Ionomycin

Annexin A11-mEmerald overexpressing H69 cholangiocytes were seeded onto 8-well chambered coverglass (Thermo Scientific, Waltham, MS) and cultured for 96 hours until confluence. 24 hours after seeding, cells were transfected with ANO1-mCherry,^{38 (link)} kindly provided by Dr. Lily Yeh Jan (University of California, San Diego, CA). Live-cell confocal microscopy was performed in Leibowitz imaging medium without phenol red (Thermo Scientific, Waltham, MS) at 37°C on a Leica TCS SP8-SMD microscope (LEICA, Wetzlar, Germany). 10 μM ionomycin (Abcam, Cambridge, United Kingdom) in Leibowitz imaging medium was added to increase intracellular Ca²⁺ levels. Cells were imaged at 15–30 second intervals for a duration of 15 minutes at 37°C in ambient air. 2D colocalization analysis was performed using Huygens Deconvolution Professional software (Scientific Volume Imaging, Hilversum, The Netherlands). Cells that responded to ionomycin treatment (defined as annexin A11-mEmerald localization shift) and expressed ANO1-mCherry were selected as ROI for the calculation of Pearson’s correlation coefficient (using Costes threshold approach^{39 (link)}).

PBMC, HBEC and PLT from healthy individuals were analysed at resting state and following phorbol 12‐myristate 13‐acetate (PMA)/ionomycin (PI) activation in order to confirm that HLA‐F is expressed and is retained in the cytoplasm under healthy physiological conditions, as previously described in other cell types.³¹, ³²PMA (Abcam) and ionomycin (Sigma‐Aldrich) were solubilised in DMSO and used at a final concentration of 0.2 and 20 μg/ml, respectively.⁴⁵ Dimethyl sulfoxide (DMSO) and unstimulated cells (UNS) were used as reference and negative controls, respectively. PI activation was performed for 24 h (PBMC), 48 h (HBEC) or overnight (PLT) at 37°C according to experimental optimization based on cell viability vs. HLA‐F surface expression (data not shown).
Incubation with thrombin receptor activator peptide 6 (Trap 6) (Sigma‐Aldrich) was used as the PLT activation control (20 μM for 10 min).⁴⁶PI efficient activation was assessed by cytometry analysis using activating markers: the CD25 (alpha chain of the IL2 receptor) for PBMC and the P‐selectin expression for the PLT; respectively using the murine IgG2 anti‐CD25 antibody clone T07774‐PE (Beckman Counter) and the anti‐CD62P recombinant antibody clone REA389‐PE (Miltenyi) with the recombinant human IgG1 as isotype (clone REA293‐PE, Miltenyi).

Cells from ELISpot plate were collected in media supplemented with GolgiStop Protein Transport Inhibitor (BD Biosciences, NJ, USA) and incubated for 6 hr at 37°C. Positive control group was treated with 50 μM PMA (Abcam, UK), 1 mg/mL Ionomycin (Abcam, UK). Cells were then washed, blocked with Fc receptor (Biolegend, CA, USA), and stained with CD3-PE (clone HIT3a, Biolegend), CD4-PE/Cyanine7 (clone RPA-T4, Biolegend), CD8-FITC (clone RPA-T8, Cell Signaling) antibodies for 2 hr at 4°C. Cells were permeabilized for 20 mins at 4°C and then stained overnight with IFN-γ-APC (clone 4S.B3, Biolegend) antibody at 4°C.

Shields and Sang M3 Insect Medium, Dispase II, vinblastine, thapsigargin, EGTA and CaCl₂ were purchased from Sigma-Aldrich. CPA, and ionomycin were purchased from Abcam, Collagenase A was purchased from Roche. Fura-2/AM was purchased from Thermo Fisher. All other materials were analytical or of the highest available grade.

Isolated CD4⁺ T cells were stimulated during 22h with latency reversal agents (LRAs) at the following concentrations: 40 nM Romidepsin (Selleckchem), 30 nM Panobinostat (Selleckchem), 1 μM JQ1 (Sigma-Aldrich), 100 nM Ingenol-3-angelate (Sigma-Aldrich), 10 nM Bryostatin-1 (Tocris Bioscience), the positive control (PMA 81 nM plus Ionomycin 1 μM, both from Abcam), or the negative control (media alone, R10). Drugs were used at concentrations previously shown to be effective at reversing latency in studies performed in CD4⁺ T cells from HIV-infected individuals as well as studies performed in latency models in vitro [23 (link),32 (link),33 (link)]. All compounds were reconstituted in DMSO at the maximum concentration of 0.006%. Moreover, in order to prevent cell death induced by the reactivation of HIV and to evaluate the reactivation effect without confounding variables, cells were pre-treated with a pan-caspase inhibitor named Q-VD-OPh (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone, Selleckchem) [51 (link),52 (link)]. Q-VD-OPh is a potent inhibitor for caspases 1, 3, 8 and 9, which are involved in the intrinsic and extrinsic apoptotic pathways, inhibiting consequently the specific cell death induced by HIV [53 (link)–56 (link)]. In all experiments, cells were treated with 10 μM of Q-VD-OPh for at least 2h prior to the addition of the latency reversal agents.

Procedures for immunostaining, fluorescence microscopy and NET quantification are described in the Supplementary Materials file. Briefly, neutrophils were stimulated with 5 µM ionomycin ((IO), Abcam, Cambridge, MA, USA) for 2–2.5 h at 37 °C in a humidified atmosphere with 5% CO₂, washed and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA). The cells were immuno-stained overnight at 4 °C with antibodies of interest, followed by incubation with secondary antibody fluor staining. The neutrophils were next counterstained and mounted with Fluoroshield^TM (Sigma Aldrich), which contains 4′,6-Diamidino-2-phenylindole (DAPI) (Sigma Aldrich). Images were acquired on a fluorescence microscope. Morphologic quantification of NETs was performed on the basis of strict morphological criteria by two investigators as described in the Supplementary Materials file.

Single-cell suspensions were prepared from tumors as previously described [52 (link)] and stimulated prior to staining with panels of antibodies for flow cytometry analysis of the T cells. In total, 2.5 × 10⁶ cells were cultivated for 3 hours in 2 mL of Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, Merck KGaA) supplemented with 10% FCS (Biosera), 100 IU/mL penicillin, 100 μg/mL streptomycin (Biosera), and 50 µM 2-mercaptoethanol and containing 81 nM phorbol 12-myristate 13-acetate, 1.34 µM ionomycin, 2 µM monensin, and 10.6 µM brefeldin A (Abcam, Cambridge, UK).