Human PBMC collection
Freshly-drawn whole blood specimens were purchased from sample collections conducted under IRB-approved protocols by Bloodworks Northwest with signed informed consent forms from all donors. PBMCs were isolated using Ficoll Premium (GE Healthcare) and stored in Cryostor10 (StemCell Technologies) in liquid nitrogen until use.
Cryopreserved PBMC thawing
Cryopreserved PBMC samples were thawed into pre-warmed AIM V media (37°C) (GIBCO), then resuspended in 5 mL of cold AIM V (4°C), and counted on the Nexcelom Cellaca MX cell counter. Individual samples were resuspended in Dulbecco's phosphate-buffered saline (DPBS) (Costar) at a concentration of 10 million cells per mL.
T cell isolation by negative selection
Thawed PBMCs were resuspended in separation buffer (DPBS containing 2% FBS, Sigma Aldrich, and 1 mM EDTA, ThermoFisher Scientific) at 50 million cells per mL. Samples were transferred to a 5 mL polystyrene round-bottom tube. Isolation Cocktail (STEMCELL Technologies) was added to each sample at 50 µL per mL, mixed, and incubated at room temperature for 5 min. RapidSpheres (STEMCELL Technologies) were vortexed for 30 seconds and 40 µL per mL were added to each sample. Separation buffer was added to samples to fill a total volume of 2.5 mL. After mixing the sample gently by pipetting, the tubes were placed into the EasySep™ magnet (STEMCELL Technologies) and samples were incubated at room temperature for 3 min. Isolated T cells were collected by decanting the enriched cell suspension into a new tube.
Drug treatment of isolated T cells
T cells were centrifuged for 5 min at 400g and resuspended in growth medium (RPMI 1640 (Gibco), 20% FBS, 1X Pen/Strep, 50 ng/mL IL-2, 5 ng/mL IL-7, 5 ng/mL IL-15). Drugs were added to cells at the indicated concentrations in Nunc™ 96-well polystyrene round-bottom microwell plates (Thermo Fisher Scientific) in a total volume of 200 µL/well. Plates were incubated at 37°C with 5% CO2 for 4, 24, or 72 hours following drug or control treatment.
T cell activation
In T cell activation experiments, T cells were incubated with Human T-Activator CD3/CD28 Dynabeads (Gibco) at a ratio of 1 bead per 2 cells. No beads were added to negative control conditions.
Extracellular flow cytometry staining
T cells were washed and resuspended in 200 µL FACS wash buffer (2% BSA and 0.5 mM EDTA in DPBS) and transferred to a new 96-well round-bottom plate, then centrifuged at 450g for 5 min, and cell pellets were resuspended to a final volume of 100 µL with a viability mix containing 1:400 FVS510 viability dye (BD, 1 µg/mL) in FACS wash buffer and incubated for 30 min at 4°C protected from light. After an additional wash with 200 µL FACS wash buffer, samples were resuspended to a final volume of 100 µL with extracellular antibodies in FACS wash buffer (table S12). Samples were then mixed and incubated for 30 min at 4°C, protected from light prior to data acquisition.
Intracellular flow cytometry staining
For experiments using intracellular staining (including Ikaros and Aiolos), cells labeled by extracellular staining described above underwent two additional washes using 200 µL FACS wash buffer, then were resuspended in 200 µL Foxp3 Fixation/Permeabilization working solution (BioLegend) for 30 min at room temperature, protected from light. Samples were centrifuged at 750g for 5 min at room temperature. This was repeated twice by discarding the supernatant and resuspending the pellets in 200 µL 1X permeabilization buffer and centrifuging at 400g for 5 min at room temperature. Intracellular antibodies (table S13) were added in 100 µL total volume, and cells were incubated for 30 min at room temperature, protected from light. Cells were then washed twice in 200 µL 1X permeabilization buffer, then were resuspended in 200 µL FACS wash buffer prior to data acquisition.
Flow cytometry compensation and processing
Compensation matrix generation and spectral unmixing was completed by creating single color controls using healthy resting PBMCs (table S14). Single color controls were stained with the same protocol. Spectral unmixing was calculated in the SpectroFlo instrument software (Cytek, Version 2.1.1). Flow Cytometry Standard (FCS) files from the Cytek Aurora spectral cytometer were exported in FCS 3.1 format. FCS files were manually gated to remove doublets, debris, and dead cells using FlowJo (BD Biosciences, Version 10.8.1).
TEA-seq
TEA-seq profiling was conducted on T cells isolated from a healthy female donor aged 58 years. CD3+ T cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) using negative selection as described above. To accommodate the TEA-seq protocol, thawing and treatment was carried out in a staggered-start reverse time course so that all samples could be collected simultaneously for processing. Samples collected following the viable CD3+ T Cell sort were centrifuged at 400g for 5 min at 4°C and resuspended in PBS. Each sample was then individually stained with a panel of 55 oligo-conjugated antibodies (below) as previously described (17). Each sample was also stained with a unique Cell Hashing Antibody (table S16) to enable sample pooling and reduce well-to-well effects (57). Following a 30 min incubation, each sample was washed 3 times with PBS + 2% BSA to remove unbound antibodies, counted, and pooled. The hashed cell pool was then tagmented, and single cell GEX, ATAC, and ADT libraries were made as previously described (17). Hash tag-derived oligo (HTO) primers were added to the pre-amplification PCR mix, and HTO libraries were prepared from the captured cell hashing antibodies using the same method as for ADT libraries. Final libraries were sequenced to target 80,000 reads per cell for GEX, 70,000 for ATAC, 50,000 for ADT, and 10,000 for HTO libraries on an Illumina NovaSeq 6000.