N4 ova peptide

Thy1.1⁺ OT-I cells (1.0 × 10⁴) were transferred i.v. and mice were infected with 10⁴ CFU Listeria monocytogenes strains expressing OVA APL epitope (LM-OVA APLs) (15 (link)) 24 h later. Secondary effectors were generated in 4 week post-infection mice by immunizing with 50 µg N4 OVA peptide (GenScript) emulsified in IFA (Gibco) in both hind foot pads. Five days later, draining popliteal LNs were collected and pooled for analysis. Serum was analyzed using Ready-Set-Go ELISA kit according to manufacturer’s instructions (eBiosciences).

Single cell suspensions were stained with anti-CD3, anti-CD8, anti-CD19, anti-CD25, anti-CD44, anti-CD45RB, anti-CD62L, anti-CD69, anti-CD122, anti-CD127, anti-CD11c, anti-PD-1, and anti-Thy1.1 or appropriate isotype control (BD Biosciences or Biolegend) for 15 min at room temperature. For intracellular marker and cytokine staining, cells were incubated for 5 h at 37 C in the presence of 1 μM N4 OVA peptide (GenScript) and 10 μg/ml GolgiPlug (BD Biosciences) and stained for intracellular IL-2, TNF, and IFN-γ following manufacturer’s instructions (BD Biosciences). Assessment of Nur77 (eBiosciences) and hnRNPLL (Clone TR75-89, Cell Signaling Technology) expression was performed using the FoxP3/Transcription Factor Staining Buffer Set (eBiosciences). hnRNPLL was detected with anti-rabbit F(ab)’2 secondary reagent (Cell Signaling Technology). Data were analyzed using FlowJo software (Tree Star).