Involvement of CD70 and CD80 intracytoplasmic domains in the co-stimulatory signal required to provide an antitumor immune response

Victorine Douin-Echinard1, Jean-Marie Péron1, Valérie Lauwers-Cancès2, Gilles Favre1 and Bettina Couderc1

1 Laboratoire d’Innovation Thérapeutique et Oncologie Moléculaire, CPTP, Inserm U563, Institut Claudius Regaud, 20–24 rue du pont St Pierre, 31052 Toulouse, France 2 Laboratoire d‘Epidémiologie et Santé Communautaire, Faculté de Médecine, 31073 Toulouse, France

Correspondence to: B. Couderc; E-mail: couderc_b{at}icr.fnclcc.fr
Transmitting editor: I. Pecht


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
CD70 and CD80 are co-stimulatory molecules which belong to the tumor necrosis factor family and the B7 family respectively. When they are co-expressed by gene-modified TS/A tumor cells, they provide an efficient protective and long-lasting T-dependent antitumor response. We first showed that when CD70 and CD80 were delivered in the tumor environment by gene-modified fibroblasts, but were not expressed by the tumor cells themselves, no antitumor response was observed. We next assessed whether the intracytoplasmic domains of CD70 and CD80 contribute to enhance the co-stimulatory activity necessary to induce effective T cell–tumor cell interactions and T cell-dependent antitumor response. TS/A cells were gene-modified to express different combinations of deleted (CD70{Delta} and CD80{Delta}) or full-length CD70 and CD80 co-stimulatory molecules. In vitro, the CD80 intracytoplasmic domain was required to regulate CD80 membrane redistribution by interacting with the actin cytoskeleton. The loss of the CD70 intracytoplasmic domain did not alter its ability to relocate on the surface membrane, but failed to co-stimulate T cell proliferation. In vivo experiments in syngeneic BALB/c mice showed that the CD70/CD80-TS/A and the CD70{Delta}/CD80-TS/A tumors were rejected via CD8 T cells, whereas CD70/CD80{Delta}-TS/A and CD70{Delta}/CD80{Delta}-TS/A tumors were not. The mice that rejected CD70{Delta}/CD80-TS/A tumors showed decreased protection against injection of parental TS/A cells when compared to mice which rejected CD70/CD80-TS/A tumors. These results showed that the intracytoplasmic domain of CD80 was critical for the effector phase of CD8 T cell-dependent tumor rejection and that the CD70 intracytoplasmic domain could mediate proliferative or surviving signals required for optimal effector/memory CD8 T cell generation.

Keywords: co-stimulatory molecule, gene therapy, in vivo animal model, tumor immunity


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
A major focal point of cancer research is how tumors escape immune recognition and destruction. Advances in our understanding of T lymphocyte activation and co-stimulation have provided new strategies for developing tumor-specific immunotherapy. T cell activation depends on the interaction of several receptors on the T cell surface with their ligands on the antigen-presenting cells (APC). These interactions induce an antigen-specific signal delivered through the TCR and a second non-antigen specific ‘co-stimulatory signal’ delivered by accessory receptors following their engagement with specific ligands expressed mostly by the APC. These interactions lead to the formation of a tight interface between a T cell and an APC (13) by the clustering of receptors and signaling molecules at the T cell–APC interface in discrete geometrical patterns (46).

CD80 is a co-stimulatory molecule which belongs to the Ig superfamily (B7 family) (7). It is a monomeric surface glycoprotein expressed by activated B cells, T cells, monocytes and dendritic cells (DC) (810). Engaging CD80 through its ligand CD28 expressed by T cells simultaneously with TCR activation leads to the generation of T cell proliferation and cytokine production (i.e. IL-2) (7,1113). CD28-mediated signals increase expression of Bcl-XL and promote survival of TCR-activated T cells (14). Previous reports have shown that the triggering of both adhesion (LFA-1) and co-stimulatory (CD28) molecules by their specific ligands, respectively ICAM-1 and B7 molecules, is necessary and sufficient to induce movement of the T cell cortical actin cytoskeleton toward the newly formed T cell–APC interface. This active accumulation of receptor pairs and other cytoskeleton-linked molecules by cortical actin polarization at the T cell–professional APC (DC) interface requires active processes in both T cell and DC, and is necessary for T cell activation and proliferation (3,5,15,16). Furthermore, Doty and Clark (17,18) have shown that the cytoplasmic tail of CD80 interacts with the actin cytoskeleton, influences its subcellular location and affects the ability of CD80-transfected Reh cells to co-stimulate T cell proliferation in vitro. These in vitro studies suggest that CD80 could play an active role in the APC to provide an effective co-stimulatory signal to the CD28-expressing T cells.

CD70 is a type II transmembrane glycoprotein that belongs to the tumor necrosis factor (TNF) family (1921). Its specific interaction with its ligand CD27 has been shown to support clonal expansion of both antigen-stimulated CD4 and CD8 T lymphocyte populations (19,20,2224), and to enhance the generation of cytolytic T cells (20,25). CD70–CD27 interactions also participate in the generation and long-term maintenance of T cell memory, in particular of CD8 T cells (26). The CD27 signaling pathway appears different from that of CD28, as, in contrast to CD28, CD27 employs TRAF molecules to induce downstream signals, in particular TRAF-2 and -5 (2729).

In a previous study, we showed that the expression of only one molecule (CD80 or CD70) by gene-modified tumor cells was not sufficient to induce an effective antitumor response in vivo. However, CD80 was able to cooperate with CD70 to induce tumor rejection and protective immunity when co-expressed by two low immunogenic tumor cells, the TS/A mammary adenocarcinoma cell line and the B16.K1 melanoma cell line, injected into syngeneic mice (30).

The aim of this study was to show, using in vivo models, that the two intracytoplasmic domains of CD70 and CD80 have critical functions to activate the T cell-mediated immune response induced by CD28–CD80 and CD27–CD70 interactions. We evaluated whether the loss of CD70 and/or CD80 intracytoplasmic domains affected the cooperation between the two co-stimulatory molecules required to induce efficient tumor rejection. We generated original models of double-transfected tumor cells expressing various combinations of full-length (CD70 and CD80) and deleted forms (CD70{Delta} and CD80{Delta}) of the two co-stimulatory molecules. Since both molecules were required for the induction of an effective and long-lasting immune response, our models allowed us to characterize the specific contribution of each molecule to the induction of the immune response in vivo.

We have shown that transfection of TS/A tumor cells by the deleted forms of CD70 and CD80 did not impair the expression level of these molecules. However, in vitro, under antibody treatment, the CD80 intracytoplasmic domain, but not that of CD70, was necessary for the molecule to become redistributed on surface TS/A cells by presenting intact actin cytoskeleton.

The CD70{Delta}/CD80{Delta} TS/A cells s.c. injected in immunocompetent syngeneic BALB/c mice formed tumors that were not rejected. These results showed that the loss of CD70 and/or CD80 intracytoplasmic domains led to an incomplete antitumor immune response. To evaluate the relative contributions of CD70 and CD80 intracytoplasmic domains, we compared the abilities of CD70/CD80{Delta}-TS/A- and CD70{Delta}/CD80-TS/A-transfected cells to induce an immune antitumor response in vivo. We showed that the CD80 intracytoplasmic domain, but not that of CD70, was necessary to induce CD8 T cell-dependent primary tumor rejection. The co-expression of full-length CD70 molecules and CD80 molecules was required to co-stimulate T cell expansion, and to induce long-lasting antitumor responses. Understanding the role of CD28/CD80 and CD27/CD70 interactions in the regulation of these processes during T cell activation and antitumor response appears to be a critical issue in designing protocols improving antitumor immunity.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
Animals
Female BALB/c mice were purchased from CERJ Janvier (Le Genest Saint-Isle, France). All mice were used for experiments at 6–8 weeks old according to European Community guidelines.

