Recombinant human il 2

For studies with human T cells, PBMCs were collected from consenting volunteers in accordance with regulatory and institutional requirements. MACS (Stemcell Technologies) purified CD8⁺ T cells were negatively selected from individual normal healthy donor whole blood at >90% purity. CAR domains employing human sequences were used to produce a third-generation, codon-optimized retroviral CAR construct containing the 5F9 human GUCY2C-specific scFv and human sequences of the GM-CSF signal peptide, CD8α hinge region, CD28 transmembrane and intracellular domains, and 4-1BB (CD137) and CD3ζ intracellular domains producing 5F9.h28BBz (Supplementary Fig. S4). CAR-encoding amphotropic γ-retrovirus production was similar to that with murine T cells, but replaced pCL-Eco with the pCL-Ampho packaging plasmid (Imgenex). Retroviral transduction occurred on day 3 or 4 post-activation with ImmunoCult CD3/CD28 Activator (Stem Cell Technologies). Cells underwent flow sorting for GFP-enrichment on day 7, followed by experimental use on day 10. Throughout the duration in culture, human CD8⁺ T cells were maintained in ImmunoCult-XF media (Stemcell Technologies) supplemented with 100 U/mL recombinant, human IL2 (NCI Repository).

Cryopreserved healthy donor human PBMCs (purchased from iXcells Biotech) were thawed then rested for five hours at 37°C. After resting, cells were cultured in complete T cell media supplemented with 5 ng/mL each of recombinant human IL-7 and IL-15 (PeproTech) and 10 U/mL recombinant human IL-2 at a concentration of 1 × 10⁶ CD3⁺ cells/mL and stimulated with 6.25 μL/mL of ImmunoCult Human CD3/CD28 T cell activator (StemCell) for two days. On day 2, T cells were stained for assessment by flow cytometry as described and cytokine production was assessed following overnight restimulation with ImmunoCult in the presence of Brefeldin A and monensin.