Expression of the Murine CD27 Ligand CD70 In Vitro and In Vivo

Mice

C57BL/6 mice were bred in the facilities of The Netherlands Cancer Institute and CLB (Amsterdam, The Netherlands) under specific pathogen-free conditions. Mice used for experiments were between 6 and 8 wk of age at the start of the experiment. All animal experiments were conducted according to institutional and national guidelines and were approved by the Experimental Animal Committees of the respective institutes. Mice were infected intranasally with influenza virus strain A/NT/60/68 as described elsewhere (19).

Generation of anti-mCD70 mAb

Armenian hamsters (Cytogen, West Roxbury, MA) were injected three times i.p. with 10⁷ irradiated (50 Gray) syngeneic fibroblasts transfected with the mCD70 cDNA, termed AR-mCD70 cells (29), at weekly intervals. Two weeks after the third injection, serum was tested for mCD70 reactivity. A booster injection (i.p., 10⁷ cells) was given and 3 days thereafter, hamsters were sacrificed. Splenocytes were fused with SP2/0 murine myeloma cells as described (6, 29). Fourteen days after fusion, hybridoma supernatants were tested on AR-mCD70 cells and the 771 murine B lymphoma cell line (30) for the presence of anti-mCD70 Ab by flow cytometric analysis using PE-labeled goat anti-hamster Ig as second step reagent. After limiting dilution, two stable clones (3B9, 6D8) were obtained. Both mAbs were purified from culture supernatant using protein A bound to Sepharose CL-4B beads (Sigma-Aldrich, St. Louis, MO). F(ab′)₂ was generated and conjugated to biotin using standard procedures.

Abs and Fc proteins

mAbs to mouse B220 (RA3.6B2), CD3ε (145-2C11; 500A2), CD4 (GK1.5), CD8 (2.43), CD16/CD32 (2.4G2), MHC class II (M5/114.15.2), CD27 (LG.3A10; Ref. 6), CD40 (FGK-40), Thy-1.1 (50 AD 22/15), and DEC-205 (NLDC-145) were purified from hybridoma supernatant and conjugated to FITC and biotin according to standard procedures. mAb to CD40 (HM-40.3) and CD28 (37.51), as well as B7-2-FITC (GL-1), CD4-PerCP (RM4-5), CD8a-PerCP (53-6.7), CD11c-PE (HL3), CD19-FITC (1D3), and CD3-allophycocyanin (145-2C11) were purchased from BD PharMingen (San Diego, CA) and goat anti-mouse IgM F(ab′)₂ was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). PE-labeled goat anti-hamster Ig was from Southern Biotechnology Associates (Birmingham, AL). F(ab′)₂ of hamster-anti hCD97 mAb 1D2 (a kind gift from Dr. J. Hamann, Academic Medical Center, Amsterdam, The Netherlands) were labeled with biotin and served as control in flow cytometry and immunohistology. Recombinant receptor Fc proteins were kindly provided by Dr. R. Goodwin (Immunex, Seattle, WA).

Cells and cell culture

771 is a cell line derived from a B lymphoma induced by neonatal inoculation of a C57BL/10 mouse with MCF 1233 murine leukemia virus (30). Expression of mCD70 on this line originally permitted the cDNA cloning (29). ARHO Armenian hamster fibroblasts (6) and mCD70-transfected ARHO cells (AR-mCD70) (29) have been described previously. Single cell suspensions were obtained by grinding tissues through nylon sieves, or flushing femurs and tibias with a needle. Erythrocytes were removed by lysis with ammonium chloride solution. For T cell purification, murine lymph node single cell suspensions were depleted of MHC class II⁺, B220⁺, IgM⁺ cells using anti-MHC class II, anti-B220 mAb, goat anti-rat IgG, and rat anti-mouse IgM MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), and MACS (BS) deletion columns following the manufacturer’s instructions. For B cell purification, CD19⁺ cells were positively selected from splenocytes using goat anti-CD19 MACS microbeads beads and MACS (MS⁺) separation columns following the manufacturer’s instructions. Purity of the resulting populations was tested by immunofluorescence with anti-CD19 and anti-CD3 mAbs and exceeded 95%. DC were generated in vitro as described (31). In short, bone marrow suspensions were depleted of CD4⁺, CD8⁺, B220⁺, and MHC class II⁺ cells using theindicated mAbs and goat anti-rat IgG MACS microbeads and MACS (BS) depletion columns. Cells were seeded at 10⁶/ml and grown in medium containing recombinant mouse GM-CSF (1 ng/ml; BD PharMingen) and IL-4 (1 ng/ml; BD PharMingen). DC were harvested at day 8. Cells were grown in IMDM containing 10% heat inactivated FCS, penicillin, streptomycin, and LPS (serotype 026:B; Sigma-Aldrich), recombinant IL-2, IL-4, IL-12, IFN-γ (BD PharMingen), or IL-10 (Endogen, Woburn, MA). The D1 dendritic cell (DC) line was cultured as described (32).

