Increases Tumor Cell Infiltration Overexpression Delays Tumor Growth ...

The Journal of Immunology

Intratumoral CC Chemokine Ligand 5 Overexpression Delays Tumor Growth and Increases Tumor Cell Infiltration1 Elise Lavergne,2 Christophe Combadie`re,2 Mutsunori Iga, Alexandre Boissonnas, Olivia Bonduelle, Maud Maho, Patrice Debre´, and Behazine Combadiere3 Chemokines participate in the antitumor immune response by regulating the movement and positioning of lymphocytes as well as effector functions and may thus be candidates for use in antitumor therapy. To test whether CCL5, a chemokine involved in the recruitment of a wide spectrum of immunocompetent cells, can control tumor growth, we forced its expression at mouse tumor sites. Tumor growth was reduced in mice with s.c. syngeneic CCL5-EL-4 compared with EL-4-injected mice, whereas both reduced tumor growth and incidence were observed in mice with OVA-expressing EG-7 transfected with CCL5 compared with EG-7-injected mice. Significant antitumor effects were observed soon after intratumoral injection of DNA plasmid coding for chimeric CCL5-Ig. Importantly, quantitative RT-PCR assays showed that the amount of CCL5 expression at the tumor site determined the effectiveness of the antitumor response, which was associated with infiltration of increased numbers of NK, CD4, and CD8 cells at the tumor site. This effect was lost in mice deficient for T/B lymphocytes (RAG-2 knockout) or for CCR5 (CCR5 knockout). Together, these data demonstrate the antitumor activity of intratumoral CCL5 overexpression, due to its recruitment of immunocompetent cells, and the potential usefulness of chimeric CCL5-Ig DNA as an agent in cancer therapy. The Journal of Immunology, 2004, 173: 3755–3762.

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ne possible new approach to anticancer therapies involves the redistribution and activation of immunocompetent cells into the vicinity of developing tumors. Chemokines may be good candidates for this task (1). They are proinflammatory cytokines that mainly signal through a family of G protein-coupled receptors to coordinate the deployment and activation of leukocytes at injury sites (2). The chemokine superfamily is divided into four subgroups (CC, CXC, CX3C, and C) according to the number and spacing of the first two closely paired and highly conserved cysteines of the N-terminal domain (3). Many CC and CXC chemokines have been tested for their antitumor effect. Although some chemokines promote cancer metastasis by regulating the spread of tumor cells (4, 5) or promote tumor growth by modulating neovascularization (6 – 8), others may favor adaptive antitumor immune responses by recruiting subsets of leukocytes. Animal experiments (9) and clinical reports indicate that chemokines may help the immune system to recognize and to kill tumor cells: more specifically, CCL2 reduces tumor growth by increasing monocyte/macrophage infiltration, and adenovirus-me-

Laboratoire d’Immunologie Cellulaire, Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 543, Hoˆpital Pitie´-Salpeˆtrie`re, Paris, France Received for publication December 5, 2003. Accepted for publication July 2, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 E.L. and A.B. were recipients of the fellowship from the French Ministry of Research and Technology and La Fondation pour la Recherche Me´dicale. M.I. was a recipient from Objectifs Recherche de Vaccin SIDA. This work was supported by grants from Association pour la Recherche sur le Cancer and from La Ligue Nationale Contre le Cancer. 2

E. L. and C.C. contributed equally to this work.

diated gene transfer of CCL3 induces dendritic cells (DC)4 to accumulate at the tumor site (10, 11). Modifications of chemokine genes, including CCL1 (12), CCL3 (4), CCL5 (13), CCL19 (14), CCL20 (15), CCL21 (16, 17), CX3CL1 (18, 19), and XCL1 (12), activate tumor-specific and nonspecific immunity: they increase local recruitment of monocytes/macrophages, DC, and T cells to the tumor site in melanoma; in ovarian, breast, and cervical carcinomas (20, 21); in adenocarcinoma (22, 23); and in sarcoma, fibrosarcoma, and glioma (24, 25). We recently reported that increased tumor expression of either CX3CL1 or chimeric CX3CL1-Ig induces antitumor activity via NK cell recruitment (26). The role of chemokines in tumor growth nonetheless remains complex and requires further investigation. The immune system’s rejection of cancer cells results from a series of events that include T lymphocyte activation, expansion, and infiltration of the tumor, and efficient cytotoxic effector functioning. Several CC chemokines are known to attract naive and effector T cells. Because receptors for CCL5 are expressed on effector Th1 CD4, CD8, and NK cells, and DC (12, 27–31), and because these immunocompetent cells are implicated in tumor immune surveillance, we investigated the role of CCL5 in the control of tumor growth. We and others have shown that activated/effector CD4 and CD8 cells that express CCR5 migrate to inflammatory sites after Ag priming in secondary lymphoid organs (32–34). Whereas naive cells are CCR5⫺CCR7⫹, effector T cells are CCR5⫹CCR7⫺ after Ag-induced cell differentiation, and they migrate out of the lymphoid organs in response to CCL5 (33). In addition, CCL5 is a chemoattractant for memory T lymphocytes in vitro (35). Together with CCL2 and CCL3, it affects the magnitude and polarity of the T cell response (28). It has also been suggested that a cell-mediated Th1 type response is promoted when T cell activation sites contain large quantities of CCL3, CCL4, and CCL5 (28).

