Anti cd8a clone 53 6.7

Tumors and lymph nodes of the mice in each group were collected 7 days after the last injection, digested and grinded to single cell suspension. Tumor cell suspension was harvested for tumor-infiltrating T cell (TIL) analysis. TILs were stained with antibodies (Biolegend, USA): anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), and anti-CD8a (clone 53-6.7) for 30 min. To detect intracellular cytokine IFN-γ, tumor cell suspension was incubated with ionomycin (1 mg mL⁻¹; Abcam), phorbol 12-myristate 13-acetate (PMA; 50 ng mL⁻¹, Abcam) and bleomycin (1 mg mL⁻¹; Abcam) for 4–6 h. After that, cells were fixed, permeated and stained with anti-IFN-γ (clone XMG1.2). Popliteal and inguinal lymph node cell suspension were harvested to detect the maturation of DCs. DCs were stained with surface antibodies: anti-CD11c (clone N418), anti-CD80 (clone 16-10A1), and anti-CD86 (clone GL-1) for 30 min. Cells were then washed and resuspended in fresh PBS, and analyzed by flow cytometry. All flow cytometry antibodies were purchased from Biolegend.

Splenocytes were prepared from non-tumor bearing BALB/c mice and resuspended at 1 × 10⁷ cells per mL in RPMI containing 2% fetal calf serum. Target splenocytes were pulsed with either 1 μg/mL HA_518-526 (IYSTVASSL), UNC45a_730-738 mutant (IYEVVRSLV), UQCRC2_405-413 wildtype (SYMPPSTVL) or UQCRC2_405-413 mutant (SYMAPSTVL) peptides for 1 h at 37°C. After washing, cells pulsed with two different peptides were labeled with 2.5 μM of either CFSE (Invitrogen, VIC, Australia) (CFSE^hi) or Violet-Tag (Biolegend, CA, USA) (Violet^hi) for 10 min at 37°C . Control unpulsed cells were labeled with 0.25 μM CFSE (CFSE^lo) or Violet-Tag (Violet^lo). Cells were resuspended to 1x10⁸/mL. 1 × 10⁷ peptide pulsed cells and 1 × 10⁷ unpulsed cells were injected intravenously into tumor-bearing mice treated with ICPB. dLNs were collected 18–20 h later and single-cell suspensions were prepared. Cells were stained with anti-CD8a (clone 53.67, Biolegend, CA, USA) for 20 min at room temperature. Cells were washed and analysis was performed on a BD LSRFortessa (San Jose, CA, USA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Percent-specific lysis was calculated as: (1−(R_BALB)/R_AB1HA-BALB) × 100, where R = %CFSE^lo/%CFSE^hi.

The primary antibodies used for flow cytometry were as follows: anti‐F4/80 (clone BM8, BioLegend), anti‐CD11c (clone N418, BioLegend), anti‐MHC II (clone 10‐3.6, BioLegend), anti‐PDCA1 (clone 927, BioLegend), anti‐Gr1 (clone RB6‐8C5, BioLegend), anti‐CD4 (clone GK1.5, BioLegend), anti‐CD8a (clone 53‐6.7, BioLegend), anti‐IFN‐γ (clone XMG1.2, BioLegend), anti‐CD44 (clone IM7, BioLegend), anti‐CD80 (clone 16‐10A1, BioLegend), anti‐CD62L (clone MEL‐14, BioLegend), anti‐CD11b (clone M1/70, BioLegend), anti‐CD86 (clone GL‐1, BioLegend), anti‐I‐A/I‐E (clone M5/114.15.2, BioLegend), DC Marker (clone 33D1, BioLegend), anti‐Siglec‐H (clone 551, BioLegend), anti‐CD3ε (clone 145‐2C11, BioLegend), anti‐CD19 (clone 6D5, BioLegend), anti‐Ly‐6G (clone 1A8, BioLegend) and anti‐CD335 (clone 29A1.4, BioLegend). Mononuclear cells were isolated from the spleen and draining lymph nodes. A total of 1 × 10⁶ cells were incubated with 1·5 mg mL⁻¹ antibody for 30 min at 4°C. For intracellular staining, cells were fixed and permeabilized using fixation/permeabilization buffer (eBioscience) for 30 min at 4°C. The cells were then washed and stained for 30 min at 4°C with antibodies diluted with permeabilization buffer. The cell suspensions were analysed on an LSRII flow cytometer (BD Biosciences), and the data were analysed using FlowJo.