Biochemical analysis

771 cells (3 × 10⁶/sample) were labeled with ¹²⁵I (Amersham International, Amersham, U.K.) by the glucose/lactoperoxidase method and lysed in 1% Nonidet P-40 buffer, containing 10 mM triethanolamine-HCl (pH 7.8), 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 20 μg/ml ovomucoid trypsin inhibitor, 1 mM N-α-p-tosyl-l-lysine chloromethyl ketone, and 20 μg/ml leupeptin. After centrifugation, the supernatant was divided. One part was precleared with pooled human serum (5%) and protein G-Sepharose (Pharmacia, Uppsala, Sweden), the other part was precleared with normal hamster serum (10%) and protein A-Sepharose. mCD70 was subsequently precipitated with mCD27-Fc, anti-mCD70 mAb, or an irrelevant control mAb and complexes were adsorbed onto protein G- or A-Sepharose, eluted, and electrophoretically separated by 5–15% SDS-PAGE, and visualized by autoradiography.

Flow cytometry

Cells (3 × 10⁵) were seeded in 96-well round-bottom plates and incubated with anti-CD16/CD32 mAb (5 μg/ml) in FACS buffer (PBS, 0.5% BSA, 0.02% azide). After washing, cells were stained with the indicated mAbs and a subsequent incubation with allophycocyanin-labeled streptavidin (BD PharMingen) was performed to detect biotin-conjugated mAbs. All incubation steps were for 30 min at 4°C. Cell staining was analyzed on a FACSCalibur using CellQuest software (BD Biosciences, Mountain View, CA). Viable cell populations were gated on the basis of forward and side scatter and, where indicated, on the basis of a histogram profile.

Proliferation assay

For proliferation assays, purified murine T cells (10⁵/well) were stimulated with Con A (Sigma-Aldrich) at the indicated concentrations and were cultured with mCD70-transfected or mock-transfected cells (5 × 10⁴ cells/well). [³H]Thymidine incorporation was measured after 4 days of culture. [³H]Thymidine (0.4 μCi; Amersham International) was present for the last 16 h of culture.

Real-time PCR

Single cell suspensions of purified T cells (1 × 10⁶/ml) were stimulated with immobilized anti-CD3 mAb (145-2C11, 10 μg/ml). Single cell suspensions of purified B cells (1 × 10⁶/ml) were stimulated with anti-CD40 mAb (HM-40.3, 5 μg/ml) and LPS (10 μg/ml). After the indicated time of culture, cells (2 × 10⁶) were collected and RNA was isolated with RNazol (Cinna/Biotecx Laboratories, Friendswood, TX). Single-strand cDNA was prepared in a 20-μl reaction volume with 500 ng of oligo(dT)_12–18 and 100 U of Superscript II (Life Technologies, Rockville, MD). Murine CD70 cDNA was quantitated with the LightCycler System (Roche, Mannheim, Germany; www.lightcycler.com, technical note no. LC 10/2000). Using a FastStart DNA Master SYBR Green I kit and 1 μl of cDNA, a 20-μl real-time PCR was set-up amplifying mCD70 or hypoxanthine phosphoribosyltransferase (HPRT) from the cDNA derived from each sample. Real-time kinetic quantification allows measurement during the log-linear phase of a PCR. Analysis of this phase allows accurate determination of the starting concentration, using the equation: number of amplified molecules = initial number of molecules × efficiency constant (E)^{cycle number}. In our approach, a standard curve of 771 cDNA was prepared to determine the amount of CD70 cDNA (E = 0.986) and HPRT cDNA (E = 0.982) in the T and B cell samples. The samples were normalized by dividing the amount of CD70 cDNA by the amount of cDNA of the housekeeping gene HPRT. The following primers were used: mCD70 (+ strand primer, 5′-GGATGCCGGAGGAAGGTCGCCC-3′; − strand primer, 5′-CAAGGGCATATCCACTGAACTC-3′); and HPRT (+ strand primer, 5′-TATGGACAGGACTGAACGTCTTGC-3′; − strand primer, 5′-GACACAAACATGATTCAAATCCCTGA-3′). Crossing points were calculated by the second derivative method using LightCycler System software. Real-time PCR was performed on a LightCycler PCR apparatus (Roche).

