Ficoll hypaque gradient

Venous blood was collected in EDTA tubes and PBMCs were isolated using a Ficoll-Hypaque gradient according to standard protocol (Axis-Shield Diagnostics, Dundee, UK). PBMCs were labeled with CellTrace Violet (ThermoFisher Scientific, Bleiswijk, the Netherlands) and were stimulated with phytohemagglutinin (PHA, 5 µg/ml, ThermoFisher Scientific), ConA (10 µg/ml), anti-CD3 (0.5 µg/ml, Sanquin, Amsterdam, the Netherlands) or anti-CD3/CD28 stimulator beads (0.5 bead per PBMC) with or without recombinant human IL-2 (1, 10 or 100 IU/ml, R&D Systems, Minneapolis, MN, USA) or IL-15 (1, 10 or 100 µg/ml, R&D Systems) for the indicated time-points. In some experiments, tofacitinib (Pfizer, New York, NY, USA; 200 or 1000 µM) was added to the anti-CD3/CD28 stimulated conditions. Cells were cultured in Iscove’s modified Dulbecco’s medium (ThermoFisher Scientific) supplemented with heat-inactivated fetal calf serum, Glutamax (ThermoFisher Scientific), 2-mercaptoethanol, penicillin, and streptomycin. For retroviral viral transduction, PBMCs from a healthy donor were isolated and T cell blasts were generated by culturing PBMCs with 1 µg/ml PHA (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 72 h in RPMI-1640 medium supplemented with 10% human serum (#H4522, Sigma-Aldrich) and expanded in culture with 100 IU/ml recombinant human IL-2 (R&D Systems).

Whole blood samples (5 ml) from syphilis patients and healthy donors were used for peripheral blood mononuclear cells (PBMC) isolation. PBMC were purified from peripheral blood by centrifugation using a Ficoll-Hypaque gradient (Axis-Shield). Because resting cells do not normally produce cytokines, cells were stimulated in vitro in order for the respective cytokine genes to be activated for intracellular cytokine staining. Phorbol myristate acetate (PMA) and ionomycin are unspecific stimulator that trigger a strong production of cytokines in vitro and are widely used to evaluate intracellular cytokine production from various T lymphocyte subpopulations [18] (link). Monensin is used to prevent the intracellular transport of cytokines from Golgi apparatus for enhancing the sensitivity of the detection. Accordingly, PBMC were seeded into 24-well culture plates (Corning) at 2×10⁶ cells/well and stimulated ex vivo with PMA (50 ng/ml) (Sigma) and ionomycin (1 µg/ml) (Sigma) for 4 hours. Monensin (2 uM) (eBioscience) was then added at the start of stimulation. CSF (10 ml) was centrifuged at 4°C immediately after spinal tap, and cells were stimulated as described above.

Venous blood samples were collected into sterile 2 ml empty tubes (serum) and 10 ml tubes containing 100 IU preservative-free heparin (PBMC). Samples were centrifuged within 2 hours after collection to separate serum/plasma and aliquots were stored at -20°C. Where sufficient blood volume was available, PBMCs were isolated from the remaining heparin tube cell pellet by centrifugation over a Ficoll-Hypaque gradient (Lymphoprep, Axis-Shield, Oslo, Norway) and cryo-preserved in 50% heat-inactivated (HI) foetal calf serum (FCS) and 7.5% DMSO. Cells were kept under liquid nitrogen vapour phase conditions during storage at IMR, transport to and storage at the Telethon Kids Institute (formerly Telethon Institute of Child Health Research, ICHR) in Perth, Western Australia.