Generation of DCs under conditions of mTORC1 and mTORC2 inhibition results in DCs that remain CD86^lo but express IL-12 on LPS stimulation. RAPA- and CTR-DCs were generated as described in “DC generation and treatment.” BM-derived Torin1-DCs were generated from B6 mice with GM-CSF and IL-4 in the presence 50nM Torin1, a dual inhibitor of mTORC1 and mTORC2. After purification on day 7 of culture, these DCs were incubated for 18 hours in media alone (Unstim) or with 0.1 μg/mL LPS. Intracellular staining for IL-12p40, combined with surface staining for CD86, reveals that DCs generated under mTORC1 and mTORC2 inhibition have increased IL-12p40 production in CD86^loCD11c⁺ cells. Numbers on dot plots indicate percentage CD11c⁺ cells, and data are representative of 3 experiments performed.