Retroviral constructs
The construction and characterization of the DFG human CD80 (DFGhCD80) and the DFG human CD70 (DFGhCD70) retroviral vectors have previously been described (31,32). These MFG-based retroviral vectors express these molecules along with a neor or zeor selectable marker, encoding resistance to G418 (Gibco/BRL, Cergy Pontoise, France) and zeocyn (Cayla, Toulouse, France) respectively. The control vector (mock) consists of the MFG retroviral vector with an IRES neor sequence. The cDNAs encoding the deleted forms of CD80 (CD80{Delta}) and CD70 (CD70{Delta}) molecules were generated by PCR cloning from the respective full-length cDNAs. To delete the 17 C-terminal amino acids of CD80, a translation stop codon was introduced at the 3' end of the cDNA sequence corresponding to the end of the transmembrane domain to generate cytoplasmic-domain-deleted CD80. To obtain a CD70 cytoplasmic-domain-deleted form, we deleted the 16 N-terminal amino acids and introduced a new translation initiation codon upstream of the beginning of the transmembrane cDNA sequence. The following primers were used: CD80 sense primer (8S), 5'-CCATGGGCCACACACGG AGG-3' and CD80{Delta} anti-sense primer (8{Delta}AS): 5'-CGGGAT CCTCACTATCT GCATCTTGGGGCAAAGC-3'; CD70{Delta} sense primer (7{Delta}S): 5'-CATGCCATGGT CCTGCGGGCTGCTTTGG TC-3' and CD70 anti-sense primer (7AS): 5'-CGGGATCCC TAATCAGCAGCAG-3'. The forward primers all contained NcoI sites and the reverse primers all contained BamHI sites. These PCR products were cloned into the MFG-based retroviral vector containing an IRES neor or IRES zeor sequence to obtain the expression vectors DFG CD70{Delta} and DFG CD80{Delta}. All constructs were sequenced to confirm sequence fidelity.

Antibodies
The mAb used to test co-stimulatory molecule cell-surface expression by flow cytometry analysis were FITC-conjugated mouse anti-human CD80 mAb (MAB 104) purchased from Immunotech (Marseille, France) and phycoerythrin (PE)-conjugated mouse anti-human CD70 mAb (Ki-24) purchased from BD Biosciences PharMingen (Le Pont de Claix, France). The mAb used to phenotype naive and memory T cells were PE-conjugated rat anti-mouse CD8 (KT15) (Immunotech), CyChrome-conjugated rat anti-mouse CD44 (IM7), FITC-conjugated rat anti-mouse CD62L (MEL-14) and isotype controls (BD Biosciences PharMingen). After staining with the appropriate mAb, FACS analysis was conducted on a Coulter XL 4C (Beckman Coulter, Roissy CDG, France) coupled with System II acquisition software by using the WIN MDI 2.8 software.

Immunofluorescence staining for microscopy analysis was performed using mouse IgG anti-human CD70 mAb, mouse IgG anti-human CD80 mAb (MAB 104) (Immunotech), rabbit biotinylated F(ab')2 anti-mouse IgG (Dako, Glostrup, Den mark) and avidin, Neutravidine–FITC conjugate (Molecular Probes, Leiden, The Netherlands).We used anti-mouse CD3{epsilon} mAb (500A2) (BD Biosciences PharMingen) to perform in vitro lymphocyte stimulation. Rat anti-mouse CD8 (53-6.72) and rat anti-mouse CD4 (GK1.5) used in depletion experiments were purified from ascitic fluids of hybridoma cell lines obtained from the ATCC (Manassas, VA).

Cells
TS/A is a tumor cell line established by P. Nanni (Bologna, Italy) derived from a spontaneous mammary adenocarcinoma of the BALB/c strain (33) and kindly provided by P. Lollini (Bologna, Italy). TS/A tumor cells were transfected with combinations of the different retroviral vectors carrying the different forms of co-stimulatory molecules (CD70 or CD70{Delta} and CD80 or CD80{Delta}) using Lipofectamine (Gibco/BRL) as previously described (30). The stably transfected cells CD70{Delta}/CD80{Delta}-TS/A, CD70/CD80{Delta}-TS/A and CD70{Delta}/CD80-TS/A were selected using G418 (1 mg/ml) and zeocyn (0.1 mg/ml). These cell lines and the stably transfected CD70-TS/A, CD80-TS/A and CD70/CD80-TS/A cell lines (30) were cultured in RPMI 1640 medium (Gibco/BRL), supplemented with 2 mM glutamine and 10% FCS (Life Technologies, Cergy Pontoise, France). BALB/c fibroblasts are syngeneic to BALB/c mice and were a gift from J. E. Gairin (Toulouse, France). BALB/c cells were co-transfected with DFGhCD70 IRES zeor and DFGhCD80 IRES neor using Lipofectamine. The stably transfected BALB/c CD70/CD80 cells were selected using G418 (0.5 mg/ml) and zeocyn (0.1 mg/ml). After cell selection, the expression of CD70 and CD80 was checked by flow cytometry as described previously (30). MM45T.Li is a hepatocarcinoma tumor cell line purchased from the ATCC. These two cell lines were cultured in Dulbecco’s modified Eagle’s growth medium (Gibco/BRL) supplemented with 10% FCS. All cell lines were tested periodically for mycoplasma using a DNA hybridization probe (Stratagene, La Jolla, CA) or ‘Molli’ technique (34).

RT-PCR
Cells were cultured in a T-75 cm2 culture flask for 72 h until subconfluence. Total RNA was isolated from a cell suspension as described by Chomczynski and Sacchi (35) from 5 x 106 cells using TRIzol reagent as described by the supplier (Life Technologies) and transcribed into cDNA using the Ready-To-Go kit (Amersham Pharmacia Biotech, Piscataway, NJ) and Random Primers purchased from Life Technologies. Amplification of cDNA was conducted with 1 U/100 µl of Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA) by 30 three-temperature cycles consisting of denaturation at 94°C (60 s), annealing at 65°C (60 s) and elongation at 72°C (60 s). The following primers were used: 8{Delta}AS, CD80 anti-sense primer (8AS): 5'-CACTGTTATACAG GGCGTACAC-3'; 8{Delta}S, CD70 sense primer (7S): 5'-CATGCC ATGGCGGAGGAGGGTTCGGGCTG-3'; 7{Delta}S and 7{Delta}AS anti-sense primer. PCR products and the {phi}X174 DNA/HaeIII mol. wt marker (Promega, Lyon, France) were separated by electrophoresis through 2% agarose (Boehringer Mannheim, Mannheim, Germany) Tris–borate–EDTA (Interchim, Monluçon, France) gels and visualized by staining with ethidium bromide (Promega, Lyon, France).

