αifnγ xmg1.2

All experiments were performed according to US National Institutes of Health guidelines and were approved by the IACUC of The Cincinnati Children’s Hospital. C57BL/6J mice were obtained from Jackson. Deletion of Gimap5 in Gimap5^flox/floxCd4^cre-ert2 mice was induced by the administration of tamoxifen chow (40mg/kg body weight; Harlan Laboratories Teklad Diets). Gimap5^sph/sph mice were generated as previously described (6) and bred in-house in the vivarium of Cincinnati Children’s Hospital. All mice were maintained under specific pathogen-free conditions.
All antibodies used for flow cytometry were purchased from eBioscience or Biolegend unless otherwise noted. Purified α-mouse-CD3 (17A2) and α-mouse-CD28 (37.51) antibodies (Biolegend) were used for mouse T cell activation. A fixable viability stain was purchased from eBioscience. 7-AAD was purchased from BD. PMA, ionomycin, and Brefeldin A were obtained from Sigma. αIFNγ (XMG1.2) and mIL-23 were obtained from eBioscience. TGFβ1, mIL-4, and hIL-2 were purchased from Miltenyi Biotech. αIL-4, IL-6, and mIL-1β, mIL-12, and TGFβ3 were obtained from PeproTech.

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).