Profiling Cytokine Secretion in Tumor Infiltrating CD8+ T Cells

We measured cytokine and cytolytic granule production (IFNγ, GZMB and TNFα) of CD8⁺ T cells from mouse tumour samples and tumour organoid-T cell co-culture. Briefly, CD8⁺ T cells were placed into the 24-well plate at 1 × 10⁶ cells/well, stimulated with 1 μM ionomycin and 50 ng ml⁻¹ phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 4 h, in the presence of 5 μg ml⁻¹ Brefeldin A (BFA), with the purpose to amplify the expression of intracellular cytokines^{61 (link)}. The cells were stained with APC/Cy7-conjugated anti-CD8a (Biolegend) for 15 min and then fixed by 4% PFA. After washing, cells were stained with PerCP/Cy7-conjugated anti-GZMB, APC-conjugated anti-IFNγ and PE-conjugated anti-TNFα (Biolegend) for 15 min. In flow cytometric analyses, T cells stained with isotype control antibodies were used as negative controls for gating the cytokine or granule-producing cells. For T cells from tumour organoid-T cell co-culture, tumour organoids were first digested into single cells with TrypLE Express at 37°C before APC/Cy7-conjugated anti-CD8a staining. In the IFNγ and TNFα secretion assay (Biolegend), T cells were stimulated with ionomycin and PMA without the presence of BFA. The media were collected for ELISA assay.