Immunofluorescence microscopy
The transfected TS/A cells were plated at a concentration of 105 cells/ml and cultured in RPMI 10% FCS for 48 h in a final volume of 2 ml on 20-mm2 micro cover glass (Erie Scientific, Portsmouth, NH) in six-well plates. After 48 h, the culture medium was removed and slides were washed 3 times with ice-cold staining buffer (5% FCS in PBS). Cells were stained at 4°C for 45 min with the primary antibody, anti-CD70 mAb (5 µg/ml) or anti-CD80 mAb (2 µg/ml) diluted in staining buffer. After two further washings with ice-cold staining buffer, cells were incubated for 45 min at 4°C with goat biotin-conjugated F(ab')2 anti-mouse IgG (1/50), washed and incubated with streptavidin–FITC (1/100) for 40 min at 4°C. The cells were washed 3 times at 4°C. Cells on cover glass were incubated at 37°C with 2 ml RPMI 10% FCS in six-well plates for 1 h. For the experiments using the actin-destabilizing drug cytochalasin B (CCB; Sigma, St Quentin Fallavier, France), CCB (10 µg/ml) was added to the culture medium RPMI just before the 37°C incubation (36). Cells were tested for viability (>60%) by Trypan blue dye exclusion before being fixed. Stained cells were then fixed in ice-cold PBS 3.7% paraformaldehyde. Disruption of the actin cytoskeleton was confirmed by staining permeabilized treated cells with rhodamine–phalloidine (1/60) for 40 min at room temperature (data not shown) (37). The cells were mounted on slides and single-color analysis was performed at x1300 magnification on a Zeiss Axioskop microscope equipped with epifluorescence filters.

Proliferation assays
T cells from the inguinal and axillary lymph nodes of BALB/c mice bearing 12- to 15-day-old TS/A tumors were prepared as previously described (30), and were brought to a concentration of 106 cells/ml in RPMI 1640 medium with 2 mM L-glutamine (Gibco/BRL) supplemented with 10% heat-inactivated FCS, 5 x 10–5 M ß-mercaptoethanol, 10 mM HEPES buffer, 0.1 mM non-essential amino-acids and non-essential vitamins (Sigma). T cells (105) were stimulated in vitro with 0.25 µg/ml of anti-mouse CD3{epsilon} and 8 x 104 mitomycin C (Mit-C; 0.3 mg/ml; Ameticyne; Sanofi, Gentilly, France)-treated transfected tumor cells (1 h 30 min at 37°C) in a final volume of 200 µl/well in 96-well round-bottomed plates. During the last 18 h of a 4-day culture, the cells were pulsed with 0.5 µCi/well of [3H]thymidine (Amersham Life Science, Little Chalfont, UK) and assayed for [3H]thymidine incorporation using a ß counter. The results from four separate wells were averaged and are reported as mean ± SD. Statistical analysis for comparison between stimulations and mock-TS/A control cells was conducted with Student’s t-test.

IFN-{gamma} cytokine production
CD8 T cells from BALB/c mice were enriched from pools of spleen cells using the Miltenyi Biotec CD8 T cell isolation kit as described by the supplier (Miltenyi Biotec, Paris, France). Briefly, spleen cells were incubated with a cocktail of biotin-conjugated rat mAb [anti-CD4 (L3T4), anti-CD45R (B220), anti-DX5, anti-CD11b (Mac-1) and anti-Ter-119 mAb]. After removal of unbound mAb, the cells were incubated with anti-biotin mAb conjugated to super-paramagnetic MicroBeads. The antibody-coated cells were removed by retaining them on a MACS column by a magnet. The unbound cells consisted of >80% CD8 T cells with no detectable CD4 T cells. The CD8 T cells or total lymph node cells (105 cells/well) were stimulated with various numbers of Mit-C-treated transfected TS/A cells (1 x 105, 3 x 104 or 104 cells/well) in 200 µl of RPMI supplemented as described above per well in 96-well plates. After 3 days, supernatants were collected and levels of IFN-{gamma} were assayed by sandwich ELISA (R & D Systems, Abingdon, UK).

In vivo studies
The mice used in the experiments were ear-tagged and randomized before injection with tumor cells. For establishment experiments, mice were injected s.c. with 105 tumor cells: wild-type, mock-, CD70/CD80-, CD70{Delta}/CD80{Delta}-, CD70/CD80{Delta}- or CD70{Delta}/CD80-transfected TS/A cells (twice the minimal tumorigenic dose of parental tumor cells), or 105 TS/A cells and 105 transfected BALB/c fibroblasts according to the experiment in 0.1 ml volume of PBS in a shaved area of the right flank. Tumor size was determined by measuring the two larger perpendicular tumor diameters with a caliper twice a week. For protection experiments, mice that did not develop tumors after 30 days received a subsequent challenge of 1.5 x 105 TS/A parental tumor cells or 105 MM45T.Li cells. Statistical analysis of protection experiments was carried out with Fisher’s exact test used to evaluate differences between groups and the CD70/CD80-TS/A positive control group. P values less than 0.05 were considered significant.

For in vivo CD4 or CD8 depletion experiments, BALB/c mice received six i.p. injections of 150 µg of anti-CD4 or anti-CD8 mAb from ascites or IgG rat control antibodies (Sigma) in 500 µl of PBS. Antibodies were given on days –2, 1, 4, 8, 11 and 15 with respect to the s.c. injection of 105 transfected TS/A cells. Depletion was checked on day 11 by flow cytometry using FITC-conjugated anti-CD4 or FITC-conjugated anti-CD8 mAb purchased from BD PharMingen. The results are expressed as mean size (mm2) of tumors from groups of four to five mice each ± SD. To perform the statistical analysis for depletion experiments, the tumor size was log transformed. Analysis was carried out by repeated measures ANOVA variance analysis and Tukey multiple comparison test. Difference was estimated significant at P < 0.05.

Phenotypic analysis of effector/memory CD8 T cells
BALB/c mice which had rejected the transfected CD70/CD80 or CD70{Delta}/CD80 TS/A cells or control naive BALB/c mice of the same age were s.c. injected 25–30 days after the initial injection with 3 x 105 TS/A parental tumor cells in both flanks. After 12 days, lymph node cells were harvested and cultured (106 cells/ml) with 0.25 µg/ml anti-CD3{epsilon} and Mit-C-treated parental or transfected TS/A cells (5 x 105 cells/ml) in 24-well plates. After 3 days, cells were collected, stained with PE-conjugated anti-CD8, FITC-conjugated anti-CD62L and CyChrome-conjugated anti-CD44 mAb, and analyzed by flow cytometry after gating on live cells. Marker expression levels were determined after gating on positive CD8 T cells.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
Direct expression of CD70 and CD80 by gene-modified TS/A tumor cells is required to induce an in vivo antitumor response
We tested whether the co-stimulatory signal delivered through the engagement of CD70 and CD80 by their ligands CD27 and CD28 expressed by T cells could be spatially dissociated from the TCR–antigen signal delivered to T cells by the TS/A MHC class I+ MHC class II tumor cells via their MHC class I–antigen complex.

We investigated if BALB/c CD70/CD80-transfected fibroblasts mixed with TS/A tumor cells induced an antitumor response against TS/A tumor growth in syngeneic mice. The BALB/c mice were injected s.c. with a mixture of 105 CD70/CD80 BALB/c fibroblasts plus 105 TS/A cells. As controls, mice were injected with 105 TS/A alone, a mixture of 105 TS/A and 105 mock-transfected BALB/c fibroblasts or 105 TS/A CD70/CD80 cells. The data revealed (Fig. 1) that unlike CD70/CD80-TS/A, tumor growth of the parental TS/A cells was not inhibited when the mice were co-injected with syngeneic CD70/CD80-transfected fibroblasts as compared with the tumor growth induced by the injection of TS/A cells alone or TS/A cells co-injected with mock BALB/c fibroblasts. We showed that CD80 and CD70 could not efficiently activate an antitumor response in vivo by interacting with their counter-receptors CD28 and CD27 when they are not directly co-expressed by tumor cells expressing the tumor antigen–MHC I complexes.



