α t bet 4b10

To assess direct binding of Eomes to the Pdcd1 promoter, we used a modified version of Cut & Run (Skene et al., 2018 (link)) with a qPCR readout. EL4 cells were nucleofected using an Amaxa cell line Nucleofector kit L (Lonza) with either pCMV-myc T-bet or pCMV-Eomes FLAG plasmids. Following a 48 hr incubation to induce recombinant protein expression, 100,000 EL4 cells were used in the Cut&Run assay. For primary mouse cells, CD8⁺ splenocytes from d45 Armstrong (T_MEM) or d45 clone 13 (T_EX) mice were isolated and 10,000 cells were used in the Cut&Run assay. α-myc (Invitrogen) or α-FLAG (Invitrogen) antibodies were used to IP myc-T-bet or Eomes-FLAG proteins in EL4 cells. α-T-bet (4B10, eBioscience) or α-Eomes (Dan11mag, eBioscience) was used to IP T-bet or Eomes from primary mouse splenocytes. Immunoprecipitated DNA was subjected to two rounds of nested PCR using the following oligo pairs: I–forward 5′-actctaacatgccacaaaaccatag, reverse 5′-cttccagttttatacctgatcgaag (Cruz-Guilloty et al., 2009 (link)), Pdcd1–5’-ccttgctcctcaccacactgc, reverse 5′-cagagcagatcatgaggactg (Kao et al., 2011 (link)), and Il4–forward 5′-gagttaaagttgctgaaaccaagg, reverse 5′-attttccaattggtctgatttcac (Cruz-Guilloty et al., 2009 (link)).

Single cell suspensions were made from the draining lymph nodes and brought up to 5×10⁶ cells/ml in RPMI supplemented with 10% FCS. Cells were cultured with PMA/Ionomycin (BD Pharmingen) for 4 hours, Fc receptors were blocked (αCD16/CD32, BD Pharmingen), and surface markers were stained with αCD3 (145-2C11, BD Pharmingen), αCD4 (RM4-5, BD Pharmingen), αCD8α (53-6.7, BD Pharmingen), αCD11b (M1/70, BD Pharmingen); αCD11c (N418, eBioscience); αCD19 (1D3, eBioscience); αLy6G (RB6-8C5, eBioscience); αCD25 (PC61.5, eBioscience), αCD39 (24DSM1, eBioscience), αCD73 (TY/23, BD Pharmingen), and/or αCTLA-4 (UC10-4F10-11, BD Pharmingen) with corresponding isotype controls (eBioscience or BD Pharmingen). Cells were fixed and permeabalized (Cytofix/Cytoperm, BD Pharmingen) for intracellular staining with the following antibodies: αFoxp3 (FJK-16s, eBioscience); αIFN-γ (XMG1.2, eBiosciences); αTbet (4B10, eBioscience) and the corresponding isotype controls IgG2aκ, IgG1κ (eBioscience). Alternatively, cells were cocultured for 72 hours with soluble leishmania antigen (SLA, L. (V.) panamensis freeze-thaw lysate). Supernatants were harvested and IFN-γ, IL-10, IL-13, IL-17, TNF-α and TGFβ were analyzed by sandwich ELISA following the manufacturer’s protocol (eBioscience).