Ionomycin

For detection of intracellular cytokines, cells after coculture were activated with PMA (50 ng/ml; Sigma) and ionomycin (1 μM;Merk) for 6 h in the presence of GolgiPlug (1 μl/ml BD Biosciences) for the final 4 h. Cytokines in coculture supernatants were analyzed by LEGENDplex Th cytokine panel (BioLegend).
For activation of eTAC, cDC and pDC, purified subsets were cultured at 5 × 103 cells per well in the presence of LPS (100 ng/ml; Sigma), poly(I:C) (25 μg/ml;Sigma) and R848 (25 μg/ml; Invivogen), or with PMA (50 ng/ml; Sigma) and ionomycin (1 μM;Merk). Culture supernatants were harvested after 24 h and analyzed by a custom LEGENDPlex human inflammation panel (BioLegend).

Human Tumor Necrosis Factor-α (TNF-α) (PeproTech, Cranbury, NJ, USA, 300-01A) was used at a final concentration of 10 ng/mL for 16 h (hours) with a vehicle control containing Phosphate-buffered saline (PBS) to build the cell line. Phorbol 12-myristate 13-acetate (PMA) (MilliporeSigma, Burlington, MA, USA, P8139) and Ionomycin (MilliporeSigma, Saint Louis, MO, USA, I0634) were used for treatments marked “PMA” (10 ng/mL of PMA and 0.1 µM of Ionomycin) with DMSO treatment as the corresponding control at the indicated time points in different experiments (16 h for western blots, 16 h for flow cytometry, and 0.5, 1, 4, 16 h for the RT-qPCR and ChIP-qPCR time courses).

Intracellular staining was visualized using a published immunofluorescence protocol (Subramanian et al., 2011 (link)). Briefly, 2 × 10⁶ cells/mL were resuspended in complete medium (RPMI-1640 containing 10% FCS, 1 mM/L pyruvate, 200 μg/mL penicillin, 200 U/mL streptomycin, 4 mM/L L-glutamine, and 5 × 10^-5 mol/L 2-β-ME), with PMA (50 ng/mL), ionomycin (500 ng/mL), and Brefeldin A (10 μg/mL, Sigma–Aldrich) for 4 h. For intracellular IL-10 detection, a modification was followed for the immunofluorescence staining protocol (Yanaba et al., 2008 (link)). Briefly, isolated leukocytes or purified cells were resuspended (2 × 10⁶ cells/mL) in complete medium and cultured with LPS (10 μg/mL) in addition to PMA (50 ng/mL), ionomycin (500 ng/mL), and Brefeldin A (10 μg/mL; all reagents from Sigma-Aldrich) for 4 h. Fc receptors were blocked with anti-FcR mAb (2.3G2, BD Pharmingen) before cell surface staining, fixed, and permeabilized with the Fixation/Permeabilization buffer (eBioscience), according to the manufacturer’s instructions. Permeabilized cells were washed with 1× Permeabilization Buffer (eBioscience) and stained with APC-conjugated anti-IL-10 mAb (JES5-16E3, eBioscience). Isotype matched mAb served as negative controls to demonstrate specificity and to establish background IL-10 staining levels.

Synovial mononuclear cellular fractions at a concentration of 10⁶/mL were stimulated with 0.1 μg/mL lipopolysaccharide (LPS) or 10 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin (all from Sigma). Synovial fibroblasts were cultured to confluency (5×10⁴/mL), treated for 3 h with IL-1β (10 ng/mL, Peprotech), washed and cultured for another 24 h. GM-CSF levels in supernatants were determined by BD Biosciences OptEIA ELISA according to the manufacturer's instructions. For intracellular cytokine staining, CD4+ T cells were stimulated with PMA and ionomycin for 5 h with 10 μg/mL Brefeldin A (Sigma) added after 1 h. Cells were then harvested, fixed and permeabilised and stained using the FoxP3/transcription factor staining buffer set (eBioscience) according to the manufacturer's instructions. Cells were preincubated with 2% mouse and rat serum (both Sigma) prior to antibody labelling.

PBMCs were TCR-stimulated for 16 h with soluble 2 μg/mL anti-CD3 and 2 μg/mL anti-CD28 (both Sanquin), 10 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin (both Sigma-Aldrich), or left untreated. To measure intracellular cytokines, 10 μg/mL brefeldine A (Sigma-Aldrich) was added 1 h after addition of anti-CD3/CD28 or PMA/ionomycin. After incubation and cellular staining, cells were acquired on a Fortessa Cell Analyzer.

J-Lat full-length cells (clone 8.4, NIH AIDS Reagent Program catalog no. 9847) were used as a model for latent HIV infection (35 (link)). For the VIP-SPOT assay, 10⁴ J-Lat cells per well were seeded in plates precoated with 10 μg/ml of the anti-p24 capture antibody (clone 39/5.4A, catalog no. ab9071; Abcam) and cultured for 48 h in the presence of different latency reversing agents (LRAs) prepared in culture medium, namely, 20 nM PMA plus 0.5 μg/ml ionomycin (catalog no. P1585 and I3909; Sigma-Aldrich), 10 ng/ml TNF (CellGenix), 1 μM vorinostat (SAHA), 10 nM and 100 nM panobinostat, 5 nM and 40 nM romidepsin, and 100 nM bryostatin (all from Santa Cruz Biotechnologies). Cells cultured under the same stimulus conditions were prepared in parallel in 48-well plates at a ratio of 10⁶ cells per well for further cytometry analysis of GFP expression and viability and for determination of cell-associated HIV RNA, as previously described (36 (link)). Briefly, one-step reverse transcription ddPCR (Bio-Rad) was used to quantify HIV RNA with a primer/probe set targeting the viral 5′ long terminal repeat.
For primary cells, latency reversing agents that were alternative to coated anti-CD3/anti-CD28 in the VIP-SPOT assay were tested as follows: 10 μg/ml PHA (L1668; Sigma-Aldrich), PMA at 2 nM or 100 nM combined with 0.5 μM ionomycin, 5 nM romidepsin, and 100 nM bryostatin.