Tim 3 apc

The following antibodies were used in this study for T cell phenotyping: CD3-PerCP (clone SK7/Cat# 347344), CD25-APC AF700, CD4-Krome Orange (13B8.2/A96417), CD8-Pacific Blue (B9.11/A82791), CD3-APC (Beckman Coulter Inc. Brea, CA), Rat Anti-Mouse IgG1-APC (X56/550874) (BD Biosciences, San Jose, CA). PD-1-Percp Cy7 and TIM3-APC (BD Biosciences, San Jose, CA) were used as markers of T cell exhaustion. MUC1 antigen expression by tumor cells was measured using anti-MUC1, (Santa Cruz Biotechnology. Inc., Dallas, TX). CAR molecules were detected using Goat anti-human F(ab’)2 antibody conjugated with AlexaFluor647 (109–606-097) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Cells were stained with saturating amounts of antibody (~5uL) for 20 min at 4 °C, washed (PBS, Sigma-Alrich, St. Louis, MO), and then acquired on Gallios™ Flow Cytometer (Beckman Coulter Inc., Brea, CA). Analysis was performed using Kaluza® Flow Analysis Software (Beckman Coulter Inc.).

Fresh peripheral blood samples were collected from healthy volunteers. The human lung cancer cell line A549 was derived from the passaged cell line preserved in the immunology laboratory of Zunyi Medical University. Tim-3 blocking antibody (AB_1877089) and PD-1 blocking antibody (AB_2820104) were purchased from Biolegend Inc., USA. The cell factors rhIL-4, rhGM-CSF, rhIFN-γ, rhTNF-α, and CD3McAb were purchased from Peprotech Inc., USA. Flow cytometry antibodies CD3-PE-Cy5, CD56-RD1, CD4-FITC, CD8-PC-Cy7, Tim-3-APC, and PD-1-PE were purchased from BD Inc., USA. The 1640 medium was purchased from Thermo Scientific Inc., USA. The GTT-551 H3 medium was from TaKaRa Company, Dalian, China.

Surface staining was performed with directly conjugated monoclonal antibodies for 20 min at room temperature for human samples. The cells were washed and resuspended in PBS before flow cytometric analysis. The monoclonal antibodies used were anti‐human CD4–Percp‐Cy5.5/APC‐H7, CD8–APC (allophycocyanin)‐R700/V500, PD‐1–PE (phycoerythrin)–Cy7, TIM‐3–APC, TIGIT–BV605, 2B4–AF700, and CD160–PE (BD Bioscience San Diego, CA, USA). Intracellular staining was carried out by using a fixation/permeabilization kit (BD Bioscience) after resuspension according to the manufacturer's instructions. Ki67–PE (BD Pharmingen) was added and incubated for 20 min at room temperature.
The surfaces of mouse samples were stained with direct‐conjugated monoclonal antibodies for 30 min at 4 °C. After incubation, the cells were washed and resuspended in phosphate‐buffered saline before flow cytometry analysis. The monoclonal antibodies used were anti‐mouse CD3–Percp, CD4–APC‐H7, CD8–FITC (fluorescein), CD44–PE–Cy7, and CD62L–APC (BD Bioscience San Diego, CA, USA). Detailed antibody information was listed in Table S3 (Supporting Information).