Genome-Wide DNA Methylation Analysis Identifies Aberrant Epigenetic Changes in CD8+ T Cells from Chronic Lymphocytic Leukemia Patients
Authors: Wu Jiazhu , Xu Xiaojing , Pei Lirong , Lee Eun-Joon , Shull Austin , Awan Farrukh , Wang Xiaoling , Fan Lei , Jianyong Li , Wei Xu , Shi Huidong 
Published: October 2019

Abstract
Background CD8+ T cells from chronic lymphocytic leukemia (CLL) patients have been demonstrated to exhibit a number of alterations in global gene expression profiles when compared with healthy controls. It has been shown that CD8+ T cells from CLL patients have increased expression of T-cell exhaustion markers like PD-1. CLL-induced functional defects in T cells are thought to directly contribute to the failure of antitumor immunity and are considered a hallmark of this disease. Nevertheless, the molecular regulation of T-cell dysfunction in CLL patients still remains poorly understood.
Methods In the present study, CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of patients with CLL (n=10) and healthy donors (n=5), and analyzed by genome-wide DNA methylation profiling using Illumina Infinium 450K methylation array. The differentially methylated genes (KLRG1, CCR6 and TCRA) identified by the 450K array analysis were validated by bisulfite pyrosequencing in additional CLL and healthy control samples. DNA methylation in the first intron, distal upstream, and proximal promoter regions of PD-1 was also examined by pyrosequencing. Luciferase reporter assays were used to determine the effects of DNA methylation on the enhancer activity of a PD-1 upstream sequence. To investigate whether CLL cells can directly alter the methylation of the candidate genes in CD8+ T cells, healthy PBMCs were cultured alone or co-cultured with purified allogeneic CLL cells for 72 hours. In parallel, healthy PBMCs were cultured in CD3mAb-coated plates containing CD28mAb or treated with PMA/ionomycin for 72 hours. Cultured PBMCs were then harvested for flow cytometrc analysis and CD8+ T cells purification. Multicolor flow cytometry was used to characterize T-cell subsets and expression of PD-1, KLRG1 and TCRα/β. Bisulfite pyrosequencing was used to determine the methylation changes of KLRG1, CCR6, TCRA, and PD-1 in CD8+ T cells after co-culture with CLL cells or after T-cell activation.
Results The Illumina 450K methylation array analysis identified 312 differentially methylated CpG sites (Student t-test, p<0.05, average methylation difference >0.25) between CD8+ T cells from CLL and healthy controls with 199 hypermethyated and 113 hypomethylated CpG sites that are associated with 206 genes. Interestingly, 4 out of the 7 most significant CpG sites (FDR<0.05) were located in the 3’-end of the TCRA gene. Bisulfite pyrosequencing confirmed the decrease in the methylation levels of CpG sites associated with KLRG1, CCR6 and TCRA in CD8+ T cells from CLL patients as compared to healthy donors. Previous studies have demonstrated the increased expression of exhaustion markers such as PD-1 on the cell surface of CD8+ T cells from CLL patients. We identified a differentially methylation region (DMR) in the distal upstream region of the PD-1 promoter in CD8+ T-cells. This particular DMR shows consistently lower methylation levels in CD8+ T cells from CLL patients as compared to healthy controls. We cloned the DMR sequence into a luciferase reporter vector pGL4.23 with a minimal promoter and demonstrated enhanced luciferase activities from the cloned sequence, suggesting the presence of potential enhancer activity from this region. We observed that co-cultures with allogeneic CLL cells lead to increased expression of TCRα/β and PD-1 in CD8+ T cells from healthy donors. The methylation level of one CpG site from the 3’-end of TCRA was reduced by 50% after co-culture with CLL cells, though no methylation change in the DMR of PD-1 was observed. T-cell activation by CD3/28mAb or PMA/Ionomycin also resulted decrease in the methylation level of the CpG site at the 3-end of TCRA, yet to a lesser extent.
Conclusion For the first time, our investigation demonstrates the genome-wide DNA methylation profiles of CD8+ T cells isolated from CLL patients and determined that recurrent epigenetic changes in PD-1, KLRG1, CCR6, and TCRA in CD8+ T cells occur in CLL patients. Our methylation data suggest that the exhaustion phenotype observed in CLL patient CD8+ T cells maybe associated with altered DNA methylation profiles, an event previously seen in antigen-specific CD8+ T cells that undergo chronic viral infection-induced epigenetic changes.

Disclosures
Awan: Boehringer Ingelheim: Consultancy; Lymphoma Research Foundation: Research Funding. Wang:NIH/NIMHD: Research Funding. Shi:NIH/NCI: Research Funding; Georgia Research Alliance: Research Funding.