Increases Tumor Cell Infiltration Overexpression Delays Tumor

Intratumoral CC Chemokine Ligand 5 Overexpression Delays Tumor Growth and Increases Tumor Cell Infiltration

This information is current as Elise Lavergne, Christophe Combadière, Mutsunori Iga, of September 24, 2021. Alexandre Boissonnas, Olivia Bonduelle, Maud Maho, Patrice Debré and Behazine Combadiere J Immunol 2004; 173:3755-3762; ; doi: 10.4049/jimmunol.173.6.3755

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 Downloaded from 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: 3755Ð3762. http://www.jimmunol.org/ ne possible new approach to anticancer therapies in- diated gene transfer of CCL3 induces dendritic cells (DC)4 to ac- volves the redistribution and activation of immunocom- cumulate at the tumor site (10, 11). Modifications of chemokine O petent cells into the vicinity of developing tumors. Che- genes, including CCL1 (12), CCL3 (4), CCL5 (13), CCL19 (14), mokines may be good candidates for this task (1). They are CCL20 (15), CCL21 (16, 17), CX3CL1 (18, 19), and XCL1 (12), proinflammatory cytokines that mainly signal through a family of activate tumor-specific and nonspecific immunity: they increase G protein-coupled receptors to coordinate the deployment and ac- local recruitment of monocytes/macrophages, DC, and T cells to tivation of leukocytes at injury sites (2). The chemokine super- the tumor site in melanoma; in ovarian, breast, and cervical car- cinomas (20, 21); in adenocarcinoma (22, 23); and in sarcoma, family is divided into four subgroups (CC, CXC, CX3C, and C) by guest on September 24, 2021 according to the number and spacing of the first two closely paired fibrosarcoma, and glioma (24, 25). We recently reported that increased tumor expression of either CX3CL1 or chimeric and highly conserved cysteines of the N-terminal domain (3). CX3CL1-Ig induces antitumor activity via NK cell recruitment Many CC and CXC chemokines have been tested for their antitu- (26). The role of chemokines in tumor growth nonetheless remains mor effect. Although some chemokines promote cancer metastasis complex and requires further investigation. by regulating the spread of tumor cells (4, 5) or promote tumor The immune system’s rejection of cancer cells results from a growth by modulating neovascularization (6Ð8), others may favor series of events that include T lymphocyte activation, expansion, adaptive antitumor immune responses by recruiting subsets of leu- and infiltration of the tumor, and efficient cytotoxic effector func- kocytes. Animal experiments (9) and clinical reports indicate that tioning. Several CC chemokines are known to attract naive and chemokines may help the immune system to recognize and to kill effector T cells. Because receptors for CCL5 are expressed on tumor cells: more specifically, CCL2 reduces tumor growth by effector Th1 CD4, CD8, and NK cells, and DC (12, 27Ð31), and increasing monocyte/macrophage infiltration, and adenovirus-me- 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 Laboratoire d’Immunologie Cellulaire, Institut National de la Sante«et de la Recherche sites after Ag priming in secondary lymphoid organs (32Ð34). Me«dicale, Unite«543, Hoˆpital Pitie«-Salpeˆtrie`re, Paris, France Whereas naive cells are CCR5ϪCCR7ϩ, effector T cells are ϩ Ϫ Received for publication December 5, 2003. Accepted for publication July 2, 2004. CCR5 CCR7 after Ag-induced cell differentiation, and they mi- The costs of publication of this article were defrayed in part by the payment of page grate out of the lymphoid organs in response to CCL5 (33). In charges. This article must therefore be hereby marked advertisement in accordance addition, CCL5 is a chemoattractant for memory T lymphocytes in with 18 U.S.C. Section 1734 solely to indicate this fact. vitro (35). Together with CCL2 and CCL3, it affects the magnitude 1 E.L. and A.B. were recipients of the fellowship from the French Ministry of Re- search and Technology and La Fondation pour la Recherche Me«dicale. M.I. was a and polarity of the T cell response (28). It has also been suggested recipient from Objectifs Recherche de Vaccin SIDA. This work was supported by that a cell-mediated Th1 type response is promoted when T cell grants from Association pour la Recherche sur le Cancer and from La Ligue Nationale activation sites contain large quantities of CCL3, CCL4, and Contre le Cancer. CCL5 (28). 2 E. L. and C.C. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Behazine Combadiere, Labo- ratoire d’Immunologie Cellulaire, Institut National de la Sante« et de la Recherche Me«dicale, Unite« 543, Faculte«deMe«decine Pitie«-Salpe«trie`re, 91 Boulevard de 4 Abbreviations used in this paper: DC, dendritic cell; KO, knockout; HEK, human l’Hoˆpital, 75013 Paris, France. E-mail address: [email protected] embryonic kidney; PEI, polyethylenimine.

