To study the immune cells in distant tumors, right tumors were harvested from mice in different groups (n = 5) and stained with Viobility 405/520 Fixable Dye (Miltenyi), CD45.2 APC-CY7 (Biolegend, Clone: 104), CD3e FITC (Biolegend, Clone: 17A2), CD8a PE-vio615 (Miltenyi, Clone: REA601), PD-1 PE (Biolegend, Clone: 29F.1A12), TIM3 APC (Miltenyi, Clone: REA602), CD4 VioBlue (Miltenyi, Clone: REA604), and Foxp3 Alexa Fluor 700(Biolegend, Clone: MF-14.1A12) antibodies, according to the manufacturer's protocols.
Briefly, tumor tissues were cut into small pieces and digested with collagenase and DNase. Then, cell suspension was filtered through a 75-μm cell mesh and resuspended in PBS (pH 7.4) with 0.5% FBS for further analysis. Flow cytometric analysis was performed using a FACS LSRFortessa flow cytometer (BD).
Tumor-infiltrating cytotoxic T lymphocytes (CTL) and helper T cells were CD45+CD3+CD4−CD8+ and CD45+CD3+CD4+CD8−, respectively. Then, the expressions of PD-1 and TIM-3 in cytotoxic T lymphocytes were analyzed. Further, CD4+ helper T cells were classified into regulatory T cells (Tregs) (CD3+CD4+Foxp3+) and effective T cells (CD3+CD4+Foxp3−).
Briefly, tumor tissues were cut into small pieces and digested with collagenase and DNase. Then, cell suspension was filtered through a 75-μm cell mesh and resuspended in PBS (pH 7.4) with 0.5% FBS for further analysis. Flow cytometric analysis was performed using a FACS LSRFortessa flow cytometer (BD).
Tumor-infiltrating cytotoxic T lymphocytes (CTL) and helper T cells were CD45+CD3+CD4−CD8+ and CD45+CD3+CD4+CD8−, respectively. Then, the expressions of PD-1 and TIM-3 in cytotoxic T lymphocytes were analyzed. Further, CD4+ helper T cells were classified into regulatory T cells (Tregs) (CD3+CD4+Foxp3+) and effective T cells (CD3+CD4+Foxp3−).