1755 Characterization of the CCL21-mediated melanoma-specific immune responses and in situ melanoma eradication Laura Novak, Olga Igoucheva, Stephanie Cho, Introduction and Vitali Alexeev The incidence of cutaneous malignant melanoma is increasing at a faster rate than any other cancer, both Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, globally and in the United States (1). Immunotherapy has Pennsylvania emerged as one of the most promising treatments of this melanocytic malignancy. Various vaccine therapies to achieve immune-based melanoma rejection have been Abstract successfully tested in preclinical animal models and several Previous studies have shown that secondary lymphoid clinical trials. However, clinical studies have shown that chemokine, CCL21, can be used for modulation of tumor- existing therapeutic approaches are generally ineffective or specific immune responses. Here, using B16F0 melanoma prohibitively expensive. cells stably expressing CCL21 under the control of Recently, chemokines have attracted considerable interest cytomegalovirus and ubiquitin promoters, we showed that within the field of tumor immunotherapy. Chemokines are CCL21-activated immune responses depend on the small proteins that are involved in immune and inflamma- amount of melanoma-derived chemokine, which, in turn, tory responses. One of them, secondary lymphoid chemo- depends on the strength of the promoter. We showed that kine CCL21 (also known as SLC or C6kine), is prominently ubiquitin promoter–driven expression of CCL21 enabled expressed in high endothelial venules and within T-cell + À + massive infiltration of tumors with CD4 CD25 , CD8 zones of secondary lymphoid organs, where it recruits + T lymphocytes, and CD11c dendritic cells, and conse- naive T cells and antigen-presenting cell (APC), such as quent activation of cellular and humoral immune dendritic cell, via a mechanism known as chemoattraction. responses sufficient for complete rejection of CCL21- This mechanism is based on CCL21-mediated activation of positive melanomas within 3 weeks in all tumor-inoculated the G protein–coupled receptor CCR7, which is expressed mice. Mice that rejected CCL21-positive tumors acquired in maturing dendritic cell and T lymphocytes. Interaction protective immunity against melanoma, which was trans- between the ligand (CCL21) and the receptor (CCR7) ferable to naive mice via splenocytes and central memory triggers a cascade of intracellular events that promotes T cells. Moreover, melanoma-derived CCL21 facilitated changes in expression of adhesion molecules and cytoskel- immune-mediated remission of preestablished, distant etal rearrangement. These changes enable directional wild-type melanomas. Overall, these results suggest that migration of the CCR7+ cells along the CCL21 gradient (2). elevated levels of tumor-derived CCL21 are required for It has thus been suggested that this chemokine may the activation of strong melanoma-specific immune promote recruitment of APC and T lymphocytes to a tumor responses and generation of protective immunologic mass to facilitate antigen recognition and enhance tumor- memory. They also open new perspectives for the specific immune responses. In support of this concept, it development of novel vaccination strategies against was recently shown that the presence of CCL21 in the melanoma, which use intratumoral delivery of the opti- vicinity of a tumor induces infiltration of tumors with mized CCL21-encoding vectors in conjunction with DNA- + based vaccines. [Mol Cancer Ther 2007;6(6):1755–64] dendritic cell and CD8 T cell and leads to immune- mediated inhibition of melanoma (3), lung (4), and colon (5) carcinomas in experimental animals. Splenocytes isolated from these CCL21-treated mice showed greatly enhanced CTL activity against tumor cells (5). The chemoattractive properties of CCL21 were also used in the development of Received 11/15/06; revised 4/9/07; accepted 5/1/07. the dendritic cell–based vaccines. Using adenoviral gene Grant support: Jefferson Medical College (V. Alexeev) and American Skin transfer, CCL21 was expressed in tumor-associated anti- Association medical student grant (L. Novak). gen–pulsed dendritic cell. Treatment of tumor-bearing The costs of publication of this article were defrayed in part by the mice with these genetically altered dendritic cells resulted payment of page charges. This article must therefore be hereby marked in tumor growth inhibition and remission in some of the advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. experimental tumors (3, 6). However, in these various Requests for reprints: Vitali Alexeev, Department of Dermatology and experimental tumor models, the utilization of CCL21 as a Cutaneous Biology, Jefferson Medical College, Thomas Jefferson protein, even at a very high dosage or in combination with University, 233 South 10th Street, BLSB, Room 326, Philadelphia, PA19107. Phone: 215-503-4835; Fax: 215-503-5788. dendritic cell–based and DNA-based vaccines (5–8), E-mail: [email protected] resulted in temporal remission of tumors or in their Copyright C 2007 American Association for Cancer Research. complete obliteration in 20% to 60% of experimental doi:10.1158/1535-7163.MCT-06-0709 animals. Mol Cancer Ther 2007;6(6). June 2007 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. 