In this study, we examined whether forcing tumor cells to pro- establishment required injection of at least 200,000 EL-4 cells (data not duce CCL5 might promote antitumor immune response and T cell shown). attraction at the tumor site. We studied the quantitative and qual- To evaluate the cells recruited to the tumor site and to detect CCL5 expression, the tumor mass was surgically removed between days 10Ð14, itative effects of CCL5 production on tumor growth and immune as indicated. The tumor and proximal lymph nodes were dilacerated and cell recruitment. We approached this question by investigating two passed through a 70-␮m cell strainer (BD Biosciences, San Diego, CA). different gene therapy strategies: inoculating mice with CCL5- Lymphocytes were collected and washed in PBS-2% FCS buffer. Red cells transfected tumor cells and directly injecting DNA coding for 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 CCL5 fused with Ig into the tumor. analyzed after immunoﬂuorescence staining.

Materials and Methods RT-PCR analyses Mice Intratumoral mRNA expression of CCL5-Ig was detected by RT-PCR. To- Wild-type C57BL/6 females were obtained from Elevage Janvier (Le Gen- tal RNA was extracted from each tumor with the QIAamp RNA Blood est, Saint Isle, France). C57BL/10 RAG-2 knockout (KO), C57BL/6 per- Mini kit (Qiagen, Courtaboeuf, France), and cDNA was generated with forin KO, and C57BL/6 CCR5 KO mice were purchased from Charles Promega’s reverse transcription system. PCR amplification took place un- River (Saint-Aubin les Elbeufs, France) and bred in our animal facility der the following conditions: 94¡C for 3 min, followed by 35 cycles of (Nouvelle Animalerie Commune of Pitie«-Salpeˆtrie`re, Paris, France). All 94¡C for 30 s, 56¡C for 30 s, and 72¡C for 20 s. Primer sequences for mice were housed under specific pathogen-free conditions and used for CCL5-Ig detection were as follows: forward primer, 5Ј-GTG CCC ACA experiments at 6Ð10 wk of age. All experiments complied with local an- TCA AGG AGT AT-3Ј, and reverse primer, 5Ј-GCT GTG TGT ACT TCC imal experimentation and ethics committee guidelines. ACG TT-3Ј. Control HPRT primers were as follows: forward primer, 5Ј- CTT TGC TGA CCT GCT GGA TT-3Ј, and reverse primer, 5Ј-TAT GTC Downloaded from Tumor cell lines CCC CGT TGA CTG AT-3Ј. For the quantitative RT-PCR, we used PreDeveloped TaqMan Assay All cell lines used in this study were obtained from the American Type Reagents (primers and probes) for detection of human CCL5 and the Ap- Culture Collection (Manassas, VA) and were derived from C57BL/6 (H- plied Biosystems TaqMan Master Mix (with uracyl-N-glycosylase) accord- b 2 ) mice. The dimethylbenzanthracene-induced thymoma, EL-4, and its ing to the manufacturer’s instructions (Applied Biosystems, Cheshire, chicken OVA peptide-expressing derivative, EG-7, were maintained in U.K.). Control HPRT real-time PCR was used to standardize the results. RPMI 1640 (Invitrogen Life Technologies, Paisley, Scotland) supple- mented with 10% heat-inactivated FCS (Seromed, Berlin, Germany), 2 http://www.jimmunol.org/ Flow cytometry mM L-glutamine, 1000 U/ml penicillin, 1 mg/ml streptomycin, 250 ng/ml ␮ amphotericin B (Invitrogen Life Technologies), and 3 M 22-ME (Sigma- Cell surface Ags were characterized with a standard staining method and Aldrich, St. Louis, MO). We used Transfast (Promega, Madison, WI), in the following mAbs: FITC-conjugated anti-mouse CD3 (clone 145-2C11), accordance with the manufacturer’s instructions, to transfect EL-4 and PE-conjugated pan-NK (clone DX5), PerCP/Cy5.5-conjugated anti-mouse EG-7 cell lines with pBlast-human CCL5 plasmid (InvivoGen, Toulouse, CD8 (clone Ly-2), biotin-conjugated anti-mouse CD4 (clone L3T4) plus ␮ France) and 10 g/ml blasticidin (InvivoGen) to select transfectants, which allophycocyanin-streptavidin, PE-conjugated anti-H-2Kb (clone AF6-88.5), ␮ were then maintained with 5 g/ml blasticidin. Parental cell lines trans- PE-conjugated anti-mouse CD49 (BD Pharmingen, Le Pont de Claix, fected with the empty plasmid pBlast were used as control tumor cell lines. France), and PE-conjugated anti-human CCL5 (clone 2D5; R&D Systems, We verified with flow cytometry that neither EL-4 nor EG-7 transfection Abingdon, U.K.).

