Yim, K. H. W. | Search bioRxiv

bioRxiv2024

Intercellular epigenomic signaling during B cell maturation.

B cell maturation is crucial for effective adaptive immunity. It requires a complex signaling network to mediate antibody diversification through mutagenesis. B cells also rely on queues from other cells within the germinal center. Recently, a novel class of intercellular signals mediated by extracellular vesicles (EVs) has emerged. Studies have shown B cell EV-mediated signaling is involved in immune response regulation and tumorigenesis. However, the mechanistic role of B cell EVs is not yet established. We herein study the biological properties and physiological function of B cell EVs during B cell maturation. We use emerging technologies to profile B cell EVs surface marker signatures at the single particle level, molecular cargo, and physiological roles in B cell maturation. EV ncRNA cargo, characterized by RNA-seq, identified an EV-mediated novel non-coding RNA regulatory network for B cell maturation. A previously uncharacterized micro-RNA (miR-5099) in combination with a set of long non-coding RNA carried within B cell EVs is shown to be important for antibody diversification. The physiological role of EVs in B cell maturation is investigated using EV blockade assays and complementation studies using diverse EV sources further confirmed the physiological role and mode of action of EVs in B cell maturation.

immunology↗

bioRxiv2021

Single cell label-free probing of chromatin dynamics during B lymphocyte maturation

Large-scale intracellular signalling during developmental growth or in response to environmental alterations are largely orchestrated by chromatin within the cell nuclei. Chemical and conformational modifications of the chromatin architecture are critical steps in the regulation of differential gene expression and ultimately cell fate determination. Therefore, establishing chemical properties of the nucleus could provide key markers for phenotypic characterisation of cellular processes on a scale of individual cells. Raman microscopy is a sensitive technique that is capable of probing single cell chemical composition - and sub-cellular regions - in a label-free optical manner. As such, it has great potential in both clinical and basic research. However, perceived limitations of Raman spectroscopy such as low signal intensity and the difficulty in linking alterations in vibrational signals directly with ensuing biological effects have hampered advances in the field. Here we use immune B lymphocyte development as a model to assess chromatin and transcriptional changes using confocal Raman microscopy in combination with microfluidic devices and correlative transcriptomics, thereby linking changes in chemical and structural properties to biological outcomes. Live B lymphocytes were assessed before and after maturation. Multivariate analysis was applied to distinguish cellular components within each cell. The spectral differences between non-activated and activated B lymphocytes were then identified, and their correlation with known intracellular biological changes were assessed in comparison to conventional RNA-seq analysis. Our data shows that spectral analysis provides a powerful tool to study gene activation that can complement conventional molecular biology techniques and opens the way for mapping the dynamics in the biochemical makeup of individual cells.

biophysics↗