Flow Cytometry

All analysis was performed using DuraClone IM (Immune Monitoring) panels: Basic (CD45, CD3, CD4, CD8, CD19, CD14, CD16, CD56), B cell (IgD, CD21, CD19, CD27, CD24, CD38, IgM, CD45), T cell (CD45, CD3, CD4, CD8, CD45RA, CD197/CCR7, CD27, CD28, CD279/PD1, CD57), Granulocyte (CD45, CD294, CD16, CD33, CD11b, CD274, CD3, CD19, CD56, CD14, CD62L, CD15), T cell Receptor (CD45, CD3, CD4, CD8, TCRγδ, TCRαβ, HLA-DR, TCRVδ1, TCRVδ2), Regulatory T cell/Treg (CD45, CD3, CD4, CD45RA, CD25, CD39, Foxp3, Helios), and Dendritic cell/DC (CD45, HLA-DR, CD14, CD19, CD20, CD56, CD16, CD1c, Clec9A, CD123). For peripheral blood analysis, 100 μl of blood was added to the appropriate tubes and cells were processed according to the manufacturer's instructions. In brief, blood was incubated with the antibodies for 15 min in the dark, followed by red blood cell lysis using VersaLyse (Beckman Coulter) for 15 min in the dark. Cells were then washed twice in PBS prior to data acquisition. Treg cell analysis was performed according to the manufacturer's protocol. Briefly, 50 μl of peripheral blood was incubated for 15 min with DuraClone IM Treg Tube 1, followed by a wash in 3 ml of PBS and centrifugation at 500 × g for 5 min. Cells were then resuspended in 50 μl of 100% fetal calf serum followed by 5 μl of fixative reagent. After a 15 min incubation, cells were resuspended in 400 μl of permeabilizing reagent and immediately transferred to DuraClone Treg Tube2 for a 60 min incubation. Cells were then washed in PBS prior to data acquisition. For tumor cells and tumor-infiltrating immune cells, 1 × 10⁶ cells were added to each tube and cell staining was performed as described above for peripheral blood. Tumor cells were filtered three times using a 70 μM cell strainer to remove dead cell debris. All processed samples were then analyzed on a 13-color CytoFlex flow cytometer (Beckman Coulter). Data were analyzed using Kaluza Software (Beckman Coulter).