affected MHC class I molecules, nor did they induce expression of co- For intracellular CCL5 staining, cells were washed and permeabilized by guest on September 24, 2021 stimulatory molecules such as CD70 and CD80 (data not shown). Prolif- with 1ϫ PBS-5% FCS-0.1% saponin before intracellular staining with PE- eration assays measuring tritiated thymidine incorporation showed no sig- conjugated anti-human CCL5 (clone 2D5). nificant differences in tumor cell growth in vitro between the transfected The cells were incubated with appropriate fluorochrome-conjugated and control lines (data not shown). mAbs for 20 min at room temperature, and then washed in 1ϫ PBS and fixed for 15 min at room temperature in 1 ml of 4% paraformaldehyde. Chimeric CCL5-Ig constructs Cells were run for four-color fluorescence staining on a cytofluorometer PstI-tailed forward primer AAA ACT GCA GAT GAA GGT CTC CGC (FACSCalibur; BD Biosciences), and 10,000 live events were analyzed GGC A and NotI-tailed reverse primer ATA GGC GGC CGC GCT CAT with ProCellQuest software. CTC CAA AGA GTT were used to amplify the signal sequence and chemokine domain of CCL5 corresponding to aa 1Ð91 (enzyme recognition Chemokine binding assay

sequences are underlined). The modified mouse IgG2a Fc domain, corre- 125 sponding to aa 97Ð329 and derived from pVRC murine IL-2/Ig (36), a Binding assays were performed with I-labeled CCL4 (Amersham Bio- ϫ 4 generous gift from Dr. D. Barouch (Division of Viral Pathogenesis, Beth sciences, Piscataway, NJ) in duplicate with 5 10 CCR5-expressing Israel Deaconess Medical Center, Harvard Medical School, Boston, MA), human embryonic kidney (HEK) cells, as previously described (37). Briefly, cells were incubated in a total volume of 200 ␮l of PBS containing was amplified with NotI-tailed primer ATA AGC GGC CGC ACA TCC 125 CAG AGG GCC CAC AAT C and BglII-tailed primer GGA AGA TCT 1 mg/ml BSA and 0.01% azide (pH 7.4) with 50 pM I-labeled CCL4 and TCA TTT ACC CGG AGG CCG GGA GAA. Amplification reactions increasing concentrations of unlabeled human CCL5 (PeproTech, Rocky were performed in standard conditions with1UofPfuDNApolymerase Hill, NJ) or CCL5-Ig. After2hat37¡C, unbound chemokines were sep- (Stratagene, La Jolla, CA). PCR cycling began at 95¡C for 5 min, followed arated from cells by centrifugation in 1 ml of PBS with 10% sucrose. by 20 cycles of 95¡C for 30 s, 55¡C for 30 s, and 72¡C for 1 min, and ended Gamma emissions were then counted in the cell pellet (1272 Clinigama; with 10 min at 72¡C. PstI-NotI CCL5 and NotI-BglII Ig fragments were LKB Wallac, Saint Quentin en Yvelines, France). directly subcloned into a pVRC plasmid cloning site. Low endotoxin plasmid was prepared on a large scale (Tebu Biolaboratories, Le Perray en In vitro chemotaxis assay Yvelines, France). Flow cytometric analysis, ELISA, and immunoblotting with the cell lysate and supernatant of Chinese hamster ovary cells trans- Chemotaxis was assayed in a 96-well chemotaxis chamber (NeuroProbe, fected with this construct all confirmed the expression of the chimeric Cabin John, MD). PBMC were labeled for 30 min at 37¡C with 5-chlo- protein, also produced and purified by Tebu Biolaboratories. romethylfluorescein diacetate (Molecular Probes, Leiden, The Nether- lands) in RPMI 1640 and resuspended in HBSS-BSA (106 cells/ml). Hu- In vivo experiments man rCCL5 (PeproTech) and CCL5-Ig were placed in the lower chamber. We then seeded 50,000 human PBL onto the membrane (5-␮m pore di- Five to 10 mice per group were injected s.c. in the right flank with 200,000 ameters) and incubated the 96-well plate for3hat37¡C, 100% humidity, ␮ tumor cells in 100 l of PBS. Tumor size was measured with a caliper three and 5% CO2. The filter top surface was rinsed with PBS, and the plate was times a week, and tumor volume was estimated with the following formula: centrifuged for 2 min at 1500 rpm. Fluorescence was measured with a width ϫ length ϫ (width ϩ length)/2. Mice were sacrificed when tumor Packard Fusion microplate analyzer (Perkin-Elmer Life Sciences, Boston, volume reached ϳ12 cm3. Five days after tumor inoculation, in vivo JetPEI MA). Results are expressed as a chemotaxis index that represents the num- transfecting reagent (Qbiogene, Illkirch, France) was used to inject 10 ␮g ber of cells migrating in response to CCL5 or CCL5-Ig relative to the of CCL5-Ig DNA plasmid or control DNA at the tumor site. Reliable tumor number migrating in the absence of the chemokine. The Journal of Immunology 3757

