Il 15rα fc

Murine IL-15 was purchased from PeproTech. Mouse IL-15R α Fc chimera protein (IL-15Rα-Fc) was purchased from R&D Systems. IL-15 and IL-15Rα-Fc were complexed by incubating a mixture of 6.67 µg/ml IL-15 and 33.3 µg/ml IL-15Rα-Fc in PBS for 30 min at 37°C before injection. Mice were injected i.p. with 0.6 µg IL-15 and 3 µg IL-15Rα-Fc in 200 µl PBS every other day for a total of three injections. This dose of IL-15–IL-15Rα-Fc was chosen based on previous studies (Rubinstein et al., 2006 (link); Shifrin et al., 2016 (link)). After IL-15–IL-15Rα-Fc complex treatment, splenic CD4⁺ T cells were gated as CD45⁺ CD19⁻ NK1.1⁻ CD1d-Tet⁻ CD3⁺ CD8⁻ CD4⁺ to exclude NKT cells because NKT cells have been shown to robustly expand in response to IL-15–IL-15Rα-Fc complex (Stoklasek et al., 2006 (link); Guo et al., 2015 (link)) and because MHC-I down-regulation was inefficient in CD4⁺ NKT cells in both PBS-treated and anti-NK1.1–treated groups in these experiments (data not shown).