title: GSM9140303: AIM T cells, VAC007, Channel 3, ADT; Homo sapiens; OTHER
library_construction_protocol: AIM data: PBMCs from each vial were split in R10 across up to 8 wells of a U-bottom 96-well plate at 1e6 PBMCs per well. Pools of overlapping peptides (15mers with 11 amino acid overlap) which covered the entire SARS-CoV-2 spike S1 and S2 protein reons were added to each well at a final concentration of 1 μg/ml and plates were incubated for 24 hours at 37 °C. One well of unstimulated cells per donor was included for use as a gating control for flow cytometric cell sorting. After incubation, plates were centrifuged at 1800 rpm for 2 min, washed in FACS buffer (PBS + 0.05% bovine serum albumin + 1 mM EDTA), and centrifuged again at 1800 rpm for 2 min prior to staining with the following antibodies: FITC-CD19 (HIB19), FITC-CD14 (M5E2), PE-4-1BB (CD137; 4B4-1), PE-OX-40 (CD134; Ber-ACT35), AF700-CD8α (SK1), BV421-CD69 (FN50), BV650-CD4 (OKT4), BV785-CD3 (OKT3), TotalSeq-C0032-CD154 (CD40L; 24-31), TotalSeq-C0155-CD107a (H4A3), TotalSeq-C0088-PD-1 (EH12.2H7), TotalSeq-C0171-ICOS (C398.4A), TotalSeq-C0063-CD45RA (HI100), TotalSeq-C0148-CCR7 (G043H7), TotalSeq-C0080-CD8 (RPA-T8), TotalSeq-C0072-CD4 (RPA-T4) and one of TotalSeq-C anti-human Hashtag antibodies (1-10; LNH-94, 2M2) per donor and timepoint. All antibodies were from BioLegend. TotalSeq antibodies were prepared as per manufacturer instructions and staining was performed in a final volume of 50 μl for 30 min at 4 °C. After staining, cells were washed three times with FACS buffer, resuspended in 100 μl of FACS buffer, and transferred to a 1.5 ml RNase-free Microfuge tube. SYTOX Green was pre-diluted 1:60 in PBS + 0.04% BSA, then diluted 1:100 in each sample. Samples were stored at 4 °C until sorting. Sorting was performed on a BD FACSAria III (BD Biosciences) using an 85-micron nozzle. All AIM+ T cells (CD69+ and OX40+ and/or 4-1BB+ CD3+) from all individual samples were sorted into a single collection tube (RPMI-1640 + 1% NEAA + 1% Na Pyruvate + 2.5% HEPES + 10% FBS), washed twice in collection media and resuspended at 13,700 cells per 38.7 μl of resuspension buffer in preparation for the 10x Genomics Chromium Next GEM Single Cell 5' v2 (Dual Index) workflow.  CTV data: One vial of cryopreserved PBMCs per vaccinee at the pre-vaccination (baseline; “T0”) timepoint was thawed and washed in R10 (RPMI-1640 + 10% FBS + 1% Penicillin/Streptomycin) + Benzonase (500 U) on the day of use. 5.25 × 106 cells per donor were collected and labeled with CTV, as described (35). Briefly, cells were pelleted and washed twice with sterile PBS. After washing, cells were resuspended in 1 ml of PBS and 0.5 μl of CTV was added (1:2000 dilution). Cells were stained for 10 minutes at room temperature and the reaction was quenched using 4 ml of ice-cold FBS. Media was removed and cells were resuspended in 5 ml of RhuAB10 media (RPMI-1640 + 10% human AB serum + 1% Penicillin/Streptomycin). Cells were allowed to rest for 5 min to allow excess CTV to leach away, counted, and resuspended in fresh RhuAB10 media at 2.5e6 cells per ml. Cells (2.5e5) were added to wells of a 96-well U-bottom plate. Unstimulated control (containing DMSO) and positive control (PHA at 2 μg/ml) wells were included. The remainder of the cells were added in sequential wells so that all cells were used and stimulated with S1 + S2 peptide pools (each at 1 μg/ml) in a final volume of 200 μl. Cells were placed in a 37 °C, 5% CO2 incubator for 7 days. On day 4, the plate was centrifuged (1800 rpm for 3 min) and 100 μl of media was removed and replaced with fresh, pre-warmed RhuAB10 media. On day 7, the plate was centrifuged (1800 rpm for 2 min), and all media was removed. Cells were washed in FACS buffer and wells were merged (3 into 1) to reduce the number of wells to stain. Staining was performed for 30 min at 4 °C using a cocktail of: FITC-CD19 (HIB19), FITC-CD14 (M5E2), AF700-CD8α (SK1), BV650-CD4 (OKT4), BV785-CD3 (OKT3), and one of TotalSeq-C anti-human Hashtag antibodies (1-10; LNH-94, 2M2) per donor. TotalSeq-C antibodies were prepared as described above. After staining, cells were washed three times with FACS buffer, resuspended in 100 μl of FACS buffer, and transferred to a 1.5 ml RNase-free Microfuge tube. SYTOX Green was pre-diluted 1:60 in PBS + 0.04% BSA, then diluted 1:100 in each sample. Samples were stored at 4 °C until sorting. Sorting was performed as per AIM+, but CTVlo CD3+ T cells were sorted out. The 10X Genomics Chromium Next GEM Single Cell 5' v2 library preparation was performed as per manufacturer instructions for Gene Expression, Feature Barcode and TCR library preparations.