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 (Ⅺ). 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 Ϯ SEM of 16 mice. The CCL5-EL-4 tumor volumes were 65Ð70% of control EL-4 tumors, be- -Sig ,ء .tween days 20Ð25 after tumor inoculation nificant value of p Ͻ 0.05 (control mice with tumor size of Ͼ10 cm3 were euthanized). Downloaded from thereafter, all CCL5-EL-4 tumors ;(ء ,Statistical analysis mors until day 23 (Fig. 1b We used Prism 2.01 (GraphPad Software, San Diego, CA) for data han- continued to grow at similar rates. dling, analysis, and graphic representation. Statistical analysis used the In a second model, we used a more immunogenic tumor, EG-7 paired two-sample t test for means and the nonparametric Mann-Whitney cell lines that expressed chicken OVA stably transfected with U test. Statistical significance was set at p Ͻ 0.05. CCL5. Two CCL5-EG-7 cell lines that expressed high (CCL5high- http://www.jimmunol.org/ EG-7) or low (CCL5low-EG-7) levels of CCL5 were selected (Fig. Results 2a). All control mice developed a solid tumor within 10 days of CCL5 inhibits syngeneic tumor development inoculation. Mice injected with CCL5low-EG-7 required 25 days The H-2b lymphoma EL-4 was transfected with plasmid encoding for tumor development (Fig. 2b). Of those injected with CCL5high- full-length CCL5 or an empty control vector. Chemokine expres- EG-7, only 30% developed a tumor by day 30, and 70% remained sion was confirmed in the CCL5-EL-4 cell lines with intracellular tumor free. Thus, these data underline the effectiveness of CCL5 in staining (Fig. 1a). In addition, ELISA showed CCL5 in the super- controlling EG-7 tumor growth, although CCL5 overexpression ϳ natant of transfected cells, at a maximum level of 8nMbyday only delayed EL-4 tumor growth. by guest on September 24, 2021 4 of cell culture (data not shown). We assessed the ability of CCL5-transfected tumor cell lines to form solid tumors in CCL5-mediated antitumor activity was lost in CCR5 KO and C57BL/6 mice. In the conditions described above, all the mice RAG-2 KO mice (100%) developed a solid tumor that grew progressively under We next ascertained the receptor primarily involved in the antitu- their skin (Fig. 1b): it was measurable by day 7 and grew over a mor properties of CCL5 in our model. CCL5 binds and activates at period of 3 wk. Growth was significantly slower in the in vivo- least three chemokine receptors, namely, CCR1 (35, 38), CCR3 transfected CCL5-EL-4 tumors than in control tumors. The mean (39), and CCR5 (40). Given that the latter is the CCL5 receptor volume of CCL5-EL-4 tumors was ϳ65Ð70% smaller than that of primarily expressed on lymphoid cells, we tested its protective the control tumors between days 20Ð25, before the control EL-4- effect in CCR5 KO mice. The total loss of this protective effect in inoculated mice were euthanized (Fig. 1b). Tumors that expressed this strain demonstrates that the antitumor effect depends on CCR5 CCL5 remained significantly smaller ( p Ͻ 0.05) than control tu- (Fig. 3a). These results further suggest that CCR1 and CCR3 are

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; Ⅺ, CCL5high). The number of mice without palpable tumors was indicated as a percentage of total mice injected (n ϭ 8). In addition, tumor size remained signiﬁcantly lower in CCL5-EG-7-injected mice than in Mock-EG-7-injected mice (data not shown). Sev- enty percent of mice that were inoculated with CCL5- EG-7 never developed any measurable tumors as long as the animals were kept in our facility (Ͼ60 days), whereas mice inoculated with control EG-7 developed tumors. 3758 CCL5-Ig ANTITUMOR GENE THERAPY

