* Molecular Immunopathology Unit, DCEXS, Universitat Pompeu Fabra, Barcelona, Spain; and
Department of Biochemistry and Immunology, Facultad de Medicina, Universidad de Murcia, Spain
1Correspondence: Molecular Immunopathology Unit, DCEXS, Universitat Pompeu Fabra, Dr. Aiguader 80, 08003-Barcelona, Spain. E-mail: miguel.lopez-botet{at}upf.edu
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Key Words: CD27L • T lymphocyte • NK cell • signal transduction • cytotoxicity • human
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Studies on the biological role of this receptor-ligand pair have mainly addressed the ability of CD27 to regulate the immune response [11 12 13 14 ]. CD27 signaling involves TNF receptor-associated factors [15 ] and appears to play a costimulatory role promoting T cell expansion and differentiation as well as an enhancement of NK cell activity [16 17 18 ]. CD27–/– mice display altered, primary T cell responses and an inefficient development of T cell memory [19 ].
Conversely, CD70 has been generally envisaged as a passive element merely required for specific CD27 engagement. Yet, there is evidence that some members of the TNF family may directly regulate lymphocyte functions beyond their ability to interact with their corresponding receptors. Engagement of CD40L was shown to promote activation of p56lck, Jun-N-terminal kinase, and p38K in T lymphocytes [20
]; moreover, induction of Ca2+ mobilization and phospholipase C (PLC)
1 phosphorylation was detected [21
]. Similarly, cross-linking of CD30L costimulated T cells in response to suboptimal CD3/T cell receptor (TcR) engagement [22
]. The CD40–CD40L interaction was reported to activate NK cell-mediated cytotoxicity [23
]. Anti-CD70 monoclonal antibodies (mAb) induced proliferation in a subset of B cell leukemias [24
]. In the same line, an anti-CD70 mAb was reported to stimulate NK and 
T cell-mediated redirected killing, thus indirectly supporting that CD70 might be a signaling molecule [25
]. In the present study, we extended the latter observations, addressing in more detail whether CD70 may play an active regulatory role in T cells.
Our results indicate that cross-linking of CD70 with specific mAb triggered T cell-mediated cytotoxicity, cytokine production, and intracellular-free calcium (Ca2+)i mobilization in 
ß+ T cell clones (TCC); moreover, CD70 aggregation induced mitogen-activated protein kinase (MAPK) phosphorylation. Specific activation via CD70 was confirmed in the Jurkat T cell line stably transfected with the TNF-like molecule. It is remarkable that interaction of CD70+ Jurkat cells with CD27-transfected COS-7 cells specifically promoted CD70-mediated signaling, detectable in different reporter gene expression systems. Altogether, these data strongly support that similarly to other members of the TNF family, CD70 plays a dual regulatory role, thus indicating that a bidirectional communication may be established between CD70+ and CD27+ lymphocytes during the immune response.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R)+ P815 mastocytoma, as described previously [29
]. The antiphosphorylated extracellular-regulated kinase (anti-P-ERK)1/2-specific antibody was purchased from Promega (Madison, WI) and the antipan-ERK antibody, from Zymed (San Francisco, CA). The antipan-Akt and antiphospho-Akt serine 473 were from Cell Signaling (Beverly, MA). Peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Pierce (Rockford, IL). F(ab')2 sheep anti-mouse IgG (SAM) and anti-FLAG M2 mAb were purchased from Sigma Chemical Co. (St. Louis, MO).
Cytotoxicity assays
NK and TCC were tested in a standard 4-h 51Cr-release assay against the murine P815 mastocytoma cell line as described [29
]. Briefly, 5 x 103 51Cr-labeled target cells were preincubated for 15 min at 37°C with mAb and further incubated with different concentrations of effector cells for 4 h at 37°C. Where indicated, effector cells were pretreated with the MAPK/ERK kinase 1-selective inhibitor PD98059 (Promega) or dimethyl sulfoxide (DMSO; solvent) for 1 h at 37°C. Specific lysis was calculated as described previously, and only experiments where spontaneous release did not exceed 20% of the maximum isotope release were considered.
