Isolation of Tissue-Infiltrating Leukocytes

Peripheral blood mononuclear cells (PBMCs) were isolated using a standard Ficoll procedure. Tissue-infiltrating leukocytes were dissociated from tissue specimens and collected as described previously [27 (link)]. Briefly, specimens were cut into small pieces and digested in RPMI 1640 (Gibco) supplemented with 100 μg/ml Liberase TL (Roche), 100 μg/ml DNase I (Sigma-Aldrich) and 20% FBS (Gibco) for 30 min at 37°C. The homogenates were filtered through a 150-mm cell strainer and separated by Ficoll density gradient centrifugation (Axis-Shield). The isolated PBMCs were washed with Hanks’ balanced salt solution and resuspended in RPMI 1640 supplemented with 10% FBS.