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 (Ⅺ) 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 Ϯ 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 Downloaded from expression at day 0 before tumor inoculation (left panel). Results are representative of CCL5 expres- -Signif ,ء .sion in tumors harvested from three mice icant value of p Ͻ 0.05. http://www.jimmunol.org/

not key players in this phenomenon. We also observed that the Tumor-secreted CCL5 induces recruitment of CD4, CD8, and control EL-4 tumors were smaller in CCR5 KO than in control NK cells, and DC mice (Fig. 3a). The absence of CCR5 may cause physiopatholog- We investigated the cells recruited into the tumors and the ipsilateral by guest on September 24, 2021 ical differences relevant to this tumor type. However, the reason lymph nodes. Organs and tumors were harvested between days 10Ð for this discrepancy is unknown. The continuing similarity of 14, and the cell contents were analyzed by flow cytometry with mAbs CCL5-EL-4 and control EL-4 tumor growth shows that the anti- directed against CD3, CD4, CD8, and NK cells. Typically, lymph tumor effect of CCL5 requires CCR5 expression. To confirm the nodes from control mice contained 2 Ϯ 0.2% CD49bϩCD3Ϫ NK role of CCR5-expressing immunocompetent cells in CCL5-medi- cells, 15 Ϯ 1.6% CD4ϩCD3ϩ cells, and 13.6 Ϯ 1.5% CD8ϩCD3ϩ T ated tumor delay, we conducted adoptive transfer experiments ϩ cells (Fig. 4a). T and NK cell levels in the lymph nodes did not differ from C57BL/6 and CCR5 KO mice (Fig. 3b). When CCR5 in the mice injected with control EL4 and those receiving CCL5-EL-4 splenocytes were adoptively transferred into CCR5 KO mice in- (Fig. 4a). In contrast, representative flow cytometric analyses of the oculated with CCL5-EL4 tumor cells, the CCL5-mediated delay of lymphocytes infiltrating the tumor site showed increased levels of NK tumor growth was restored. These data strengthen the evidence of (Fig. 4b, left panels)andT(right panels) cell infiltration. As Fig. 4c the crucial role of CCR5-expressing immune cells in CCL5-me- shows, lymphocyte-associated tumor cell content at the tumor site in diated antitumor growth. the mice injected with CCL5-EL-4 tumor cells was composed of To determine whether immune effector lymphocytes were in- 26 Ϯ 5% DX5ϩCD3Ϫ cells, 13 Ϯ 2% CD4ϩCD3ϩ cells, and 18 Ϯ volved in the inhibition of the solid CCL5-expressing tumor 2% CD8ϩCD3ϩ cells ( p Ͻ 0.005), compared with 5 Ϯ 2% growth, we injected CCL5-expressing EL-4 and EG-7 cells into DX5ϩCD3Ϫ cells, 7 Ϯ 1.5% CD4ϩCD3ϩ cells, and 4 Ϯ 1% RAG-2 KO mice, which lack T and B lymphocytes (Fig. 3b). CD8ϩCD3ϩ cells for the control EL-4-injected mice. We also ob- Interestingly, CCL5’s protective effect disappeared completely. To served a significant increase in DC migration into the tumor site in test whether plasmid remained in the tumor cells several days after mice inoculated with CCL5-EL-4 tumor cells (2.4 Ϯ 0.4%) compared injection and could express CCL5, we then removed solid tumors with control tumor cells (0.6 Ϯ 0.3%; p Ͻ 0.005; n ϭ 10). Thus, the from three mice and analyzed CCL5 expression with flow cytom- CCL5-producing tumors contained significantly more NK, CD8, and etry (Fig. 3c). CCL5-EL-4 cells recovered from the tumor mass at CD4 cells, and DC, than did the control tumors. day 25 after tumor inoculation still expressed CCL5 levels similar To test whether the cytotoxic potencies of these recruited cells were to those expressed at day 0, before injection. This finding indicates involved in the CCL5-mediated antitumor effect, we conducted sim- that CCL5 expression remains stable in RAG-2 KO mice and thus ilar experiments in perforin KO mice. As shown in Fig. 4e, we found rules out the loss of in vivo CCL5 expression as an explanation for only a partial loss of the antitumor effect (65Ð70% tumor regression in the disappearance of the protective effect (Fig. 3c). Taken together, C57BL/6 mice compared with 40% in the perforin KO mice). We also these results suggest that CCL5’s antitumor effect depends on im- noted that the cytolytic function of NK and CD8 cells did not differ in munocompetent host lymphocytes and thus that lymphocytes that the cells isolated from mice with control EL-4 and CCL5-EL-4 tu- express CCR5 may play a crucial role in this CCL5-mediated tumors (data not shown). These results suggest that increased lympho- mor growth delay. cytes recruited to the tumor site must have some cytotoxic potency. The Journal of Immunology 3759

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 (1.5Ð2.5 cm3). Distal lymph nodes (a) and tumors (b–d) were surgically removed, and single-cell suspensions were prepared. Forward-scatter and side-scatter pa- rameters determined the tumor cells excluded from the analysis. NK, CD4, and CD8 cells, and DC, were estimated in mice with tumors of similar size (ϳ1.5Ð2.5 cm3). Representative ﬂow cytometric analyses are shown in b. Percentages of cells are indicated in each quadrant. a, c, and d, Percentage Ϯ SEM was indicated for each group (f, control, n ϭ 10; Ⅺ, CCL5, n ϭ 10) and obtained from two independent experiments. e, Perforin KO mice were injected s.c. with

