PBMC isolation and dead cell removal

PBMCs were isolated from blood samples using Lymphoprep (StemCell Technologies) density gradient centrifugation according to the manufacturer’s instructions. Single-cell suspensions were then washed with Dulbecco’s PBS (Sigma) and frozen in aliquots containing 5–10 million cells in 90% (vol/vol) heat-inactivated FCS (Gibco) and 10% (vol/vol) DMSO (Sigma-Aldrich). On the day of the experiment, the cells were thawed for 1 min, transferred to wash buffer (PBS supplemented with 2% (vol/vol) FCS and 2 mM EDTA) and centrifuged at 500g for 5 min. Resuspended cells were passed through a 30-μm filter and counted before live-cell magnetic-activated cell sorting (MACS) enrichment with the dead cell removal kit (Miltenyi Biotech), per the manufacturer’s instructions. Cell pellets were resuspended in microbeads and incubated at room temperature for 15 min. Each stained sample was passed through an LS column and rinsed with binding buffer (all from Miltenyi Biotec) before centrifugation. Cell pellets were resuspended in wash buffer and counted for antibody staining by cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq).

PBMCs were isolated using Leucosep tubes (Greiner Bio-One) with Histopaque 1077 (Sigma) by centrifugation at 800g for 15 min at room temperature. PBMCs at the interface were collected, rinsed twice with autoMACS running buffer (Miltenyi Biotech) and cryopreserved in FBS with 10% DMSO. All samples were processed within 4 h of collection. Purified PBMCs were thawed at 37 °C, transferred to a 50-ml tube, and ten volumes of prewarmed thawing medium (IMDM; Gibco, 12440-053) with 50% (vol/vol) FCS (not heat inactivated; PAN-Biotech, P40-37500) and 0.1 mg ml⁻¹ DNase I (Worthington, {"type":"entrez-nucleotide","attrs":{"text":"LS002139","term_id":"1321652585","term_text":"LS002139"}}LS002139)) were added slowly and dropwise, followed by centrifugation at 500g for 5 min. The pellet was resuspended in 1 ml of FACS buffer (PBS; Sigma, D8537) with 3% (vol/vol) heat-inactivated FCS, and the viability of each sample was assessed by counting in an improved Neubauer chamber using Trypan blue. Pools of four samples were generated by combining 0.5 million live cells per individual (2 million live cells in total). The pools were washed twice in FACS buffer (10 ml and 2 ml, respectively) followed by centrifugation for 5 min at 500g. The pellet was then resuspended in 35 μl of FACS buffer and the viability of each pool was assessed.

Peripheral whole blood was collected in EDTA tubes and processed fresh via Ficoll-Paque Plus separation (GE Healthcare, 17144002). The blood was first diluted with 5 ml 2 mM EDTA-PBS (Invitrogen, 1555785-038), before 10–20 ml of diluted blood was carefully layered onto 15 ml of Ficoll in a 50-ml falcon tube. If the sample volume was less than 5 ml, blood was diluted with an equal volume of EDTA-PBS and layered onto 3 ml Ficoll. The sample was centrifuged at 800g for 20 min at room temperature. The plasma layer was carefully removed and the PBMC layer collected using a sterile Pasteur pipette. The PBMC layer was washed with three volumes of EDTA-PBS by centrifugation at 500g for 10 min. The pellet was suspended in EDTA-PBS and centrifuged again at 300g for 5 min. The PBMC pellet was collected and the cell number and viability assessed using Trypan blue. Cell freezing medium (90% FBS and 10% DMSO) was added dropwise to PBMCs slowly on ice and the mixture cryopreserved at −80 °C until further full-sample processing.