Cd107a pe

100μL of cells were added to a flat-bottom 96-well tissue culture plate. The following was added to cells: 100μL of 200 μM N11 HMPV peptide or NP366 for irrelevant control diluted 1:10 in RPMI/10% FBS, 6μL CD107a-PE, and 22μL BFA (BD Cat. #51-2301KZ)/Monensin (BD Cat. #2092KZ). In addition, 1:1000 PMA/ionomycin instead of peptide was added to one aliquot of cells for a positive control. Cells were incubated for 5 h at 37 °C.
For both conditions: After either tetramer staining or peptide stimulation, cells were stained with Live/Dead dye 1:1000 in PBS for 12 min, washed 1 × with PBS, and blocked with αCD16/32 Fc block (Tonbo Biosciences Cat. #70–0161-M001) 1:100 in FACS buffer for 10 min. For surface staining, cells were stained with surface antibody 1:100 in BD Brilliant Stain Buffer (BD Cat. #566,349) buffer for 30 min at 4 °C. Cells were spun down at 1500 rpm for 3 min and washed 1 × with FACS buffer.

For binding ability of CHI3L1 to receptor, NSCLC cell line H1299 was harvested and thoroughly mixed with His-tagged rCHI3L1 or nCHI3L1 Ab for 1 h. Subsequently, cells were incubated with FITC-labeled 6x-His Tag antibody at 4 °C for 1 h and analyzed by FACS. To determine PD-1 and TIM-3 presentation on CD8⁺ T cells, cells were stained using anti-human PD-1-BB515 (BD Bioscience) and TIM-3-BV421 (BD Bioscience) antibodies. After cell surface staining, intracellular CD107a was stained with CD107a-PE (BD Bioscience) antibodies to detect T cell activities.
Allograft tumor tissues were digested with 0.1 mg/mL collagenase (Sigma-Aldrich) and 1 mg/ml dispase II (Sigma-Aldrich) at 37 ℃ for 30 min and meshed. Resulting single-cell suspension were stained with CD4, CD8, CD11b, CD25, CD86, CD206, CTLA4 and Foxp3 antibody (BD Bioscience). The data were recorded by CytoFLEX (Beckman Coulter). The detailed antibodies conditions are listed in Table S2.

NK cell activation, degranulation, and cytokine secretion in PBMC from healthy donors were analyzed by co-culturing of 200,000 AML cells or patient-derived AML cells with 500,000 PBMC at an E:T ratio of 2.5:1 with indicated treatment (1 µg/mL). To determine NK cell degranulation, GolgiPlug and GolgiStop (BD Biosciences) were added to the co-cultures and cells were harvested after 4 hours (h), stained for CD107a-PE (clone: H4A3, BD Pharmingen) and analyzed by FACS. NK cell activation of cells harvested at 24 h and 72 h determined by measuring the expression of CD69-PE (clone: FN50, BD Pharmingen) and CD25-PE (clone: BC96, BioLegend) by flow cytometry. NK cells within PBMC were characterized as CD3^-CD56⁺CD16⁺ subset. To investigate cytokine secretion by NK cells, supernatants from 24 h co-culture were collected and analyzed for the secretion of granzyme A, granzyme B, perforin, granulysin, TNF, IL-2, IFNγ, and IL-10 secretion using Legendplex (BioLegend) according to the manufacturer’s protocol. All experiments were performed as biological replicates from different donors.

2×10⁵ CD4⁺ T cells were cultured alone or stimulated with 50 ng/mL IL-7 or IL-15 for 4 days. Four hours before analysis, CD107a-PE (BD Biosciences) was added to the culture and cells were either left un-stimulated or stimulated with 2 µg/mL functional grade anti-human CD3 monoclonal antibody (Invitrogen); BD GolgiStop (BD Biosciences) was added for the last 3 h of culture. Cells were then stained with CD4-FITC, CD28-APC, and 7-AAD (BD Biosciences) and CD107a expression was quantified by flow cytometry.

Sarcoma cells lines were co-cultured with PBMCs from at least 3 donors at selected 10:1 E:T ratio in the presence of increasing concentrations of pAXL×CD3ε (0.1 µg/mL, 1 µg/mL, and 2.5 µg/mL) or negative control, and were incubated for 72 h at 37 °C and 5% CO₂. T cells were stained using anti-human CD4 (SK3) FITC (#345768), CD8 (SK1) APC-Cy7 (#641400), CD25 APC (#555434), CD69 PE (#555531), CD3 (UCHT1) PerCP-Cy5.5 (#560835), CD45 (HI30) BV510 (#563204) and CD107a PE (#555801) (BD Biosciences), for 4 h at 37 °C and 5% CO₂. T cells were selected as CellTrace™ Violet-positive, gated for CD4-, CD8- or CD3-positive, and CD69-, CD25- or CD107a-positive cells. The intracellular production of cytokines and cytolytic enzymes was investigated adding brefeldin A 10 mg/mL. After 4 h, cells were incubated with surface antibodies and treated using FIX&PERM^® kit (Nordic MUbio, Susteren, The Netherlands), according to the manufacturer’s guidelines. Subsequently, cells were incubated with anti-TNFα PE-Cy™7 (Mab11) (#560678), anti-IFNγ PE (#559327) and anti-Granzyme B (AlexaFluor^®647) (#560212) (BD Biosciences) for 15 min at RT in the dark. Samples were finally washed in PBS 1X and analyzed by a flow cytometer.