Anti mouse cd8a antibody

In order to understand the effect of CD8⁺T cells on CD4⁺T-cell response, we performed antibody blocking experiments in vitro. The single spleen cell suspensions were incubated in 24-well flat-bottom plates (1 ml/well, 2 × 10⁶ cells/ml). For CD8⁺T cells blocking experiments, spleen T cells were treated with 5 mg/ml of anti-mouse CD8a antibody (BioLegend, 100735, Canada) or isotype control antibody IgG2a for 1 h at 37 °C. Then Tat-TPI, TPI (100 mg/ml) and PBS were added, respectively, and incubated at 37 °C, 5 % CO₂. After 18 h incubation, T cells were restimulated for 6 h at 37 °C in 5 % CO₂ with 2 μl Leukocyte Activation Cocktail (BD Biosciences). Finally, cells were collected for flow cytometry detection of Th1 expression. The experiments were repeated four times.

The spleens of mice were harvested and mechanically dissociated into fragments. Each fragment was ground up using a syringe plunger on a 70‐µm pore‐size cell strainer to prepare single‐cell suspensions. CD8 ⁺ T cells were sorted with anti‐mouse CD8a antibody (Biolegend). Enriched CD8⁺ T cells were then cultured in activation medium, consisting of 50 U interleukin‐2 (rIL‐2) and mouse T cell activator CD3/CD28 (Life Technology, 11452D), for activation and expansion. To analyze the ability of T‐cells to kill tumor cells, 4T1 cells were cocultured with activated CD8⁺ T cells in the presence of DMSO (control) or 6J1. After 60 h, the wells containing the 4T1 cells were washed with PBS twice to remove the T cells, and any surviving tumor cells were quantified with the CCK8 assay.

Flow cytometry was performed using the following fluorochrome-conjugated antibodies: CD3e Monoclonal Antibody (145-2C11) (Thermo Fisher Scientific, Waltham, USA), PE/Cyanine7 anti-mouse CD4 Antibody (BioLegend, San Diego, USA), anti-mouse CD8a Antibody (BioLegend, San Diego, USA), CD25 Monoclonal Antibody (PC61.5) (Thermo Fisher Scientific, Waltham, USA), PE/Cyanine7 anti-mouse TCR β chain Antibody (BioLegend, San Diego, USA), Purified anti-mouse/human IL-5 Antibody (BioLegend, San Diego, USA), Ki-67 Monoclonal Antibody (SolA15)(Thermo Fisher Scientific, Waltham, USA), and FOXP3 Monoclonal Antibody (FJK-16s) (Thermo Fisher Scientific, Waltham, USA). A Foxp3 Transcription Factor Staining Set was purchased from eBioscience (Thermo Fisher Scientific, Inc.). A Zombie Aqua Fixable Viability Kit was obtained from BioLegend. Collagenase-IV, 17β-estradiol (E2), DNase I, sesame oil, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA).