Monocyte-derived DCs were generated from PBMCs as previously described with some modifications . Briefly, CD14+ monocytes were enriched by positive selection using CD14 Microbeads (Miltenyi Biotec). Monocytes were cultured in the presence of 20 ng/mL GM-CSF (Immunex, Seattle, WA, USA) and 20 ng/mL IL-4 (R&D systems) in RPMI1640 supplemented with 2.5% fetal calf serum. Medium was replaced by fresh medium containing cytokines 3 days later. On day 6, cells were harvested and used for subsequent experiments. The concentration of IL-12p70 and IL-10 was measured by ELISA Kit (eBioscicence) according to the instruction provided by the manufacturer. Statistical significance was evaluated
by Student’s t-test; p values less than 0.05 are considered significant. This article is dedicated to EGFR inhibitor the memory of Lloyd J. Old, M.D. We thank Drs. T. Takahashi and J. B. Wing for critical reading of the manuscript, and L. Wang, C. Brooks, E. Krapavinsky, E. Ritter, and D. Santiago for technical support. This study was supported by Grant-in-Aid for Scientific Research on Priority Areas (No. 17016031, H. Shiku, and No. 20015019, H. Nishikawa) and Grants-in-Aid for Scientific Research (B) (No. 23300354, H. Nishikawa), the Cancer Research Institute Investigator
Award (H. Nishikawa) and Cancer Vaccine Collaborative Grant for AZD3965 cost Immunological Monitoring (S. Gnjatic, G. Ritter and L.J. Old), Cancer Research Grant from Foundation of Cancer Research Promotion (H. Nishikawa), Takeda Science Foundation (H. Nishikawa), Kato Memorial Bioscience Foundation (H. Nishikawa), the Sagawa Foundation for Florfenicol Promotion
of Cancer Research (H. Nishikawa), and Senri Life Science Foundation (H. Nishikawa). MH is a research fellow of the Japan Society for the Promotion of Science. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Figure S1. (A) Preparation of NY-ESO-1 and 146HER2 proteins complexed with cholesteryl pullulan (CHP): Recombinant NY-ESO-1 and 146HER2 proteins for clinical use were prepared, and the nano-particles consisting of CHP and the NYESO-1 protein, and CHP and the HER2 complex were formulated. (B) Study design of the clinical trial. (C) Patient characteristics in this study. Figure S2. (A) DCs were prepared from four healthy individuals as described in Materials and Methods. TNF-⟨ (100 ng/ml), LPS (1 mg/ml), or OK-432 (1 ìg/ml) was added in the culture of 1 × 105 immature DCs on day 6. After 48 h, supernatant was collected and cytokine production was analyzed with ELISA. (B) Summary of cytokine secretion in from four healthy individuals.