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Address correspondence and reprint requests to Dr. Behazine Combadiere, Laboratoire d’Immunologie Cellulaire, Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 543, Faculte´ de Me´decine Pitie´-Salpe´trie`re, 91 Boulevard de l’Hoˆpital, 75013 Paris, France. E-mail address: [email protected] Copyright © 2004 by The American Association of Immunologists, Inc.

4 Abbreviations used in this paper: DC, dendritic cell; KO, knockout; HEK, human embryonic kidney; PEI, polyethylenimine.

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CCL5-Ig ANTITUMOR GENE THERAPY

In this study, we examined whether forcing tumor cells to produce CCL5 might promote antitumor immune response and T cell attraction at the tumor site. We studied the quantitative and qualitative effects of CCL5 production on tumor growth and immune cell recruitment. We approached this question by investigating two different gene therapy strategies: inoculating mice with CCL5transfected tumor cells and directly injecting DNA coding for CCL5 fused with Ig into the tumor.

establishment required injection of at least 200,000 EL-4 cells (data not shown). To evaluate the cells recruited to the tumor site and to detect CCL5 expression, the tumor mass was surgically removed between days 10 –14, as indicated. The tumor and proximal lymph nodes were dilacerated and passed through a 70-␮m cell strainer (BD Biosciences, San Diego, CA). Lymphocytes were collected and washed in PBS-2% FCS buffer. Red cells were eliminated with cell lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), and cells were washed in complete medium. They were analyzed after immunofluorescence staining.

Materials and Methods

RT-PCR analyses

Mice Wild-type C57BL/6 females were obtained from Elevage Janvier (Le Genest, Saint Isle, France). C57BL/10 RAG-2 knockout (KO), C57BL/6 perforin KO, and C57BL/6 CCR5 KO mice were purchased from Charles River (Saint-Aubin les Elbeufs, France) and bred in our animal facility (Nouvelle Animalerie Commune of Pitie´ -Salpeˆ trie`re, Paris, France). All mice were housed under specific pathogen-free conditions and used for experiments at 6 –10 wk of age. All experiments complied with local animal experimentation and ethics committee guidelines.

Tumor cell lines All cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA) and were derived from C57BL/6 (H2b) mice. The dimethylbenzanthracene-induced thymoma, EL-4, and its chicken OVA peptide-expressing derivative, EG-7, were maintained in RPMI 1640 (Invitrogen Life Technologies, Paisley, Scotland) supplemented with 10% heat-inactivated FCS (Seromed, Berlin, Germany), 2 mM L-glutamine, 1000 U/ml penicillin, 1 mg/ml streptomycin, 250 ng/ml amphotericin B (Invitrogen Life Technologies), and 3 ␮M 22-ME (SigmaAldrich, St. Louis, MO). We used Transfast (Promega, Madison, WI), in accordance with the manufacturer’s instructions, to transfect EL-4 and EG-7 cell lines with pBlast-human CCL5 plasmid (InvivoGen, Toulouse, France) and 10 ␮g/ml blasticidin (InvivoGen) to select transfectants, which were then maintained with 5 ␮g/ml blasticidin. Parental cell lines transfected with the empty plasmid pBlast were used as control tumor cell lines. We verified with flow cytometry that neither EL-4 nor EG-7 transfection affected MHC class I molecules, nor did they induce expression of costimulatory molecules such as CD70 and CD80 (data not shown). Proliferation assays measuring tritiated thymidine incorporation showed no significant differences in tumor cell growth in vitro between the transfected and control lines (data not shown).

