Cd160

To investigate the functions of SRSF2 in the immune response of TILs against autologous tumor cell lines, TILs transfected with SRSF2 siRNA were co-cultured with autologous tumor cell lines. Then, flow cytometry was performed with an anti-CD8 antibody (Biolegend, 344704) and anti-IFN-γ antibody (BD Biosciences, 554552) on BD AccuriC6 (BD Biosciences). To examine the expression level of the immune checkpoints in the TILs and PBMCs, the frequency of immune checkpoint + and CD8 + TILs was assessed using flow cytometry on BD AccuriC6 (BD Biosciences). The antibodies against the immune checkpoints used for flow cytometry included PD-L1 (BD Biosciences, 561787), BTLA (BD Biosciences, 564802), CTLA-4 (BD Biosciences, 561717), LAG3 (BD Biosciences, 565617), and CD160 (BD Biosciences, 562351). All data were analyzed by FlowJo software.

Cryopreserved PBMCs were thawed in a 37 C water bath as quickly as possible and were washed twice with RPMI 1640 medium to remove the dimethyl sulfoxide. The samples were used for flow cytometry analysis. The PBMCs were immunolabelled using antibodies against the following surface proteins: CD3, CD56, CD28, and CD160 (BD Biosciences, Franklin Lake, USA) according to the manufacturer's instructions. Flow cytometry was carried out using Fluorescence Activated Cell Sorting flow cytometers (Beckman Coulter FC50, Life Sciences, Indianapolis, USA). Data acquisition was carried out using Cell Quest software (BD Biosciences, Franklin Lake, USA).

For flow cytometry, fluorochrome-conjugated antibodies against the following antigens were used: CD56 (clone NCAM16.2; conjugated to BV605) (B159; APC), CD3 (SK7; BUV395) (HIT3a; PE), CD107a (H4A3; APC), IFN-γ (B27; PerCP/Cy5.5), TNF (MAb11; PE), CD69 (FN50; BV421 and BV711), CD11a (HI111; BV711), NKG2D (149810; PE-CF594), NKp30 (p30-15; BV421), TACTILE (6F9; BV711), TRAIL (RIK-2; BV421), BTLA (J168-540; PE-CF594), Phospho-Lck (4/LCK-Y505; AF647), Phospho-LAT (J96-1238.58.93; PE), Phospho-SLP-76 (J141-668.36.58; PE), Phospho-MEK1/2 (O24-836; PE), Phospho-ERK1/2 (20A; BV421), Phospho-SHP-2 (L99-921; AF647) (all BD Biosciences), CD160 (BY55; PE/Cy7), NKp46 (9E2; PE/Cy7),NKp44 (P44-8; PE), DNAM-1 (11A8; APC), 2B4 (C1.7; PerCP/Cy5.5), NKp80 (5D12; PE) (all BioLegend), NKG2C (134591; APC) (R&D Systems), and VZV gE:gI (SG1-1; conjugated in-house to DyLight 488) (Meridian Life Science). Matched isotype control antibodies were also used where appropriate.