KIR2DL4 expression in T cells and NK3.3 cells. (A) KIR2DL4 transcription was quantified by real-time PCR in CD4⁺CD28⁺ and CD4⁺CD28⁻ T cells. The results are shown as KIR2DL4 transcript numbers after normalization for 1 × 10⁷ β-actin transcripts and represent mean ± sd from three independent experiments. Expression was higher in CD4⁺CD28⁻ than in CD28⁺ T cells (P=0.02). (B) KIR2DL4 transcription in T cell lines and a NK3.3 cell line; results are presented as mean ± sd from four independent experiments. Expression in HUT78 was higher than in Jurkat cells but lower than in NK3.3 cells (P<0.001). (C) Cells were stained with PE-labeled anti-KIR2DL4 mAb (solid lines) and analyzed by flow cytometry. Staining with PE-IgG served as control (dashed lines). Histograms representative of three independent experiments show unimodal distributions of low (HUT78) and high (NK3.3) KIR2DL4. (D) KIR2DL4 transcription was not different in KIR2DL4^low (lower 30% in mean fluorescence intensity) and KIR2DL4^int (upper 30%) HUT78 cells, suggesting that KIR2DL4 in HUT78 expression was global and not clonally distributed. Results are shown as transcripts relative to 1 × 10⁷ β-actin transcripts and presented as mean ± sd from two independent experiments (P=0.8).

The effects of DNA demethylation on KIR2DL4 transcription. (A) KIR2DL4 transcription in Jurkat, HUT78, and NK3.3 was measured by real-time PCR after DNMT inhibition. Cells were harvested after 12, 24, 48, and 72 h culture in the presence or absence of 1 μM 5-Aza-dC. The results are shown as KIR2DL4 transcript numbers after normalization for 1 × 10⁷ β-actin transcripts and represent mean ± sd from three independent experiments. (B) DNA was purified from the cells after 72 h DNMT inhibition. KIR2DL4 methylation was measured and calculated as described in Figure 2B. (C) Promoter activity of a methylated and unmethylated KIR2DL4 promoter construct (–6 bp to –262 bp) was assessed after transfection into HUT78 cells. pGL3-basic vector and pGL3-promoter vector were used as negative and positive controls. Firefly luciferase activities were normalized to pRL-TK Renilla luciferase activity, which served as an internal control. Methylation decreased KIR2DL4 promoter activity significantly (P=0.0004). The results represent mean ± sd from three independent experiments.

Nucleosome remodeling of the KIR2DL4 promoter. (A) Schematic representation of the nucleosome map. The translation initiation codon, the position of primer used for the CHART-PCR, and the XmnI and PstI cleavage sites relative to the first nucleotide of the translation initiation codon are indicated. (B) Nuclei from Jurkat, HUT78, and NK3.3 were digested with PstI; genomic DNA were purified and subjected to real-time PCR with primers indicated in A. Results were normalized to uncut DNA and are expressed as a percent accessibility. Data shown are the mean ± sd from three independent experiments.

Induction of KIR2DL4 transcription by DNA demethylation does not involve histone acetylation or methylation. Cells were cultured in the absence (shaded bars) or presence of 1 μM 5-Aza-dC (solid bars) for 72 h, at which time, KIR2DL4 was fully expressed in treated HUT78 T cells. ChIP assays were performed with antiacetyl-H3 antibody (A), antiacetyl-H4 antibody (B), antidimethyl-Lys 4 antibody (C), or antidimethyl-Lys 9 antibody (D); and KIR-specific sequences were amplified. DNA demethylation in HUT78 induced transcription without changes in histone acetylation and methylation patterns.

CpG methylation of the KIR2DL4 core promoter. (A) The methylation patterns of CpG sites in the KIR2DL4 core promoter in Jurkat, HUT78, NK3.3, CD4⁺CD28⁺, and CD4⁺CD28⁻ cells were analyzed by bisulfite sequencing. Genomic DNA was treated with sodium bisulfite and amplified with primer pairs encompassing the +18 to –228 region. The amplification product was subcloned and sequenced. Each row represents an individual subclone. Closed symbols indicate methylated CpG sites; open symbols indicate unmethylated CpG sites. The nucleotide position of CpG sites is indicated relative to the first nucleotide of the translation initiation codon. (B) DNA methylation statuses at CpG –24/–34, –47, and –223/–228 were quantified for Jurkat cells and HUT78 cells with low and intermediate KIR2DL4 cell surface expression and CD4⁺CD28⁺ and CD4⁺CD28⁻ T cells by methylation-specific real-time PCR using 3′-locked nucleic acid primers specific for the methylated and unmethylated sequences after bisulfite treatment. Results are shown as mean ± sd of triplicate copy numbers of methylated DNA/copy numbers of methylated + unmethylated DNA and are representative of three experiments. KIR2DL4 promoter methylation of the CpG dinucleotides at –24/–34 and –47 bp was lower in HUT78 and in CD4⁺CD28⁻ than in Jurkat cells and CD4⁺CD28⁺ T cells (P<0.001). (C) Recruitment of the DNMT1 to KIR2DL4 promoter was assayed by ChIP with antibody to DNMT1. The recovered DNA was quantified by real-time PCR using primers specific for the KIR2DL4 promoter. Results are calculated as the ratio of DNA_{specific IP} – DNA_{control IP}:DNA input and are shown as mean ± sd of triplicate precipitations.

Influence of age on KIR2DL4 transcription and methylation status in CD4⁺ and CD8⁺ T cells. CD4 and CD8 T cells and NK cells were isolated from 20- to 30 (light gray boxes)- and 70- to 80 (dark gray boxes)-year-old individuals, and KIR2DL4 transcription levels (A) and methylation levels (B) were analyzed by real-time PCR. Results are shown as box plots of 13 young and 12 old individuals. CD8 T cells from young individuals had lower transcription and higher methylation levels than those from the elderly. (C) CD4⁺ and CD8⁺ cells from eight young and eight elderly, healthy individuals were cultured in the absence or presence of 1 μM 5-Aza-dC for 72 h before KIR2DL4 transcription was quantified. Transcription levels were expressed as transcripts relative to 1 × 10⁷ β-actin transcripts. Before DNMT inhibition, relative transcripts were below 100 in CD4 and in CD8 T cells from young individuals. CD8 T cells from elderly individuals had increased relative copy numbers with a median of 550. Upon DNMT inhibition, KIR2DL4 transcription increased in all individuals but significantly more so in CD4 and CD8 T cells from the elderly. Results are shown as box plots with medians, 25th and 75th percentiles as boxes, and 10th and 90th percentiles as whiskers.

Histone acetylation and methylation of the KIR2DL4 promoter in T cells. (A) Chromatin was precipitated with antiacetyl-H3 and antiacetyl-H4. (B) H3 dimethyl-Lys 4, H3 trimethyl-Lys 4, and H3 dimethyl-Lys 9 were precipitated with specific antibodies. The recovered DNA was subjected to real-time PCR for KIR2DL4 sequences. Results shown are mean ± sd of three independent experiments.