Infused T cells lack characteristic features of CD4⁺ Tregs. (A-B) Intracellular cytokine expression was examined on CD4-gated PBMCs after 6 hours of stimulation with P/I. IFN-γ and IL-2 production were evaluated on pretreatment, week-1, and week-4 samples of PBMCs. Numbers indicate the percentage of cells in each quadrant. Representative data from 1 of 15 patient samples analyzed are shown. (C) Intracellular IFN-γ expression was examined after a 6-hour stimulation of infusion TILs with P/I. Representative data from 1 of 15 patient samples analyzed are shown. (D) DNA methylation assays of week-4 PBMC sample from 1 patient in the CD8⁺ young TIL trial. Cells were sorted into CD4⁺CD25H (high), CD4⁺CD25M (medium), and CD4⁺CD25L (low), and the frequency of FoxP3⁺ cells in each population is shown. The methylation status of 11 CpG dinucleotides within intron 1 of the FOXP3 gene shown previously to be undermethylated in CD4⁺ Tregs were analyzed. The percentages of the 11 CpGs that were methylated, evaluated as described in “Methods,” are shown in heat maps, and the average percentages of the 11 CpGs that were methylated re indicated below the heat maps. (E) DNA methylation analysis of a sample of infusion TILs. DNA isolated from CD4⁺CD25⁺, CD4⁺CD25⁻, and CD8⁺CD25⁺ T cells that were sorted using magnetic beads as described in “Methods” was subjected to methylation analysis.