B95 8

Human B-lymphoma cell lines (Farage, SU-DHL-4, OCI-LY-10, OCI-LY-7, Toledo), and EBV-producing marmoset B-cell line B95-8 were purchased from ATCC (American Type Culture Collection. Manassas, VA, USA). Cells were cultured in RPMI-1640 (Gibco, Shanghai, China) and humidified atmosphere of 95% air and 5% CO₂ at 37 °C. HEK-293 T was cultured in DMEM (Gibco, Shanghai, China). EBV-infected human B-lymphoma cell models were established using the method previously described [11 (link)]. Briefly, EBV virion was prepared from EBV-producing B95-8 cells. SU-DHL-4 and OCI-LY-10 cells were exposed to the supernatant of B95-8 cells at 37 °C for acute EBV infection (within 4 days) or long-term EBV infection (for 3 weeks). SU-DHL-4 and OCI-LY-10 cells cultured in EBV-free growth medium were used as negative controls.

EBV LMP2A-positive cell lines, including C666-1, CNE-2Z (Human NPC cell lines, obtained from Taisheng Bio-Tech Co., Ltd., Guangzhou, China), B95-8 (EBV transformed lymphocyte, ATCC: CRL-1612) and EBV-negative melanoma A375 (ATCC: CRL-1619), were used for evaluation of Z_LMP2A-N affibody cytotoxicity and cell binding affinity. C666-1, CNE-2Z and B95-8 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin, while A375 cells were cultured in DMEM medium supplemented in the same manner as EBV-positive cells. All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

KSHV and EBV-negative (BJAB and DG75 from American Type Culture Collection [ATCC], Manassas, VA), KSHV-positive (BC3 and BCBL1 from ATCC) B-lymphoma cells, EBV-positive cells (B95.8 from ATCC and EBV-transformed primary B cell lines LCL in vitro generated in the lab[41 ]), iSLK (1mg/ml puromycin, 250ug/ml G418, a gift from Shou-Jiang Gao at University of South California) and iSLK-Bac16 (K-iSLK, 1mg/ml hygromycin, 250ug/ml G418 and 1ug/ml puromycin, a gift from Shou-Jiang Gao at University of South California) and iSLK.219 (400 μg/mL hygromycin, 250 μg/mL G418, and 4 μg/mL puromycin[42 (link)]) cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (Gibco-BRL). HUVEC (ATCC), and HEK293 (ATCC) cells, were maintained in DMEM supplemented with 10% FBS. All cell lines were incubated at 37°C in a humidified environmental incubator with 5% CO₂. 293 cells were transfected with 1 mg/ml polyethyleneimine (PEI) at a ratio of 1μg plasmid DNA: 3μl PEI. iSLK and iSLK-Bac16 were transfected with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s recommendations. Cells were cultured 24hrs before transfection with cell confluence reaching 60–70%.

In addition to the LCLs established in our laboratory, seven other lines were used, the characteristics of which are mentioned in Table 3. B95-8 and the four BL lines (Jijoye, Namalwa, P3HR1, and Raji) were purchased from the ATCC (Catalog numbers CRL 1612—ECACC 85011419, CCL-87, CRL-1432, HTB-62, and CCL-86 respectively, Manassas, VA, USA). The extranodal NK/T cell lymphoma lines (MEC04 and SNK6) were kindly provided by Marion Travert (Inserm U955, Hôpital Henri Mondor, Créteil, France). All lines were grown in RPMI1640 medium with glutaMAX (ThermoFisher Scientific, Illkirch-Graffenstaden, France; catalog number 61870-010) supplemented with 10% fetal bovine serum (FBS; Eurobio Scientific, Les Ulis, France; catalog number CVFSVF00-0U) and 1% penicillin-gentamicin at 37 °C in a humidified 5% CO₂ atmosphere. MEC04 and SNK6 cell lines were cultured under the same conditions and supplemented with 100 U/mL of human IL-2 (Sigma-Aldrich, Saint-Quentin Fallavier, France; catalog number I7908).

Human B-lymphoma cell lines Farage (EBV+), DB (EBV-), EBV-producing marmoset B-cell line B95–8, and murine B-lymphoma cell line A20 were obtained from American Type Culture Collection (Manassas, VA, USA). Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood with ficoll using density gradient centrifugation. Then ficoll medium interface was carefully removed, washed with salt-buffered solution, then centrifuged, leaving purified PBMCs. To freeze, freshly isolated PBMCs were resuspended to 5 × 10⁶ cells/mL in freezing medium containing 10% DMSO and 40% fetal bovine serum (FBS) in RPMI-1640 medium, and placed inside a freezing container at − 80 °C overnight. The following day, samples were moved to a liquid nitrogen tank for long-term storage. Cells were cultured in humidified atmosphere of 95% air and 5% CO₂ at 37 °C. Anti-human PD-L1 antibody and anti-human PD-1 antibody were from Innovent (Suzhou, China). Anti-mouse PD-L1 antibody Invivomab was from Bio X Cell (West Lebanon, NH, USA).