Figure 4.

Low DNA methylation at the PD1 promoter is associated with PD1 expression in Vδ2 T cells. (A) Seven days after stimulation, hydralazine was added to one well of CBMC culture (15µM), while a second well was left untreated as a negative control. PD1 expression by Vδ2 T cells was monitored 3 days after hydralazine treatment (N=9). The scatter plot shows paired values for individual specimens. Two weeks after stimulation, Vδ2 cells were sorted from PBMC or CBMC (> 97% purity) and their genomic DNA was used for methylation analysis. After bisulfite conversion, an amplicon that spans the whole regulatory region CR-C (Conserved Region C, 550 bp) or a shorter 297 bp amplicon at the proximal end of CR-C (boxed in panel B) in the PD1 promoter were generated by PCR. 36–44 amplicons were analyzed for each sample and each time point by Sanger sequencing. (B) The bar graph shows, for each CG pair in the CR-C region, the fraction of methylated cytosine averaged for 9 adults and 12 neonates. Error bars represent SD. Only p<0.001 (***) and p<0.0001 (****) are shown in the chart. (C) The plot shows the inverse correlation between methylation levels at positions −837 and −842 and the proportion of neonatal PD1+ Vδ2 T cells. (D) For 5 CB samples, promoter methylation was monitored 14 and 28 days after stimulation. The methylation levels for the region between −845 and −669 are shown in the bar graph as means + SD. Differences between means were analyzed using unpaired or paired Student’s t-test.