Forty-two hours after peptide stimulation, cultures were pre-purified with EasySep Human T Cell Isolation Kit (using 20% of the recommended reagents, Stemcell Technologies, # 17951). T cells were stained with hashtag TotalSeq-B antibodies and subjected to FACS. Live, single CD3+ CD8+ or CD3+ CD4+ cells that were CD69hi and/or CD137+ were collected. After FACS, cells were counted and pooled into one sample across T cell subsets and donors at similar cell numbers, if possible. The pooled sample was stained with diluted TotalSeq-B cell surface marker antibodies (BioLegend) in PBS containing 2% FBS for 30 minutes at 4C. Cells were washed twice with PBS containing 2% FBS, and cell viability was determined with Trypan Blue which was consistently >90%. Cells were subjected to high-throughput single cell capture via the Chromium controller (10x genomics), Chromium Next GEM Single Cell 3' Kit v3.1 (Single Index, 10x genomics, #PN-1000121) and gel beads (10x genomics, #1000376). About 20,000 cells per pooled samples were targeted for cell capture. 3' gene expression as well as antibody-derived tags (ADT) for TotalSeq-B antibodies including hashtag antibodies and cell surface marker (3' Feature Barcode Library Kit, 10x genomics, #PN-1000079) collective known as CITE-seq, were generated from each captured sample.