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All animal studies were performed according to National Institutes of Health (NIH) guidelines for the use and care of live animals and were approved by the Animal Care and Use Committees of the National Cancer Institute (NCI) under the protocol 20-073. C57BL/6 CD4-Cre transgenic, RAG^−/− mice and Ifngr KO (JAX:003288) mice were obtained from the Jackson Laboratory and bred in our institute animal facility under specific pathogen–free conditions. Sptlc1^foxl/flox mice were generated in our laboratory as previously described (34 (link)). Sptlc1^foxl/floxCD4-Cre (KO) conditional KO mice were generated by crossing Sptlc1^floxl/flox mice to CD4-Cre transgenic mice. Sptlc1^foxl/flox or Sptlc1^+/+CD4-Cre were used as control mice (WT). All mice used for experiments were aged 8 to 12 weeks, and both female and male mice were used for experiments. Primers for qPCR were obtained from IDT (Integrated DNA Technologies, USA). Cytokines mIL-4, mIL-12, hTGF-β1, IL-7, and IL-1β were purchased from PeproTech, USA; hIL-2 was obtained from R&D Systems; and IL-6 and IL-23 were purchased from BioLegend, USA. Antibodies for activation and cytokine neutralization were procured from Bio X Cell, USA. All chemicals were purchased from Sigma-Aldrich unless otherwise mentioned.

A mouse CD4⁺ T cell isolation kit (Miltenyi Biotec or BioLegend) was used to isolate CD4⁺ T cells from LN and spleen. The isolated CD4⁺ T cells were stimulated for 3 d with plate-bound anti-hamster IgG (12.5 µg/ml), soluble anti-CD3ɛ (145-2C11; BD PharMingen), and anti-CD28 (37.51; BD PharMingen). On day 2, cells were transferred to a fresh plate, without or with the addition of 75 U/ml hIL-2 (Peprotech) and 10 µg/ml anti–mIL-2 (Bio X Cell). The culture conditions for Th0 (no differentiation) were 2.5 µg ConA; for Th1, Th2, and iT reg, 0.5 µg/ml anti-CD3 and 1 µg/ml anti-CD28; and for Th17, 0.25 µg/ml anti-CD3 and 1 µg/ml anti-CD28. For skewing, the following cytokines were added to the culture: Th0, 2.5 µg/ml anti–mIL-4 (Bio X Cell) and 2.5 µg/ml anti–mIFN-γ (Bio X Cell); Th1, 10 ng/ml mIL-12 (PeproTech) and 2.5 µg/ml anti–mIL-4; Th2, 50 ng/ml mIL-4 (PeproTech) and 2.5 µg/ml anti–mIFN-γ; Th17, 0.5 ng/ml mTGFβ, 20 ng/ml mIL-6 (both Peprotech), 2.5 µg/ml anti–mIFN-γ, and 2.5 µg/ml anti–mIL-4; and iT reg, 5 ng/ml mTGFβ, 5 µg/ml anti–mIFN-γ, and 5 µg/ml anti–mIL-4. The cell culture medium was RPMI 1640 for Th1, Th2, and iT reg or IMDM for Th17 cell differentiation and supplemented with 5% FCS, β-mercaptoethanol, and penicillin/streptomycin (Invitrogen).

Rag2^−/− γc^−/− recipient mice 5 wk after adoptive transfer were injected i.p. with recombinant mIL-12 (Peprotech) at 50 µg/kg body weight. Mice were analyzed 48 h after injection.