Recombinant murine il 12

Allophycocyanin (APC)-conjugated PBS57-loaded CD1d tetramers and unloaded CD1d tetramers were obtained from the NIH Tetramer Core Facility (Atlanta, GA). APC-, Phycoerythrin (PE)– and FITC–conjugated monoclonal antibodies (mAbs) against murine NK-, B- or T cell-specific markers, including NK1.1, MHC II, CD11c, B220, CD1d (1B1), CD4, CD8 and TCRβ, and Annexin V were purchased from BD Biosciences (San Diego, CA). The human CD1d-specific 42.1 mAb and anti-mouse IL-12Rβ1 (CD212)-specific antibody were also from BD Biosciences. The GATA3 mAb was from Biolegend (San Diego, CA). Propidium Iodide (PI) and antibodies for T-bet and RORγt were from eBioscience (San Diego, CA). Antibodies specific for phosphorylated JNK1/2, total JNK1/2, JNK1 or JNK2, were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Recombinant murine IL-12 was from Peprotech (Rocky Hill, NJ). The CD1d ligand α-galactosylceramide (α-GalCer) was obtained from Enzo Life Sciences (Farmingdale, NY).

Naïve (CD44-) CD8+ T cells were enriched from naïve IFNγ^−/− mice using Naïve CD8a+ T cell isolation kits (Miltenyi). Purity was consistently greater than 90%. Cells were cultured on plate-bound anti-CD3 (2μg/ml - Biolegend) and anti-CD28 (2μg/ml - Biolegend) in 96 well flat-bottomed plates with 10ng/ml recombinant murine IL-12 (Peprotech) and 50U/ml human IL-2 (Peprotech) for 2 days. Cells were removed from anti-CD3/28, cultured a further 2 days with IL-2 and IL-12, before 1 day with IL-2 alone. CD8s were re-stimulated with PMA and ionomycin and stained for intracellular GM-CSF as before.

Naive OT-1 or P14 cells, respectively, from spleen were purified using naive CD8a⁺ T Cell Isolation Kit (Miltenyi Biotec) and subsequently separated by AutoMACS (Miltenyi Biotec). Purity of the cells (95%) was confirmed by flow cytometry. For in vitro stimulation, 2×10⁵ naive CD8⁺ T cells were resuspended in D-MEM supplemented with 10% FCS (fetal calf serum), 2 mM GlutaMAX (GIBCO), 10 mM HEPES (GIBCO), 100 U/mL Penicillin/Streptomycin (GIBCO), and 50 μM 2-Mercaptoethanol and stimulated with anti-CD3 (clone 145.2C11, Biolegend), anti-CD28 (clone 37.51, Biolegend) (1 mg/ml) and recombinant murine IL-12 (Peprotech) in 96-well plates pre-coated with 100 μg/mL goat anti-hamster IgG (H+L) secondary antibody (Jackson Immunoresearch).

Female C57BL/6J and C3H/HeJ mice, 6–12 weeks old, were obtained from the Jackson Laboratory. Mice were housed and maintained under pathogen-free conditions in microisolator cages. The Institutional Animal Care and Use Committee at the University of Arkansas approved all experimental procedures. Animal care complied with the recommendations of The Guide for Care and Use of Laboratory Animals (National Research Council).
Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640, Dulbecco’s Phosphate Buffered Saline (PBS), and Fetal Bovine Serum (FBS) were obtained from HyClone Laboratories. Chitosan glutamate 200–600 kDa, 75–90% deacetylated (Protosan G 213) was purchased from Novamatrix. Recombinant murine IL-12 was purchased from PeproTech.
The C57BL/6 syngeneic transitional cell carcinoma MB49 was kindly provided by Dr. Jeffrey Schlom, Laboratory of Tumor Immunology and Biology, National Cancer Institute. The C3H syngeneic transitional cell carcinoma MBT-2 was kindly provided by Dr. Yi Luo, Department of Urology, University of Iowa. The C57BL/6 syngeneic melanoma B16-F10 was purchased from the American Type Culture Collection. MB49 and B16-F10 cells were maintained in DMEM while MBT-2 cells were maintained in RPMI 1640. Both media were supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin.