Cd56 pe cy7 hcd56

PBMCs were incubated with 1 ng/mL IL-15 overnight at a cell density of 2x10⁶ cells/mL and then co-cultured with selinexor- or leptomycin B-treated lymphoma cell lines at an E:T ratio of 5:1 for 4 hours at 37°C. Immediately before co-culture, 0.17 µg/mL α-CD107a (LAMP)-eFluor660 (clone eBioH4A3, Invitrogen) was added to PBMCs. After 1-hour of co-culture, GolgiStop (per manufacturer recommendations, BD Biosciences) was added. Following 4-hours of incubation, cells were incubated with 10% human serum at 4°C for 15 minutes before surface staining with antibodies against CD3-PerCP (clone UCHT1, Biolegend), CD56-PE/Cy7 (HCD56, Biolegend) and NKG2A-FITC (REA110, Miltenyi Biotech) in FACS buffer (PBS, BSA 1%, Sodium Azide 0.05%) at 4°C for 30 minutes. Cells were then permeabilized and fixed with BD Cytofix/Cytoperm (BD Biosciences) per manufacturer recommendations and stained with anti-IFNγ-BV421 (BD Biosciences) at 4°C for 30 minutes. Cells were then washed twice with 1X Perm/Wash buffer and immediately assessed by flow cytometry using a BD FACS Aria II (BD Biosciences) and FACSDiva software (BD Biosciences) as above.

We used flow cytometry with hierarchical gating strategies adapted from established guidelines for analysis of human PBMCs and suitable for cryopreserved samples (18 (link), 19 (link)). Our panel offered survey phenotyping of T cell subsets, including Tregs, as well as B cells and NK cells (see Figure S1 in Supplementary Material). The following fluorochrome-conjugated monoclonal antibodies were used: CD3 BV605 (OKT3, Biolegend), CD4 Alexa 700 (RPA-T4, Biolegend), CD8α V500 (SK1, BD Biosciences), CCR6 PE-Cy7 (G034E3, Biolegend), CXCR3 PE (G025H7, Biolegend), CD127 APC (A019D5, Biolegend), CD25 BV421 (M-A251, Biolegend), CD19 V500 (HIB19, BD Biosciences), CD56 PE-Cy7 (HCD56, Biolegend), CD20 PE (2H7, BD Biosciences), CD14 V450 (MFP9, BD Biosciences), and CD16 FITC (3G8, BD Biosciences). We also used the commercially available IOTest^® Beta Mark Kit (Beckman Coulter) for quantitative determination of the human TCR Vβ repertoire in CD4⁺ and CD8⁺ cells [nomenclature from Ref. (20 (link))].