Elena R. Bovolenta

The epigenetic landscape of individual cells determines their identity and function, yet current methods for profiling chromatin modifications at single-cell resolution remain low-throughput, costly, or limited in parametric depth. Here we present EpiFlow, a spectral flow cytometry-based platform that enables the simultaneous quantification of 16 epigenetic markers, including histone post-translational modifications, DNA methylation, and hydroxymethylation, at the single-cell level. We demonstrate that EpiFlow is robust across species from yeast to mammals and resolves biologically meaningful epigenetic transitions during the cell cycle, stem cell differentiation, germinal centre B cell maturation, diabetic liver remodelling, and seizure-induced chromatin reprogramming. High-dimensional integration of EpiFlow data enables cell-type classification based solely on epigenetic profiles in liver, brain, blood, and cancer. Furthermore, EpiFlow detects on-target and off-target/indirect effects of epigenetic drugs in a high-throughput-compatible format. Collectively, these results establish EpiFlow as a broadly applicable platform for single-cell epigenetic analysis in basic, pharmaceutical, and translational research.

The B cell response in the germinal centre (GC) reaction requires a unique bioenergetic supply. Although mitochondria are remodelled upon antigen-mediated B cell receptor stimulation, mitochondrial function in B cells is still poorly understood. To gain a better understanding of the role of mitochondria in B cell function, here we generate mice with B cell-specific deficiency in Tfam, a transcription factor necessary for mitochondrial biogenesis. Tfam conditional knock-out (KO) mice display a blockage of the GC reaction and a bias of B cell differentiation towards memory B cells and aged-related B cells, hallmarks of an aged immune response. Unexpectedly, blocked GC reaction in Tfam KO mice is not caused by defects in the bioenergetic supply but is associated with a defect in the remodelling of the lysosomal compartment in B cells. Our results may thus describe a mitochondrial function for lysosome regulation and the downstream antigen presentation in B cells during the GC reaction, the dysruption of which is manifested as an aged immune response.

Significance: The ability of the T cell receptor (TCR) to convey signals of different intensity is essential for the generation of a diverse, protecting, and self-tolerant T cell repertoire. We provide evidence that pre-TCR signaling during the first stage of T cell differentiation, thought to only check for in-frame rearrangement of TCRβ gene segments, determines the degree of diversity in a signaling intensity–dependent manner and controls the diversity of the TCR repertoire available for subsequent thymic positive and negative selection. Pre-TCR signaling intensity is regulated by the transmembrane region of its associated CD3ζ chains, possibly by organizing pre-TCRs into nanoclusters. Our data provide insights into immune receptor signaling mechanisms and reveal an additional checkpoint of T cell repertoire diversity.