Anti cd69 percp

Cell surface CAR expression was assessed using flow cytometry by labelling with anti-MYC antibody (Clone 9B11, Cell Signaling, Danvers, MA, USA) at 4 °C for 45 min and detection of intracellular mCherry expression. Confirmation of the peptide-MHC complex on the cell surface of target cells were performed with staining on ice for one hour, with a recombinant scFv tetramer-Atto-488.
For analysis of T cell activation, CAR T cells and tumour cells were co-incubated for 10 h in T cell media at a 1:1 ratio at 37 °C and 5% CO₂, in triplicate. Positive control for CAR stimulation was provided using plate bound unconjugated MYC-tag (Clone 9B11, Cell Signaling, Danvers, MA, USA), pre-coated overnight 4 °C at in PBS. Cells were then incubated with anti-CD25 APC (Clone PC61, BioLegend, San Diego, CA, USA), anti-CD69 PerCP (Clone H1.2F3, BD Biosciences, East Rutherford, NJ, USA), anti-CD4 FITC (Clone GK1.5, produced at WEHI Bundoora), and anti-CD8 PE antibodies (Clone 53-6.7, BD Pharmingen, East Rutherford, NJ, USA) at 4 °C for 30 min. Cells were analysed on a Fortessa X20 (BD Biosciences, East Rutherford, NJ, USA) and analysis performed on FlowJo software (TreeStar, Ashland, OR, USA). For analysis, cells were gated on live cells, CD4 and CD8, then CD25 and CD69.

Splenocytes were treated with ACK buffer for red cell lysis and washed with RPMI with 10% FBS. The spleen, heart, lymph node, and liver cells were stained with H2K^k-TEWETGQI multimer for 10 min at RT. The cell surface was stained for 30 min at 4°C. The following antibodies were used for surface staining: anti-CD3 APCcy7 (clone 145-2C11, BD), anti-CD8 PERCP or anti-CD8 PACIFIC BLUE (clone 53-6.7, BD), anti-CD11a FITC (clone 2D7, BD), anti-CD11c APCcy7 (clone HL3, BD), anti-CD44 FITC (clone IM7, BD), anti-CD62L PE (clone MEL-14, BD), anti-CXCR3 PERCP/Cy5.5 (clone 173, BioLegend), anti-CD27 FITC (clone LG3A10, BD), anti-CD4 PEcy7 (clone RM4-5, BD), anti-KLRG1 FITC (clone 2F1, eBioscience), anti-CD49d PEcy7 (clone R1-2, BD), anti-CD69 PERCP (clone H1.2F3, BD), anti-CD43 PEcy7 (1B11, BioLegend), anti-CD95 PEcy7 (clone JO2, BD), anti-CD25 FITC (clone LG3A10, BD), anti-CD127 PE (clone SB/199, BD), anti-CD122 FITC (clone TM-β1, BD), anti-CD38 PE (clone 90, BD), anti-β7 PERCP (clone FIB27, BioLegend), anti-CD31 FITC (clone MEC 13.3, BD), anti-CD272 PE (clone 8F4, eBioscience), anti-PD-1 FITC (clone J43, eBioscience), anti-CTLA-4 PE (clone UC10-4B9, eBioscience), and anti-CCR7 PE (clone 4B12, BD). At least 500,000 cells were acquired on a BD FACS Canto II flow cytometer and analyzed with FlowJo 8.7.

The following monoclonal antibodies were used to characterize MoDC: anti-CD86 FITC, anti-CD1a PE, anti-HLA-DR PERCP, anti-CD83 APC, anti-CD14 APC-H7, anti-CD80 FITC, anti-CD40 PE, anti-HLA-I APC, anti-CCR7 PE-Cy7, anti-CCR5 APC-H7 from BD Biosciences, and anti-BT3A.1 PE from BioLegend. To evaluate γδ T lymphocytes phenotype we used anti-Vδ2 FITC, anti-CD3 PE, anti-CD69 PERCP, anti-CD45RA PE-Cy7, anti-CD27 APC, anti-CD16 PACIFIC BLU, anti-CD25 APC-H7 (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide, and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 4% paraformaldehyde, and analyzed using a FACS Canto II (Becton Dickinson). Since the presence of 2 purified populations, the gating strategy was performed as follow: dead cells were excluded by scatter characteristics; MoDC were identified by morphological parameters (FSC vs SSC); gated cells were then analyzed for the expression of the molecules described above. T lymphocytes were first gated by using morphological parameters; in this gate Vγ9Vδ2 T cells were identified as Vδ2+CD3+. Analysis was carried out by using Facs Diva software (Becton Dickinson). The histogram overlays were performed by FlowJo software (TreStar, Olten, Switxìzerland).