Expression Of PD-1 Gene And Regulation Of Methylation In CD8 ~ + T Cells In Chronic Lymphoblastic Leukemia

Part ⅠPrognostic significance and PD-1 expression of CD8+T cells in chronic lymphocytic leukemiaObjectives:Chronic lymphocytic leukemia (CLL) cells interact with and are supported by different populations of accessory cells from microenvironment, such as mesenchymal stromal cells, monocyte-derived nurselike cells and T cells. Patients with CLL display immune deficiency are observed. The aim of this study is to investigate whether the absolute number of CD4+ and CD8+T cells in the peripheral blood of CLL patients is altered and associated with clinical characteristics, and analyze PD-1 expression and of T cells and T cell subsets by dividing T cell compartments.Methods:The population for retrospective study consisted of a cohort of 234 untreated CLL patients and 10 age-matched healthy donors and correlated our findings with clinical outcome data. PD-1 surface expression was tested by flow cytometry (FCM) in liquid nitrogen stored peripheral blood CD8+T cells from CLL patients (n=22) and compared to age-matched healthy donors (n=10). A total of 11 PBMC samples from CLL patients (nine untreated cases and two relapse cases) and 11 PBMC samples from age-matched healthy donors were freshly obtained in our department and FCM was performed to investigate the distribution of T cell subpopulations as well as the expression of inhibitory molecule PD-1 expression.Results:Compared with age-matched healthy donors, the median number of CD8+T cells in CLL patients cohort was increased (p=0.020), while no difference was observed in CD4+T cells (p=0.108).54 patients (23.1%) showed an inversion in the CD4+/CD8+ ratio based on a cutoff ratio of 1.0. Borderline significant association between inverted CD4+/CD8+ratio and advanced Binet C stage (p=0.039) was observed, while no significant association with immunoglobulin heavy chain variable (IGHV) gene mutation status (p=0.298), CD38 expression (p=0.054), ZAP-70 expression (p=0.098), or TP53 mutation or del(17p) (p=0.105) was observed. Moreover, patients with the inverted CD4+/CD8+ratio had inferior time to first treatment (TTFT) and overall survival (OS) when compared with patients with the normal CD4+/CD8+ratio (p=0.031 and p=0.039). There was no difference between CLL patients and donors regarding the distribution of naive (CD45RA+/CCR7+), central memory (CD45RA-/CCR7+), effector memory (CD45RA-/CCRT) and terminally differentiated (CD45RA+/CCR7-) T cell subpopulations. PD-1 expression was increased in all the CD4+T cell subpopulations (p<0.001) as well as the naive, effector memory and terminally differentiated subpopulations of CD8+T cells.Conclusions:Some CLL patients presented with inverted CD4+/CD8+ ratio, which may be attributed to the relative increase of CD8+T cells. Inverted CD4+/CD8+ ratio predicts poor prognosis. PD-1 was increased in CLL CD8+T cells and was not associated with the distribution of T cell subpopulations.PartⅡThe methylation level and regulatory mechanism of PD-1 in CD8+T cells from chronic lymphocytic leukemiaObjectives:Higher level of PD-1 on CD8+T cells from CLL patients has been reported in previous studies as well as observed in our preliminary study. To identify the potential mechanism of increased expression of PD-1, here we investigated whether the methylation level altered in CD8+T cells from CLL patients and regulated the expression of PD-1.Methods:We purified CD8+T cells from CLL patients (n=15) and healthy donors (n=10) and tested methylation status of multiple CpG sites of PD-1 across the 1st intron (CR1), gene promoter (CR2), and distal upstream (CR3) via bisulfite pyrosequencing. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure mRNA levels of PD-1. A 677bp fragment encompassing the PD-1 distal upstream region was cloned into vector pGL4.23 with a minimal promoter in luciferase report assay. The reporter plasmid DNA was methylated or not by CpG Methyltransferase M.SssI before transfected into HEK293 cells or Jurkat cells. Jurkat cells were either stimulated with PMA and ionomycin (PMA/Ion) or not. Firefly luciferase was normalized to Renilla luciferase to quantify luciferase activities, comparing with vector pGL4.23. Primary PBMC from healthy donors were cocultured with CLL cells with the ratio of 1/4 or 4/1, or activated with anti-CD3 and -CD28 antibodies, or stimulated by PMA/Ion. Following 72h incubation, PD-1 surface expression was measured by FCM and the methylation level was measured by bisulfite pyrosequencing. Purified naive CD8+T cells were stimulated with anti-CD3 and anti-CD28 and IL2 for six days, then cells were harvested for pyrosequencing and qRT-PCR analysis.Results:The quantitative pyrosequencing analysis demonstrated that the first intron region (CRl) was highly methylated, while the proximal promoter region (CR2) was completely unmethylated in CD8+T cells from both CLL and healthy donor samples. The methyaltion level of CRl of CD8+T cells from CLL was slightly decreased although did not reach statistical significance. CR3 region was significantly hypomethylated in CD8+T cells from CLL patients (p<0.050). The methylation level of CR3 region was inversely correlated with PD1 mRNA levels (r=-0.529,p=0.043). We observed that the relative luciferase activity of the unmethylated reporter construct was significantly increased in both HEK293 and Jurkat cells (p<0.010 and p<0.010), while the methylated reporter construct was not (p=0.018 and p=0.636). We observed a significant decrease in DNA methylation at CpG sites 4 (p=0.002) and 5 (p=0.002) of CR3 region in CD8+T cells following PMA/Ion stimulation along with the increased expression of PD-1. When naive CD8+T cells were activated by anit-CD3/anti-CD28 antibodies in the presence of IL2 for 6 days, we observed decreased methylation in this region in the activated naive CD8+T cells.Conclusions:CLL-specific hypomethylation of the CR3 region of PD-1 in CLL CD8+T cells was observed and inversely correlated with PD-1 mRNA. CR3 region may regulate PD-1 expression as an enhancer and the ability could be waned after being methylated.Part ⅢScreening and identification of genome-wide DNA methylation analysis of CD8+T cells from chronic lymphocytic leukemiaObjectives:Based on our preliminary study, the distal upstream enhancer of PD-1 in CLL CD8+T cells was hypomethylated, we aim to explore further epigenetic changes of CD8+T cells from CLL patients in this study.Methods:We purified CD8+T cells from CLL patients (n=15) and healthy donors (n=10) and performed a genome-wide DNA methylation array using the Illumina 450K methylation array platform. Differentially methylated CpG sites and correlated genes were selected by statistical analysis. The methylation level was measured by bisulfite pyrosequencing and selected genes’ expression levels were measured by qRT-PCR.Results:The genome-wide DNA methylation analysis showed that a total of 312 CpG sites associated with 206 genes were deemed differentially methylated between CLL and control CD8+T cells, of which 199 were hypermethylated and 113 were hypomethylated (p<0.05; an average methylation difference>0.25), which associated with 206 genes including immune regulation genes such as TCRA, HLA-DQB1, HLA-DQA1, CCR6, HIVEP3, NFATC1 and KLRG1. Bisulfite pyrosquencing analysis was performed on CpG sites from three candidate loci associated with TCRA, CCR6 and KLRG1 chosen from the array cohort. We observed decreased methylation of one CpG site near 3’UTR of TCRA, six CpG sites from CCR6 promoter and four CpG sites from KLRG1 promoter. The median methylation level of KLRG1 was inversely correlated with mRNA level but not CCR6.Conclusions:In the context of CLL, CD8+T cells exhibit global methylation alteration apart from phenotypical changes. The "exhaustion" of CD8+T form CLL may be involved with epigenetic alterations.