Cell isolation and flow cytometry analysis

Bone marrow cells, splenocytes, peripheral white blood cells and draining lymph node cells were prepared as described previously⁵⁹. For isolation of infiltrated cells in alloskin grafts and transplanted tumor, skin grafts at day 7 and tumor at day 10 were retrieved and minced into small pieces with a scalpel and then digested for 1 h at 37 °C in RPMI 1640 medium containing 30 U/ml collagenase (type IV; Sigma-Aldrich). Collagenase pretreated tissues were then ground with the plunger of a 5-ml disposable syringe and passed through a 70 μm nylon cell strainer. Cells were collected after centrifugation at 300 × g for 10 min and re-suspended in FACS staining buffer for cell surface marker staining. Cells were stained with optimized Ab dilutions. For surface marker staining, the following Ab–fluorochrome combinations were used: PE-Cy5–anti-mCD11b (M1/70), PE or PE-Cy5–anti-mGr1 (RB6-8C5), PE or FITC–anti-mLy6C (AL-21), PE–anti-mLy6G (1A8), PE-Cy5–anti-mCD4 (RM4-5), PE or FITC–anti-mCD8a (53-6.7). For Intracellular iNOS and cytokine staining, FITC–anti-miNOS (6/iNOS/NOS TypeII), PE–anti-mIFN-γ (XMG1.2) mAb and PE–anti-mIL-2 (JES6-5H4) mAb were used and the method was previously described^14,60. Abs were purchased from eBioscience, BioLegend, or BD Pharmingen. Samples were analyzed on a Beckman Coulter Epics XL benchtop FCM (Beckman Coulter) with FlowJo (Tree Star, San Carlos, CA) software.