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Fig. 1. Direct expression of CD70 and CD80 molecules by gene-modified tumor cells is required to induce tumor rejection. BALB/c mice were injected s.c. on day 0 with 105 TS/A tumor cells, TS/A and mock-BALB/c fibroblasts, TS/A and CD70/CD80-BALB/c fibroblasts or CD70/CD80-TS/A tumor cells. Tumor growth was monitored twice a week as described in Methods. The results are expressed as mean size (mm2) of tumors from groups of five mice each ± SD. Data are from one experiment representative of two others.

 
Elaboration of tumor cell lines which expressed different combinations of full-length or intracytoplasmic domain-deleted CD70 and CD80 co-stimulatory molecules
In order to determine the respective roles of the intracytoplasmic domain of the two co-stimulatory molecules CD70 and CD80 in the induction of an effective T cell-dependent antitumor response, we constructed tumor cells which express co-stimulatory molecules deleted for their intracytoplasmic domain.

The TS/A tumor cells were stably co-transfected with MFG-derived vectors encoding either full-length CD70 (CD70) or a cytoplasmic-deleted form of CD70 (CD70{Delta}) and full-length CD80 (CD80) or a cytoplasmic-deleted form (CD80{Delta}) along with a neor or zeor selectable marker. We thus generated CD70{Delta}/CD80-TS/A, CD70/CD80{Delta}-TS/A and CD70{Delta}/CD80{Delta}-TS/A cells. After selection with the appropriate antibiotics (G418 and zeocyn), the stably transfected cells were checked for expression of the different combinations required, by RT-PCR using specific primers. As confirmed in Fig. 2, the CD70{Delta}- and CD80{Delta}-transfected cells only expressed mRNA encoding the cytoplasmic-deleted form of the respective co-stimulatory molecules. Indeed, no amplification by PCR occurred when we used the couple of primers specific for the wild-type cDNA in the CD70{Delta}-or CD80{Delta}-transfected cells. In contrast, when cells were transfected with wild-type CD70 or CD80 cDNAs, each couple of primers (wild-type or deleted) gave rise to an amplification band by PCR (Fig. 2).



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Fig. 2. Verification of the deletion of CD70 and CD80 intracytoplasmic domains by RT-PCR. Total mRNA from mock- (lanes 1 and 2), CD70/CD80- (lanes 4 and 5), CD70{Delta}/CD80{Delta}- (lanes 6 and 7), CD70{Delta}/CD80- (lanes 8 and 9) and CD70/CD80{Delta}- (lanes 10 and 11) transfected TS/A tumor cells were extracted and used for RT-PCR as described in Methods. To analyze CD70 and CD80 deletions, pairs of specific primers were designed to amplify the deleted and/or the full-length cDNA of each co-stimulatory molecule (A). For CD70 (B), two pairs of specific primers were used, the sense primer 7S and the anti-sense primer 7AS (lanes 1, 4, 6, 8 and 10), which amplify only the wild-type CD70 cDNA, or the sense primer 7{Delta}S and the anti-sense primer 7AS (lanes 2, 5, 7, 9 and 11), which amplify both the wild-type CD70 cDNA (CD70) and the deleted form of CD70 cDNA (CD70{Delta}). The CD80 deletion (C) was analyzed by using two pairs of specific primers, the sense primer 8S and the anti-sense primer 8AS (lanes 1, 4, 6, 8 and 10), which amplify only the wild-type CD80 cDNA, or the sense primer 8S and the anti-sense primer 8{Delta}AS (lanes 2, 5, 7, 9 and 11), which amplify both the wild-type CD80 cDNA (CD80) and the deleted form of CD80 cDNA (CD80{Delta}). PCR products and the {phi}X174 DNA/HaeIII mol. wt marker (lane 3) were analyzed by electrophoresis through 2% agarose Tris–borate–EDTA gels and visualized by incubation with ethidium bromide.

 
CD70{Delta}/CD80-TS/A, CD70/CD80{Delta}-TS/A and CD70{Delta}/CD80{Delta}-TS/A cell clones were chosen by flow cytometry to express high amounts of either deleted or wild-type CD70 and CD80 molecules (Fig. 3). The deletion of the cytoplasmic domains of CD70 or CD80 did not alter the membrane expression level of these molecules as CD70{Delta}/CD80-, CD70/CD80{Delta}- and CD70{Delta}/CD80{Delta}-transfected TS/A cells showed high mean fluorescence intensity for the two co-stimulatory molecules when analyzed by flow cytometry (Fig. 3).



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Fig. 3. Expression of CD70 and CD80 on transfected TS/A tumor cells. The different transfected TS/A cell lines, CD70-TS/A, CD80-TS/A, CD70/CD80-TS/A, CD70{Delta}/CD80-TS/A, CD70/CD80{Delta}-TS/A or CD70{Delta}/CD80{Delta}-TS/A, were stained with PE-conjugated anti-CD70 (A, B, C, G and H) or FITC-conjugated anti-CD80 (D, E, F, I and J) mAb. Data are shown as histograms which represent the relative cell number (y-axis) as a function of fluorescence intensity (x-axis) with arbitrary fluorescence units (log). Each frame consisted of 10,000 cells.

 
Antibody-induced membrane CD70 and CD80 redistributions are differently affected by the loss of their intracytoplasmic domains and by disorganization of the actin cytoskeleton
We wanted to assess whether the delivery of a co-stimulatory signal to T cells could be affected by the loss of the CD80 or CD70 intracytoplasmic domains, and could be related to different surface membrane redistribution properties to reflect T cell and tumor cell interactions.

The CD80 intracytoplasmic domain has been shown to be required for membrane re-localization of CD80 via interaction with the actin cytoskeleton and to play a role in the CD28-dependent T cell activation process in vitro (17). Using immunofluorescence assays, we followed the membrane redistribution of full-length and deleted CD80 or CD70 molecules on transfected TS/A cells after induction by specific antibodies. TS/A cells which presented distinct patched fluorescence staining following incubation with the specific antibody were scored as positive for clustering, whereas homogeneous diffuse membrane staining distribution was scored as negative.

As shown in Fig. 4, CD70 and CD80, either full-length or deleted, presented the same diffuse membrane distribution profile at time 0. After 1 h of incubation at 37°C, CD70- or CD80-stained CD70/CD80-TS/A cells showed respectively 100 and 83% of the cells with relocalization of CD70 or CD80 from a diffuse pattern to a clustered pattern (Fig. 4A and B). The cells expressing the deleted form of CD80 showed reduced formation of mAb-induced CD80 clusters (50.33%, Fig. 4A). In contrast, CD70 clustering was not affected by the loss of its intracytoplasmic domain—100% of the CD70{Delta}/CD80{Delta}-TS/A cells showed a CD70-clustered membrane distribution after the 1 h incubation at 37°C (Fig. 4B). CD70{Delta} and CD80{Delta} molecules showed the same distribution profiles in the CD70{Delta}/CD80 and CD70/CD80{Delta}-TS/A cells (data not shown).



