Young (2 to 3 months) and old (22 to 24 months) mice were euthanized using isoflurane overdose. Then, spleens were harvested and mashed into a 70-μm cell strainer. Lysis of red blood cells was performed using 300 μl of Ammonium-Chloride-Potassium (ACK) buffer for 1 min (Lonza, Basel Switzerland). Then, CD4+ T cells were purified from total splenocytes using a magnetic microbeads negative selection kit (EasySep Mouse CD4+ T Cell Isolation Kit, STEMCELL Technologies), according to the manufacturer’s instructions. In all processing steps, wide-bore pipette tips were used, and centrifugation condition did not exceed 400g, to minimize physical damage to the cells caused by shearing forces.

FACS sorting. To enhance purity and discard dead cells, CD4 T cells were further purified using FACS. Cells were sorted as eFluor780-Live cells+ (Fixable Viability dye, eBioscience), PerCP/cy5.5-conjugated anti-CD8 (53-6.7; BioLegend), phycoerythrin (PE)–conjugated anti-CD19 (6D5; BioLegend), PE-conjugated anti-CD11b (M170; BioLegend), PE-conjugated anti-NK1.1 (PK136; BioLegend), BV421-conjugated anti-CD4+ (GK1.5; BioLegend), and fluorescein isothiocyanate (FITC)–conjugated anti-TCRb+ (H57-597; BioLegend) using the FACSAria III instrument (BD Biosciences, San Jose, CA). To minimize physical stress to the cells, a 100-μm nozzle and 20-psi sheet fluid pressure were used. Cells were sorted into RPMI (Life Technologies)–enriched media with 10% fetal bovine serum, 10 mM Hepes, 1 mM sodium pyruvate, 10 mM nonessential amino acids, 1% penicillin-streptomycin-nystatin, and 50 μM 2-mercaptoethanol (2-ME). After sorting, CD4 T cells in all samples reached >99% purity. Before the loading on Chromium Controller Instrument (10x Genomics), cells were suspended in phosphate-buffered saline (PBS) supplemented with 0.04% bovine serum albumin, counted four times under a light microscope, and passed through a 40-μm strainer (for extended protocol, see “Cell preparation guide,” 10x Genomic protocol; https://support.10xgenomics.com/single-cell-gene-expression/sample-prep). To reach 2000 to 3000 recovered cells, ~5000 cells per sample were loaded to the 10x Genomics platform.

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