B cells mature in the germinal centre to produce high-affinity antibody-secreting memory and plasma cells. Through the germinal centre, B cells undergo a global loss of DNA methylation. While DNA methylation has been characterized using bisulfite sequencing of isolated human B cell subsets, the mechanisms by which demethylation occurs after activation has not been explored in a human ex vivo context.
We first modelled B cell maturation in vitro through the activation of primary human naïve B cells co-cultured with CD40-L/IL-21 expressing feeder cells. Over a time course, cultured cells were collected for long-read whole genome methylation sequencing of native DNA using Oxford Nanopore Technologies. Using 0.5X coverage genome skimming we quantified the global change of both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). We observed a 10% global loss of 5mC over time that is decoupled from the changes to global 5hmC levels.
Subsequent sequencing at 20X coverage allowed for identification of differentiation and time-dependent changes in methylation across the genome. Differentially methylated regions (DMRs) were determined through comparison of sorted B cell subsets at a given time point in culture. To identify methylation dynamics over time, DMRs were identified between naïve B cells and cultured B cells. Long-read sequencing allowed for the characterisation of the inter- and intra-molecular heterogeneity of methylation modifications. This study provided deeper insights into the dynamics of B cell demethylation after activation in an ex vivo context.