KIR2DL3 expression in T cells is controlled by DNA methylation. CD4 and CD8 T cells were isolated from 20- to 30-year-old persons (n = 13; □) and 70- to 85-year-old persons (n = 12; ▩), activated by CD3/CD28 beads for 72 hours, and then cultured in the absence or presence of 1 μM 5-Aza-dC for an additional 72 hours. CD158b/j surface expression was analyzed by flow cytometry (A) and KIR2DL3 transcripts were quantified by real-time PCR and are given relative to 10⁷ β-actin transcripts (B). Methylation of CpG sites in the KIR2DL3 core promoter in CD158b/j⁻ Jurkat cells and CD158b/j⁺ HUT78 cells were analyzed by bisulfite sequencing. Each row represents an individual subclone; nucleotide positions are relative to the translation initiation codons. Closed symbols indicate methylated, open symbols unmethylated, CpG (C). DNA methylation levels at CpG −170/−160, −120/−116, −50, and −27/−23 were quantified for Jurkat and HUT78 cells by methylation-specific real-time PCR with the use of 3′-locked nucleic acid primers (D). Results are shown as mean ratio ± SD of methylated DNA versus total copies and are representative of 3 experiments.