Chimeric CCL5-Ig constructs PstI-tailed forward primer AAA ACT GCA GAT GAA GGT CTC CGC GGC A and NotI-tailed reverse primer ATA GGC GGC CGC GCT CAT CTC CAA AGA GTT were used to amplify the signal sequence and chemokine domain of CCL5 corresponding to aa 1–91 (enzyme recognition sequences are underlined). The modified mouse IgG2a Fc domain, corresponding to aa 97–329 and derived from pVRC murine IL-2/Ig (36), a generous gift from Dr. D. Barouch (Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA), was amplified with NotI-tailed primer ATA AGC GGC CGC ACA TCC CAG AGG GCC CAC AAT C and BglII-tailed primer GGA AGA TCT TCA TTT ACC CGG AGG CCG GGA GAA. Amplification reactions were performed in standard conditions with 1 U of Pfu DNA polymerase (Stratagene, La Jolla, CA). PCR cycling began at 95°C for 5 min, followed by 20 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and ended with 10 min at 72°C. PstI-NotI CCL5 and NotI-BglII Ig fragments were directly subcloned into a pVRC plasmid cloning site. Low endotoxin plasmid was prepared on a large scale (Tebu Biolaboratories, Le Perray en Yvelines, France). Flow cytometric analysis, ELISA, and immunoblotting with the cell lysate and supernatant of Chinese hamster ovary cells transfected with this construct all confirmed the expression of the chimeric protein, also produced and purified by Tebu Biolaboratories.

In vivo experiments Five to 10 mice per group were injected s.c. in the right flank with 200,000 tumor cells in 100 ␮l of PBS. Tumor size was measured with a caliper three times a week, and tumor volume was estimated with the following formula: width ⫻ length ⫻ (width ⫹ length)/2. Mice were sacrificed when tumor volume reached ⬃12 cm3. Five days after tumor inoculation, in vivo JetPEI transfecting reagent (Qbiogene, Illkirch, France) was used to inject 10 ␮g of CCL5-Ig DNA plasmid or control DNA at the tumor site. Reliable tumor

Intratumoral mRNA expression of CCL5-Ig was detected by RT-PCR. Total RNA was extracted from each tumor with the QIAamp RNA Blood Mini kit (Qiagen, Courtaboeuf, France), and cDNA was generated with Promega’s reverse transcription system. PCR amplification took place under the following conditions: 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 20 s. Primer sequences for CCL5-Ig detection were as follows: forward primer, 5⬘-GTG CCC ACA TCA AGG AGT AT-3⬘, and reverse primer, 5⬘-GCT GTG TGT ACT TCC ACG TT-3⬘. Control HPRT primers were as follows: forward primer, 5⬘CTT TGC TGA CCT GCT GGA TT-3⬘, and reverse primer, 5⬘-TAT GTC CCC CGT TGA CTG AT-3⬘. For the quantitative RT-PCR, we used PreDeveloped TaqMan Assay Reagents (primers and probes) for detection of human CCL5 and the Applied Biosystems TaqMan Master Mix (with uracyl-N-glycosylase) according to the manufacturer’s instructions (Applied Biosystems, Cheshire, U.K.). Control HPRT real-time PCR was used to standardize the results.

Flow cytometry Cell surface Ags were characterized with a standard staining method and the following mAbs: FITC-conjugated anti-mouse CD3 (clone 145-2C11), PE-conjugated pan-NK (clone DX5), PerCP/Cy5.5-conjugated anti-mouse CD8 (clone Ly-2), biotin-conjugated anti-mouse CD4 (clone L3T4) plus allophycocyanin-streptavidin, PE-conjugated anti-H-2Kb (clone AF6-88.5), PE-conjugated anti-mouse CD49 (BD Pharmingen, Le Pont de Claix, France), and PE-conjugated anti-human CCL5 (clone 2D5; R&D Systems, Abingdon, U.K.). For intracellular CCL5 staining, cells were washed and permeabilized with 1⫻ PBS-5% FCS-0.1% saponin before intracellular staining with PEconjugated anti-human CCL5 (clone 2D5). The cells were incubated with appropriate fluorochrome-conjugated mAbs for 20 min at room temperature, and then washed in 1⫻ PBS and fixed for 15 min at room temperature in 1 ml of 4% paraformaldehyde. Cells were run for four-color fluorescence staining on a cytofluorometer (FACSCalibur; BD Biosciences), and 10,000 live events were analyzed with ProCellQuest software.

Chemokine binding assay Binding assays were performed with 125I-labeled CCL4 (Amersham Biosciences, Piscataway, NJ) in duplicate with 5 ⫻ 104 CCR5-expressing human embryonic kidney (HEK) cells, as previously described (37). Briefly, cells were incubated in a total volume of 200 ␮l of PBS containing 1 mg/ml BSA and 0.01% azide (pH 7.4) with 50 pM 125I-labeled CCL4 and increasing concentrations of unlabeled human CCL5 (PeproTech, Rocky Hill, NJ) or CCL5-Ig. After 2 h at 37°C, unbound chemokines were separated from cells by centrifugation in 1 ml of PBS with 10% sucrose. Gamma emissions were then counted in the cell pellet (1272 Clinigama; LKB Wallac, Saint Quentin en Yvelines, France).