Immunohistology

For light microscopy, acetone-fixed cryosections of lung were incubated in methanol containing 0.02% H₂O₂, followed by incubation with 2% BSA in PBS. Next, they were incubated overnight at 4°C with biotinylated 3B9 anti-mCD70 F(ab′)₂ or biotinylated anti-Thy-1.1, diluted in PBS with 1% BSA. After thorough washing in cold PBS, sections were incubated with Vectastain ABC regent (Vector Laboratories, Burlingame, CA) for 30 min and developed with 0.05% 3,3′-diaminobenzidine with 0.01% H₂O₂ in 0.1 M phosphate buffer. After counterstaining with hematoxylin, sections were coverslipped for observation. For confocal microscopy, cryostat sections were fixed in acetone and incubated with anti-CD16/CD32 mAb (5 μg/ml). Sections were subsequently incubated with biotinylated 3B9 anti-mCD70 F(ab′)₂ at saturating concentrations for 2 h at room temperature. After thorough washing in cold PBS, sections were incubated with FITC-labeled mAb and streptavidin-conjugated Alexa Fluor 594 (Molecular Probes, Eugene, OR) overnight at 4°C. Stained sections were washed in cold PBS and coverslipped using antifading mounting medium (DAKO, Glostrup, Denmark). Omission of incubation with biotinylated anti-mCD70 mAb was used as negative control. Suspensions of cells isolated from lung-draining lymph nodes were analyzed for mCD70 expression as follows: Cells were pipetted onto cover slips, which had been coated with poly-l-lysine (50 μg/ml in PBS, 30 min, 37°C) and incubated for 30 min at 37°C. They were fixed for 5 min with methanol kept at −20°C. All subsequent incubations were at room temperature, as outlined for tissue sections. Coverslips were mounted in Vectashield (Vector Laboratories) and viewed under a Leica TCS NT confocal laser-scanning microscope (Deerfield, IL).

Generation and characterization of mAbs directed against mCD70.

To generate mAbs to characterize CD70 protein expression in the mouse, Armenian hamsters were immunized with syngeneic fibroblasts transfected with the mCD70 cDNA (29). Eight percent of the supernatants of the resulting hybridomas were reactive in immunofluorescence with the mCD70-expressing B cell line 771 and the hamster fibroblast transfectant AR-mCD70. The selected mAbs, 3B9 and 6D8, could be used to isolate mCD70 from cell surface-iodinated 771 B cells. As was shown for the murine CD27-Fc protein (29), the anti-mCD70 mAbs not only precipitated monomeric mCD70, but also the SDS-resistant dimeric and trimeric forms of mCD70 (Fig. 1⇓A). Flow cytometric analysis showed that 3B9 and 6D8 blocked each other’s binding to 771 cells, indicating that they react with the same epitope (data not shown). The mAbs did not cross-react with human CD70 (data not shown).

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FIGURE 1.

Characterization of anti-mCD70 mAbs. A, Immunoprecipitation of mCD70 with anti-mCD70 mAb 3B9 and 6D8, and mCD27-Fc protein. Murine CD70 was immunoprecipitated from a Nonidet P-40 lysate of cell surface-iodinated 771 B lymphoma cells. Proteins were separated by SDS-PAGE under reducing conditions and detected by autoradiography. The outer right lane shows the immunoprecipitate of a negative control mAb. B, Functional effect of anti-mCD70 mAb. [³H]Thymidine incorporation by purified murine T cells, activated for 4 days with Con A in the indicated concentrations is enhanced by mCD70-transfected hamster fibroblasts (AR-mCD70), but not by mock-transfected hamster fibroblasts (AR-mock). This costimulation is blocked by anti-mCD70 mAb 3B9 (5 μg/ml), but not by a negative control mAb (5 μg/ml).

