CCL19 as an adjuvant for intradermal gene gun immunization in a Her2/neu mouse tumor model: improved vaccine efficacy and a role for B cells as APC

Cancer Gene Therapy (2012) 19, 880–887 & 2012 Nature America, Inc. All rights reserved 0929-1903/12 www.nature.com/cgt ORIGINAL ARTICLE CCL19 as an adjuvant for intradermal gene gun immunization in a Her2/neu mouse tumor model: improved vaccine efficacy and a role for B cells as APC T Nguyen-Hoai1,2, O Hohn3, MD Vu1,2, G Baldenhofer1,2, MS Sayed Ahmed1,2, B Dörken1,2, S Norley3, M Lipp2, A Pezzutto1,2 and J Westermann1,2 The aim of this study was to evaluate the efficacy of the chemokine CCL19 (ELC) as an adjuvant for intradermal gene gun delivery of Her2/neu DNA and to investigate the role of B cells in CCL19-mediated enhancement of immune responses. Balb/c mice were immunized intramuscularly (i.m.) on days 1 and 15 with plasmid DNA (pDNA) (100 mg DNA) or intradermally (i.d.) by gene gun delivery (1–2 mg DNA). Administration of pDNA encoding Her2/neu (pDNA(Her2/neu) was compared with pDNA(Her2/neu) plus pDNA(CCL19), pDNA(CCL19), mock vector or uncoated gold particles/phosphate-buffered saline (PBS). Tumor challenge was performed subcutaneously on day 25 with syngeneic Her2/neu þ tumor cells (D2F2/E2). Intradermal immunization by gene gun led to an enhancement of tumor protection by the DNA vaccine as compared with i.m. immunization. The protective effect of the vaccine was further enhanced by coadministration of pDNA(CCL19) both after i.m. and i.d. immunization. Tumor protection was associated with Her2/neu-specific T cell and humoral immune responses. Experiments in B-cell-deficient mMT mice showed that B cells are crucial for CCL19-mediated enhancement of tumor rejection, most likely as antigen-presenting B cells. DNA vaccines against Her2/neu may play a future role in the treatment of Her2/neu-positive breast cancer patients in a clinical situation of minimal residual disease. Cancer Gene Therapy (2012) 19, 880–887; doi:10.1038/cgt.2012.78; published online 26 October 2012 Keywords: CCL19; CCR7; chemokines; Her2/neu; DNA vaccination; gene gun INTRODUCTION DNA vaccination using plasmid DNA (pDNA) is a method that was initially performed in mice by intramuscular injection1 and was subsequently used with success in both infectious and malignant disease models.2 Gene gun immunization by delivery of DNAcoated gold particles to the skin was reported to be more efficient in generating antigen-specific T cell and antibody responses in mice, although a TH2-bias of the emerging immune response was also observed.3 The higher efficiency of gene gun DNA delivery to the skin compared with i.m. application is most likely to be explained by the high density of antigen-presenting cells (APC) such as dendritic cells (DC) in epidermal sites. Gene gun particle bombardment causes local injury ( ¼ inflammation/danger signal) and results in direct transfection of DC and non-immune cells such as keratinocytes. The latter may also express and secrete the antigen into the environment where it is taken up by DC (crosspriming). This process results in efficient T-cell priming within secondary lymphatic tissues.4–7 Although DNA vaccines have been very successful in inducing antigen-specific immune responses in mice,8–10 immunogenicity in humans and large animals has so far been largely disappointing.3,9,11 The reasons are diverse, but are mostly related to topics such as dosing, application route, tissue distribution and differences in toll-like receptor (TLR) expression patterns between mice and humans.12 New experimental approaches aimed at amplifying the immune responses induced by DNA vaccines are therefore warranted. Several approaches have been pursued to improve the immunogenicity of DNA vaccines including the coexpression of immunomodulatory molecules such as cytokines and chemokines.10,13–16 Chemokines such as CCR7-ligands Ebstein–Barr-Virus-inducedmolecule-1-ligand-chemokine (ELC/CCL19) and secondary lymphoid-tissue chemokine (SLC/CCL21) are key regulators of innate and adaptive immune responses, since they orchestrate migration of DC and T cells into secondary lymphatic tissues, thereby organizing immune synapse formation.17–20 CCL19 is constitutively expressed by cells distributed throughout the T-cell zones of lymph nodes, spleen and Peyer’s patches. Additionally, recent studies suggest that DC themselves are able to secrete CCL19 during activation and migration.21 Whereas immature steady-state DC residing in the tissues do not express the cognate CCL19 receptor, CCR7 is induced upon activation of DC, that is, by external infectious stimuli via TLR.17 Upregulation of CCR7 that possibly coincides with autocrine CCL19 (and CCL21) production, leads to migration of activated mature DC into regional lymph nodes following a CCL19/CCL21 gradient. Since CCR7 is not only expressed by mature DC, but also by particular subsets of T- and B cells such as naive T cells, central memory T cells and activated B cells, CCR7-ligands increase the chance of interaction between DC, T- and B cells in secondary lymphatic 1 Department of Hematology, Oncology and Tumor Immunology Charité—University Medicine Berlin, Campus Berlin-Buch, Campus Benjamin Franklin and Campus VirchowKlinikum, Berlin, Germany; 2Max Delbrück Center for Molecular Medicine, Berlin, Germany and 3Center for HIV and Retrovirology, Robert Koch Institute, Berlin, Germany. Correspondence: Dr J Westermann, Department of Hematology, Oncology and Tumor immunology, Charité—University Medicine Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: Received 6 June 2012; revised 20 September 2012; accepted 22 September 2012; published online 26 October 2012 CCL19 improves Her2/neu DNA vaccination by gene gun T Nguyen-Hoai et al 881 tissue and thus regulate primary (or secondary) adaptive immune responses. We have previously shown in a Her2/neu mouse tumor model that CCL19 is able to amplify both a TH1-polarized antigen-specific immune response and tumor protection by an intramuscular DNA vaccine.22 We chose a prophylactic tumor vaccination model since therapeutic vaccination has been largely disappointing during the past decades, whereas vaccines able to induce protective immunity in the situation of minimal residual disease are possibly more appropriate for clinical application.23 In this study, we asked (1) whether the adjuvant activity of CCL19 can also be observed after intradermal gene gun delivery of DNA, (2) which application route (intramuscular or intradermal) should be preferred for the further preclinical development of a Her2/neu-CCL19 DNA vaccine and (3) which role can be attributed to B cells (which may also express CCR7) in our system. MATERIALS AND METHODS Cell lines The mouse mammary tumor cell line D2F2 is derived from a spontaneous mammary tumor that arose in a Balb/c background from the hyperplastic alveolar nodule cell line D2. D2F2/E2 is transfected with the human Her-2/ neu expression vector pCMV/E2 and the selectable pDNA pRSV/neo. The Her2/neu-expressing cell line D2F2/E2 was maintaine (...truncated)