Apc anti mouse cd8α antibody

DSPE-PEG2000 was purchased from NOF Corporation (Kanagawa, Japan), SL9 peptide (SVYDFFVWL) and SL9-FITC (SVYDFFVWL-FITC) were synthesized by Hefei Bankpeptide Company Ltd. (Hefei, China), and cholesterol-CpG ODN was purchased from Sangon Company (Shanghai, China). BCA kit was purchased from Beyotime (Beijing, China). The ouse PBMC isolation kit was purchased from Solarbio (Beijing, China).
Murine melanoma B16F10 cells were obtained from the Institute of Basic Medical Science, Chinese Academy of Medical Science (Beijing, China) and the cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin under an atmosphere of 5% CO2 at 37 °C. Purified anti-mouse CD16/32 antibody, APC anti-mouse CD8α antibody, and the mouse IFN-γ ELISA kit were purchased from Biolegend (Beijing, China). T-Select H-2Kb TRP-2 Tetramer-SVYDFFVWL-PE was purchased from MBL (Beijing agent, China).

MC38 tumour-bearing mice were randomly divided into four groups and treated with PBS, α-PD1, PGN_4.9 nanoadjuvant, and α-PD1 + PGN_4.9 nanoadjuvant. PGN_4.9 nanoadjuvant was intravenously injected into the C57BL/6 mice (equivalent to 2 mg kg⁻¹ IMDQ) on days 3, 9 and 15. Simultaneously, mouse α-PD1 (100 μg/mouse, BioXcell) was administered intraperitoneally on days 0, 3, 6 and 9 (n = 11 mice for the PBS group; n = 10 mice for other groups). The tumour volumes were measured by electronic calipers every 2 days, and the survival curves were recorded according to Kaplan–Meier analysis. Besides, the tumour samples were stained with PerCP/Cy5.5 anti-mouse CD45 (103132, Biolegend, clone number: 30-F11, Dilution 1:100), FITC anti-mouse CD3 antibody (100204, Biolegend, clone number: 17A2, Dilution 1:50), PE anti-mouse CD4 antibody (100512, Biolegend, clone number: RM4-5, Dilution 1:80), APC anti-mouse CD8α antibody (100712, Biolegend, clone number: 53-6.7, Dilution 1:80), and BV421 anti-mouse CD25 (102043, Biolegend, clone number: PC61, Dilution 1:200) according to the manufacturer’s instructions. The proportions of CD3⁺CD8⁺ cytotoxic T lymphocytes and CD4⁺CD25⁺ regulatory T lymphocytes were obtained by flow cytometry.