Programmed Death-1 (PD-1) is an inhibitory cell surface receptor of the immunoglobulin superfamily expressed on activated lymphocytes, monocytes and dendritic cells. Although PD-1 function is best characterized in T-cells, it is known that PD-1 also suppresses the immune response of B lymphocytes through protein phosphatase recruitment and dephosphorylation of signaling molecules downstream of the B-cell receptor (BCR). Recent studies have found that PD-1 expression is elevated at the mRNA as well as the protein levels in B cells obtained from chronic lymphocytic leukemia (CLL) patients compared to those from healthy controls. Using genome-wide DNA methylation sequencing, we identified PD-1 as one of the significantly hypomethylated genes in CLL compared to normal B-cell samples. Three differentially methylated regions (DMRs) were discovered in the first intron, proximal promoter and up-stream enhancer regions. We validated these DMRs in 43 CLL and 7 normal control samples using bisulfite pyrosequencing. The pyrosequencing analysis further confirmed that all three regions were significantly hypomethylated in CLL patient samples (p<0.001). These epigenetic changes resulted in the overexpression of PD-1 in primary CLL B cells, which was confirmed by real-time quantitative PCR. In B cells isolated from healthy controls, flow cytometry analysis showed that only approximately 1% expressed PD-1, whereas PD-1 positive B cells in CLL patients ranged from 5% to 64%. No correlation between PD-1 status and IGHV mutations or CD38 expression was observed. To elucidate the mechanisms of epigenetic regulation of PD-1 expression, we studied five non-Hodgkin's lymphoma cell lines including Mec-1, Granta 519, RL, Raji and DB. Bisulfite pyrosequencing results showed that only the up-stream enhancer is differentially methylated in these cell lines. Treatment of lymphoma cell lines with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors can up-regulate PD-1 expression in RL, Raji and DB cell lines in which the up-stream enhancer region is hypermethylated; however, the same treatments decreased the PD-1 expression in Mec-1 cells, which are demethylated in the PD-1 enhancer region and express PD-1 on the cell surface. Chromatin immunoprecipitation (ChIP) analysis revealed that H3K4me3 and H3K4me1 modifications were significantly enriched in the promoter and enhancer regions in PD-1 positive Mec-1 cells, respectively. However, H3K27me3 modification was enriched in both promoter and enhancer regions in PD-1 negative RL cells, while enrichment of H3K3me3 and H3K4me1 modification was significantly decreased. These results suggest that coordinated regulation of enhancer activity by DNA methylation and histone modification is crucial for PD-1 expression in CLL B cells. Furthermore, we mapped the nucleosome occupancy in the enhancer regions using a high-resolution, single-molecule approach. The nucleosome mapping results revealed nucleosome-depleted regions in Mec-1 cells, but not in RL cells. This novel finding demonstrates the complexity of epigenetic regulation of PD-1 expression. To determine the function of PD-1 in CLL, we co-cultured the Mec-1 cell line and primary CLL B-cells with a hepatocyte cell line Huh7.5 that overexpresses exogenous PD-1 ligand, PD-L1. Surprisingly, unlike PD-1 positive normal B cells, we did not observe increased apoptosis in the co-cultured CLL B cells, suggesting that PD-1 has a different functional role in CLL compared to normal B cells. In summary, we present here the novel finding that DNA hypomethylation in the enhancer region of PD-1 leads to aberrant overexpression of PD-1 on CLL B-cell surfaces and that in CLL PD-1 may have a different function than in normal B cells.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.