In vitro chemotaxis assay Chemotaxis was assayed in a 96-well chemotaxis chamber (NeuroProbe, Cabin John, MD). PBMC were labeled for 30 min at 37°C with 5-chloromethylfluorescein diacetate (Molecular Probes, Leiden, The Netherlands) in RPMI 1640 and resuspended in HBSS-BSA (106 cells/ml). Human rCCL5 (PeproTech) and CCL5-Ig were placed in the lower chamber. We then seeded 50,000 human PBL onto the membrane (5-␮m pore diameters) and incubated the 96-well plate for 3 h at 37°C, 100% humidity, and 5% CO2. The filter top surface was rinsed with PBS, and the plate was centrifuged for 2 min at 1500 rpm. Fluorescence was measured with a Packard Fusion microplate analyzer (Perkin-Elmer Life Sciences, Boston, MA). Results are expressed as a chemotaxis index that represents the number of cells migrating in response to CCL5 or CCL5-Ig relative to the number migrating in the absence of the chemokine.


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FIGURE 1. CCL5 overexpression in EL-4 tumor cells reduces tumor development. a, Flow cytometric analyses of CCL5 expression in CCL5-transfected EL-4 (CCL5-EL-4) and control empty plasmid-transfected EL-4 cells (Mock-EL-4). b, C57BL/6 mice were injected s.c. with 200,000 Mock-EL-4 (f) or CCL5-EL-4 cells (䡺). All mice developed a tumor that we measured, as described in Materials and Methods, and expressed in cubic centimeters. Each data point represent the mean tumor volume ⫾ SEM of 16 mice. The CCL5-EL-4 tumor volumes were 65–70% of control EL-4 tumors, between days 20 –25 after tumor inoculation. ⴱ, Significant value of p ⬍ 0.05 (control mice with tumor size of ⬎10 cm3 were euthanized).

Statistical analysis We used Prism 2.01 (GraphPad Software, San Diego, CA) for data handling, analysis, and graphic representation. Statistical analysis used the paired two-sample t test for means and the nonparametric Mann-Whitney U test. Statistical significance was set at p ⬍ 0.05.

Results CCL5 inhibits syngeneic tumor development The H-2b lymphoma EL-4 was transfected with plasmid encoding full-length CCL5 or an empty control vector. Chemokine expression was confirmed in the CCL5-EL-4 cell lines with intracellular staining (Fig. 1a). In addition, ELISA showed CCL5 in the supernatant of transfected cells, at a maximum level of ⬃8 nM by day 4 of cell culture (data not shown). We assessed the ability of CCL5-transfected tumor cell lines to form solid tumors in C57BL/6 mice. In the conditions described above, all the mice (100%) developed a solid tumor that grew progressively under their skin (Fig. 1b): it was measurable by day 7 and grew over a period of 3 wk. Growth was significantly slower in the in vivotransfected CCL5-EL-4 tumors than in control tumors. The mean volume of CCL5-EL-4 tumors was ⬃65–70% smaller than that of the control tumors between days 20 –25, before the control EL-4inoculated mice were euthanized (Fig. 1b). Tumors that expressed CCL5 remained significantly smaller ( p ⬍ 0.05) than control tu-

FIGURE 2. CCL5 overexpression in EG-7 tumor cells reduces tumor development. a, Flow cytometric analyses of CCL5 expression in CCL5-transfected EG-7 (CCL5-EG-7) and control empty plasmid-transfected EG-7 cells (Mock-EG-7). Two EG-7 cell lines that expressed different amounts of CCL5 were isolated. The level of CCL5 expression remained stable in the in vitro culture. b, C57BL/6 mice were injected s.c. with 200,000 Mock-EG-7 (f) or CCL5-EG-7 (‚, CCL5low; 䡺, CCL5high). The number of mice without palpable tumors was indicated as a percentage of total mice injected (n ⫽ 8). In addition, tumor size remained significantly lower in CCL5-EG-7-injected mice than in Mock-EG-7-injected mice (data not shown). Seventy percent of mice that were inoculated with CCL5EG-7 never developed any measurable tumors as long as the animals were kept in our facility (⬎60 days), whereas mice inoculated with control EG-7 developed tumors.