Cell stimulation, immunoprecipitation, and Western blot analysis
Transiently transfected COS-7 cells were lysed in buffer lysis containing 1% Brij-96, protease, and phosphatase inhibitors. Lysates were incubated with 10 µg/ml QA32 for 2 h at 4°C, and immunocomplexes were isolated with protein G-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden). For antibody cross-linking, 106 cells/sample were stimulated for different periods of time at 37°C with 1 µg QA32, SpVT3b, or a mouse IgG (control) followed by the addition of 5 µg SAM; in every case, the effect of SAM alone was tested. Phorbol 12-myristate 13-acetate (PMA) was used at 100 ng/ml. Cells were then treated with lysis buffer containing 1% Nonidet P-40 in the presence of protease and phosphatase inhibitors. Whole cell lysates and immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. Membranes where then blocked with 5% nonfat dried milk in Tris-buffered saline/Tween 20 (TBST; 10 mM Tris, pH 7.8, 150 mM NaCl, with 0.05% Tween detergent) and incubated overnight at 4°C with the indicated antibodies in TBST containing 1% nonfat dried milk. Labeling was detected with horseradish peroxidase-conjugated secondary antibodies followed by visualization by a chemoluminescence kit (Pierce).
Flow cytometry analysis
For immunofluorescence staining, cells were preincubated with saturating concentrations of different mAb, followed by washing and labeling with fluorescein isothiocyanate-conjugated F(ab')2 goat anti-mouse Ig antibody (Dakopatts, Glostrup, Denmark). Samples were analyzed on a FACScan flow cytometer (Becton Dickinson) with Cell Quest software in the presence of 10 µg/ml propidium iodide to exclude dead cells from analysis.
Detection of (Ca2+)i mobilization
Measurement of (Ca2+)i was performed using a FACScan (Becton Dickinson) and the calcium-sensitive fluorescent probe Fluo-3 (Molecular Probes, Eugene, OR), as described previously [30
]. Briefly, 5 x 106-labeled cells were stimulated in a final volume of 1.5 ml with F(ab')2 of QA32 (40 µg/ml), SpVT3b (10 µg/ml), or a control IgG1 (10 µg/ml). Where indicated, 20 µg/ml polyclonal sheep anti-mouse IgG (SAM) was used as cross-linker.
Detection of TNF- secretion
K14B06 TCC (250,000 cells/well) were cultured with P815 murine cells (100,000 cells/well) for 18 h in 96 microtiter plates in the presence or absence of anti-CD3, anti-CD70, anti-CD29, or IgG1 isotype-control mAb at a final concentration of 2 µg/ml. Supernatants were collected, and human TNF- secretion was quantified using the OptEIA human TNF- set (PharMingen, San Diego, CA).
Plasmids and transfections
The coding sequence of human CD70 tagged with a FLAG epitope at the C terminus was subcloned into pCDNA3 (Invitrogen, Carlsbad, CA). The plasmid pCDM8-CD27 was a kind gift from Dr. Brian Seed (Massachusetts General Hospital and Harvard Medical School, Boston, MA). The pJFE14-CD94 plasmid was generated as described [31
]. PCDM8 vector was purchased from Invitrogen. COS-7 cells were transiently transfected by electroporation. Briefly, 3 x 106 cells/0.2 ml complete medium were pulsed in a Bio-Rad gene pulser at 975 µF and 200 V. After electroporation, cells were removed immediately, added to 10 ml medium, and plated. Transfection of the Jurkat cell line with pCDNA3-CD70/FLAG vector or with the empty pCDNA3 was performed using electroporation. Briefly, 107 cells/0.5 ml incomplete medium were pulsed at 1200 µF and 280 V in the presence of 15 µg plasmid DNA. Stable transfectants were selected in 1 mg/ml G418 (Calbiochem, La Jolla, CA). CD70/FLAG+ clones were obtained by culture under limiting dilution conditions and were selected by flow cytometry analysis.
