Genome Wide DNA Methylation Landscape Reveals Glioblastoma Mediated Epigenetic Modification in Tumor Infiltrating CD4+ T-Cells

Abstract

Abstract INTRODUCTION CD4+ helper T (Th) cells initiate and maintain adaptive immune responses and play a critical role in orchestrating effective antitumor immune response. Although recent immunotherapeutic strategies have shown promising results against glioblastoma, the full potential of this modality has yet to be achieved. One of the major limitations of immunotherapy is the poor efficacy of antiglioblastoma T-cell response in the tumor microenvironment. We hypothesized that glioblastoma modulates antitumor T-cell response by epigenetic modification of tumor infiltrating Th cells (TIThC). METHODS To investigate the influence of glioblastoma on TIThCs, we isolated CD4+ T-cells from the tumor and peripheral blood (PB) of 5 steroid naïve patients with newly diagnosed glioblastoma and performed whole-genome bisulfite sequencing (WGBS) as well as RNAseq and identified differentially methylated and expressed genes between the two cell populations. RESULTS Our results show that glioblastoma mediated epigenetic modifications define the molecular characteristics of glioblastoma infiltrating CD4+ T-cells. Tegmentation based WGBS revealed more than 25 000 regions that are methylated differentially in pairwise comparison of TIThC and PB CD4+ T-cells. Methylation status correlated with the gene expression profile with more than 20 000 differentially expressed genes in TIThCs compared to PB. Of the CD4 lineage specific genes, TBX21, GATA3, RORC, and FOXP3, TBX21, GATA3, and FOXP3 showed differential methylation and expression level in TIThC; whereas, RORC only showed difference in methylation status but not in gene expression level. There was a significant difference in overall and CD4 lineage specific methylation and gene expression profile between patients. Pathway analysis of differentially methylated regions and differentially expressed genes indicated several pathways of tumor induced deregulation, including those involved in T-cell activation, lymphocyte differentiation, regulation of immune effector process, and cytokine production. CONCLUSION Glioblastoma multiforme (GBM) regulates antitumor immune response by significant epigenetic reprogramming of TIThC; thus, influencing their lineage specific differentiation and function.