Mice, infections, and adoptive transfers

Female C57BL/6 were originally purchased from the Jackson Laboratory (Harbor, ME) and mostly bred in house. Female aged mice were received at 20 to 21 months of age from the National Institutes of Aging and maintained in the same specific pathogen–free conditions for at least 1 month before infection. In most of cases, aged and young subject bedding was cross-contaminated weekly to control for different microbial environments. Young OT-I and dLck-Cre Tgfbr2^fl/fl OT-I lymphoid tissues (KO, CD45.1⁺) were shipped from N.Z. (University of Texas, Health Science Center at San Antonio). All mice were housed in a specific pathogen–free environment and used under conditions fully reviewed and approved by the institutional animal care and use committee guidelines at the Mayo Clinic (Rochester, MN). For primary influenza virus infection, influenza A/PR8/34 strain [~100 plaque-forming units (pfu) per mouse] was diluted in fetal bovine serum (FBS)–free Dulbecco’s modified Eagle’s medium (DMEM) (Corning) on ice and inoculated in anesthetized mice through intranasal route as described before (72). X-31 strain was prepared identically, and 2.8 × 10⁵ pfu was administered per mouse for secondary challenge. During the secondary challenge phase, experimental mice were treated with FTY720 (1 μg/g) daily starting 1 day before rechallenge. For adoptive transfers, 1 × 10⁴ OT-I cells from lymph nodes of 8- to 10-week-old females were transferred intravenously, and mice were infected with PR8-SIINFEKL (33)(150 pfu) 1 day later.