Recombinant human rh il 2

The ability of the modified DCs to activate primary antigen-specific immune response was evaluated in a 5-day co-culture of mature DCs (0.18×10⁶ cells) and naive spleen cells (1.8×10⁶ cells) in the presence of recombinant human (rh)IL−2 (200 U/ml, Immunotools). In order to determine the percentage of CD107a⁺ cells, the degranulation assay was performed. Primarily stimulated spleen cells were incubated for 2 hours with MC38 cells in the presence of monoclonal anti−CD107a antibodies conjugated with APC (clone 1D4B, BioLegend) together with ionomycin (1 µg/ml, Sigma-Aldrich), phorbol-12-myristate-13-acetate (50 ng/ml, Sigma-Aldrich) and rhIL-2 (200 U/ml). Afterwards, cells were harvested and stained with anti−CD45 Brilliant Violet 605 (clone 30-F11), anti−CD4 FITC (clone RM4-5), anti−CD8a APC-Fire (clone 53-6.7) and anti−NK1.1 PE-Dazzle 594 (clone PK136) (all from BioLegend). For the elimination of the dead cells, DAPI dye was used. The flow cytometry analysis was performed using the LSRFortessa flow cytometer with Diva software (BD Biosciences).

Allogeneic co-cultures were started 2-4 days after crypt isolation. At this time point, grown organoids were harvested from the 24-well-plates with cold PBS, washed twice with cold PBS and resuspended in BCM. Approximately 100 organoids were mixed with 2.5 x 10⁵ magnetically enriched T cells (cf. 2.4.1/.2) in BCM in a 48-well-plate and incubated for 30 min at 37 °C. After that, organoids and T cells were harvested again from the well with cold PBS, spun down, resuspended in 50 μl of a 1:1 mixture of PBS and Matrigel (Corning) and plated in a 48-well-plate. Lastly, after polymerization of the Matrigel dome, 300 μl of CCM + 10 ng/ml rmIL-7 (Immunotools) + 10 ng/ml rmIL-15 (Immunotools or R&D systems) + 100 IU/ml recombinant human (rh) IL-2 (Immunotools) were added per well.