The effects of the anti-mCD70 mAb on CD27-CD70 interaction were tested in two assays. Firstly, CD27 is known to be down-modulated after contact with CD70 (20, 33). Anti-mCD70 mAb 3B9 and 6D8 were found to specifically inhibit this down-modulation (data not shown). A second well-established effect of CD27-CD70 interaction is enhancement of [³H]thymidine incorporation by activated T cells. As shown in Fig. 1⇑B, mCD70 transfectant AR-mCD70 incremented [³H]thymidine incorporation of Con A-stimulated purified murine T cells. This was inhibited by mAb 3B9 (Fig. 1⇑B) as well as 6D8 (not shown). Thus, addition of anti-mCD70 mAb 3B9 and 6D8 could block early and late consequences of CD27 triggering, indicating that these anti-mCD70 mAb prevent functional interaction between mCD70 and CD27.

Analysis of mCD70 expression on in vitro-activated cells

In humans, CD70 is absent from resting lymphocytes, but can readily be induced upon in vitro activation. Concordantly, bone marrow cells, thymocytes, splenocytes, and lymph node cells, freshly isolated from naive mice, showed no cell surface expression of mCD70, according to flow cytometry (data not shown). Expression of mCD70 upon T and B cell activation in vitro was analyzed by real-time PCR at the mRNA level and by flow cytometry at the protein level. Purified T and B cells were unstimulated, or incubated for 2 days with anti-CD3 mAb, or anti-CD40 mAb with LPS, respectively. In unstimulated cells, no PCR product was obtained with mCD70 primers, indicating a total lack of mCD70 RNA expression in resting T and B cells (not shown). In stimulated T cells, CD70 mRNA was detectable, but it was present at much lower levels than in stimulated B cells (Fig. 2⇓A). In accordance with the difference in mCD70 mRNA expression between in vitro activated T and B cells, cell surface mCD70 protein expression also differed. On activated T cells, only a very low level of mCD70 was found, barely detectable by flow cytometry, whereas mCD70 was easily detectable at the cell surface of activated B cells (Fig. 2⇓B). Expression of mCD70 on activated T cells was not more pronounced at other time points after activation, nor when T cells were additionally stimulated with anti-CD28 mAb, IL-2, IL-4, IL-10, IL-12, or IFN-γ (data not shown).

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FIGURE 2.

Murine CD70 mRNA expression by in vitro-stimulated cells. Purified T and B cells were stimulated with anti-CD3 mAb (immobilized, 10 μg/ml), or anti-CD40 mAb (5 μg/ml) and LPS (10 μg/ml), respectively, and collected 2 days after stimulation. Real-time PCR and flow cytometry were performed using cells from the same experiment. A, The result of the PCR analysis. The relative amount of CD70 mRNA, expressed in arbitrary units, was determined by real-time PCR with HPRT as an internal standard. B, The mean fluorescence intensity of activated T and B cells stained with anti-mCD70 mAb, relative to the control mAb (1D2, hamster anti-hCD97).

The regulation of expression of mCD70 on B cells was studied in more detail, because literature data are partially conflicting on this issue (25, 26). Purified B cells were stimulated with anti-IgM Ab, anti-CD40 mAb, and the T cell-independent stimulus LPS, either alone or in combination, and mCD70 expression was analyzed in time. Anti-IgM Ab or anti-CD40 mAb alone both induced cell surface expression of mCD70 to a similar extent, which reached maximum levels of ∼50–70% positive cells at day 4 (Fig. 3⇓A). These agents acted synergistically in mCD70 up-regulation, allowing for expression of mCD70 on almost all B cells, already at 2 days after activation. LPS alone was much less potent in up-regulating mCD70 cell surface expression, but acted in synergy with anti-CD40, allowing for maximal expression at day 2 after stimulation on ∼70% of B cells (Fig. 3⇓B).