Reporter assays
Jurkat and Jurkat-CD70/FLAG cells were electroporated (950 µF, 260 V) with luciferase (Luc) reporter plasmids (0.5 µg/106 cells) containing three tandem copies of the distal nuclear factor of activated T cells (NFAT)-activated protein-1 (AP-1) site of the murine interleukin-2 promoter (3xNFAT-Luc) or the human TNF- promoter [32
] and Renilla luciferase expression plasmid (Promega; 0.1 µg/106 cells) for normalization of transfection efficiency [33
]. Twenty-four hours later, cells were stimulated with different mAb in the presence of the FcR-bearing P815 cell line or with COS-7 cells transiently transfected with the indicated expression plasmids. After 8 h stimulation, cells were lysed in 5x lysis buffer (Promega) and assayed using the dual luciferase reporter kit (Promega). Luciferase activity was read on a luminometer and expressed as relative light units per s (RLU/s).
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To address this hypothesis, a first set of experiments was performed using a CD70-specific mAb, QA32. As shown in Figure 1A , the QA32 mAb recognized in fluorescence-activated cell sorter (FACS) analysis COS-7 cells transiently transfected with a plasmid encoding for the FLAG-tagged CD70 molecule. Moreover (Fig. 1B) , QA32 mAb immunoprecipitated from surface-labeled COS-7 transfectants the CD70 monomer, together with additional specific bands of higher molecular weight, thus supporting that the TNF-related molecule assembles forming different oligomers; these data are consistent with some discrepancies in early biochemical descriptions of CD70 [1 , 2 ].
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Figure 1. The QA32 mAb specifically recognizes CD70. (A) COS-7 cells were transiently transfected with empty pCDNA3 vector or pCDNA3-CD70/FLAG vector. Forty-eight hours after transfection, cells were stained by indirect immunofluorescence with the QA32 mAb or P3X63 myeloma supernatant [negative control (Control)]. (B) Cell lysates from COS-7 cells transiently transfected with empty pCDNA3 vector (lane 1) or pCDNA3-CD70/FLAG vector (lane 2) were immunoprecipitated (IP) with QA32, analyzed by 9% SDS-PAGE under nonreducing conditions, and immunoblotted [Western blot (WB)] with the anti-FLAG M2 mAb.
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TCC (Fig. 2
), although to a lesser extent than anti-CD3 mAb. Anti-CD70 mAb induced cytotoxicity of ß CD8+ TCC as well, whereas no response was obtained in most CD70+ CD4+ ß TCC tested, which otherwise killed P815 cells in the presence of anti-CD3 mAb (Fig. 2)
.
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Figure 2. Anti-CD70 mAb triggers T cell-mediated cytotoxicity in redirected lysis assays. Different CD70+ TCC ( ; A), CD8+ ß TCC ( ; B), or CD4+ ß TCC (•; B) were assayed against the P815 FcR-bearing murine mastocytoma cell line in the presence of anti-CD70 (QA32), anti-CD3 (SpVT3b), or a mouse IgG (negative control) at a final concentration of 1.8 µg/ml. Lysis of P815 in the presence of mouse IgG by TCC was less than 4% in every experiment (not shown). Effector/target ratio was 2:1 in most of the cell clones tested. Lines were used in the graphic to pair results from single TCC. (C) The cytolytic activity of a TCC induced via CD3 and CD70 is compared at different effector/target ratios. No significant lysis was detected in the presence of mouse IgG (negative control).
