Magnisort human t cell enrichment kit

Human T-cells were purified from frozen buffy coats and isolated by negative selection using the MagniSort Human T Cell Enrichment Kit (Thermofisher). T cells were seeded into wells of a 48 well plate at a density of 1 × 10⁶ cells/well and activated at a 1:1 ratio with Human T activator CD3/CD28 conjugated Dynabeads (ThermoFisher). Cells were cultured in a humidified CO2 incubator at 37 °C for 6 days in either organoid media or RPMI plus 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 10 mM Hepes buffer, 69 mM L-arginine and 50 μg beta-mercaptoethanol. After 6 days in culture, cells were stained with fluorochrome-conjugated monoclonal antibodies: anti-CD3 (SK7), anti-CD4 (OKT-4) and anti-CD62L (DREG-56) were obtained from eBioscience; and anti-CD8 (RPA-T8) was obtained from BD Biosciences. Cells were stained with 7-aminoactinomycin (7AAD, eBioscience) as a viability marker. Flow cytometry was performed on a LSRII flow cytometer. (BD Biosciences), and the data were analyzed using FlowJo software version 10 (FlowJo, LLC).

Expression kinetics were determined by measuring ICs at defined time points after T-cell stimulation ex vivo. Briefly, peripheral blood mononuclear cells were isolated from blood collected in heparin tubes by density gradient centrifugation using Lymphoprep (Alere Technologies AS, Oslo, Norway). Next, T cells were isolated by negative selection using the MagniSort Human T cell Enrichment Kit (Thermo Fisher Scientific, Waltham, MA., USA) or the EasySep™ Human T cell isolation kit (Stemcell Technologies, Vancouver, Canada) (results were found to be similar for both kits, purity ≥92%) according to manufacturer’s instructions. After T-cell enrichment, 0.5 × 10⁶ T cells were added to 1 mL of Roswell Park Memorial Institute (RPMI) culture medium with HEPES and L-Glutamine (Lonza, Basel, Switzerland) supplemented with gentamycin (Lonza), in round-bottomed polypropylene tubes. Gibco Dynabeads™ Human T-activator anti-CD3/anti-CD28 (ThermoFisher, Waltham USA) were added to T cells in a ratio of 1:5. Stimulated T cells were then incubated at 37 °C, 5% CO2 and collected after 1,2,3,4, 18, 42, 66 and 90 h and stained for CD8, CD45RA, CD25, PD-1, VISTA, and CD40L. In parallel, unstimulated T cells were also assessed for the same ICs including CD28 at each time point (See additional Table 2 for the antibodies used). Staining was performed as described above.

Human T-cells were isolated from human frozen blood buffy coats using a MagniSort Human T Cell Enrichment Kit (ThermoFisher) and quantified pre- and post-enrichment by flow cytometry gated on CD3 positivity. CD4+ and CD8+ populations were measured by flow cytometry. T-cells were seeded into 24-well plates at 1 × 10⁶ cells per well in either RPMI or OGM in a humidified CO2 incubator at 37 °C. For activation, Dynabeads Human T activator CD3/CD28 (ThermoFisher) were added at a 1:1 bead:cell ratio per manufacturer’s instructions.