mors until day 23 (Fig. 1b, ⴱ); thereafter, all CCL5-EL-4 tumors continued to grow at similar rates. In a second model, we used a more immunogenic tumor, EG-7 cell lines that expressed chicken OVA stably transfected with CCL5. Two CCL5-EG-7 cell lines that expressed high (CCL5highEG-7) or low (CCL5low-EG-7) levels of CCL5 were selected (Fig. 2a). All control mice developed a solid tumor within 10 days of inoculation. Mice injected with CCL5low-EG-7 required 25 days for tumor development (Fig. 2b). Of those injected with CCL5highEG-7, only 30% developed a tumor by day 30, and 70% remained tumor free. Thus, these data underline the effectiveness of CCL5 in controlling EG-7 tumor growth, although CCL5 overexpression only delayed EL-4 tumor growth. CCL5-mediated antitumor activity was lost in CCR5 KO and RAG-2 KO mice We next ascertained the receptor primarily involved in the antitumor properties of CCL5 in our model. CCL5 binds and activates at least three chemokine receptors, namely, CCR1 (35, 38), CCR3 (39), and CCR5 (40). Given that the latter is the CCL5 receptor primarily expressed on lymphoid cells, we tested its protective effect in CCR5 KO mice. The total loss of this protective effect in this strain demonstrates that the antitumor effect depends on CCR5 (Fig. 3a). These results further suggest that CCR1 and CCR3 are

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FIGURE 3. The antitumor effect of CCL5 is determined by intact immune system and CCR5 expression. CCR5 KO (a and b) and RAG-2 KO (c and d) mice were injected s.c. with 200,000 CCL5-EL-4 (䡺) or Mock-EL-4 (f). All mice developed a palpable tumor. Tumor volumes were measured and expressed in cubic centimeters. Each data point represents the mean tumor volume ⫾ SEM of 10 mice. b, Five million splenocytes from either C57BL/6 mice or CCR5 KO mice were adoptively transferred at day 2, into CCR5 KO mice that were inoculated with CCL5-EL-4 cells. d, Mock-EL-4 or CCL5-EL-4 tumors were harvested from mice with palpable tumors, and cell suspensions were stained for intracellular CCL5 expression at day 25 after tumor inoculation (right panel) and compared with CCL5 expression at day 0 before tumor inoculation (left panel). Results are representative of CCL5 expression in tumors harvested from three mice. ⴱ, Significant value of p ⬍ 0.05.

not key players in this phenomenon. We also observed that the control EL-4 tumors were smaller in CCR5 KO than in control mice (Fig. 3a). The absence of CCR5 may cause physiopathological differences relevant to this tumor type. However, the reason for this discrepancy is unknown. The continuing similarity of CCL5-EL-4 and control EL-4 tumor growth shows that the antitumor effect of CCL5 requires CCR5 expression. To confirm the role of CCR5-expressing immunocompetent cells in CCL5-mediated tumor delay, we conducted adoptive transfer experiments from C57BL/6 and CCR5 KO mice (Fig. 3b). When CCR5⫹ splenocytes were adoptively transferred into CCR5 KO mice inoculated with CCL5-EL4 tumor cells, the CCL5-mediated delay of tumor growth was restored. These data strengthen the evidence of the crucial role of CCR5-expressing immune cells in CCL5-mediated antitumor growth. To determine whether immune effector lymphocytes were involved in the inhibition of the solid CCL5-expressing tumor growth, we injected CCL5-expressing EL-4 and EG-7 cells into RAG-2 KO mice, which lack T and B lymphocytes (Fig. 3b). Interestingly, CCL5’s protective effect disappeared completely. To test whether plasmid remained in the tumor cells several days after injection and could express CCL5, we then removed solid tumors from three mice and analyzed CCL5 expression with flow cytometry (Fig. 3c). CCL5-EL-4 cells recovered from the tumor mass at day 25 after tumor inoculation still expressed CCL5 levels similar to those expressed at day 0, before injection. This finding indicates that CCL5 expression remains stable in RAG-2 KO mice and thus rules out the loss of in vivo CCL5 expression as an explanation for the disappearance of the protective effect (Fig. 3c). Taken together, these results suggest that CCL5’s antitumor effect depends on immunocompetent host lymphocytes and thus that lymphocytes that express CCR5 may play a crucial role in this CCL5-mediated tumor growth delay.