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(Fig. 3
) production as well as CD25 expression (not shown) were induced in +, ß+ CD8+, and CD4+ TCC when the anti-CD70 mAb was presented on the surface of the FcR-bearing P815 cells, as for rADCC assays. To further explore the issue, experiments were performed measuring different intracellular signaling events upon QA32-mediated engagement of CD70 in the CD8+ CD70+ CD27– K14 TCC (Fig. 4A
). As shown in Figure 4B
, (Ca2+)i mobilization was detected upon CD70 cross-linking, and similar results were obtained with two different CD70+ CD4+ ß TCC (data not shown). In this assay, the intensity and duration of CD70 signaling were variable in different clones but always of a lower magnitude than the response induced via CD3, which was detectable without cross-linking. As previously reported for signaling via CD40L, which also induced (Ca2+)i mobilization [21
], phosphorylation of PLC1 was detected on engagement of CD70 (data not shown). Moreover, an early transient induction of ERK phosphorylation was detectable, consistent with MAPK activation (Fig. 5A
); the MAPK pathway has been proposed to regulate cell-mediated cytotoxicity. In this regard, treatment of the K14 cytolytic T lymphocyte (CTL) clone with a pharmacological inhibitor of MAPK (PD98059) partially reduced QA32-induced lysis of the P815 cell line (Fig. 5B)
, thus further supporting that this pathway may contribute to CD70-dependent signaling. The doses of PD98059 required to inhibit cytolytic activity were comparable with those used by previous studies [34
, 35
], and the results are consistent with the concept that MAPK partially contributes to triggering cell-mediated cytotoxicity [34
]. An involvement of phosphatidylinositol 3-kinase (PI-3K) was also indirectly suggested by the substantial reduction of CD70-induced cytotoxicity observed in the presence of Wortmannin (3–10 µM) or LY294002 (50–100 µM; data not shown).
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Figure 3. Engagement of CD70 stimulates TNF production in TCC. Different CD70+ TCC CD4+ () or CD8+ TCC (•) were incubated at a final concentration of 250,000 cells/well with p815 murine cell line (100,000 cells/well) for 18 h in the presence or absence of anti (A)-CD3 (SpVT3b), anti-CD70 (QA32), anti-CD29 (TS1/16), or IgG1 isotype control mAb at a final concentration of 2 µg/ml. Supernatants were collected, and human TNF- secretion was quantified using the OptEIA human TNF- set (PharMingen).
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Figure 4. Cross-linking of CD70 induces (Ca2+)imobilization. (A) The K14B06 ß CD8+ TCC was stained by indirect immunofluorescence with anti-CD70 (QA32), anti-CD3 (SpVT3b) mAb, or the P3X63 myeloma supernatant as a negative control (Control). (B) K14B06 cells were loaded with Fluo-3/AM fluorescent probe and stimulated with F(ab')2 fragments of anti-CD70 (QA32) mAb, anti-CD3 (SpVT3b), or a mouse IgG1 (control) followed by the addition of SAM. Arrows mark the timing of antibody addition. In every sample, (Ca2+)i was analyzed by flow cytometry, plotting time versus relative fluorescence.
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Figure 5. MAPK activation is involved in CD70-mediated signaling. (A) Detection of tyrosine-phosphorylated p42/44 MAPK in K14 cells after cross-linking with anti-CD70 mAb. K14 cells were stimulated as described in Materials and Methods. Cell lysates (106 cells/lane) were analyzed by immunoblotting [Western blot (WB)] with antibodies to active MAPK (anti-P-ERK1/2) and reprobed with antibodies to pan-ERK (anti-pan-ERK1/2) to check for comparable loading. (B) Inhibition of CD70-induced cytotoxicity by PD98059. K14 cells were pretreated for 1 h at 37°C with PD98059 (30 µM, 60 µM, 90 µM) or with DMSO and were tested against the P815 cell line in a standard 4-h 51Cr-release assay in the presence of anti-CD70 mAb (QA32). No significant lysis was induced in the presence of an isotype-matched mouse IgG preparation (negative control). XL, crosslinking.
