Louisa Katherine James

Droplet-based single-cell RNA sequencing (scRNA-seq) frequently encounters significant challenges from contamination of cell-free mRNAs, known as “ambient mRNAs”, which can substantially distort single-cell transcriptome data interpretation to a large extent. In this study, we investigate the impact of ambient mRNA contamination on differential gene expression and biological pathway enrichment analyses, using two independent scRNA-seq datasets: ten peripheral blood mononuclear cells (PBMCs) samples from dengue-infected patients and forty-two scRNA-seq samples of human fetal liver tissues. We apply two independent ambient mRNA correction approaches – CellBender (automate correction) and SoupX (using a predefined set of potential ambient mRNA genes). We demonstrate that ambient mRNA transcripts appear among differentially expressed genes (DEGs), subsequently leading to the identification of significant ambient-related biological pathways in unexpected cell subpopulations before ambient mRNA contamination correction. In contrast, after suitable correction, we observe a reduction in ambient mRNA expression levels, resulting in improved DEG identification and leading to the highlight of biologically relevant pathways specific to cell subpopulations. Our study underscores the importance of understanding and applying appropriate corrections for ambient mRNA contamination to enhance the reliability and accuracy of scRNA-seq data analyses, thereby improving the robustness of data interpretation in droplet-based scRNA-seq datasets.

Epstein-Barr virus (EBV) is strongly associated with multiple sclerosis (MS). It is likely to play a causal role in the pathogenesis of MS, possibly via triggering autoimmunity through molecular mimicry, autoantigenic presentation or immune dysregulation. Alternatively, evidence supports a direct role for EBV in driving MS disease activity via latent-lytic infection cycling either within the CNS or the periphery. We highlight the recent immunological and virological findings supporting the role of active EBV infection in MS, supporting an evaluation of anti-EBV strategies as potential treatments for MS. Anti-EBV strategies include CNS penetrant small molecule anti-viral agents targeting latent and lytic infection, and immunotherapies. Immunotherapies include EBV-specific autologous or allogeneic cytotoxic T cells (CTLs) and therapeutic EBV vaccines and/or immune checkpoint inhibitors to rejuvenate and boost endogenous EBV-targeted CTL responses. In parallel, several licensed MS disease-modifying therapies may work via mechanisms targeting EBV directly or indirectly. B-cell depleting therapies have been shown to have anti-EBV activity; additionally, new strategies to target intrathecal B cells, plasmablasts and plasma cells are being explored, including high-dose anti-CD20 therapy, cladribine, proteasome inhibitors, BTK inhibitors, CNS-penetrant anti-CD20/CD19 monoclonal antibodies and CD19-targeted CAR T cells. Innovative trial designs for proof-of-concept studies to test EBV antivirals and immunotherapies in MS are needed to catalyse a wave of drug development targeting EBV as a therapeutic strategy to prevent or treat MS.

Epstein–Barr virus (EBV) is a ubiquitous herpesvirus with well-established oncogenic potential, contributing to various malignancies and immune-mediated diseases. Its capacity to infect and immortalize B-cells forms the basis for the generation of lymphoblastoid cell lines (LCLs), which serve as vital models in immunology, virology, and translational research. While conventional LCLs are produced by exogenous EBV infection of peripheral blood mononuclear cells, spontaneous lymphoblastoid cell lines (S-LCLs) can emerge without deliberate viral inoculation, particularly in EBV-seropositive individuals. This review highlights multiple methodologies used to establish S-LCLs, including the use of cyclosporin A, CpG DNA, checkpoint kinase inhibitors, and cytokine modulation, and presents findings from diverse clinical contexts such as autoimmune diseases, post-transplant lymphoproliferative disorders, and cancer. We discuss the biological mechanisms underpinning EBV latency and reactivation, emphasizing the viral transcriptional programmes that drive B-cell transformation and persistence. Additionally, we explore how cytokines, particularly IL-10, support S-LCL survival, and how sodium butyrate and antiviral agents like acyclovir can influence EBV reactivation and replication. The review also considers the diagnostic and therapeutic relevance of LCLs, including their potential as antigen-presenting cells, vaccine platforms, and models for cellular immunotherapies such as CAR T-cells and virus-specific cytotoxic T lymphocytes. By evaluating the generation, molecular features, and immunological significance of S-LCLs, this review underscores their value in modelling EBV-driven disease and advancing novel therapeutic strategies.

Optimized transfer RNA (tRNA) codon use can speed up antibody generation Antibodies are critical for human health, providing long-lived and exquisitely specific immunity to pathogens. Plasma cells secrete tens of thousands of antibody molecules every second and sustain this output continuously for several decades. Plasmablasts and plasma cells [collectively, antibody-secreting cells (ASCs)] are generated following the activation and differentiation of B cells, which involves complete reprogramming of the transcriptional machinery and remodeling of the endoplasmic reticulum and Golgi apparatus. ASCs have co-opted the unfolded protein response (UPR), a stress response, to accommodate the exceptional rate of protein synthesis and secretion required. On page 205 of this issue, Giguere et al. (1) reveal that mRNA encoding antibody genes is enriched with codons recognized by modified transfer RNA (tRNA). This codon optimization is accompanied by an increase in tRNAs with complementary modifications, which serves to enhance antibody biosynthesis by ASCs. This has wider implications for therapeutic protein production, vaccine design, and beyond.

Summary: The ability of B cells to generate antibodies and provide long-lived protective immunity is the cornerstone of vaccination and has contributed to the success of modern medicine. The nine different antibody subclasses produced by humans have effector functions that differ according to antigen type and route of exposure. Expression of the appropriate isotype is critical for effective humoral immunity, and it is becoming clear that subclass specificity is to some extent reflected at the cellular level. Understanding the mechanisms that govern the induction, expansion, and maintenance of B cells expressing different antibody subclasses informs the strategic manipulation of responses to benefit human health. This article provides an overview of the mechanisms by which the different human antibody subclasses regulate immunity, presents an update on how antibody subclass expression is regulated at the cellular level and highlights key areas for future research.

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.

The synthesis of serine from glucose is a key metabolic pathway supporting cellular proliferation in healthy and malignant cells. Despite this, the role that this aspect of metabolism plays in germinal center biology and pathology is not known. Here, we performed a comprehensive characterization of the role of the serine synthesis pathway in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional serine synthesis pathway is a metabolic hallmark of B cell activation and the germinal center reaction. Inhibition of phosphoglycerate dehydrogenase (PHGDH), the first and rate-limiting enzyme in this pathway, led to defective germinal formation and impaired high-affinity antibody production. In addition, overexpression of enzymes involved in serine synthesis was a characteristic of germinal center B cell–derived lymphomas, with high levels of expression being predictive of reduced overall survival in diffuse large B cell lymphoma. Inhibition of PHGDH induced apoptosis in lymphoma cells, reducing disease progression. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in lymphoma.