We.p. receptors and an enhanced proliferation state of effector T cells was seen only upon intracutaneous but not intravenous or intraperitoneal immunization. Tumor growth was prevented only by intracutaneous DC vaccination. Our results indicate that under suboptimal conditions the route of DC vaccination crucially determines the efficiency of tumor defense. DC-based strategies for immunotherapy of cancer should take into account the immunization route in order to optimize tissue targeting of tumor antigen specific T cells. Introduction Dendritic cells play a central role in the initiation of immune responses. Since tumor antigen-bearing DC are capable to induce protective T cell mediated immune responses several strategies have been developed using DC vaccines for cancer therapy [1]C[3]. The most promising strategy might be the generation of DC from blood or bone marrow-derived precursors, as these can be fully characterized and an array of parameters can be manipulated to optimize the DC vaccine. These include DC subset differentiation, activation status as well as antigen delivery and presentation. It has been described that injection of mature DC pulsed with tumor associated antigens (TAA) or tumor lysate were more potent in generating antigen-specific T cell responses compared to immature DC [4],[5]. DC vaccination was used in patients with different types of cancer [6]C[11] and in phase 1 trials it was shown that generated peptide-pulsed DC induced antigen-specific immune responses [4], [12]. Unfortunately immune responses were transient and clinical outcomes have been poor. The best results were obtained in patients with melanoma at cutaneous or lymphatic sites [13]. Recently the FDA has NIC3 approved the first DC-based vaccine against human metastatic prostate cancer [14]. It was shown that different injection routes of DC result in activation of T cells in different lymphoid organs. For example i.v. injected DC efficiently enter the spleen [15] whereas subcutaneously (s.c.) injected DC access peripheral LN draining the injection area [16]C[19]. It also was described that this magnitude of the primary immune response directly correlates with the infiltration of DC and antigen-specific naive T cells into individual LN [20]. This was demonstrated in a mouse model of tumor control by injection of varying numbers of bone marrow-derived DC. The number of injected DC correlated with the number of DC infiltrating the draining LN, with the number of antigen specific CD8+ T cells in the same LN and the decrease in tumor size. Furthermore it was described that T cells activated by DC isolated from different tissues express different homing receptors necessary for NIC3 entering peripheral organs. T cells activated with DC isolated from mesenteric LN (mLN) express the small intestine homing receptors chemokine receptor 9 (CCR9) and 47 integrin. In contrast T cells activated with Langerhans cells isolated from the epidermis express the skin homing receptors E-selectin ligand (E-lig) [21]. We and others have previously shown that this peripheral tissue microenvironment has an impact on the capacity of the DC to induce homing receptors on T cells [22]C[24]. Therefore the microenvironment of CSF2RB the DC origin licenses DC to induce homing receptors on T cells in the draining lymph nodes. This was further shown by Calzascia and colleagues by transplanting tumors expressing two different tumor antigens into different sites, intracerebral and subcutaneous, in the neck [25]. Both sites are drained into the cervical LN where T cell homing receptors were analyzed. In this cervical LN, DC were found to have immigrated from the two different tumor sites presenting the respective tumor antigens. These activated DC induced the homing receptor profiles on T cells corresponding to their tissue of origin, the brain and the skin, in the same lymph node. Therefore multiple imprinting programs occurred in the same LN. The authors concluded that the identity of a given LN is not essential in determining homing receptor imprinting but rather the site of NIC3 antigen uptake by DC [25]. In contrast to this another study showed that this microenvironment of a given LN also has an impact around the induction of homing receptor imprinting on T cells [23]. This was done by transplanting peripheral LN (pLN) into the gut mesenteries. These transplanted LN fail to support the generation of gut-homing T cells, even though gut-derived DC enter the transplants and primary T cells [23]. All these reports.