Nkp46 fitc

Spleens were harvested from all animals at experimental end-point and placed in RPMI 1640 media (Lonza). Single-cell suspensions were prepared by mechanically pressing the cells through a 70 μm cell-strainer. Red blood cells were lysed using RBC lysis buffer (Roche), and cells were washed twice with PBS. Splenocytes were frozen at −80 °C in RPMI 1640 media supplemented with 20 % (v/v) heat-inactivated FBS (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin and 10 % (v/v) DMSO (Sigma) until analysis by flow cytometry. Cells were stained with labeled antibodies for 60 min at RT in the dark to analyze the immune cell subpopulations. First, a gate on CD45⁺ cells (CD45-PE-Cy7, 1:50, #25–0451-82, Invitrogen) was used to isolate hematopoietic cells. Further gates were set on CD3⁺ (CD3-APC, 1:50, #47–0031-80, Invitrogen) and CD8a⁺ (CD8a-645, 1:50, #64–0081-80, Invitrogen) cells to study cytotoxic T cells. NK cells were studied by gating on NKp46⁺ cells (NKp46-FITC, 1:50, #560756, BD Bioscience). Myeloid-derived suppressor cells (MDSCs) were defined as CD11b⁺ (CD11b-PE, 1:3, #PNIM2581U, IOTest) and Gr-1⁺ (Gr-1-Alexa Fluor 700, 1:50, #56–5931-80, Invitrogen). Gating strategy is presented in Fig. S1. The data were acquired on LSR Fortessa Flow cytometer (BD Biosciences) and analyzed using the Flow Jo software.