Rea1162

Anti-NK1.1, anti-CD8α, or IgG (all 10 mg/kg) antibodies were injected intraperitoneal in LLC tumor-bearing mice treated with RT+L19–IL2+anti-PD-L1, 3 days before treatment start and then every 3 days until the end of treatment (6 injections in total). Depletion of CD8⁺ T cells and NK cells was monitored by flow cytometry on peripheral blood collected via puncture of the saphenous vein using non-competing antibodies that recognize a different chain (anti-CD8β-BV510 (H35-17.2, BD Biosciences)) or a different epitope (anti-NK1.1-APC (REA1162, Miltenyi Biotec)). An overview of the experimental setup is shown in figure 4A.

Single-cell suspensions from murine bone marrow, spleen, PBMCs and RAW264.7 cells were stained with mAb against mouse CD45 (30F11; Miltenyi Biotec; 1:50), mouse CD11b (M1/70.15.11.5; Miltenyi Biotec; 1:50), mouse Ly-6C (REA796; Miltenyi Biotec; 1:50), mouse CD86 (PO3.3; Miltenyi Biotec; 1:10), mouse F4/80 (BM8; Biolegend; 1:10), mouse CD206 (C068C2; Biolegend; 1:50), mouse Qa-2 (695H1-9-9; SONY; 1:50), mouse CD38 (REA616, Miltenyi Biotec; 1:50), mouse SPiDER- β-Gal (SG03-10; Dojindo Molecular Technologies; 1:500), mouse NK1.1 (REA1162, Miltenyi Biotec; 1:50), mouse CD69 (REA937, Miltenyi Biotec; 1:50), mouse CD3 (REA641, Miltenyi Biotec; 1:10). After 20 min incubation at 4 °C in the dark, cells were washed and resuspended in staining buffer for the FACS analysis as previously established [50 (link)]. Additional staining for Saβ-galactosidase was performed after the above-mentioned staining and incubated for 15 min at 37 °C in the dark without washing before FACS analysis. For each test, cells were analyzed using FACS Verse Flow Cytometer (BD Biosciences).