Tcrαβ

Approximately 500,000 cells were washed once with FACS buffer [PBS (Sigma, P3813) containing 1% fetal bovine serum], pelleted, and antibodies were added in appropriate amounts. After incubation for 20 min at 4°C in the dark, the cells were washed with FACS buffer. The GD2.CAR was detected with the 1A7 idiotypic antibody (19 (link)) followed by PE-conjugated rat anti-mouse IgG. The cells were stained with CD25, CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD62L, TCR γ/δ, CD56, CD28, PD-1, LAG-3, and CD95 monoclonal antibodies (Becton Dickinson, Franklin Lakes, NJ) TCR α/β (Biolegend, #306718) for phenotyping, and with Pro5® CLG Pentamer (ProImmune Ltd., Oxford, UK) for EBV A^*02:01 LMP-2 (426–434) specific T cells. To stain for apoptosis, 100,000 cells were removed from culture, resuspended in 1X binding buffer (Becton Dickinson BD) with 5 μL of annexin V (Becton Dickinson BD,) and 4 μL of 7-AAD (Becton Dickinson BD,), and incubated at room temperature for 15 min. The cells were then immediately assessed after incubation without a washing step.

For detecting the anti-PD-L1 scFv, a PD-L1-Fc Fusion protein was used (Sino Biological). For detecting the anti-CD19 scFv, a monoclonal anti-FMC63 antibody was used (Acro Biosystems). For T cell phenotype characterization, the following antibodies were used: mouse anti-human CD3 (clone: UCHT1), TCRαβ (clone: IP26), CD4 (clone: RPA-T4), CD8 (clone: RPA-T8), CD45RA (clone: HI100), CD62L (clone: DREG-56), CCR7 (clone: G043H7) from BioLegend. To evaluate cytotoxicity, Live/Dead Fixable Yellow (Thermo Fisher Scientific) and Annexin V (BioLegend) were used to stain the effector and tumor cells in the co-culture. To examine the degranulation, CD107a (BioLegend) and Granzyme B (BD) were used in the assay. FACS data were acquired on Attune (Thermo Fisher Scientific) and analyzed with Attune software.

Single-cell suspension from LMCs was prepared, the cellular phenotype was determined by flow cytometry. The following monoclonal antibodies were used: Gr-1, CD11b, Ly6C, F4/80, CD11c, MHC-II, CD2, CD3, TCRαβ, CD4, CD8, α-GalCer mouse CD1d complex (L363), NK1.1 (PK136), FoxP3 and CD25 (Biolegend). Briefly, LMCs were suspended in PBA and stained with antibody cocktail from 30 min at 4 °C and washed with PBA. To determine the intracellular expression of FoxP3, LMCs were fixed (fixation buffer; Biolegend) and permeabilized (Perm/Wash buffer; BD Pharmingen, San José, CA, USA). After permeabilization, cells were washed twice and then stained with anti-human perforin mAb. Cells were washed and resuspended in 1% paraformaldehyde.
The percentage of RAW macrophages expressing CD3 and transmembrane TNF (tmTNF) was assessed by flow cytometry, following the same protocol to LMC’ cellular phenotype.
Fluorochrome-labeled isotype-matched control antibody was used to evaluate background staining. After incubation with antibodies, cells were washed twice in PBA and data collected using a FACs CyAn and analyzed with FlowJo software. 100,000 events were acquired per sample.