Fig. 2: Pre-activated PBMCs co-cultured with CCA organoids and IL-2 show patient-specific killing.

CCA2 organoids were co-cultured with anti-CD3/CD28 pre-activated PBMCs for 7 days. Bright field imaging (a) shows that CCA2 organoids lose their regular morphology and become fragmented, especially upon addition of IL-2. Flow cytometry cell count for EpCAM-positive and CD45- and DAPI-negative cells (b) demonstrates that CCA2 organoids sustain significant cell death in the presence of 50 IU/ml (10 ng/ml, mid) or 100 IU/ml (20 ng/ml, high) IL-2, but not with 25 IU/ml (5 ng/ml, low) IL-2. CCA1 (c) and CCA3 (d) organoids do not show a significant decrease in live cells in the same co-culture setting (n ≥ 3 biological replicates for each organoid line; n = 2 different PBMC batches). Flow cytometry cell count for live (DAPI-negative) CD4⁺ and CD8⁺ T cells and CD56⁺ NK cells demonstrates that the number of live cells is comparable between solo culture and co-culture and addition of 100 IU/ml IL-2 increases the number of live cells (e). Flow cytometry for intra-cellular staining of IFN-γ and TNF-α in non-pre-activated and 3-day pre-activated PBMC cultures, and co-cultures of CCA organoids with pre-activated PBMCs, either with or without 100 IU/ml IL-2 (f). The percentage of cytokine-positive CD4⁺ and CD8⁺ T cells was determined after 19 h of (co-)culture in the presence of brefeldin and monensin (n = 3 technical replicates). Scale bar: 500 µm (a). All values with error bars represent mean with SEM. *p < 0.05, **p < 0.01.