Anti inkt microbeads

Human CD14⁺ monocytes and iNKT cells were isolated from fresh PBMC by positive selection using CD14 MicroBeads and anti-iNKT MicroBeads (Miltenyi Biotech, Germany), respectively, according to manufacturer’s instructions. iNKT expansion was performed as described (48 (link)) with modifications. Briefly, iNKT were co-cultured with CD14⁺ monocytes at 1:1 ratio in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Gibco), 10 µg/ml gentamicin (Gibco), and 0.25 µg/ml fungizone (Gibco) in the presence of 20 ng/ml GM-CSF (Peprotech), and 20 ng/ml IL-4 (R&D Systems), and 100 ng/ml αGC (Cayman Chemical). Half of the medium was replaced and 20 U/ml IL-2 (Chiron, Emeryville, USA) was added every day from day 2 to day 21 in order to reach around 1×10⁶ cells. ILT2 expression was evaluated and iNKT cells were phenotypically validated by flow cytometry before the iNKT activation assay. Purity of the iNKT cell population was systematically higher than 90%. The iNKT cells used in the activation assay with DC-10 cells were isolated and maintained in culture with the presence of IL-2 and αGC till the autologous mDC and DC-10 cells were differentiated.

Healthy donor PBMCs were obtained from the UCLA/CFAR Virology Core Laboratory and were used to generate the PBMC-Tc and PBMC-iNKT cells.
To generate PBMC-Tcon cells, PBMCs were stimulated with CD3/CD28 T-activator beads (ThermoFisher Scientific) and cultured in C10 medium supplemented with human IL-2 (20 ng/mL) for 2–3 weeks, following the manufacturer’s instructions.
To generate PBMC-iNKT cells, PBMCs were enrich for iNKT cells using anti-iNKT microbeads (Miltenyi Biotech) and MACS-sorting, followed by stimulation with donor-matched irradiated αGC-PBMCs at the ratio of 1:1 and cultured in C10 medium supplemented with human recombinnat IL-7 (10 ng/mL) and IL-15 (10 ng/mL) for 2–3 weeks. If necessary, the resulting PBMC-iNKT cells could be further purified using Fluorescence-Activated Cell Sorting (FACS) via human iNKT TCR antibody (Clone 6B11; BD Biosciences) staining.

iNKT cells represent a very rare population of T cells, being present at a frequency ranging between 0.01 and 1% (ref. 15 (link)). First, we performed magnetic separation using anti-iNKT microbeads (Miltenyi Biotec) followed by staining of the labelled cells. A total of 50 million frozen PBMC was used as starting material. After centrifugation, the cells were resuspended in 400 μl of cold buffer solution (PBS, pH 7.2, 0.5% BSA, 2 mM EDTA) and 100 μl anti-iNKT microbeads were added. Samples were incubated for 15 min in the fridge. At the end of the incubation time, 50 μl of Anti-iNKT-PE antibody (Miltenyi Biotec, Clone 6B11, Catalogue Number 130–098–128) were used for the staining and the cells were kept in the fridge for 5 min. After a washing step, magnetic separation using a MACS separator (Miltenyi Biotec) and MACS columns (Miltenyi Biotec) was carried out. To enrich the purity of the separated fraction, an additional separation was performed over a second column and cells were stained with Fluorogold (Life Technologies) to label dead cells. Second, FACS sorting was performed, retrieving 10,215 cells.