Tumor-secreted CCL5 induces recruitment of CD4, CD8, and NK cells, and DC We investigated the cells recruited into the tumors and the ipsilateral lymph nodes. Organs and tumors were harvested between days 10 – 14, and the cell contents were analyzed by flow cytometry with mAbs directed against CD3, CD4, CD8, and NK cells. Typically, lymph nodes from control mice contained 2 ⫾ 0.2% CD49b⫹CD3⫺ NK cells, 15 ⫾ 1.6% CD4⫹CD3⫹ cells, and 13.6 ⫾ 1.5% CD8⫹CD3⫹ T cells (Fig. 4a). T and NK cell levels in the lymph nodes did not differ in the mice injected with control EL4 and those receiving CCL5-EL-4 (Fig. 4a). In contrast, representative flow cytometric analyses of the lymphocytes infiltrating the tumor site showed increased levels of NK (Fig. 4b, left panels) and T (right panels) cell infiltration. As Fig. 4c shows, lymphocyte-associated tumor cell content at the tumor site in the mice injected with CCL5-EL-4 tumor cells was composed of 26 ⫾ 5% DX5⫹CD3⫺ cells, 13 ⫾ 2% CD4⫹CD3⫹ cells, and 18 ⫾ 2% CD8⫹CD3⫹ cells ( p ⬍ 0.005), compared with 5 ⫾ 2% DX5⫹CD3⫺ cells, 7 ⫾ 1.5% CD4⫹CD3⫹ cells, and 4 ⫾ 1% CD8⫹CD3⫹ cells for the control EL-4-injected mice. We also observed a significant increase in DC migration into the tumor site in mice inoculated with CCL5-EL-4 tumor cells (2.4 ⫾ 0.4%) compared with control tumor cells (0.6 ⫾ 0.3%; p ⬍ 0.005; n ⫽ 10). Thus, the CCL5-producing tumors contained significantly more NK, CD8, and CD4 cells, and DC, than did the control tumors. To test whether the cytotoxic potencies of these recruited cells were involved in the CCL5-mediated antitumor effect, we conducted similar experiments in perforin KO mice. As shown in Fig. 4e, we found only a partial loss of the antitumor effect (65–70% tumor regression in C57BL/6 mice compared with 40% in the perforin KO mice). We also noted that the cytolytic function of NK and CD8 cells did not differ in the cells isolated from mice with control EL-4 and CCL5-EL-4 tumors (data not shown). These results suggest that increased lymphocytes recruited to the tumor site must have some cytotoxic potency.


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FIGURE 4. Local increased secretion of CCL5 induces recruitment of T and NK cells, and DC, to the tumor site. Mice were sacrificed at different days after tumor inoculation and paired for tumor volume (1.5–2.5 cm3). Distal lymph nodes (a) and tumors (b– d) were surgically removed, and single-cell suspensions were prepared. Forward-scatter and side-scatter parameters determined the tumor cells excluded from the analysis. NK, CD4, and CD8 cells, and DC, were estimated in mice with tumors of similar size (⬃1.5–2.5 cm3). Representative flow cytometric analyses are shown in b. Percentages of cells are indicated in each quadrant. a, c, and d, Percentage ⫾ SEM was indicated for each group (f, control, n ⫽ 10; 䡺, CCL5, n ⫽ 10) and obtained from two independent experiments. e, Perforin KO mice were injected s.c. with CCL5-EL-4 (䡺) or Mock-EL-4 (f). Tumor volumes were measured and expressed in cubic centimeters. All mice developed a palpable tumor. Each data point represents the mean tumor volume ⫾ SEM of six mice. ⴱ, Significant value of p ⬍ 0.05. ⴱⴱ, Significant value of p ⬍ 0.005.

Functional characterization of CCL5-Ig chimeric molecule It is nonetheless difficult to use transfected tumor cells in cancer therapy. For that reason, we generated a DNA construct that coded for a sequence fusing CCL5’s chemokine domain with an Ig N-terminal domain, called pVRC-CCL5-Ig. We chose to fuse CCL5 to the Ig Fc domain, thereby extending the period of cytokine efficacy by the Ig half-life (41). We then investigated whether this new chimeric molecule maintained its receptor specificity and functions. The chimeric CCL5-Ig was first tested in a conventional binding assay with CCR5-transfected HEK cells (Fig. 5a). CCL5-Ig potently competed with receptor-bound radiolabeled CCL4 and showed 1–2 log less affinity for CCR5 than did rCCL5. CCL5-Ig was CCR5 specific and did not interact with other chemokine receptors tested, including CX3CR1, CCR2, or CXCR2 (data not shown). To test the functional potency of CCL5-Ig further, we compared CCL5 and CCL5-Ig in chemotaxis assays with human PBMC. Cells responded to both ligands dose dependently, according to the expected bell curve. Maximal responses were observed at ⬃1 nM for CCL5 and ⬃10 nM for CCL5-Ig (Fig. 5b). Although CCL5-Ig had less affinity and sensitivity than CCL5, it remained CCR5 specific and fully functional. Intratumoral DNA injections of chimeric CCL5-Ig provide strong antitumoral activity We then determined the in vivo antitumor activity of the CCL5-Ig by injecting plasmid coding for it or empty vector into growing tumors at day 5 after tumor inoculation (Fig. 6). Because in vivo transfection of naked DNA provides inefficient protein expression, DNA associated with polyethylenimine (PEI) particles was injected in vivo. Because changes in the stability and size of DNA/ PEI particles that rely in part on glucose concentration have proved to affect the efficiency of in vivo gene transfer (42), we chose to study tumor growth after injection of DNA/PEI complexes in two different formulations, with high (8%) and low (5%) glucose levels (Fig. 6). We found that both formulations induced a significant decrease in tumor size in all mice injected with pVRC-CCL5-Ig compared with the mice receiving the control plasmid (Fig. 6).