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Figure 6. Expression of functional CD70 by transfection of the Jurkat T cell line. (A) Jurkat cells were stably transfected with the empty pCDNA3 vector or pCDNA3–CD70/FLAG vector. After staining with anti-CD70 (QA32) mAb or P3X63 myeloma supernatant (negative control), samples were analyzed by flow cytometry. (B) CD70 cross-linking specifically increases (Ca2+)i in the CD70+ Jurkat cells. As described in Figure 4
, cells were loaded with Fluo-3/AM fluorescent probe and stimulated with anti-CD70 (QA32), anti-CD3 (SpVT3b), or a mouse IgG1 (control), followed by the addition of SAM. (Ca2+)i was analyzed by flow cytometry and is represented as fluorescence relative units against time.
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Figure 7. Cross-linking of CD70 induces phosphorylation of MAPK and Akt in Jurkat-CD70/FLAG transfectants. CD70+ Jurkat cells were stimulated as described in Figure 5
. Whole cell lysates were analyzed in 10% SDS-PAGE under nonreducing conditions and probed in Western blots (WB) with (A) phospho-p42/44 MAPK (anti-P-ERK1/2)-specific antibody and (B) antiphospho-Akt (anti-P-AKT) serine 473 mAb. Membranes were reprobed with antipan-ERK and antipan-Akt antibodies to check for comparable loading.
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R-bearing P815 cells, and induction of Luc activity was subsequently measured. As shown in Figure 8A
, expression of luciferase was detected in both reporter systems when cells were specifically engaged by the anti-CD70 mAb, thus supporting that signaling led to levels of transcription factor activation sufficient for promoting gene expression. It is of note that the activity of the NFAT-AP-1-dependent promoter requires the participation of both transcription factors [36
]. Moreover, CD70-dependent activation was observed using another reporter under the control of a NF-
B-dependent promoter (data not shown).
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Figure 8. CD27-mediated engagement of CD70 induces transcriptional activity. Jurkat-CD70/FLAG cells were transiently transfected with two different plasmids containing the Luc reporter gene under the control of a NFAT-binding sequence or the human TNF- promoter. (A) Cells were stimulated with the P815 cell line in the presence of anti-CD70 (QA32), anti-CD3 (SpVT3b), or a mouse IgG1 (control); in parallel, samples were activated with 20 nM PMA plus 1 µM ionomycin (Io), and luciferase activity was assessed after 8 h. (B) Cells were stimulated with COS-7 transiently transfected with pCDM8-CD27, the empty pCDM8 vector, or pJFE14-CD94. Induction of luciferase activity over that detected in samples stimulated with the empty vector is represented.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CD70 expression is inducible upon T and NK cell activation; conversely, CD27 is also displayed by T and NK cells, regulating their functional activity [11 , 16 , 17 , 25 ]. Thus, receptor-ligand coexpression may complicate a selective functional analysis of CD70 in T cell populations. By studying a CD70+ CD27– TCC (i.e., K14B06), we circumvented that limitation and observed that cross-linking of CD70 with specific mAb stimulated cytotoxicity, cytokine production, (Ca2+)i mobilization and MAPK phosphorylation. Such functional properties of CD70 were transferred by stable transfection of a CD70– CD27– leukaemia T cell line (Jurkat), in which evidence for induction of PI-3K activity was also obtained. Stimulation of CD70+ Jurkat cells, with a CD70-specific mAb or with COS-7 cells transiently transfected with CD27, induced transcriptional activity detectable in different reporter gene expression systems.
The ability to mediate agonistic effects triggering lymphocyte functions is a property common to mAb specific for activating receptors coupled to protein tyrosine kinase (PTK) pathways. Yet, as other members of the TNF family, CD70 is a type II transmembrane protein, which lacks well-defined cytoplasmic motifs recruiting these signaling molecules; CD70 does not contain any transmembrane-charged residue, enabling noncovalent interactions with immunoreceptor tyrosine-based activation motif-bearing adaptor molecules. Although it is still unclear how the TNF-like receptors mediate such "reverse signaling", it is of interest that a casein kinase I motif present in these molecules has been proposed to be involved [37 ].
