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Animal studies were approved by the Vanderbilt Institutional Care and Animal Use Committee (IACUC) and were performed in accordance with Vanderbilt IACUC guidelines. All animals were housed under pathogen-free conditions at the Vanderbilt Animal Care Facility. C57BL/6 mice, athymic nude mice, CD40 knockout, and NSG mice were purchased from Jackson Labs. Tumor xenografts were established in 8–10 week-old male or female mice. For the in vivo melanoma model, mice received 3 × 105 of tumor cells in 100 μl of serum-free DMEM medium by subcutaneous injection in the lower back. Mouse body weight was assessed once a week, and tumor measurements were taken twice a week with micro-calipers. Tumor volume was estimated as 0.5*length*width*width. Treatment began when tumors reached ~100mm3 volume on average or at day 10 post tumor cell inoculation and continued until tumors in the experiment exceeded 15 mm in diameter or became perforated.
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