Cd3 apc cy7 hit3a

A minimum of 350,000 freshly isolated PBMCs were incubated with an Fc receptor-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and the cells were stained with fluorochrome-conjugated antibodies specific for the surface molecules of interest. For B cells the following antibodies were used: CD19 (PerCP/Cy5.5; HIB19), CD27 (FITC; 323), CD38 (BV421, HIT2) and CD11c (PE/Cy7; Bu15), all from BioLegend (San Diego, CA, USA). For T cells, the following antibodies were used: CD3 (APC/Cy7; HIT3a), CD4 (PerCP/Cy5.5; RPA-T4), CD25 (PE; M-A251), CD127 (APC; A019D5), CXCR5 (AF488; J252D4), PD-1 (BV605; EH12.2H7), CXCR3 (PE/Cy7; G025H7) and CCR6 (BV421; G034E3) from BioLegend. Where applicable, corresponding isotype controls were used. Absolute counts of T and B cell subpopulations were measured using TruCount beads (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry data acquisition was performed using a FACS Canto II flow cytometer (BD Biosciences), and data analysis was conducted using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Peripheral blood samples were first stained for surface epitopes [αCD8 AF700 (53-6.7), αCD28 BV510 (CD28.2), αCD69 FITC (FN50), αCD160 PE-Cy7 (By55), αPD-1 PerCP-Cy5.5 (EH12.2H7) from BioLegend] including a live/dead staining reagent (LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV excitation from ThermoFisherScientific) for 30 min at 4°C. Afterwards, lymphocytes were fixed and red blood cells (RBCs) were lysed (1-step Fix/Lyse solution from eBioscience). Fixation and permeabilisation of cells was performed using the Foxp3/Transcription Factor Buffer Set (ThermoFisherScientific) according to the manual. To block unspecific binding of antibodies, cells were incubated for 10 min at 4°C with CohnII and subsequently, antibodies directed against intracellular epitopes (GzmB AF647 (GB11), CTLA-4 PE (L3D10), Ki67 AF488 (Ki-67), CD3 APC-Cy7 (HIT3a) from BioLegend, Perforin BV421 (δG9) from BD) were added and further incubated for 20 min at 4°C. Samples were recorded using the LSRII (BD) and analyzed using the FlowJo X 10.0.7r2 Treestar software. Gates were set according to fluorescence minus one (FMO) controls. Gating strategy is depicted in Supplementary Figure 5.

Rosette Sep T cell Purification (StemCell technologies, Vancouver, Canada) was employed as instructed by incubation of blood for 30 min with tetrameric antibody mixture against CD14, CD19, CD20/MS4A1, CD36, CD56, CD66b, CD123, GYPA, and CD16/FCGR3A which binds non-T cells to erythrocytes. Density-gradient centrifugation with Lymphocyte Separation Medium (Cellgro, Manassas, VA) was used to isolate the unstimulated T cells. T cell purity is routinely >93% by this method as determined by CD3 APC/Cy7 HIT3a (Biolegend) staining detected on a Beckman Coulter Gallios Cytometer. RNA was then prepared by Qiagen AllPrep Kit (Valencia, CA) from 3 million T cells with DNAse-I treatment. Roughly 2ug of total RNA was submitted to sequencing, and OD260/280 ratios were approximately 2.1.