Murine il 12

B16-F1 and B16-F10 melanoma cell lines were purchased from the American Type Culture Collection (ATCC). A highly aggressive subclone of B16-F10 (B16-M1) was generated by isolating and expanding a metastatic clone that arose in a C57BL/6 mouse 6 weeks after surgical removal of an established primary tumor. The B16-OVA melanoma cell line was kindly provided by E.M. Lord (University of Rochester Medical Center, Rochester, NY). The expression of OVA by B16-OVA cells was confirmed by real time PCR. All melanoma cells were cultured in DMEM (Thermo Scientific) supplemented with 10% heat inactivated FBS, 2mM L-Glutamine and 250 IU of penicillin/streptomycin.
CD4⁺ T cells were isolated using anti-CD4 conjugated magnetic Dynabeads (Life Technologies) according to the manufacturer’s protocol. Where indicated, CD4⁺ T cells were differentiated into T_H1 helper cells by activation with plate-bound anti-CD3ε (clone 2C11; 0.25 μg/mL) and anti-CD28 (clone 37.51; 0.25 μg/mL) antibodies (BD Biosciences) and cultured for six days in DMEM supplemented with 10% heat inactivated FBS, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, nonessential amino acids and essential vitamins (Cambrex), in the presence of murine IL-12 (10 ng/mL) (eBioscience), anti-mouse IL-4 antibody (clone 11C.11; 10 μg/ml) and 10 U/mL recombinant human IL-2 (Biological Resources Branch of the National Cancer Institute).

Fluorochrome-conjugated antibodies recognizing NK1.1 (PK136, BD Biosciences), NKp46 (29A1.4, BD Biosicences), CD3 (145-2C11, Biolegend), and IFN-γ (XMG1.2, Biolegend) were used to identify NK cell production of IFN-γ. Viability was assessed by staining cells with Zombie Yellow Stain (Biolegend) or Fixable Yellow Dead Stain (Life Technologies). NK cells were stimulated with 10ng/ml murine IL-12 (Peprotech), 10 or 100ng/ml murine IL-15 (Peprotech), 50ng/ml murine IL-18 (MBL), 10ng/ml PMA (Sigma), and/or 500ng/ml calcimycin (Sigma). Where indicated, NK cells were cultured in low-dose IL-12 (1ng/ml) and IL-18 (1ng/ml). Antibody stimulation was performed by culturing enriched NK cells in plates coated with 20µg/ml of purified anti-NK1.1 (PK136, BioXcell), anti-Ly49D (4E4, courtesy of W.M. Yokoyama and prepared by the Rheumatic Diseases Core Center), or IgG control (BioXcell). Intracellular IFN-γ production was assayed using BD Cytofix/Cytoperm™ (BD Biosciences) as recommended, with brefeldin A added to cells after 1hr of culture to inhibit secretion of IFN-γ. For in vivo priming of NK cells, wt mice were injected intra-peritoneally with 300µg of polyinosine-polycytidylic acid [poly(I:C) HMW; InvivoGen] or control PBS and splenocytes harvested 14–16h later.