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Fig. 4. Membrane redistribution of CD70 and CD80 induced by antibodies. Comparison of the abilities of CD80 and CD80{Delta} (A) or of CD70 and CD70{Delta} (B) co-stimulatory molecules to redistribute on the gene-modified TS/A surface membrane under anti-CD70 mAb or anti-CD80 mAb treatment. The CD70/CD80-TS/A and CD70{Delta}/CD80{Delta}-TS/A cells were labeled for fluorescence microscopy at 4°C as described Methods, fixed (0 h) or transferred to 37°C for 1 h with or without 10 µg/ml of CCB, and then fixed and mounted on slides for fluorescence microscopy analysis. Mock-TS/A cells were used as control samples for staining and did not display any detectable fluorescence. Data represent the percentage of cells with clustered staining distribution on the surface membrane. At least 100 cells were analyzed for each sample and scored as diffuse or clustered. The results are expressed as the mean ± SD of two separate experiments. (C) Images were collected on an Axioskop fluorescence microscope at x1300 magnification.

 
CCB is known to prevent F-actin formation and surface receptor clustering (17,36). To test whether the membrane redistribution of CD80 and CD70 could be related to possible interactions of CD70 or CD80 cytoplasmic domains with the actin cytoskeleton, we examined the effects of inhibiting the formation of transfected TS/A filamentous actin with CCB before inducing membrane redistribution of full-length or deleted forms of CD70 or CD80.

As shown in Fig. 4(B), disorganization of the actin cytoskeleton by CCB did not alter the ability of CD70 or CD70{Delta} to be redistributed under mAb treatment on surface membrane after 1 h at 37°C. On the other hand, treatment of CD80-expressing cells for 1 h with CCB reduced the number of cells with CD80-clustered formation (43.7%). This result was comparable with what was observed at 1 h for the CD80{Delta}-expressing cells (50.3%) and the CCB-treated CD80{Delta} cells (40%) (Fig. 4A).

These data showed that CD70 and CD80 were relocated after 1 h incubation at 37°C at the surface membrane under mAb activation. The deletion of the intracytoplasmic domain of CD70 or the destabilization of the actin cytoskeleton did not alter its redistribution. The intracytoplasmic domain deletion of CD80 or the inhibition of the actin cytoskeleton reduced the ability of CD80 to undergo membrane redistribution.

The co-stimulatory signal required in vitro to co-stimulate T cell proliferation is impaired by the loss of the CD70 intracytoplasmic domain
In order to evaluate if the deletion of the CD70 or CD80 intracytoplasmic domain could modulate the co-stimulatory signal involved in T cell proliferation, we evaluated [3H]thymidine uptake by lymph node cells when stimulated for 4 days with anti-CD3{epsilon} and non-replicative CD70{Delta}/CD80{Delta}-TS/A, CD70/CD80{Delta}-TS/A or CD70{Delta}/CD80-TS/A cells compared with mock-TS/A, CD70/CD80-TS/A, CD70-TS/A or CD80-TS/A cells (Fig. 5). The proliferation rate of lymph node cells stimulated by CD70{Delta}/CD80{Delta}-TS/A was not significantly increased compared to the CD70-TS/A group, whereas CD70/CD80-TS/A and CD70/CD80{Delta}-TS/A cells induced the strongest levels of T cell proliferation. Co-expression of CD80 with CD70 by gene-modified TS/A cells without the CD70 intracytoplasmic domain failed to stimulate T cell proliferation. The co-stimulatory signal involved in T cell proliferation induced by CD70/CD80-TS/A cells resided mainly in CD70 full-length expression associated with the full-length CD80 molecule or the deleted form (Fig. 5).



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Fig. 5. Co-stimulatory activities of the deleted forms of CD70 and CD80 to induce T cell proliferation. Comparison of the co-stimulatory capacity of mock-, CD70-, CD80-, CD70/CD80-, CD70{Delta}/CD80-, CD70/CD80{Delta}- or CD70{Delta}/CD80{Delta}-transfected TS/A cells to stimulate [3H]thymidine uptake by optimally stimulated lymph node cells. Lymphocytes were harvested from BALB/c mice bearing 12-day-old TS/A tumors and stimulated for 4 days with Mit-C-treated transfected tumor cells and anti-mouse CD3{epsilon}. After 3 days, cultures were pulsed with [3H]thymidine and assayed for thymidine incorporation. The results are expressed as the mean ± SD of four separate wells. Data are from one experiment representative of two others. Asterisks denote points significantly different (*P < 0.05 and ** P < 0.01 by Student’s t-test) from the mock-TS/A control stimulation.

 
The tumor rejection process required an intact CD80 intracytoplasmic domain
To test the ability of the deleted forms of CD70 and CD80 molecules to elicit an effective antitumor response, we compared the tumor growth of the mock-TS/A-, CD70/CD80-TS/A- and, in order to exclude a clone-specific response, three different clones of CD70{Delta}/CD80{Delta}-TS/A-transfected cells after s.c. injection into syngeneic BALB/c mice (Fig. 6A). Even if a delay in tumor growth rate of CD70{Delta}/CD80{Delta}-TS/A cells was observed with regard to the mock-TS/A growth rate, the expression of the deleted forms of CD70 and CD80 by TS/A cells did not induce tumor rejection as full-length CD70 and CD80 did (Fig. 6A).



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Fig. 6. Impaired antitumor responses induced by the loss of the intracytoplasmic domains of CD70 or CD80. (A) Subcutaneous tumor growth of 105 mock-TS/A, CD70/CD80-TS/A and three different clones of CD70{Delta}/CD80{Delta}-TS/A cells, clone 8, clone 9 and clone 11, were monitored in syngeneic BALB/c mice. (B) BALB/c mice were s.c. injected with 105 mock-TS/A, CD70/CD80{Delta}-TS/A, CD70{Delta}/CD80-TS/A or CD70/CD80-TS/A (triangles) and tumor size was measured twice a week. The results are expressed as mean size (mm2) of tumors from groups of five mice each ± SD. Data represent one experiment representative of two others. (C) The tumor rejection after s.c. injection of 105 mock-TS/A, CD70/CD80-TS/A, CD70{Delta}/CD80-TS/A, CD70/CD80{Delta}-TS/A or CD70{Delta}/CD80{Delta}-TS/A cells was monitored for 30 days. Data represent the percentage ± SD of mice which rejected transfected-TS/A from four separate experiments. Asterisks denote values significantly different (ns, non significant; **P = 0.01 and ***P < 0.001) from the CD70/CD80-TS/A control group by Fisher’s exact test (C).

 
To analyze which transfected co-stimulatory molecule is mainly involved in the tumor rejection, the tumor growth of CD70/CD80{Delta}-TS/A- and CD70{Delta}/CD80-TS/A-transfected cells was monitored after s.c. injection into BALB/c mice (Fig. 6B). CD70/CD80{Delta}-TS/A cell injection induced the development of tumors with a growth rate similar to what was observed with CD70{Delta}/CD80{Delta}-TS/A cell injection (Fig. 6A and B), whereas CD70{Delta}/CD80-TS/A cells were rejected by the injected mice (Fig. 6B).