It is conceivable that receptor-induced aggregation of TNF-like molecules may contribute to the activation of Src tyrosine kinases, promoting subsequent downstream events that regulate cytotoxicity, cytokine production and gene transcription. In this regard, p56lck activation was reported to be induced via the CD40L molecule [20 ] and, moreover, we observed a reduction of CD70 rADCC by the Src inhibitor PP2 (data not shown), indirectly supporting that hypothesis. In contrast to mAb specific for known triggering receptors coupled to tyrosine kinase pathways (i.e., TcR/CD3), it is of note that anti-CD70 mAb did not activate T cells when immobilized onto a plastic surface. These observations revealed qualitative differences between the activation pathways and suggested that lateral mobility of the CD70L on the plasma membrane may be critical for signaling, possibly favoring CD70 oligomerization and PTK activation. A detailed structure-function analysis of the CD70–CD27 pair might eventually allow us to evaluate this hypothesis.
Induction of Akt phosphorylation via CD70 indirectly suggested that PI-3K activity is also involved in signaling. PI-3K has been shown to regulate NK- and CD95/CD95L-independent T cell-mediated cytotoxicity [38 , 39 ]; in fact, we observed that two different PI-3K inhibitors (i.e., Wortmannin and LY294002) partially reduced CD70-dependent rADCC (data not shown). Conversely, PI-3K appears to play an important role in regulating cell survival [40 ]. Thus, the reciprocal communication established between CD70+ and CD27+ lymphocytes may not only regulate the development of their effector functions [41 ] but could modulate cell survival and differentiation as well.
Despite that lysis was triggered in rADCC assays via CD70, attempts to detect killing of the CD27low+ DAUDI cell line by the CD70+ K14B06 TCC were unsuccessful (data not shown). Thus, it is unlikely that its interaction with CD27 may be sufficient to induce cell-mediated cytotoxicity under physiological conditions, leading to "fratricide" killing between CD70+- and CD27+-activated lymphocytes during the immune response. It is rather conceivable that physiological costimulatory effects induced by the CD70/CD27 interaction may be magnified by mAb-mediated engagement under the rADCC assay conditions.
A number of observations point out the putative importance of the CD27/CD70 pair in the development of the immune response. Moreover, defects in expression and/or function of CD27 [42 ] and CD70 [43 , 44 ] have been reported in lymphocytes from some common variable immunodeficiency patients, and it has been proposed that these alterations may contribute to the pathogenesis of the immune dysfunction. CD27 gene targeting revealed the relevance of this costimulatory molecule for the generation of T cell memory; CD27–/– mice developed a deficient secondary response to antigenic challenging [19 ]. To what extent the lack of CD70 engagement indirectly contributes to the immune alterations observed in CD27–/– mice cannot be discerned.
In addition to the difficulty for discriminating the functional importance of CD70-mediated signaling relative to its passive role as CD27L, it is of note that other members of the TNF family expressed in activated lymphocytes appear to share a similar signaling capacity, and thus redundancy may blur their individual relevance. Yet, despite the putative functional overlap of the TNF-related molecules, variability of their expression pattern and ligand distribution may determine subtle differences in their specific roles during the immune response. The development of more refined experimental approaches would be required to address this complex issue.
Received October 28, 2003; revised March 8, 2004; accepted March 9, 2004.
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B and stress-activated protein kinase/c-Jun N-terminal kinase via TRAF2, TRAF5, and NF-B-inducing kinase J. Biol. Chem. 273,13353-13358-mediated B cell depletion Immunity 15,801-812[CrossRef][Medline]/+ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody J. Immunol. 144,3238-3247[Abstract]-induced lymphocyte polarization and chemotaxis J. Immunol. 163,4001-4012-Opioid receptors expressed by Jurkat T cells enhance IL-2 secretion by increasing AP-1 complexes and activity of the NF-AT/AP-1-binding promoter element J. Immunol. 159,5431-5440[Abstract]This article has been cited by other articles:
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