All mice were bred and maintained on the C57BL/6 genetic background. For experiments, mice were used at between 6 and 12 weeks of age. All animals were housed in a specific pathogen–free facility at University of California, San Francisco (UCSF) and were treated according to protocols that were approved by UCSF animal care ethics and veterinary committees and are in accordance with National Institutes of Health (NIH) guidelines. The OT1 TCR transgene was crossed to CskAS transgenic and Nur77-GFP reporter mice (33, 67). The lightning allele (LL or CD45-low) was similarly crossed to mice harboring the CskAS transgene (30). To assess cell populations, spleen and lymph nodes were isolated from mice, and organs were dissociated in complete medium [RPMI supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, nonessential amino acids (Gibco), penicillin and streptomycin (Gibco), 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 10 mM Hepes]. Red blood cells were removed using ammonium-chloride-potassium (ACK) lysis. Cells were washed and resuspended in fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS) supplemented with 2% FBS and 2 mM EDTA] and stained using fluorescently labeled antibodies. Cells were analyzed using a BD Fortessa, and quantification was carried out using FlowJo software.

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