Tks 1

For the establishment of the B16F10-4-1BBL, B16F10-OX40L and B1610-GFP strains, parental B16F10 cells were transduced with retroviral vectors pCL-4-1BBL, pCL-OX40L and pBabe-eGFP, respectively. The pCL vector encodes an antibiotic resistance gene G418, whereas the pBabe-eGFP vector confers selectivity to puromycin. Resistant clones for G418 were analyzed by flow cytometry using anti-4-1BBL (eBioscience clone RM134L) and anti-OX40L (eBioscience clone TKS-1) clones. Puromycin-resistant clones were also analyzed by flow cytometry.

All the cell lines were derived from the poorly immunogenic mouse melanoma cell line B16F10. Cell cultures were transduced using a retrovirus and selected with G418 or puromycin. The pCL vectors encode G418 resistance and the pBabe vector encodes puromycin resistance. The G418-resistant clones were analyzed by flow cytometry using antibodies anti-OX40L (eBIOSCIENCES clone RM134L) and anti-4-1BBL (eBIOSCIENCES clone TKS-1). We have chosen high-expression clones to establish the cell lines B16-0X40L and B16-41BBL. Next, we transduced B16-4-1BBL, B16-OX40L, and B16-GM-CSF (kindly provided by Dr. Glen Dranoff, Harvard, USA) using the retroviral vector pBabe-eGFP, which also harbors a puromycin selection cassette. Clones were analyzed using flow cytometry to select cells with a high level of GFP expression and also a high expression level of immunomodulators like 4-1BBL and OX40L. The B16-GM-CSF was also analyzed by flow cytometry for GFP and GM-CSF using a quantitative assay to determine secreted GM-CSF by ELISA (duo set ELISA kit, R&D).

Agonistic monoclonal antibody (Ab) against 4‐1BB (3E1) was generated from nude mice that were injected intraperitoneally with a subcloned hybridoma to induce ascites formation 22. The Ab was purified from ascites fluid by affinity column chromatography with protein G Sepharose (Sigma‐Aldrich, St. Louis, MO, USA). Recombinant 4‐1BB Fc (r4‐1BB FC) was purchased from Adipogen (Seoul, Korea). Antagonistic monoclonal Ab against 4‐1BBL (TKS‐1) was purchased from e‐Bioscience (San Diego, CA, USA). Rat immunoglobulin G (Rat IgG) and human IgG1 were purchased from Sigma‐Aldrich and were used as the control.