[CANCER RESEARCH 59, 3340 –3345, July 15, 1999] Advances in Brief Biodistribution and Vaccine Efficiency of Murine Dendritic Cells Are Dependent on the Route of Administration 1 Andreas A. O. Eggert, Marco W. J. Schreurs, Otto C. Boerman, Wim J. C. Oyen, Annemiek J. de Boer, Cornelis J. A. Punt, Carl G. Figdor, and Gosse J. Adema 2 Tumor Immunology Laboratory [A. A. O. E., M. W. J. S., A. J. d. B., C. G. F., G. J. A.] and Departments of Nuclear Medicine [O. C. B., W. J. C. O.] and Medical Oncology [C. J. A. P.], University Hospital Nijmegen St. Radboud, 6525 EX Nijmegen, the Netherlands Abstract Dendritic cells (DCs) are professional antigen-presenting cells, well equipped to initiate an immune response. Currently, tumor antigen- derived peptide loaded DCs are used in clinical vaccination in cancer patients. However, the optimal dose and route of administration of a DC vaccine still remain to be determined. Using indium-111-labeled DCs, we investigated whether the route of administration does affect the biodistri- bution of DCs in lymphoid organs and whether it influences the outcome of DC vaccination in the B16 mouse melanoma tumor model. The results demonstrate that i.v. injected DCs mainly accumulate in the spleen, whereas s.c. injected DCs preferentially home to the T-cell areas of the draining lymph nodes. Using tyrosinase-related protein-2-derived pep- tide-loaded DC vaccination in a fully autologous B16 melanoma tumor model, we observed a delay in tumor growth, improved survival as well as increased antitumor cytotoxic T-cell reactivity after s.c. vaccination as compared to i.v. vaccination. These data demonstrate that optimal induc- tion of antitumor reactivity against the autologous melanocyte differenti- ation antigen tyrosinase-related protein-2-derived peptitde occurs after s.c. vaccination and correlates with the preferential accumulation of DCs in the T-cell areas of lymph nodes. Introduction DCs 3 constitute a family of APCs defined by their morphology and their unique capacity to initiate a primary immune response (1, 2). They originate from bone marrow and are present as immature APCs in nonlymphoid tissues. On activation by antigenic challenge and/or inflammation, the DCs mature and migrate to the secondary lymphoid tissues where they present the processed antigen to T cells. There they interact with T cells and present the processed antigen in MHC molecules (3). Because of these properties, mature DCs are thought to be ideal for generating a primary immune response against cancer, viral infections, and other diseases. In recent years, techniques have been developed to generate large numbers of functionally potent DCs by culturing bone marrow (4, 5) or peripheral blood monocytes in the presence of GM-CSF and/or other cytokines, such as IL-4 (6), tumor necrosis factor, stem cell factor, and FLT3 ligand (7, 2). The avail- ability of class I-restricted peptides derived from tumor-associated antigens, such as tyrosinase, TRPs, gp100, and MART-1/Melan-A offered the possibility to use the DCs as APCs to generate tumor- reactive CTLs in vitro (8). Similarly, DCs are now also used in vivo in antimelanoma vaccinations (9). In the murine B16 melanoma, the homologous of the melanocyte differentiation antigens are identified and can now be used to investigate DC vaccination strategies in more detail (10, 11). In these DC vaccination models, one of the crucial functions of the DCs is their ability to migrate specifically into T-cell areas of secondary lymphoid organs. Herein, we compared the bio- distribution of 111 In-labeled DCs after different routes of administra- tion in time. Besides major differences in biodistribution, we also provide evidence that the efficiency of DC vaccination is dependent on the route of DC administration. The implications of these findings for DC vaccination will be discussed. Materials and Methods Mice. Male C57BL/6 (H-2b) mice were purchased from Charles River Wiga (Sulzfeld, Germany) and held under specified pathogen-free conditions in the Central Animal Laboratory, Nijmegen University (Nijmegen, the Neth- erlands). For experimental purposes, mice 6 – 8 weeks of age were used. Reagents. Cells are cultured in Iscove’s modified DMEM with glutamax supplemented with 10% heat-inactivated FCS (Life Technologies, Inc., Breda, the Netherlands), 50 M -mercaptoethanol, antibiotics, and antimycotics (Life Technologies, Inc.), unless mentioned otherwise. Murine recombinant GM-CSF (rmGM-CSF) and murine recombinant IL-4 (rmIL-4) were kindly provided by Dr. G. Zurawski (DNAX Research Institute, Palo Alto, CA). The following monoclonal antibodies were used: CD45R/B220 (RA3– 6B2), CD4 (MT-4), CD8 (LYT-2), I-A b (17/227), and the activating anti-CD40 mono- clonal antibody FGK-45 (kindly provided by Dr. Rolink, Basel Institute for Immunology, Basel, Switzerland; Ref. 12). The mTRP2 (VYDFFVWL) pep- tide and irrelevant peptide OVA (SIINFEKL) were synthesized with a free COOH terminus by F-moc peptide chemistry using ABIMED Multiple Syn- thesizer or by T-boc chemistry on a Biosearch SAM2 peptide synthesizer. The peptides were 90% pure, as indicated by high-performance liquid chroma- tography. Peptides were dissolved in DMSO and stored at -20°C. DC Culture. DCs were generated according to Inaba et al. (4), with modifications. Briefly, femurs were dissected, placed in 70% alcohol for 1 min, and washed with PBS. Marrow was flushed and passed through nylon mesh to remove debris. After washing, lymphocytes, granulocytes, and I-A- positive cells were removed by immunomagnetic depletion against the CD45R, CD4, CD8, and I-A-antigens. Remaining cells were cultured overnight, and the nonadherent cells were seeded at 2  10 5 cells/ml and 4 ml/well in the presence of 20 ng/ml rmGM-CSF and rmIL-4 in 6-well plates (Costar, Bad- hoevedorp, the Netherlands). On day 4, the cultures were refreshed by adding 1 ml of culture medium supplemented with GM-CSF and IL-4 (10 ng/ml). At day 7, nonadherent and loosely adherent proliferating DC aggregates were collected and replated in fresh medium, cytokines (1  10 6 cells/ml), and 500 l of hybridoma supernatant of activating anti-CD40 antibody FGK 45. [ 111 In]oxinate Labeling, Administration, Gamma Camera Imaging and Biodistribution Analysis. Bone marrow DCs were labeled with [ 111 In]oxi- nate (Mallinckrodt Medical, Petten, the Netherlands) in 0.1 M Tris-HCl (pH 7.0) for 20 min at room temperature. Cells were washed three times with PBS, and the labeling efficiency was calculated as the percentage of the activity that remained associated with the cell pellet. To determine the stability in vitro, DC samples were kept in medium without cytokines at 37°C. After different time periods, cells were spun down, the supernatant was transferred to other tubes, cell pellets were resuspended in the same volume, and the emitted radioactivity Received 2/15/99; accepted 6/1/99. 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 Supported by the European Community, ERB FMRX CT960053. 2 To whom requests for reprints should be addressed, at Department of Tumor Immunology, University Hospital Nijmegen St. Radboud, Philips van Leydenlaan 25, 6525 EX Nijmegen, the Netherlands. Phone: 31-24-3617600; Fax: 31-24-3540339; E- mail: g.adema@dent.kun.nl. 3 The abbreviations used are: DC, dendritic cell; TRP, tyrosinase-related protein; p.i., postinjection; In, indium; APC, antigen-presenting cell; GM-CSF, granulocyte macro- phage colony-stimulating factor; ID, injected dose; IL, interleukin. 3340 on July 7, 2015. © 1999 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from