of CD70 mAb to these B cells promoted entry into the cell cycle (Fig. 2) and altered expression of cell surface markers, especially in combination with TLR4-, CD40-, and IL-4R-derived signals (Figs. 3 and 5). Moreover, CD70 signaling inhibited plasma cell formation and IgG secretion (Figs. 6 and 7).
Upon activation, B cells migrate toward the boundary of B and T cell zones due to altered expression of chemokine receptors (33). At this particular site, B cells interact with activated, Ag-specific CD4+ T cells, receiving costimulatory signals. Because activated B cells express CD70 (17, 18) and activated T cells highly express CD27 (34), it might be that costimulation also occurs through CD70. In addition, expression of both CD70 and CD27 has been demonstrated on germinal center B cells (35, 36). The physiological outcome, in terms of the production of protective humoral immune responses, of the bidirectional signaling via CD27-CD70 interactions remains to be resolved. Although, on one hand, CD70 ligation on B cells inhibits IgG secretion, triggering of CD27 on T cells promotes their survival and will facilitate the generation of Th cells. Because it has been demonstrated that CD70 transfectants promote B cell differentiation in responses to IL-4 and IL-10 in vitro (25), a third level of regulation of B cell differentiation by these molecules can be envisaged.
A previous study showed that addition of CD27-transfected cells to T cell-dependent human B cell cultures resulted in marked decreased IgG levels (23), which may now be explained by our observation that signaling through CD70 inhibits IgG production. Although CD70 ligation promoted cell cycle entry, in presence of LPS and to a lesser extent in presence of anti-CD40 mAb the CD70-triggered B cells underwent less cell cycle progression compared with the fraction of rapidly dividing B cells cycling B cells without CD70 stimulation. This may explain the inhibitory effect of CD70 ligation on IgG production, given that IgG isotype switching is related to cell division number (37). In contrast to the effects of CD70 signaling, signaling through CD27 on B cells results in enhanced plasma cell formation and increased IgG production (23, 24, 25). Thus, CD70- and CD27-transduced signals in B cells appear to have opposing effects in the fine-tuning of B cell responses.
Signal transduction pathways induced by CD70 signaling
Although signaling through TNF receptor family members by the TNF receptor-associated factors and death domain has been extensively studied (2), relatively little is known about the downstream signaling pathways used by TNF family members. Although the cytoplasmic domains of individual TNF family members are evolutionary conserved across species, comparison between different family members showed relatively little homology, and therefore provided no clues as to generic signaling mechanisms used. It has been reported that a casein kinase I consensus sequence is conserved in the cytoplasmic domains of human and mouse TNF, CD40L, CD95L, CD30L, and 4-1BBL, and of human, but not mouse CD70 (38). However, signaling properties have been described of TNF ligand family members lacking casein kinase I motifs including OX40L, TRANCE, TRAIL, and mouse CD70 (our results). By deletion mutant studies, it was shown that the sequence RPRFER of the cytoplasmic portion of OX40L was important for induction of c-jun and c-fos (39). Furthermore, involvement of p38 MAPK was found in signaling through TRANCE (10) and TRAIL (11) on T cells. In this study, we found that signaling through murine CD70 on B cells results in phosphorylation of both PKB and ERK, being direct targets of PI3K and MEK, respectively (Fig. 4). The activation of PKB and ERK most likely associates with the response of the CD70-triggered B cell, because these pathways are critically involved in antiapoptotic signal transduction as well as cell cycle progression. Although it has been shown that BCR-induced phosphorylation of ERK is dependent on PI3K via the Ras pathway (40), we found that CD70 triggering can activate MEK in a PI3K-independent manner. This implies either a common upstream signaling intermediate or multiple independent signaling pathways initiated by CD70 signaling. Although we could show a clear effect of the PI3K and MEK inhibitors on the induction of early activation Ags by CD70 triggering, this does not directly prove that both pathways are required for the long-term effects seen on cell cycle progression and B cell differentiation. Along the same line, we cannot exclude that other pathways are required for these effects.
Role of CD70 signaling in malignancies
Besides the restricted expression of CD70 in normal tissues, strong CD70 expression was found on Hodgkin’s lymphoma, B cell chronic lymphocytic leukemia, and large B cell lymphomas (26, 27, 28). In addition to these hematological malignancies, CD70 has also been reported to be present on various other malignancies (29, 30, 31). The apparent deregulated expression of CD70 on malignant B cells suggests that CD70 might function as an agonistic receptor for malignant B cell growth (28). A possible role for this interaction in lymphomagenesis is suggested by the observations that aggressive non-Hodgkin’s lymphomas abundantly express CD70 and that blastoid transformation of mantle cell lymphoma coincides with the up-regulation of CD70 (41). In this respect, it is of interest to note the paradox that certain malignancies with a poor prognosis (e.g., Hodgkin’s disease and anaplastic larger cell lymphoma) are characterized by a high infiltration of T cells (42, 43). Potentially, these infiltrating T cells can provide the CD27 as ligand for CD70 on the tumor cell. Alternatively, some tumor cells express CD70 together with CD27, suggesting that this interaction may constitute an autocrine circuit regulating malignant cell function.
Taken together, our results provide evidence that signals through the TNF family member CD70 in B cells result in enhanced cell cycle entry while preventing differentiation into IgG-secreting plasma cells. We also suggest that CD70 signaling might be important in CD70-expressing malignancies. Therefore, modulation of the CD70 signaling pathway might be able to control the extent of (malignant) B cell responses and potentially provide therapeutic benefits to treat certain immune system disorders.
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Acknowledgments |
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Footnotes |
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1 R.A. and M.A.N. contributed equally to this work. 
2 Current address: Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands.
3 Current address: Department of Clinical Viro-Immunology, Sanquin Research at CLB, Amsterdam, The Netherlands.
4 Address correspondence and reprint requests to Dr. Marinus H. J. van Oers, Academic Medical Center, Department of Hematology, F4-224, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail address: m.h.vanoers{at}amc.uva.nl
5 Abbreviations used in this paper: TRANCE, TNF-related activation-induced cytokine; HPE, high performance ELISA; KLH, keyhole limpet hemocyanin; PKB, protein kinase B; sIg, soluble Ig; Tg, transgenic; TNP, trinitrophenol; WT, wild type.
Received for publication January 7, 2004. Accepted for publication July 16, 2004.
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) or control Ab (
). 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 ± 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 CD27–/–x CD70 Tg mice were stimulated with the indicated concentrations of (µg/ml) LPS, anti-CD40 mAb, polyclonal F(ab')2 goat anti-mouse IgM, or IL-4 (ng/ml) in presence of 5 µg/ml anti-CD70 mAb (
