Cd124

For splenic T cell sorting, magnetic bead-mediated positively selected CD4⁺ cells were stained with combinations of antibodies (0.5μg/mL per) to CD62L-PE (BioLegend, San Diego, CA) or CD62L-BB515 (BD Bioscience), CD44-APC (BioLegend, San Diego, CA), CD45Rb-APC/Cy7 (BioLegend, San Diego, CA), CD124 (1μg/mL, BD Biosciences, San Jose, CA), and CD25-BV421 (BD Biosciences, San Jose, CA). For Tregs, FoxP3-RFP reporter signal was used for FACS. Cells were washed twice in MACS buffer (Miltenyi Biotec, San Diego, CA) before sterile cell sorting using a FACSAria (BD Biosciences, San Jose, CA) with the support of the Cytometry & Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center. Analysis of all FACS data was performed using FlowJo v10 (Tree Star, Inc., Ashland, OR).

Cytokine levels were analyzed by standard sandwich ELISA performed as per manufacturer’s instructions (BioLegend, San Diego, CA), modified to utilize europium-conjugated streptavidin (Perkin-Elmer), and detected with a Victor V3 plate reader (Perkin Elmer, San Jose, CA). Blocking experiments utilized antibodies to IFNγ (10μg/mL, BioLegend, San Diego, CA), IL-21 (10μg/mL, eBioscience, San Diego, CA), IL-22 (10μg/mL, eBioscience), CSF2 (10μg/mL, eBioscience), IL-9 (1μg/mL, eBioscience), IL-13 (2μg/mL, eBioscience), IL-4 (1μg/mL, eBioscience), IL-24 (2μg/mL, eBioscience), IL-3 (1μg/mL, eBioscience), Neuropilin-1 (1μg/mL, R&D Systems, Minneapolis, MN), CD124 (1μg/mL, BD Biosciences, San Jose, CA), and corresponding isotype controls IgG1 and IgG2a (BioLegend, San Diego, CA). For CD124 blocking experiments, indicated cell types were incubated with 1μg/mL αCD124 at 4°C for 15 minutes, washed twice with PBS, then combined into co-culture. Supplementation assays were performed with recombinant mouse IL-2 and IL-4 (R&D Systems, Minneapolis, MN) at the indicated concentrations. For Luminex assays, media from indicated cultured populations were snap frozen in liquid nitrogen and sent to Eve Technologies (Calgary, Ontario, Canada) for mouse 32-plex and TGFβ 3-plex analysis.