CCL5-EL-4 (Ⅺ) or Mock-EL-4 (f). Tumor vol- Downloaded from umes were measured and expressed in cubic centimeters. All mice developed a palpable tumor. Each data point represents the mean tumor vol- Signiﬁcant value of ,ء .ume Ϯ SEM of six mice .Signiﬁcant value of p Ͻ 0.005 ,ءء .p Ͻ 0.05 http://www.jimmunol.org/

Functional characterization of CCL5-Ig chimeric molecule Tumor reduction was more efficient at high (ϳ80% tumor size ϳ It is nonetheless difficult to use transfected tumor cells in cancer reduction at day 17) than at low ( 50%) glucose concentrations. therapy. For that reason, we generated a DNA construct that coded for Glucose concentration had no effect on tumor growth after control a sequence fusing CCL5’s chemokine domain with an Ig N-terminal plasmid injections (Fig. 6). This delayed tumor growth observed domain, called pVRC-CCL5-Ig. We chose to fuse CCL5 to the Ig Fc after CCL5-Ig DNA/PEI injection was lost in RAG-2 KO mice domain, thereby extending the period of cytokine efficacy by the Ig (data not shown). Thus, intratumoral injection of CCL5-Ig DNA half-life (41). We then investigated whether this new chimeric mol- induced effective in vivo gene therapy. by guest on September 24, 2021 ecule maintained its receptor specificity and functions. Further assessment of CCL5-Ig gene expression in tumors be- The chimeric CCL5-Ig was first tested in a conventional binding gan with RT-PCR analyses of different-sized tumors from several assay with CCR5-transfected HEK cells (Fig. 5a). CCL5-Ig po- mice after injection of DNA coding for either Ig or CCL5-Ig in the tently competed with receptor-bound radiolabeled CCL4 and high-glucose DNA/PEI formulation on day 4 after tumor inoculation showed 1Ð2 log less affinity for CCR5 than did rCCL5. CCL5-Ig (Fig. 6b). No CCL5-Ig transcripts were detected in the tumors injected was CCR5 specific and did not interact with other chemokine re- with the Ig-coding DNA (Fig. 7a, upper panel). Small tumors (de- Ͻ 3 ceptors tested, including CX3CR1, CCR2, or CXCR2 (data not fined as those 0.6 cm ) were always associated with a higher ex- ϭ shown). To test the functional potency of CCL5-Ig further, we pression of CCL5-Ig transcripts (CCL5-Ig/HPRT ratio 2Ð2.3) than Ͼ 3 ϭ compared CCL5 and CCL5-Ig in chemotaxis assays with human were large tumors ( 1.3 cm ; CCL5-Ig/HPRT ratio 1.3Ð1.5) (Fig. PBMC. Cells responded to both ligands dose dependently, accord- 7a, lower panel). To confirm the quantitative effect of CCL5 on the ing to the expected bell curve. Maximal responses were observed tumor growth rate, we performed quantitative RT-PCR analyses of at ϳ1 nM for CCL5 and ϳ10 nM for CCL5-Ig (Fig. 5b). Although different-sized tumors from 10 mice after injection of DNA coding for CCL5-Ig had less affinity and sensitivity than CCL5, it remained either Ig or CCL5-Ig in both glucose DNA/PEI formulations on day CCR5 specific and fully functional. 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 Intratumoral DNA injections of chimeric CCL5-Ig provide tumors were always associated with higher expression of CCL5-Ig strong antitumoral activity transcripts than were the larger tumors, which expressed fewer CCL5-Ig transcripts (tumor range, 1Ð6.3 cm3) (Fig. 7a). This signif- We then determined the in vivo antitumor activity of the CCL5-Ig icant correlation between a high level of CCL5 mRNA expression and by injecting plasmid coding for it or empty vector into growing small tumor size ( p Ͻ 0.003) indicates that the level of CCL5 ex- tumors at day 5 after tumor inoculation (Fig. 6). Because in vivo pression determines tumor size. 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/ Discussion PEI particles that rely in part on glucose concentration have proved We investigated two gene therapy approaches to increase tumor- to affect the efficiency of in vivo gene transfer (42), we chose to induced immune response: inoculating mice with CCL5-trans- study tumor growth after injection of DNA/PEI complexes in two fected tumor cells or directly injecting DNA coding for CCL5 different formulations, with high (8%) and low (5%) glucose levels fused with Ig into the tumor. Reduced tumor growth and incidence (Fig. 6). We found that both formulations induced a significant were observed in mice with syngeneic EL-4 or OVA-expressing decrease in tumor size in all mice injected with pVRC-CCL5-Ig EG-7 lymphomas transfected with CCL5 compared with the pa- compared with the mice receiving the control plasmid (Fig. 6). rental cell lines. Strong antitumor effects were observed soon after 3760 CCL5-Ig ANTITUMOR GENE THERAPY Downloaded from

