Biotinylated anti cd44 im7

Naïve WT and Stat4^−/− OT-I or YopE TCR transgenic cells were enriched from the spleen and lymph nodes using a CD8α⁺ T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) and depleted of memory T cells with the addition of biotinylated anti-CD44 (IM7; BD Biosciences, Franklin Lakes, NJ, USA) antibody. Mice received 10⁴–10⁵ congenically distinct transgenic T cells the day before oral gavage with 2 × 10⁸ CFUs of Yptb-OVA or ΔyopM Yptb in 200 μl Phosphate Buffered Saline (PBS). To discriminate between intravascular circulating CD8⁺ T cells and tissue-resident CD8⁺ T cells, 3 μg of anti-CD8α-Allophycocyanin (APC)/R-phycoerythrin (PE) (53–6.7; Biolegend, San Diego, CA, USA) was administered intravenously 10 minutes before the mice were sacrificed. To examine the T-cell proliferation, 0.5 mg of BrdU (Thermo Fisher Scientific, Waltham, MA, USA) was administered intraperitoneally 1 hour before tissue harvest. To reactivate the Trm cells in vivo, the mice were given 10 μg of YopE peptide (SVIGFIQRM; Genemed Synthesis, San Antonio, TX, USA) intraperitoneally 1 hour before tissue harvest. FTY720 (Millipore Sigma, St. Louis, MO, USA) was given intraperitoneally at 1 mg/kg every other day from day 7 p.i. until tissue harvest on day 20 p.i..