Long-read transcriptome profiling of germinal centre B cells

Alternative splicing (AS) is a major regulatory process underpinning the development and function of the immune system. 29% of alternatively spliced genes are specific to immune cells, yet the regulation and function of AS during B cell activation remain largely unknown. The activation of naive B cells and the subsequent germinal centre (GC) reaction drive the cascade of reactions resulting in short- and long-term antibody responses, making the GC crucial for adaptive immunity. However, abnormal function of GC B cells contributes to autoimmune disease and the development of lymphomas. Hence, GC reaction needs to be tightly regulated. Previous studies have linked individual AS events in GC B cells to B cell malignancies using short-read sequencing; however, this methodology is limited in defining the complete sequence of transcript variants generated by AS. Therefore, many transcript variants remain undefined. During my PhD, I have developed a long-read sequencing methodology Oxford Nanopore Technologies (ONT) workflow to understand post-transcriptional regulation at both gene and isoform levels in human and mouse GC B cells. Because one of the challenges of ONT is the accurate computational analysis of isoforms, we developed the ‘Nexons’ pipeline to identify the differentially spliced transcript variants. Transcriptome characterisation using ONT allowed us to detect differentially expressed transcripts during antigen-mediated activation of GC B cells in human and mouse. Moreover, identification of individual isoforms with Nexons revealed differential splicing of transcripts, including potentially novel splice variants, as well as changes which were undetectable in short-read sequencing data. An in-depth analysis revealed the differential regulation of poison exons (PE) in serine/arginine-rich splicing factors (SRSF) (e.g., SRSF3 and SRSF7). Naive B cells preferentially expressed isoforms carrying PE, leading to nonsense-mediated mRNA decay, whilst the PE were preferentially removed in activated and GC B cells. Notably, we found this regulation of PE in splicing factors is conserved between human and mouse. We validate an ONT/Nexons workflow as a suitable method for the identification and quantification of transcript isoforms and highlight the SRSF family as important candidates for regulating the GC reaction.Early-Stage Researcher of The Marie-Skłodowska-Curie Innovative Training Network COSMIC, European Horizon 2020 Programme (765158) BBSR

An integrated proteome and transcriptome of B cell maturation defines poised activation states of transitional and mature B cells

Abstract During B cell maturation, transitional and mature B cells acquire cell-intrinsic features that determine their ability to exit quiescence and mount effective immune responses. Here we use label-free proteomics to quantify the proteome of B cell subsets from the mouse spleen and map the differential expression of environmental sensing, transcription, and translation initiation factors that define cellular identity and function. Cross-examination of the full-length transcriptome and proteome identifies mRNAs related to B cell activation and antibody secretion that are not accompanied by detection of the encoded proteins. In addition, proteomic data further suggests that the translational repressor PDCD4 restrains B cell responses, in particular those from marginal zone B cells, to a T-cell independent antigen. In summary, our molecular characterization of B cell maturation presents a valuable resource to further explore the mechanisms underpinning the specialized functions of B cell subsets, and suggest the presence of ‘poised’ mRNAs that enable expedited B cell responses

Polypyrimidine tract binding protein 1 regulates the activation of mouse CD8 T cells

Funder: the BBSRC Core Capability Grant to the Babraham InstituteFunder: Cambridge Commonwealth, European and International Trust studentshipAbstract: The RNA‐binding protein polypyrimidine tract binding protein 1 (PTBP1) has been found to have roles in CD4 T‐cell activation, but its function in CD8 T cells remains untested. We show it is dispensable for the development of naïve mouse CD8 T cells, but is necessary for the optimal expansion and production of effector molecules by antigen‐specific CD8 T cells in vivo. PTBP1 has an essential role in regulating the early events following activation of the naïve CD8 T cell leading to IL‐2 and TNF production. It is also required to protect activated CD8 T cells from apoptosis. PTBP1 controls alternative splicing of over 400 genes in naïve CD8 T cells in addition to regulating the abundance of ∼200 mRNAs. PTBP1 is required for the nuclear accumulation of c‐Fos, NFATc2, and NFATc3, but not NFATc1. This selective effect on NFAT proteins correlates with PTBP1‐promoted expression of the shorter Aβ1 isoform and exon 13 skipped Aβ2 isoform of the catalytic A‐subunit of calcineurin phosphatase. These findings reveal a crucial role for PTBP1 in regulating CD8 T‐cell activation