The percentages of mice which rejected the transfected TS/A cells are represented in Fig. 6(C). The co-expression of the deleted or the full-length CD70 with full-length CD80 induced the rejection of the transfected tumor cells within 15 days post-injection for >90% of the injected mice (n = 18 mice for CD70{Delta}/CD80-TS/A and n = 20 for CD70/CD80-TS/A). CD70{Delta}/CD80{Delta}-TS/A cells were rejected only in 17% of the injected mice (n = 53). When TS/A cells expressed the CD80-deleted form, even with the wild-type CD70 molecule, only 15% of the injected CD70/CD80{Delta}-TS/A mice remained tumor free (n = 20). These results showed that the CD70/CD80 tumor rejection process required the intracytoplasmic domain of CD80 molecule, but not the CD70 intracytoplasmic domain (Fig. 6C).

The specific protective antitumor response is affected by the loss of the CD70 cytoplasmic domain
Mice that did not develop tumors after the primary injection of transfected TS/A cells were challenged with wild-type TS/A cells and MM45T.Li tumor cells. Thirty days after the TS/A cell injection, only 39% of the CD70{Delta}/CD80-TS/A primary injected mice (n = 18) remained TS/A tumor free, whereas 80% of CD70/CD80-TS/A primary injected mice (n = 20) rejected tumor challenge (Fig. 7). CD70{Delta}/CD80-TS/A cells retained the ability to stimulate immune effector cells responsible for tumor rejection, but were less effective than CD70/CD80-TS/A to generate a protective, long-lasting antitumor response to a subsequent challenge of wild-type TS/A cells (P = 0.01, {chi}2-test). This protective immune response was specific as all the mice injected in the contra-lateral flank with unrelated MM45T.Li hepatocarcinoma tumor cells (H-2Kd) developed tumors (Table 1). The partial antitumor response observed for CD70/CD80{Delta}-TS/A and CD70{Delta}/CD80-TS/A (Fig. 6C and 7) showed that the loss of CD70 and CD80 cytoplasmic domains impaired the co-stimulatory signal delivered to the immune system at different stages of the antitumor response.



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Fig. 7. Protection against TS/A parental cell injection is impaired by the loss of the intracytoplasmic domain of CD70. Mice which rejected the transfected TS/A cells, CD70/CD80-TS/A, CD70{Delta}/CD80-TS/A, CD70/CD80{Delta}-TS/A or CD70{Delta}/CD80{Delta}-TS/A cells, were injected with 1.5 x 105 TS/A cells and tumor appearance was monitored for 30 days. Data represent the percentage ± SD from four separate experiments of mice which rejected parental TS/A cells out of the number of primary injected transfected TS/A mice. Asterisks denote values significantly different (**P = 0.01 and *** P < 0.001) from the CD70/CD80-TS/A control group by Fisher’s exact test.

 

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Table 1. Specific tumor protection is impaired by the loss of CD70 intracytoplasmic domain
 
CD8 T cells are necessary to mediate tumor rejection
Our previous work in nude mice has shown that the control of CD70/CD80-TS/A tumor growth observed in immunocompetent BALB/c mice was dependent on the T cells (30). We evaluated which T cell populations could be specifically involved in the antitumor immune response mediated by co-stimulatory molecule expression and affected by the loss of the CD70 or CD80 intracytoplasmic domain. We compared the tumor growth of the different transfected TS/A cell lines in CD4 or CD8 T cell-depleted BALB/c mice versus control IgG-treated mice (Fig. 8). In CD8-depleted BALB/c mice, CD70/CD80-TS/A and CD70{Delta}/CD80-TS/A cells formed tumors that were not rejected in all the injected mice (Fig. 8A). The CD8 T cells were involved in TS/A tumor growth control even if the co-stimulatory molecules were expressed without an intact intracytoplasmic domain (P < 0.05). The tumor growth of CD70/CD80-TS/A cells in CD8 T cell-deficient mice was slower than the CD70{Delta}/CD80-TS/A and mock-TS/A tumor growth in CD8-deficient mice (Fig. 8A). These data suggest that an immune cell population other than CD8 T cells could be stimulated by CD70/CD80-TS/A cells. The similarity of growth between CD70/CD80{Delta}-TS/A cells in non-depleted mice and CD70/CD80-TS/A in the CD8-depleted mice suggested that the CD80 wild-type molecule was necessary to recruit and/or activate CD8 T cells, and to induce CD70/CD80-TS/A and CD70{Delta}/CD80-TS/A tumor rejections.



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Fig. 8. Involvement of the CD4 and CD8 T cells in the antitumor immune response mediated by the co-stimulatory molecules. On day 0, 105 mock-TS/A, CD70/CD80{Delta}-TS/A, CD70{Delta}/CD80-TS/A or CD70/CD80-TS/A were s.c. injected in the control BALB/c mice (IgG). Either 105 mock-TS/A, CD70/CD80{Delta}-TS/A, CD70{Delta}/CD80-TS/A or CD70/CD80-TS/A were injected in the CD8-depleted BALB/c mice ({alpha}CD8) (A) or in the CD4-depleted BALB/c mice ({alpha}CD4) (B) as described in Methods. The results are expressed as mean size (mm2) of tumors from groups of four to five mice each ± SD except for the CD70/CD80{Delta}-TS/A IgG group, where the mean tumor size represents the mean of the four mice developing tumors out of five injected mice. (x/y) x represents the number of mice developing tumors and y the total number of injected mice for each group. Data are from one experiment representative of one other.

 
Tumor growth of CD70{Delta}/CD80-TS/A cells and CD70/CD80-TS/A cells was abrogated in control and in CD4-depleted mice, but rejection occurred later, delayed from day 11 to, 20 for the CD70{Delta}/CD80-TS/A tumors (Fig. 8B). CD70/CD80{Delta}- TS/A tumor growth in CD4 T cell-deficient mice was transiently increased compared to CD70/CD80{Delta}-TS/A tumor growth in IgG control mice (Fig. 8B).

CD70/CD80-TS/A cells stimulate IFN-{gamma} secretion by CD8 T cells better than CD70{Delta}/CD80-TS/A cells
In vivo rejection of both CD70/CD80-TS/A and CD70{Delta}/CD80-TS/A tumors was CD8 T cell dependent. We hypothesized that the lack of an antitumor memory response in the CD70{Delta}/CD80-TS/A group could be related to the lack of additional CD8 T cell stimulation after the primary antitumor response. We then compared the co-stimulatory signals provided in vitro by the CD70/CD80-TS/A and CD70{Delta}/CD80-TS/A tumor cells to total lymph node cells or to CD8 T cells isolated from mice bearing 12-day-old parental TS/A tumors. T cells sensitized in vivo to TS/A cells were stimulated in vitro with various doses of Mit-C-treated wild-type or gene-modified tumor cells without anti-CD3{epsilon}. After 3 days of culture, IFN-{gamma} secretion was quantified in culture supernatants by sandwich ELISA. As shown in Fig. 9, the stimulation of CD8 T cells or total lymph node cells by CD70/CD80-TS/A cells induced stronger IFN-{gamma} secretion than CD70{Delta}/CD80-TS/A stimulation.



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Fig. 9. CD70/CD80 co-stimulation increases IFN-{gamma} production by lymphoid cells and CD8 T cells. CD8 T cells and lymph node cells harvested from BALB/c mice bearing 12-day-old TS/A tumors were stimulated in vitro for 3 days with increased numbers of Mit-C-treated wild-type-, CD70{Delta}/CD80- or CD70/CD80-transfected TS/A tumor cells. After 3 days, IFN-{gamma} production was assessed by sandwich ELISA. The results are expressed as the mean ± SEM of three separate wells. Data are from one experiment representative of one other. Stars denote points significantly different (*P < 0.05 and **P < 0.01 by Student’s t-test) from the TS/A stimulation.