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FIGURE 3.

mCD70 expression on in vitro-stimulated B cells. Purified B cells were stimulated as indicated and analyzed by flow cytometry for the percentage of mCD70-expressing cells at the indicated time points after stimulation. The graphs show representative results of three or more experiments. A and B, Expression kinetics after stimulation with anti-IgM F(ab′)₂ (5 μg/ml), anti-CD40 mAb (HM40-3, 5 μg/ml), LPS (10 μg/ml), or a combination of both stimuli. C and D, mCD70 expression on anti-IgM or LPS/anti-CD40-stimulated B cells at day 3 after addition of IL-2 (50 U/ml), IL-4 (2 ng/ml), IL-10 (2.5 ng/ml), IL-12 (2 ng/ml), or IFN-γ (7 ng/ml).

Apart from Ag receptor signals, cytokines also control CD70 expression in humans (12, 23). We tested the influence of different cytokines on mCD70 expression on purified murine B cells activated by anti-IgM Ab or LPS with anti-CD40 mAb. Although IL-2, IL-4, or IL-12 had no effect, IFN-γ consistently inhibited the induction of mCD70 expression by anti-IgM Ab (Fig. 3⇑C), but not by LPS and anti-CD40 mAb (Fig. 3⇑D). Addition of IL-10 slightly (1.5-fold), but consistently, reduced induction of mCD70 expression by LPS/anti-CD40 mAb (Fig. 3⇑D), or LPS alone (data not shown).

Although in the human system CD70 has only been found on cells of the lymphoid lineage, in the mouse CD70 has been found on activated CD11c⁺ DC (27, 28). The availability of the murine DC line D1 (32) and culture procedures to obtain primary murine DC from bone marrow cells in vitro permitted us to test regulation of mCD70 expression on DC. As is shown in Fig. 4⇓, D1 cells (Fig. 4⇓A) and in vitro-generated immature DC (CD11c⁺, MHC class II^dull) (Fig. 4⇓B) do not express detectable mCD70 at the cell surface. However, after maturation, as induced by LPS, anti-CD40 mAb or GM-CSF (34) in particular primary DC, express high levels of mCD70.

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FIGURE 4.

mCD70 expression on DC. A, D1 cells were either untreated or stimulated with LPS (10 μg/ml) or anti-CD40 mAb (FGK-40, 30 μg/ml) and analyzed by flow cytometry for mCD70 and B7-2 expression at the indicated time points after stimulation. B7-2 was used as a marker for maturation. B, Primary CD11c⁺ DC were generated from bone marrow cells with GM-CSF and IL-4. After 8 days, cells were harvested and recultured in the presence of GM-CSF (2 ng/ml), LPS (5 μg/ml), or anti-CD40 mAb (HM40-3, 2.5 μg/ml). CD70 and MHC class II expression as a control were analyzed on CD11c⁺ cells at the indicated time points after restimulation. MHC class II and mCD70 expression was analyzed within the CD11c⁺ cell population.

Analysis of mCD70 expression in vivo

As mentioned above, we could not detect mCD70 by flow cytometry on cell suspensions derived from primary and secondary lymphoid organs of naive mice. To further document mCD70 expression in lymphoid organs of nonimmunized mice, immunohistology was performed. In multiple independent analyses of mice (10 animals of different strains), mCD70 expression in spleen and lymph nodes appeared to be extremely rare. Occasionally, some scattered cells in the red pulp of the spleen and dispersed cells in peripheral lymph nodes expressed mCD70 (data not shown). However, consistently mCD70-expressing cells were found in the medulla of the thymus (Fig. 5⇓). These cells also stained with an Ab to DEC-205, a carbohydrate receptor, which is expressed by DC and thymic epithelial cells (35).

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FIGURE 5.

mCD70 expression in the thymus of naive mice. Thymus section of naive (nonimmunized) mouse stained with FITC-conjugated NLDC-145 mAb directed at the DEC-205 receptor (green), as well as biotinylated 3B9 anti-mCD70 F(ab′)₂ (red)_. The epithelial cells of the thymic cortex are exclusively stained by DEC-205, while the medulla contains double-positive cells.