1756 CCL21-Mediated In situ Melanoma Eradication Our current data show that strong ubiquitin promoter– 18% Tris-glycine gels, transferred onto polyvinylidene driven expression of CCL21 led to elevated levels of difluoride membranes, and probed with CCL21-specific melanoma-derived CCL21, which stimulated massive antibodies (R&D Systems) diluted 1:5,000. Immunocom- infiltration of both CD4+ and CD8+ T cells into developing plexes were detected by horseradish peroxidase–labeled melanomas, activation of melanoma-specific immune secondary antibodies and visualized using SuperSignal responses, and generation of strong, melanoma-specific WestFemto Detection kit (Pierce). For the quantitative protective immunity that was transferable into naive mice ELISA assay, B16F0, B16F0-CMV-CCL21, and B16F0- + via splenocytes and CD8 central memory T cells (TCM). UB-CCL21 cells from pools and individual clones were CCL21-induced immune responses were sufficient for the seeded in 100 AL of culture medium onto 96-well plates obliteration of primary CCL21-positive tumors as well as (5 Â 105 per well) and kept in culture for 48 h. Medium obliteration of wild-type (WT) B16F0 melanomas inocu- was then collected, purified, and subjected to the mouse lated at distant sites. In contrast to previous findings, CCL21 ELISA assay (R&D Systems) as advised by the these responses were observed in all animals inoculated manufacturer. with CCL21-expressing melanomas. In vivo Tumor Inoculation and Monitoring CCL21-mediated activation of melanoma-specific im- mune responses was tested in vivo on naive C57BL6 Materials and Methods (Charles River Laboratories) and B6.CB17-Prkdcscid/SzJ Cloning of Murine CCL21 [C57BL6 background severe combined immunodeficient Murine CCL21 cDNA (Genbank accession no. (SCID); The Jackson Laboratory] mouse strains. Three MMU88322) was amplified from total RNA extracted from groups of naive (nine animals per group) and three mouse lymph nodes via reverse transcription-PCR using groups of SCID (nine animals per group) mice were 5¶-ATGGCTCAGATGATGACTCTGAGCCTC-3¶ direct and inoculated with B16F0, B16F0-CMV-CCL21, or B16F0-UB- 5¶-ATGGAGAGCAGGTTCAGGTCT-3¶ reverse primers. CCL21 melanoma. Two additional groups of C57BL6 mice The PCR product was subcloned into pCUB and pcNDA were inoculated with B16F0-CMV-Mock and B16F0-UB- 3.1 Zeo expression vectors under the control of ubiquitin Mock cells. Tumors were inoculated via i.d. injection of and cytomegalovirus (CMV) promoters, respectively. Plas- 1 Â 105 cells per animal into the right flank. Developing mids were designated as pCUB-CCL21 and pCMV-CCL21. tumors were monitored and measured with Vernier Cell Culture calipers twice weekly for 3 weeks unless otherwise stated. B16F0 mouse melanoma cells were obtained from the Tumor growth for each animal was recorded as tumor American Type Culture Collection (CRL6332). Derivative area. At the end of the first, second, and third weeks, CCL21-positive B16F0 cells were cultured in DMEM control (B16F0 inoculated) and experimental (B16F0-CMV- supplemented with 10% fetal bovine serum, 2 mmol/L CCL21 and B16F-UB-CCL21 inoculated) animals (n =3 L-glutamine, and 10 mg/mL penicillin/streptomycin. Me- per group per each time point) were euthanized and dium and supplements were purchased from Invitrogen. tumors were excised, measured, and either snap frozen Normal mouse melanocytes, Melan-a, were kindly provid- for indirect immunofluorescence or used for other assays ed by Dr. D. Bennett (St. George’s Medical School, London, as described below. For the prolonged experiments and United Kingdom). collection of splenocytes and blood sera, an additional Generation of the CCL21-Expressing Melanoma Cells four groups of naive C57BL6 mice (five animals per CCL21-expressing B16F0 melanoma cells were generated group) were inoculated with B16F0-UB-CCL21 cells as via cotransfection of parental B16F0 cells with pPur described above. (Invitrogen) and either pCUB-CCL21 or pCMV-CCL21 Indirect Immunofluorescence vectors followed by selection with puromycin (1