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 Ⅺ) glucose formulations (n ϭ 5 mice in each group). FIGURE 5. CCL5-Ig chimeric protein binds CCR5 and is chemotactic Tumor volumes were measured and expressed in cubic centimeters. Each http://www.jimmunol.org/ Significant value ,ء .for PBMC. CCL5-Ig chimeric protein purified from Chinese hamster ovary data point represents the mean tumor volume Ϯ SEM cells was tested for its potency to bind CCR5 (a) and to induce PBMC of p Ͻ 0.05 (CCL5-Ig DNA-injected mice were compared with control Ig migration (b), compared with rCCL5. a, The binding properties of chimeric DNA-injected mice for each data point). CCL5-Ig and rCCL5 were compared in radio-ligand competition binding assays that used 50,000 CCR5-expressing HEK. Data represent the mean Ϯ of two experiments run in duplicate and are expressed as percentage SD study reported that fibrosarcomas expressing CCL5 grow at a re- of maximal specific binding. b, Results are expressed as a chemotaxis duced rate, although the mechanism remains unknown (24), and 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 another reported that adenoviruses expressing a combination of Ag Ϯ and various chemokines such as CCL5 and CCL4 can induce tu- chemokine. Results are expressed as chemotactic index SD from two by guest on September 24, 2021 experiments run in triplicate. mor 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 intratumoral injection of the DNA plasmid coding for chimeric phenomenon, thereby moving closer to the development of thera- CCL5-Ig. Whereas CCL5 expression in EG-7 tumor induce an peutic approaches. CCL5’s antitumor effect depends on the ex- efficient control of tumor growth in 70% of the mice, CCL5 over- pression of CCR5 by immunocompetent host cells. We demon- expression only delayed EL-4 tumor growth. Indeed, the expres- strated that CCL5 directly affects immune cell trafficking, and that sion of an immunogenic Ag in EG-7 induced activation of T cells tumor growth is controlled by recruitment of such immunocom- that participate in the control of tumor growth (34). petent host cells as T and NK cells, and DC. In addition, we (26) We also showed that the amount of CCL5 expressed at the tu- recently reported that another chemokine, CX3CL1, mediates an- mor site determined the effectiveness of the antitumor response. titumor effects through innate response (NK cells, but not T and B Solid EL-4 and EG-7 tumors also express CCL5 mRNA (data not cells, express CX3CR1, whereas CCR5 is expressed on T/NK cells shown), but this in vivo expression seems insufficient to cause and DC). The antitumor effect was maintained in RAG-2 KO mice tumor regression of the strength observed in our model. A previous deficient for T/B cells but not in NIH III mice deficient for NK/T/B