Tumor reduction was more efficient at high (⬃80% tumor size reduction at day 17) than at low (⬃50%) glucose concentrations. Glucose concentration had no effect on tumor growth after control plasmid injections (Fig. 6). This delayed tumor growth observed after CCL5-Ig DNA/PEI injection was lost in RAG-2 KO mice (data not shown). Thus, intratumoral injection of CCL5-Ig DNA induced effective in vivo gene therapy. Further assessment of CCL5-Ig gene expression in tumors began with RT-PCR analyses of different-sized tumors from several mice after injection of DNA coding for either Ig or CCL5-Ig in the high-glucose DNA/PEI formulation on day 4 after tumor inoculation (Fig. 6b). No CCL5-Ig transcripts were detected in the tumors injected with the Ig-coding DNA (Fig. 7a, upper panel). Small tumors (defined as those ⬍0.6 cm3) were always associated with a higher expression of CCL5-Ig transcripts (CCL5-Ig/HPRT ratio ⫽ 2–2.3) than were large tumors (⬎1.3 cm3; CCL5-Ig/HPRT ratio ⫽ 1.3–1.5) (Fig. 7a, lower panel). To confirm the quantitative effect of CCL5 on the tumor growth rate, we performed quantitative RT-PCR analyses of different-sized tumors from 10 mice after injection of DNA coding for either Ig or CCL5-Ig in both glucose DNA/PEI formulations on day 15 after the tumor inoculation. Ratios of CCL5-Ig/HPRT transcripts vs tumor size were shown for 10 mice in Fig. 7b. Again, the smaller tumors were always associated with higher expression of CCL5-Ig transcripts than were the larger tumors, which expressed fewer CCL5-Ig transcripts (tumor range, 1– 6.3 cm3) (Fig. 7a). This significant correlation between a high level of CCL5 mRNA expression and small tumor size ( p ⬍ 0.003) indicates that the level of CCL5 expression determines tumor size.

Discussion We investigated two gene therapy approaches to increase tumorinduced immune response: inoculating mice with CCL5-transfected tumor cells or directly injecting DNA coding for CCL5 fused with Ig into the tumor. Reduced tumor growth and incidence were observed in mice with syngeneic EL-4 or OVA-expressing EG-7 lymphomas transfected with CCL5 compared with the parental cell lines. Strong antitumor effects were observed soon after

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FIGURE 5. CCL5-Ig chimeric protein binds CCR5 and is chemotactic for PBMC. CCL5-Ig chimeric protein purified from Chinese hamster ovary cells was tested for its potency to bind CCR5 (a) and to induce PBMC migration (b), compared with rCCL5. a, The binding properties of chimeric CCL5-Ig and rCCL5 were compared in radio-ligand competition binding assays that used 50,000 CCR5-expressing HEK. Data represent the mean of two experiments run in duplicate and are expressed as percentage ⫾ SD of maximal specific binding. b, Results are expressed as a chemotaxis index representing the number of cells migrating in response to CCL5 or CCL5-Ig relative to the number of cells migrating in the absence of the chemokine. Results are expressed as chemotactic index ⫾ SD from two experiments run in triplicate.

intratumoral injection of the DNA plasmid coding for chimeric CCL5-Ig. Whereas CCL5 expression in EG-7 tumor induce an efficient control of tumor growth in 70% of the mice, CCL5 overexpression only delayed EL-4 tumor growth. Indeed, the expression of an immunogenic Ag in EG-7 induced activation of T cells that participate in the control of tumor growth (34). We also showed that the amount of CCL5 expressed at the tumor site determined the effectiveness of the antitumor response. Solid EL-4 and EG-7 tumors also express CCL5 mRNA (data not shown), but this in vivo expression seems insufficient to cause tumor regression of the strength observed in our model. A previous

FIGURE 7. Quantitative RT-PCR expression of CCL5 at the tumor site. a, Various tumors injected with either Ig or CCL5-Ig DNA plasmids were removed, and RT-PCR specific for CCL5-Ig and HPRT was performed. b, Quantitative RT-PCR was conducted with a predeveloped TaqMan assay.


