Elisa for mouse ifn γ

Cell culture supernatants were analyzed by ELISA for mouse IFN-γ (BD Bioscience), IL-9 (BioLegend), IL-17 (BioLegend) or IFN-β (PBL Assay Science) or for human IFN-γ (BioLegend) or human IL-9 (BioLegend) according to the manufacturer’s instructions.

Naïve CD4⁺ T cells (CD4⁺CD62L^hiCD44^lo) were purified from spleen and lymph nodes. Isolated naïve CD4⁺ T cells were stimulated with plate-bound antibodies against CD3 (clone 145-2C11, 2 μg mL⁻¹, BioXcell) and CD28 (clone PV-1, 2 μg mL⁻¹, BioXcell) and polarized into effector CD4⁺ T lymphocyte subsets without cytokines (TH0 cells), or with IL-12 (20 ng mL⁻¹) for TH1 cells, or with IL-4 (20 ng mL⁻¹) for TH2 cells, or with TGF-β (2 ng mL⁻¹) for Treg cells, or with TGF-β (2 ng mL⁻¹) and IL-6 (25 ng mL⁻¹) for TH17 cells, or with TGF-β (2 ng mL⁻¹) and IL-4 (20 ng mL⁻¹) for TH9 cells. After 72 h of polarization in the presence of increasing doses of R-crizotinib, cell culture supernatants were assayed by ELISA for mouse IFN-γ (BD Biosciences), IL-17 (BioLegend, San Diego, CA, USA), and IL-9 (BioLegend) according to the manufacturer’s protocol. Likewise, after three days, total RNA from T cells was extracted with TriReagent (Ambion, Austin, TX, USA), reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen) and was analyzed by real-time quantitative PCR (RT-qPCR) with the Sybr Green method according to the manufacturer’s instructions using the 7500 Fast Real-Time PCR system (Applied Biosystems). Expression was normalized to the expression of mouse Actb. Primers designed to assess gene expression are described in Supplementary Table 1.