FIGURE 7. Quantitative RT-PCR expression of CCL5 at the tumor site. a, Various tumors injected with either Ig or CCL5-Ig DNA plas- mids were removed, and RT-PCR specific for CCL5-Ig and HPRT was performed. b, Quanti- tative RT-PCR was conducted with a predeveloped TaqMan assay. The Journal of Immunology 3761 cells. We showed that CX3CL1-expressing EL-4 and EG-7 cells 3. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and reduced tumor growth by increasing NK cell recruitment, but we their role in immunity. Immunity 12:121. 4. Menten, P., A. Saccani, C. Dillen, A. Wuyts, S. Struyf, P. Proost, A. Mantovani, observed no increase in T cell recruitment. Thus, NK cells are J. M. Wang, and J. Van Damme. 2002. Role of the autocrine chemokines MIP-1␣ involved in the control of EL-4 and EG-7 tumor cells in vivo. For and MIP-1␤ in the metastatic behavior of murine T cell lymphoma. J. Leukocyte Biol. 72:780. tumors expressing CCL5, delayed tumor growth appears to be me- 5. Muller, A., B. Homey, H. Soto, N. Ge, D. Catron, M. E. Buchanan, diated principally through the adaptive immune response, but the T. McClanahan, E. Murphy, W. Yuan, S. N. Wagner, et al. 2001. Involvement of increased NK cell recruitment in these tumors indicates that innate chemokine receptors in breast cancer metastasis. Nature 410:50. 6. Norgauer, J., B. Metzner, and I. Schraufstatter. 1996. Expression and growth- immune response may also be involved. Similar increases in T, promoting function of the IL-8 receptor ␤ in human melanoma cells. J. Immunol. NK, and DC migration were obtained after injection of CCL5-Ig 156:1132. DNA at the tumor site compared with control DNA injection. To- 7. Owen, J. D., R. Strieter, M. Burdick, H. Haghnegahdar, L. Nanney, R. Shattuck-Brandt, and A. Richmond. 1997. Enhanced tumor-forming capacity gether, these results are concordant with findings that CCR5 is for immortalized melanocytes expressing melanoma growth stimulatory activity/ expressed on NK and T effector cells (12, 27, 28). growth-regulated cytokine ␤ and ␥ proteins. Int. J. Cancer 73:94. We observed no other modifications in cell types at the tumor 8. Moore, B. B., D. A. Arenberg, K. Stoy, T. Morgan, C. L. Addison, S. B. Morris, M. Glass, C. Wilke, Y. Y. Xue, S. Sitterding, et al. 1999. Distinct CXC chemo- site. Similar experiments in which CCL5-EL-4 cells were injected kines mediate tumorigenicity of prostate cancer cells. Am. J. Pathol. 154:1503. into perforin KO mice showed loss of antitumor effect, but only 9. Vicari, A. P., and C. Caux. 2002. Chemokines in cancer. Cytokine Growth Factor Rev. 13:143. partial (tumor size reduced by 50% in perforin KO mice at day 18 10. Ueno, T., M. Toi, H. Saji, M. Muta, H. Bando, K. Kuroi, M. Koike, H. Inadera, compared with 70% in C57BL/6 mice). These results suggest that and K. Matsushima. 2000. Significance of macrophage chemoattractant protein-1 CCL5-mediated delay in tumor growth involves in part perforin- in macrophage recruitment, angiogenesis, and survival in human breast cancer. Clin. Cancer Res. 6:3282. Downloaded from mediated cytotoxicity and depends on a local increase in the num- 11. Rousseau, R. F., A. E. Haight, C. Hirschmann-Jax, E. S. Yvon, D. R. Rill, Z. Mei, ber of CTLs, such as NK, CD8, CD4 Th cells, and DC. S. C. Smith, S. Inman, K. Cooper, P. Alcoser, et al. 2003. Local and systemic Several investigations have attempted to prevent tumor devel- effects of an allogeneic tumor cell vaccine combining transgenic human lympho- tactin with interleukin-2 in patients with advanced or refractory neuroblastoma. opment by creating a microenvironment that promotes innate and Blood 101:1718. acquired immune mechanisms through the introduction of cyto- 12. Robertson, M. J. 2002. Role of chemokines in the biology of natural killer cells. kine and chemokine genes into tumor cells (4Ð8, 15). Although J. Leukocyte Biol. 71:173. 13. Paul, S., E. Regulier, Y. Poitevin, H. Hormann, and R. B. Acres. 2002. The one report showed CCL5 to reduce fibrosarcoma growth (24), el- combination of a chemokine, cytokine and TCR-based T cell stimulus for effec- http://www.jimmunol.org/ evated levels of autologous CCL5 production have been correlated tive gene therapy of cancer. Cancer Immunol. Immunother. 51:645. 14. Braun, S. E., K. Chen, R. G. Foster, C. H. Kim, R. Hromas, M. H. Kaplan, with advanced breast carcinoma (21) and with massive monocyte H. E. Broxmeyer, and K. Cornetta. 2000. The CC chemokine CK ␤-11/MIP-3␤/ recruitment there (20). Accordingly, the effect of CCL5 overex- ELC/Exodus 3 mediates tumor rejection of murine breast cancer cells through pression may be specific to the type of tumor and the type of NK cells. J. Immunol. 164:4025. 15. Eo, S. K., U. Kumaraguru, and B. T. Rouse. 2001. Plasmid DNA encoding CCR7 antitumor immune response. Several chemokines have been shown ligands compensate for dysfunctional CD8ϩ T cell responses by effects on den- to be promising anticancer therapeutic tools: CCL19 and CCL21, dritic cells. J. Immunol. 167:3592. which bind to CCR7, mediate tumor rejection by recruiting effec- 16. Vicari, A. P., S. Ait-Yahia, K. Chemin, A. Mueller, A. Zlotnik, and C. Caux. 2000. Antitumor effects of the mouse chemokine 6Ckine/SLC through angiostatic tor T cells (15, 17), and elicit tumor immunity by inducing local T and immunological mechanisms. J. Immunol. 165:1992. cells and DC accumulation (15, 43). In our model, the protective 17. Sharma, S., M. Stolina, J. Luo, R. M. Strieter, M. Burdick, L. X. Zhu, R. K. Batra, by guest on September 24, 2021 effect of CCL5 appeared to be mediated through increased recruit- and S. M. Dubinett. 2000. 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