The B16-F10 mouse melanoma cell line was originally obtained from Isaiah Fidler (MD Anderson Cancer Center) and had previously been passaged intradermally (i.d.) nine times in C57BL/6 mice to ensure reproducible growth in the skin (Malik et al., 2017 ). This line was used to generate all intradermal primary tumors and was retrovirally transduced with luciferase to produce a B16-luciferase subline that was used for intranodal and portal vein rechallenge studies. Another subline of B16 (“B16–25K”) that had been separately passaged for reproducible growth in the lungs, was administered via tail vein injection for the generation of surface lung metastases; this line did not contain luciferase. All tumor growth experiments involved inoculation of B16 cells at low (less than three) in vitro passages. B16 lines were certified to be free of rodent pathogens by IDEXX BioAnalytics (Columbia, MO).
Depleting anti-CD4 (mAb clone GK1.5) and anti-CD8 (mAb clone 2.43) were produced as bioreactor supernatants from a hybridoma cell lines (American Type Culture Collection) and administered at 200 μg/dose, i.p. Flurorescent antibodies for flow cytometry and microscopy were obtained from Biolegend, unless otherwise indicated.
Depleting anti-CD4 (mAb clone GK1.5) and anti-CD8 (mAb clone 2.43) were produced as bioreactor supernatants from a hybridoma cell lines (American Type Culture Collection) and administered at 200 μg/dose, i.p. Flurorescent antibodies for flow cytometry and microscopy were obtained from Biolegend, unless otherwise indicated.