Cd3 af700 clone okt3

Peripheral blood mononuclear cells (PBMC) from healthy subjects were isolated from heparinized whole blood by density gradient centrifugation and 1x10⁶ PBMCs were stained with viable and dead cells LIVE/DEAD™ Fixable Aqua dead cell stain kit (Life Technologies) for 15 min followed by an extracellular staining. First CCR7 PerCp Cy 5.5 was stained (clone G043H7, Biolegend) for 15 min at 37 °C followed by CD3 AF700 (clone OKT3, Biolegend), CD4 BV605 (clone RPA-T4, BD Biosciences), CD8 PB (clone SK1, Biolegend), CD45 RA FITC (clone HI100, Biolegend), CD56 APC-Cy7 (clone HCD56, Biolegend), CD19 PE-Cy7 (clone HIB19, Biolegend), CD26 APC (clone BA5b, Biolegend) for 15 min at 4°C. Non-specific binding of Fc-receptors was blocked by 2% polyclonal IgG (Flebogamma). CD26 surface expression was measured with CytoFLEX LX (Beckman Coulter) and data was analyzed with FlowJo software 10.0.08. Gating strategies are shown in the Supplementary Information.

T cell coculture was performed as previously described (78 (link)) with minor modifications. Briefly, tissue culture plates were coated with purified anti-CD3 (5 μg/ml) (clone HIT3a, BioLegend) at 4°C overnight and subsequently washed twice with 1X PBS. PBMCs from a healthy donor (Research Blood Components) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen) following the manufacturer’s protocol. PBMCs were resuspended in SFEM II (StemCell Technologies) with purified anti-CD28 (5 μg/ml) (clone CD28.2, BioLegend) and plated at a density of 1 million cells/ml. Isolated iMS1 or iMono cells were added at different ratios as indicated. The cells were left in culture for 3 to 4 days, with media replenished after 2 days. At the end of incubation, the cells were stained with CD3-AF700 (clone OKT3), CD4-APC (clone OKT4), and CD8a-PE (clone RPA-T8) (BioLegend) to determine the amount of CFSE dilution within the T cells.