FIGURE 6. Chimeric CCL5-Ig-coding DNA generates strong antitumor activity. a, Five days after EL-4 tumor engraftment, plasmid coding for chimeric CCL5-Ig and control Ig (10 ␮g) were injected at the tumor site in two conditions: DNA/PEI complexes were prepared with low (5%; ƒ) and high (8%; f and 䡺) glucose formulations (n ⫽ 5 mice in each group). Tumor volumes were measured and expressed in cubic centimeters. Each data point represents the mean tumor volume ⫾ SEM. ⴱ, Significant value of p ⬍ 0.05 (CCL5-Ig DNA-injected mice were compared with control Ig DNA-injected mice for each data point).

study reported that fibrosarcomas expressing CCL5 grow at a reduced rate, although the mechanism remains unknown (24), and another reported that adenoviruses expressing a combination of Ag and various chemokines such as CCL5 and CCL4 can induce tumor rejection (13). We thus confirmed in this study the general capacity of CCL5 to inhibit immunogenic tumor growth, and we extended this work to specify some of the cells involved in this phenomenon, thereby moving closer to the development of therapeutic approaches. CCL5’s antitumor effect depends on the expression of CCR5 by immunocompetent host cells. We demonstrated that CCL5 directly affects immune cell trafficking, and that tumor growth is controlled by recruitment of such immunocompetent host cells as T and NK cells, and DC. In addition, we (26) recently reported that another chemokine, CX3CL1, mediates antitumor effects through innate response (NK cells, but not T and B cells, express CX3CR1, whereas CCR5 is expressed on T/NK cells and DC). The antitumor effect was maintained in RAG-2 KO mice deficient for T/B cells but not in NIH III mice deficient for NK/T/B

The Journal of Immunology cells. We showed that CX3CL1-expressing EL-4 and EG-7 cells reduced tumor growth by increasing NK cell recruitment, but we observed no increase in T cell recruitment. Thus, NK cells are involved in the control of EL-4 and EG-7 tumor cells in vivo. For tumors expressing CCL5, delayed tumor growth appears to be mediated principally through the adaptive immune response, but the increased NK cell recruitment in these tumors indicates that innate immune response may also be involved. Similar increases in T, NK, and DC migration were obtained after injection of CCL5-Ig DNA at the tumor site compared with control DNA injection. Together, these results are concordant with findings that CCR5 is expressed on NK and T effector cells (12, 27, 28). We observed no other modifications in cell types at the tumor site. Similar experiments in which CCL5-EL-4 cells were injected into perforin KO mice showed loss of antitumor effect, but only partial (tumor size reduced by 50% in perforin KO mice at day 18 compared with 70% in C57BL/6 mice). These results suggest that CCL5-mediated delay in tumor growth involves in part perforinmediated cytotoxicity and depends on a local increase in the number of CTLs, such as NK, CD8, CD4 Th cells, and DC. Several investigations have attempted to prevent tumor development by creating a microenvironment that promotes innate and acquired immune mechanisms through the introduction of cytokine and chemokine genes into tumor cells (4 – 8, 15). Although one report showed CCL5 to reduce fibrosarcoma growth (24), elevated levels of autologous CCL5 production have been correlated with advanced breast carcinoma (21) and with massive monocyte recruitment there (20). Accordingly, the effect of CCL5 overexpression may be specific to the type of tumor and the type of antitumor immune response. Several chemokines have been shown to be promising anticancer therapeutic tools: CCL19 and CCL21, which bind to CCR7, mediate tumor rejection by recruiting effector T cells (15, 17), and elicit tumor immunity by inducing local T cells and DC accumulation (15, 43). In our model, the protective effect of CCL5 appeared to be mediated through increased recruitment of immunocompetent cells, specifically, NK and T cells. We also found that intratumoral injection of DNA that codes for CCL5-Ig induced a potent antitumor effect that depended on the intensity of CCL5-Ig expression in vivo. This study used several different methods to demonstrate that increasing the in vivo concentration of CCL5, either in stable transfectants or after injection with DNA coding for CCL5-Ig, decreased tumor growth. Quantitative RT-PCR after CCL5 DNA injection demonstrated a significant correlation between high levels of CCL5 mRNA expression and small tumor size ( p ⬍ 0.0001). These data are consistent with the CCL5-induced delay in EG-7 tumor growth where a high level of CCL5 expression in this cell line appeared to prevent EG-7 growth in 70% of the mice. Taken together, these data support the use of DNA coding for chimeric CCL5-Ig as a potential strategy for reducing the size of solid encapsulated tumors. The use of intratumoral CX3CL1-Ig DNA injection for reducing tumor growth in vivo, has been recently described by our group (26). The large panel of chemokines with potent antitumor activity together with recent advances in protein engineering may thus lead to therapeutic tools that induce both humoral and cellular immune responses and may be very effective in the treatment of cancer. These findings provide the first evidence of the potential use of a chimeric CCL5-Ig as anticancer therapy.

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