Golgistop

Following 14 days of ex vivo expansion as described above, MACS^® GMP ExpAct Treg Beads were removed, then CD4⁺CD25⁺CD127^lo/- and CD4⁺CD25⁺CD226^- sorted Tregs were immediately assessed for intracellular cytokine expression. Cells were either stimulated with PMA (10 µg/mL; Thermo Fisher) and ionomycin (500 nM; Thermo Fisher) or unstimulated for four hours in the presence of GolgiStop (0.66 µL/mL; BioLegend, San Diego, CA, USA). Stimulated cells underwent staining for viability and extracellular markers, including CD4-BV510, CD25-APC, CD127-PE, CD226-PE-Cy7, and TGF-β1-PerCP-eFluor 710 (Table 1), and were subsequently permeabilized as described above. Following permeabilization, cells were stained with the FOXP3-AF488 and Helios-Pacific Blue cocktail, as well as an intracellular cytokine cocktail consisting of IL-10-BV421, IL-17A-BV605, IFN-γ-BV570, TGF-β1-Alexa Fluor 647, and TNF-BV650 (Table 1). Fold change of cytokine expression levels were assessed by dividing the gMFI of the stained, stimulated sample by the gMFI of the applicable stained, unstimulated control. Differences between fold change of cytokine expression are reported as Z-scores, [Z = (Mean fold change for Treg subset – Mean fold change for all Tregs assessed)/standard deviation of the sample].

PBMC were cultured with immobilized BFP in the absence of target cells or with patient AML cells for 24 h. Monensin (GolgiStop, BD Biosciences) was added 8 h prior to flow cytometric analysis. Intracellular flow cytometry was conducted using the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) according the manufacturer’s instructions. For detection of intracellular proteins, fluorescence-conjugated granzyme B and perforin antibodies (both from BioLegend) were used in 1:25 dilutions. For assessment of cytokine secretion, PBMC were treated as described above but without adding GolgiStop to allow for analyses of cytokine secretion into supernatants by Legendplex assays (BioLegend).

After treatment with CFP or LPS, the mLNs from C57BL/6 mice were harvested. The mLN cells were further incubated at 37 °C with Golgistop™ (2 μM monensin solution; BioLegend) for 2 h in vitro. After washing with PBS, the cells were stained using the Zombie Violet Fixable Viability Kit (BioLegend, San Diego, CA, USA) and fixed with fixation buffer (BioLegend, San Diego, CA, USA) at 4 °C for 20 min after staining with surface Abs. The cells were permeabilized using permeabilization wash buffer (BioLegend, San Diego, CA, USA) and intracellular proteins were stained with Abs at 25 °C for 30 min. After washing, the cells were re-suspended in PBS and analyzed using a flow cytometer (ACEA Biosciences Inc., Santa Clara, USA).

NK degranulation activity was measured by flow cytometry at a fix 5:1 NK effector to target cell ratio. Targets were HLA-E*01:03/VL9-K562 cells pulsed with canonical and non-canonical (HIV-derived) peptides. NK cells sorted from cryopreserved PBMC and peptide-pulsed target cells were co-cultured for 4 hours in the presence of Golgi Stop, Golgi Plug and APC anti-human CD107 (Biolegend) and stained for surface markers: SK7 Amcyan anti-CD3 (BD), 5.1H11 BV785 anti-CD56 and B73.1 BV-605 anti-CD16 (Biolegend). Subsequently, cells were fixed, permeabilized (Fix&Perm Cell Permeabilization Kit, Thermofisher) and stained with 6401.1111 FITC anti-human TNF-α and B27 PE-Cy7 anti-human IFN-γ (BD). Approximately 20,000 events per samples were acquired on a Canto II BD Cytometer and analyzed using FlowJo v10 software.