 
Interestingly, the CD70/CD80-TS/A stimulation of total lymph node cells induced stronger IFN-{gamma} secretion than stimulation of CD8 T cells alone. These data show that CD70/CD80-TS/A cells directly induced a stronger co-stimulation to CD8 T cell than CD70{Delta}/CD80-TS/A cells did. CD70 intracytoplasmic domain with CD80 appeared critical to co-stimulate the IFN-{gamma} production by CD8 T cells. However, this effect seemed potentiated by stimulation of other immune cell populations.

CD70/CD80-TS/A cells promote expansion of CD44highCD62Llow effector/memory CD8 T cells
We studied by flow cytometry analysis the CD44/CD62L effector/memory marker expression (38,39) on CD8 T cells after in vitro stimulation. Lymph node cells were harvested from mice bearing 12-day-old parental TS/A tumor, and were cultured with anti-CD3{epsilon} and Mit-C-treated CD70{Delta}/CD80-TS/A or CD70/CD80-TS/A cells for 3 days. As shown in Fig. 10, lymph node cells stimulated in vitro with Mit-C CD70/CD80-TS/A cells displayed a higher percentage of CD8 T cells expressing the CD44highCD62Llow phenotype (37.7 ± 1.17%) compared to the Mit-C CD70{Delta}/CD80-TS/A stimulation (21.6 ± 2.75%). The increased percentage of effector/memory CD8 T cells in total lymph node population after CD70/CD80-TS/A stimulation resulted both in an increased number of surviving CD8 T cells, and in an increased proportion of CD8 T cells expressing high CD44 and low CD62L markers (Table 2).



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Fig. 10. Example of CD44 and CD62L surface marker modulation after in vitro stimulation of lymph node cells by CD70{Delta}/CD80-TS/A or CD70/CD80-TS/A cells. Total lymph node cells from a pool of two BALB/c mice bearing 12-day-old TS/A tumors were stimulated in vitro with Mit-C-treated CD70{Delta}/CD80-TS/A or CD70/CD80-TS/A cells and anti-CD3{epsilon}. After 3 days, cells were collected, stained with PE-conjugated anti-CD8, FITC-conjugated anti-CD62L and CyChrome-conjugated anti-CD44 mAb, and analyzed by flow cytometry after gating on live cells. Panels shown are gated on live CD8+ cells. The numbers in the upper left quadrants indicate the percentage of CD8 T cells expressing high CD44 and low CD62L markers.

 

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Table 2. Expression of CD44 and CD62L markers on CD8+ T cells
 
These results reflected the higher capacity of CD70/CD80-TS/A tumor cells to expand effector and potentially memory CD8 T cells during the primary antitumor response.

We then studied the CD44/CD62L effector/memory marker expression on CD8 T cells during the secondary antitumor responses against parental TS/A tumor cells elicited in CD70{Delta}/CD80 mice and CD70/CD80 mice. Twenty-seven days after the initial injection of the CD70/CD80-TS/A or CD70{Delta}/CD80-TS/A cells, mice which had rejected the transfected tumors were challenged with parental TS/A cells. Twelve days after, lymph node cells were harvested,and stimulated in vitro for 3 days with anti-CD3{epsilon} and Mit-C-treated TS/A cells. Lymph node cells from mice which had rejected the CD70/CD80-TS/A tumors displayed, after in vitro stimulation with parental TS/A cells, a higher percentage of CD44highCD62Llow CD8+ T cells (26.3 ± 2.29%) than lymph node cells from mice which had rejected CD70{Delta}/CD80 TS/A tumors (21.6 ± 2.76%) (Table 2). These data suggest that CD70/CD80 co-stimulation in vivo favored the in vitro expansion of CD44highCD62Llow CD8 T cells in response to the parental TS/A tumor cells and anti-CD3{epsilon} stimulation compared to CD70{Delta}/CD80 co-stimulation.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
As CD70 and CD80 belong to different co-stimulatory families, the TNF family (19) and the B7 family respectively (7), they are expected to act at different stages of the antitumor response. CD80, mostly expressed by activated APC (40), is known to be involved in the activation phase of naive T cells inducing, via CD80–CD28 interactions, IL-2 production and proliferation, and favoring cytotoxic T cell generation (41). The CD70-dependent co-stimulatory signal has been shown to be involved after antigen-specific T cell stimulation, and to induce clonal expansion of the antigen-activated CD4 and CD8 T cell by direct LT–LT cell or T–B cell interactions (24,26,42). Hendricks et al. have recently shown that CD27 is involved in the generation of T cell memory to influenza virus, most likely due to enhanced T cell survival and/or expansion of memory T cell populations (26).

The necessity of an intact intracytoplasmic domain in CD80 biological activity was controversial at the beginning of our studies. The CD80 intracytoplasmic domain was initially shown to be required to regulate in vitro CD80 redistribution and T cell co-stimulation when expressed by gene-modified CHO and Reh cells (17,18). Recently, Faas et al. have described an alternative spliced soluble form of the porcine CD80 molecule that lacks both the transmembrane and intracytoplasmic domains, but which still interacts with both porcine and human CD28 and CTLA-4. This soluble form of CD80 could inhibit the in vitro proliferation of human CD4 T cells and block IL-2 production from human phytohemagglutinin-stimulated T cells (43). These two points suggest that the intracytoplasmic domain of CD80 could be involved in an efficient CD80/CD28 T cell co-stimulation signal. In contrast, Yu et al. (1998) have shown that the intracytoplasmic domain of CD80 was not involved in vivo to induce tumor rejection of CD80 gene-modified EL4 cells (44).

In vitro, as previously shown, we confirmed that CD80 membrane redistribution in TS/A cells also required an active process involving an interaction of the CD80 intracytoplasmic domain with the actin cytoskeleton. Previous studies have shown the involvement of the actin cytoskeleton of the professional APC (DC) in the priming of naive CD4 T cells in vitro (16). Our results suggest that actin cytoskeleton interactions with the CD80 intracytoplasmic domain in a tumor cell could be implicated in the delivery of an effective CD28-dependent stimulatory signal to CD8 T cells. In vivo, we have shown for the first time that the CD80 intracytoplasmic domain was critical to induce CD8 T cell-dependent tumor rejection of the gene-modified TS/A tumors, CD70/CD80-TS/A and CD70{Delta}/CD80-TS/A. However, the expression of CD80 alone without CD70 on TS/A cells was not able to induce tumor rejection and only slowed down tumor growth (30).

We showed that the CD70 membrane redistribution induced by mAb was independent of its intracytoplasmic domain and of the actin cytoskeleton. However, its ability to deliver in vitro a co-stimulatory proliferation signal with CD80 to TCR–CD3{epsilon}-activated T cells was dependent on its intracytoplasmic domain. This result suggests a direct link between the intracytoplasmic domain of CD70 and its biological activity unrelated to the clustering. The CD70 molecule is physiologically transiently up-regulated on CD4 or CD8 T cells and B cells after stimulation (4548) to induce clonal expansion of the antigen-activated CD4 and CD8 T cells by direct T–T cell or T–B cell interactions (21,46,49,50). These lymphoid cells have different cytoskeleton characteristics compared to the adherent DC expressing CD80, which are the most potent APC to activate naive T cells (16,5153). The different requirement of the CD70 or CD80 intracytoplasmic domain to induce the membrane redistribution profiles observed in this study could be related to these morphological features.

