*2.9. DC-CD4+ T Cell Co-Cultures*

The MagniSort Human CD4 T cell Positive Selection kit was used according to the manufacturer's protocol to isolate CD4<sup>+</sup> T cells from PBMC. CD4<sup>+</sup> T cells were co-cultured with allogeneic DC that had been pre-treated with IP or HPP and stimulated with LPS as before. The cells were co-cultured at a ratio of 10:1 T cells to DC for five days with no ketoacid present. The supernatants were removed for analysis of IL-10 by ELISA prior to restimulation of the cells in complete RPMI in the presence of 50 ng/mL PMA, 500 ng/mL ionomycin, and 5 μg/mL brefeldin A for 4 h. The cells were washed in PBS and stained for viability (Zombie NIRTM Fixable Viability kit) for 15 min at room temperature. The cells were then washed again in PBS and stained with fluorochrome-conjugated antibodies for surface markers CD3 and CD8 for 15 min at room temperature. The cells were then washed and fixed (FIX & PERM™ Cell Permeabilization Kit) for 15 min at room temperature. Finally, the cells were washed and stained for intracellular markers Ki67 and IFNγ in permeabilization buffer for 15 min at room temperature. A BD LSRFortessa flow cytometer was used to acquire samples, and analysis was performed using FlowJo v.10 software.
