Cd107a apc

As the source of effector cells, we used both PBMCs and BM samples derived from MM patients at different state disease and PBMCs from healthy donors. K562 were used as target cells that were co-incubated with effector cells in complete medium at 2.5:1 effector/target (E/T) ratio for 2 h at 37 °C and 5% CO₂ [11 (link)]. In some experiments, different human cell lines such as SKO-007(J3), ARK and ARP and primary malignant plasma cells were used as targets. Thereafter, cells were washed with PBS/2% FCS and stained with the lysosomal marker CD107a/APC (BD Biosciences, San Jose, CA) and anti-CD3/APC-H7, anti-CD56/PE, anti-CD45/PE-Cy7, anti-CD16/PerCP for 45 min at 4 °C. All the samples were acquired using a FACSCanto II (BD Biosciences, San Jose, CA) and data analysis was performed using the FlowJo 9.3.2 program (TreeStar, Ashland, OR, USA).

The indicated target
cells were stained with Cell Trace Yellow following the manufacturer’s
recommended protocol (ThermoFisher). Cells were then stained with
the indicated antibody adaptors/amounts and were washed twice with
flow cytometry buffer and resuspended in cell culture media. 10,000
target cells per well were then cocultured with 50,000 mSA2 CAR T
cells (effector/target = 5:1) in a 96-well V-bottom plate. All coincubation
experiments with mSA2 CAR T cells were performed in DMEM media supplemented
with 10% FBS. For UV-cleaving experiments, wells were then exposed
to 365 nm light using a 365 nm LED (Mouser, 416-LST101G01UV01). For
small-molecule cleavage experiments, the indicated amounts of 2DPBM
were added to wells containing all cell populations. Cells were then
incubated at 37 °C for 24 h. Cells were then evaluated and stained
with fluorescently labeled antibodies recognizing T cell activation
markers: CD69-BV711 (BD Biosciences) and CD107a-APC (BD Biosciences)
and GFP were used for NFAT-induction experiments. Marker expression
was specifically evaluated on mSA2 CAR+ by gating the TagBFP+ population.
Supernatants from primary cell assays were also collected and analyzed
for IFNγ by ELISA (BioLegend).

Tumors were minced into small (1–2 mm³) pieces, and digested using a Tumor Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. The resulting supernatant was filtered through a 200-mesh sieve and washed twice with ice-cold PBS¹² . healthy donors were incubated for 5 min with Fc-block (BD Biosciences). The phenotype of MDSCs in patients was determined by a combination of surface markers including HLADR-PerCP-Cy5.5 (Clone L243, BD Biosciences), CD33-FITC (Clone P67.6, BD Biosciences), CD11b-APC (Clone CBRM1/5, BD Biosciences), and CD45-BV421 (Clone HI30, BD Biosciences). Afterward, PD-L1 membrane expression was evaluated using PD-L1 (CD274)-PE (Clone 29E.2A3, BD Biosciences).
For the mouse model, single-cell suspensions were stained with either a myeloid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD11b-APC-Cy7 (Clone M1/70, BD Biosciences), Gr-1-PE (Clone RB6-8C5, BD Biosciences), and PD-L1-APC (Clone 10F.9G2, BD Biosciences) or a lymphoid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD4-PerCP/Cy5.5 (Clone RM4-5, BD Biosciences), CD8-PE (Clone 53–6.7, BD Biosciences), CD62L-APC (Clone MEL-14, BD Biosciences), and CD107a-APC (Clone 1D4B, BD Biosciences). Flow cytometry was performed with a BD FACS Canto II flow cytometer. Data were analyzed using FlowJo software (TreeStar, Inc, Ashland, OR).

Cells were prepared in a single-cell suspension and washed with phosphate-buffered saline. After incubation with FcR Blocking Reagent (miltenyi, 130-059-901) for 10 min, NK cells were stained with fluorescence antibodies for 30 min at 4°C. The following antibodies were used in this research (all antibodies from BioLegend unless otherwise indicated): FITC-conjugated antibody to human CD3 (300306), CD56-PE (304606), CD107a-APC (328620), PD-1-APC (BD Pharmingen, 558694), Perforin-APC (353311), IFN-γ-APC (502512), CFSE, and PI (BD Pharmingen). GCMSCs were assessed for membrane expression of selected markers by using CD19-FITC, CD29-PE, CD34-FITC, CD45-FITC, CD90-PE, and CD105-PE antibodies (all antibodies above from eBioscience). For intracellular cytokine staining, NK cells were stimulated for 5 h with a 2 μl/ml cell stimulation cocktail (plus protein transport inhibitors) (eBioscience) containing phorbol 12-myristate 13-acetate, ionomycin, brefeldin A, and monensin. Then, cells were stained for surface markers, fixed, and permeabilised with eBioscience FoxP3 fixation buffer according to the manufacturer's instructions. The fixed cells were then stained with perforin, CD107a, and IFN-γ.