The co-expression of CD70 and CD80{Delta} or of CD70{Delta} and CD80{Delta} by TS/A cells generated a delay of tumor growth, but no tumor rejection. These results suggest that the full-length CD70 molecule was not able, without the wild-type CD80 molecule, to induce tumor rejection. CD70/CD80 and CD70{Delta}/CD80 tumor rejections were both dependent on the activation of CD8 effector T cells (Fig. 8A). However, in the CD8 T cell-depleted mice, CD70/CD80-TS/A tumors grew slower than the CD70{Delta}/CD80-TS/A tumors (Fig. 8A). Furthermore, CD70/CD80, but not CD70{Delta}/CD80, co-stimulation in vitro was more effective on total lymph node cells than on CD8 T cells to induce IFN-{gamma} secretion (Fig.9). These findings suggest that the full-length CD70 molecule with CD80 could be necessary to co-stimulate non-CD8 T cell populations with antitumor activities.

In CD4-depleted mice, CD70/CD80-TS/A and CD70{Delta}/CD80-TS/A tumors were still rejected, but the rejection time was delayed (Fig. 8B). We observed by immunocytoanalysis, a CD4 T cell infiltrate at day 8 post s.c. injection in tumors expressing CD70/CD80-TS/A and CD70/CD80{Delta}-TS/A compared to mock-TS/A cells (data not shown). However, in vitro, no CD4 T cell proliferation was observed when total lymph node cells were stimulated with anti-CD3{epsilon} and Mit-C CD70/CD80-TS/A cells (data not shown). NK cells express CD27 and CD28 (54,55), and so could potentially be activated by CD70/CD80 TS/A cells. In vitro experiments have shown that CD27 or CD28 triggering on NK cells by CD70 or CD80 gene-modified tumor cells stimulates NK cell proliferation and IFN-{gamma} secretion (54,56,57). Recent findings have shown that CD70 expression by gene-modified MHC I-deficient murine T lymphoma cells (RMA-S) induced in vivo NK-dependent tumor rejection (56). This primary NK-dependent antitumor response elicited by CD70 expression by tumor cells played a major role in promoting T cell antitumor immunity against rechallenge with parental MHC I-sufficient RMA tumor cells (56).

These results are consistent with a model which assigns the CD80{Delta}-ineffective primary antitumor response to decreased capacities of CD80{Delta} to undergo mAb-induced clustering by its inability to interact with the tumor cell actin cytoskeleton. The APC cytoskeleton, in particular actin microfilaments and myosin motors, is thought to play a key role in driving the redistribution of synapse molecules. Overall, the changes in the cytoskeleton likely serve to increase the contact between TCR and MHC, and provide an optimal environment for signaling molecules downstream of the TCR and perhaps of the co-stimulatory molecules such as the ligands used in our models. For tumor cells expressing CD80 without its intracytoplasmic domain (CD70/CD80{Delta}-TS/A and CD70{Delta}/CD80{Delta}-TS/A tumors), even if a slowing down of tumor growth was observed compared to mock-TS/A cells, the activation threshold appeared too low to generate effective T cell responses leading to tumor rejection.

The intracytoplasmic domain of CD70 expressed by tumor cells along with CD80 expression near tumor antigen appeared to be involved in mediating long-lasting antitumoral immunity. The mice which rejected CD70{Delta}/CD80-TS/A tumors were partially protected against a rechallenge with the wild-type TS/A cells. This incomplete immune response observed with CD70{Delta}/CD80-TS/A cells could be related to the inability of the intracytoplasmic domain-deleted CD70 to deliver a co-stimulatory signal involved in the generation of memory T cells relative to its inability to induce optimal T cell proliferation or survival signals as suggested by our in vitro data (Figs 5, 9 and 10). In vitro co-stimulation of lymph node cells with CD70 and CD80 molecules, but not CD70{Delta} and CD80 molecules, induced IFN-{gamma} secretion (Fig. 9), and contributed to the expansion of CD8 T cells displaying an effector and potentially memory CD44highCD62Llow phenotype (38,39) during the primary antitumor response (Fig. 10).

In vivo, CD70/CD80 co-expression, like CD70{Delta}/CD80 co-expression, by transfected TS/A cells induced comparable CD8 T cell-dependent tumor rejection rates during the primary antitumor response. When direct flow cytometric analysis of CD8 T cell phenotype was conducted on lymph node cells harvested from CD70/CD80 and CD70{Delta}/CD80 mouse groups, 30 days after the initial injection time, after in vivo TS/A rechallenge, no difference was observed between the two groups (data not shown). After 3 days in vitro Mit-C TS/A stimulation, lymph node cells from the CD70/CD80 mouse group displayed higher numbers of CD8 T cells expressing the effector/memory CD44highCD62Llow phenotype than lymph node cells from the CD70{Delta}/CD80 mouse group (Table 2). These data support the hypothesis that CD70/CD80 co-stimulation in vivo could improve the number of CD8 T cells potentially active against parental TS/A tumor cells by survival or expansion signals.

This work suggests that the full-length CD80 molecule along with CD70 expressed by the gene-modified tumor cells could provide, at the tumor site, additional stimulation to T cells to favor their full maturation into effector cells, whereas the CD80 molecule without its intracytoplasmic domain with full-length CD70 does not. The two co-stimulatory molecules, CD70 and CD80, acted both on T cell populations with a predominant requirement of the CD80 intracytoplasmic domain to fully activate CD8 effector T cells and a potential requirement of CD70 for the activation or expansion of memory T cells. Recent findings in 4-1BBL–/– knockout mice have shown that, during in vivo secondary response to influenza infection, the expansion level of the specific CD8 T cells is reduced to the level of a primary response (58). The LCMV-infected 4-1BBL–/– mice showed a general defect in expansion of the CD44high subset of CD8 T cells compared to wild-type mice (58). These data support the hypothesis that members of the TNF receptor family, like CD27, OX40 (59) or 4-1BB (58,60,61), could sequentially act on T cells after CD28 co-stimulation to sustain the CD28-dependent T cell activation and differentiation signals by providing survival signals increasing or stabilizing T cell effector/memory numbers later in the response. The precise molecular signals required to induce each of the co-stimulatory pathways remain to be fully elucidated. These questions are central to our understanding of how gene-modified tumor cells are efficient immune stimulators and will help to define more precisely how optimal triggering of a specific antitumor response occurs.


    Acknowledgments
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 
We thank Christiane Pages and Lourdes Gasquet for technical assistance in the animal facility. We gratefully acknowledge Dr Fatima L’Faqihi for technical assistance with flow cytometry. We thank Dr A. F. Tilkin for the careful reading of this manuscript. This work was supported by grants from the Claudius Regaud Institute (Toulouse, France), Association for Research against Cancer (ARC, grant 9719) and Conseil Régional Midi-Pyrénées. V. D.-E. was supported by the ‘Ligue Nationale contre le Cancer’.


    Abbreviations
 
APC—antigen-presenting cell

CCB—cytochalasin B

CD70{Delta}—intracytoplasmic domain-deleted CD70

CD80{Delta}—intracytoplasmic domain-deleted CD80

DC—dendritic cell

Mit-C—mitomycin C

PE—phycoerythrin

TNF—tumor necrosis factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgments
 References
 

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