Intracellular staining buffer kit

Mature CD8SP or CD4SP cells from ATOs were isolated by magnetic negative selection using the CD8+ or CD4+ Isolation Kits (Miltenyi) and sorted by FACS to further deplete CD45RO+ cells (containing immature naïve T cells and CD4ISP precursors). Purified T cell populations were plated in 96-well U-bottom plates in 200 μl AIM V (ThermoFisher Scientific, Grand Island, NY) with 5% human AB serum (Gemini Bio-Products, West Sacramento, CA). PMA/ionomycin/protein transport inhibitor cocktail or control protein transport inhibitor cocktail (eBioscience, San Diego, CA) were added to each well and incubated for 6h. Cells were stained for CD3, CD4, and CD8 (Biolegend, San Diego, CA) and UV455 fixable viability dye (eBioscience, San Diego, CA) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNγ, TNFα, IL-2, IL-4, or IL-17A (Biolegend, San Diego, CA).

For intracellular cytokine detection, ILC2 or T cells were stimulated with PMA/ionomycin/protein transport inhibitor cocktail or control protein transport inhibitor cocktail (eBioscience, Cat 00–4975-93, Cat 00–4980-03, San Diego, CA) for 6 hours. APC-labeled CD107a antibody (Biolegend, clone H4A3) was added to wells at a 1:100 dilution for the final 2 hours of culture. Cells were collected, washed, and stained for surface markers and Zombie Aqua Fixable Viability dye (Biolegend, Cat. 423101) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, Cat. 88–8824-00) and intracellular staining with antibodies against corresponding cytokines. For antigen-specific CAR-T cytokine assays, T cells were expanded for 5 days with irradiated NALM6 cells as above and 1×10⁵ CAR-T cells were co-cultured with RAJI or RAJI-CD19^KO cells at a 1:1 ratio for 6 hours with addition of APC anti-CD107a for the final 2 hours prior to fixation and staining as above.

Week 6 H1-CAR ILC2 were purified from mechanically dissociated ATOs using the Dead Cell Removal Kit (Miltenyi, Auburn CA), followed by staining with PE-anti-CD8 and anti-PE MicroBeads (Miltenyi, Auburn CA) to deplete CD8+ T and NK/ILC1 cells. 1.5×10⁵ ILC2-enriched cells were plated in 96-well U-bottom plates as per proliferation assays, above. For type 1 polarization, 20 ng/mL rhIL-12 (Peprotech) was added in addition to rhIL-7 and rhIL-2. For type 2 polarization, rhIL-25, rhIL-33, and rhTSLP were added in addition to rhIL-7 and rhIL-2 at 20 ng/ml each. On day 5, PMA/ionomycin with protein transport inhibitor cocktail (eBioscience, San Diego, CA) was added to each well for 6 hours. Cells were washed and stained for surface markers and Zombie Aqua (Biolegend, San Diego, CA) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNγ, TNFα, IL-5, and IL-13 (Biolegend, San Diego, CA).