DNA methylation is a heritable epigenetic modification that is essential for gene regulation and often associated with gene silencing. Through germinal centre (GC) maturation, B cells undergo dynamic changes to DNA methylation, including a 10-20% reduction of total 5-methylcytosine (5mC) relative to naïve B cells. These hypomethylated CpGs are maintained in post-GC B cell populations. Dysregulation of DNA methylation has been associated with immune disease, however the mechanisms and significance for this demethylation is not well understood.
To investigate this, we quantified the dynamics of DNA methylation during human B cell maturation using an ex vivo culture of peripheral naïve B cells to model T cell-dependent activation and maturation in the GC. We co-cultured B cells with CD40L-IL21 expressing feeder cells, which allowed 100- to 1000-fold expansion of B cells with high cell viability and differentiation into CD138+ plasma cells.
Whole genome methylation sequencing identified a progressive genome-wide loss of 5mC modifications over a 12-day time course, which is not localised to distal regulatory elements. Changes to 5mC were similar between undifferentiated B cells and CD138+ cells, suggesting that demethylation occurs independent of differentiation. Unexpectedly, we observed complete loss of hydroxymethylcytosine enrichment at open chromatin regions in B cells within 4 days in culture. This depletion is decoupled from the loss of 5mC suggesting that demethylation is not solely mediated by TET protein activity.
Individuals with primary immunodeficiency demonstrate impaired activation and maturation of B cells through the GC. We performed methylation sequencing of sorted PBMC cell subsets from immunodeficiency patients. Compared to healthy individuals, immunodeficiency patients demonstrate reduced changes to the methylation of post-GC switched memory B cells. However, total DNA methylation levels of unswitched memory B cells were comparable between cohorts. This scalable model therefore offers capacity to understand dynamics of GC-dependent demethylation of B cells during maturation.