Next, we examined mCD70 expression in vivo after antigenic stimulation. For this purpose, mice were infected intranasally with influenza virus. This well-characterized model is hallmarked by activation of Ag-specific T cells in lung-draining lymph nodes, followed by a transient appearance of activated T cells in the lung. In the absence of CD27, the primary T cell response in lymphoid organs and lung is significantly reduced (19). After infection, lung tissue and cell suspensions of lung-draining lymph nodes were analyzed for mCD70 expression. Lung tissue sections were prepared and stained by conventional immunohistochemistry with Abs directed at Thy-1.1 or mCD70 and examined by light microscopy (Fig. 6⇓A). In these sections, conspicuous fields of mCD70-positive cells were detected in parabronchiolar regions. These were T cells, according to Thy-1.1 staining of serial sections (Fig. 6⇓A). Such fields of mCD70⁺ cells were never observed in noninfected lung tissue (not shown). Lung tissue sections were also stained with fluorescent Abs specific for T cells (CD3), B cells (B220), and DC (CD11c) and examined by confocal microscopy. In this analysis, lung-infiltrating T and B cells were detected in significant amounts. However, DC were scarce. Double staining for mCD70 revealed that a sizeable proportion of lung-infiltrating T cells expressed mCD70 (Fig. 6⇓B). No mCD70-positive B cells or DC were found. Cells in lung-draining lymph nodes were examined for mCD70 expression by fluorescent staining of cytospins, followed by confocal microscopy (Fig. 6⇓C). This analysis revealed significant amounts of T and B cells, identified by CD3 and B220 staining, while DC (CD11c⁺) were very rare. Double staining for mCD70 indicated that a significant proportion of T and B cells in these lung-draining lymph nodes expressed mCD70 protein. The small number of DC present permitted no reliable conclusion on mCD70 expression in DC. Strikingly, both in T and B lymphocytes, the great majority of mCD70 staining appeared cytoplasmic, rather than at the cell membrane. This can be seen particularly well in B cells, which show peripheral staining for B220 which only rarely overlaps with the mCD70 staining (Fig. 6⇓C).

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FIGURE 6.

mCD70 expression in lung and lung-draining lymph nodes of infected mice. Mice were infected intranasally with influenza virus and sacrificed at day 14 (A), or day 8 (B and C) after infection. Lung and lung-draining lymph nodes were isolated. A, Light microscopic view of lung sections. Serial sections were stained with biotinylated anti-mCD70 3B9 F(ab′)₂) or anti-Thy1.1 mAb and were developed with streptavidin-peroxidase. Magnifications are indicated. B, Tissue sections of lung were stained with biotinylated anti-mCD70 (3B9 F(ab′)₂) (red) and FITC-conjugated anti-CD3 mAb (green). C, Draining lymph node cell suspensions were coated onto cover slips and stained with FITC-conjugated anti-mCD70 (3B9 F(ab′)₂) (green) and biotinylated anti-B220 mAb (red) to detect B cells. The mCD70-positive, B220-negative cells in this preparation are T cells according to anti-CD3 staining (not shown).

The immunohistology data showed that mCD70 is expressed by activated T cells in lung and lung-draining lymph nodes during an ongoing immune response to influenza virus. We next questioned whether, in contrast to in vitro-stimulated T cells, in vivo-activated T cells express CD70 at the plasma membrane. Therefore, FACS analysis of lung-infiltrating cells during an ongoing response to influenza virus was performed. At different time points after infection, lung-infiltrating cells were isolated and double-stained with anti-CD3 and anti-CD70 mAbs (Fig. 7⇓). This experiment showed clearly that mCD70 is detectable by flow cytometry at the plasma membrane of a small proportion of lung-infiltrating T cells. Maximal expression was found at the height of the response (day 8), as defined by the size of the T cell infiltrate and the number of Ag-specific T cells (19).

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FIGURE 7.

mCD70 is present at the cell surface of lung-infiltrating T cells. Mice were infected intranasally with influenza virus and sacrificed at day 3, 6, 8, or 11 after infection. Lung-infiltrating T cells were isolated as described (19 ) and double-stained with anti-mCD70-FITC (3B9 F(ab′)₂) and anti-CD3-allophycocyanin mAbs. Total cell suspensions were analyzed by flow cytometry. Mean fluorescence intensity (MFI) is indicated.