TFEB activation hallmarks antigenic experience of B lymphocytes and directs germinal center fate decisions.

Ligation of the B cell antigen receptor (BCR) initiates humoral immunity. However, BCR signaling without appropriate co-stimulation commits B cells to death rather than to differentiation into immune effector cells. How BCR activation depletes potentially autoreactive B cells while simultaneously primes for receiving rescue and differentiation signals from cognate T lymphocytes remains unknown. Here, we use a mass spectrometry-based proteomic approach to identify cytosolic/nuclear shuttling elements and uncover transcription factor EB (TFEB) as a central BCR-controlled rheostat that drives activation-induced apoptosis, and concurrently promotes the reception of co-stimulatory rescue signals by supporting B cell migration and antigen presentation. CD40 co-stimulation prevents TFEB-driven cell death, while enhancing and prolonging TFEB's nuclear residency, which hallmarks antigenic experience also of memory B cells. In mice, TFEB shapes the transcriptional landscape of germinal center B cells. Within the germinal center, TFEB facilitates the dark zone entry of light-zone-residing centrocytes through regulation of chemokine receptors and, by balancing the expression of Bcl-2/BH3-only family members, integrates antigen-induced apoptosis with T cell-provided CD40 survival signals. Thus, TFEB reprograms antigen-primed germinal center B cells for cell fate decisions.

Potentially functional variants of INPP5D and EXOSC3 in immunity B cell-related genes are associated with non-small cell lung cancer survival.

B cells are adaptive immune cells in the tumor microenvironment and play an important role in tumor development and metastasis. However, the roles of genetic variants of the immunity B cell-related genes in the survival of patients with non-small cell lung cancer (NSCLC) remain unknown. In the present study, we first evaluated associations between 10,776 single nucleotide polymorphisms (SNPs) in 220 immunity B cell-related genes and survival of NSCLC in a discovery dataset of 1,185 patients from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial. We found that 369 SNPs were significantly associated with overall survival (OS) of NSCLC in multivariable Cox proportional hazards regression analysis (P ≤ 0.05, Bayesian false discovery probability ≤ 0.80), of which 18 SNPs were validated in another independent genotyping dataset of 984 patients from the Harvard Lung Cancer Susceptibility (HLCS) Study. We then performed linkage disequilibrium (LD) analysis, followed by stepwise analysis with a multivariable Cox regression model. Finally, two independent SNPs, inositol polyphosphate-5-phosphatase D (INPP5D) rs13385922 C>T and exosome component 3 (EXOSC3) rs3208406 A>G, remained significantly associated withNSCLC OS with a combined hazards ratio (HR) of 1.14 (95% confidence interval = 1.06-1.23, P = 2.41×10-4) and 1.20 (95% confidence interval = 1.14-1.28, P = 3.41×10-9), respectively. Furthermore, NSCLC patients with the combination of unfavorable genotypes for these two SNPs were associated with a poor OS (P trend = 0.0002) and disease-specific survival (DSS, P trend < 0.0001) in the PLCO dataset. Expression quantitative trait loci (eQTL) analysis suggested that the INPP5D rs6782875 T allele was significantly correlated with elevated INPP5D mRNA expression levels in normal lung tissues and whole blood samples, while the EXOSC3 rs3208406 G allele was significantly correlated with increased EXOSC3 mRNA expression levels in normal lung tissues. Our data indicated that genetic variants in these immunity B cell-related genes may predict NSCLC survival possibly by influencing the gene expression.

Extracellular vesicles derived from antigen-presenting cells pulsed with foot and mouth virus vaccine-antigens act as carriers of viral proteins and stimulate B cell response.

Foot and mouth disease (FMD) is a highly contagious infection caused by FMD-virus (FMDV) that affects livestock worldwide with significant economic impact. The main strategy for the control is vaccination with FMDV chemically inactivated with binary ethylenimine (FMDVi). In FMDV infection and vaccination, B cell response plays a major role by providing neutralizing/protective antibodies in animal models and natural hosts. Extracellular vesicles (EVs) and small EVs (sEVs) such as exosomes are important in cellular communication. EVs secreted by antigen-presenting cells (APC) like dendritic cells (DCs) participate in the activation of B and T cells through the presentation of native antigen membrane-associated to B cells or by transferring MHC-peptide complexes to T cells and even complete antigens from DCs. In this study, we demonstrate for the first time that APC activated with the FMDVi O1 Campos vaccine-antigens secrete EVs expressing viral proteins/peptides that could stimulate FMDV-specific immune response. The secretion of EVs-FMDVi is a time-dependent process and can only be isolated within the first 24 h post-activation. These vesicles express classical EVs markers (CD9, CD81, and CD63), along with immunoregulatory molecules (MHC-II and CD86). With an average size of 155 nm, they belong to the category of EVs. Studies conducted in vitro have demonstrated that EVs-FMDVi express antigens that can stimulate a specific B cell response against FMDV, including both marginal zone B cells (MZB) and follicular B cells (FoB). These vesicles can also indirectly or directly affect T cells, indicating that they express both B and T epitopes. Additionally, lymphocyte expansion induced by EVs-FMDVi is greater in splenocytes that have previously encountered viral antigens in vivo. The present study sheds light on the role of EVs derived from APC in regulating the adaptive immunity against FMDV. This novel insight contributes to our current understanding of the immune mechanisms triggered by APC during the antiviral immune response. Furthermore, these findings may have practical implications for the development of new vaccine platforms, providing a rational basis for the design of more effective vaccines against FMDV and other viral diseases.

B cells require DOCK8 to elicit and integrate T cell help when antigen is limiting.

Dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome is characterized by a failure of the germinal center response, a process involving the proliferation and positive selection of antigen-specific B cells. Here, we describe how DOCK8-deficient B cells are blocked at a light-zone checkpoint in the germinal centers of immunized mice, where they are unable to respond to T cell-dependent survival and selection signals and consequently differentiate into plasma cells or memory B cells. Although DOCK8-deficient B cells can acquire and present antigen to initiate activation of cognate T cells, integrin up-regulation, B cell-T cell conjugate formation, and costimulation are insufficient for sustained B cell and T cell activation when antigen availability is limited. Our findings provide an explanation for the failure of the humoral response in DOCK8 immunodeficiency syndrome and insight into how the level of available antigen modulates B cell-T cell cross-talk to fine-tune humoral immune responses and immunological memory.

Epstein-Barr virus noncoding RNA EBER1 promotes the expression of a ribosomal protein paralog to boost oxidative phosphorylation.

Epstein-Barr virus (EBV) is a highly successful pathogen that infects ~95% of the adult population and is associated with diverse cancers and autoimmune diseases. The most abundant viral factor in latently infected cells is not a protein but a noncoding RNA called EBV-encoded RNA 1 (EBER1). Even though EBER1 is highly abundant and was discovered over forty years ago, the function of EBER1 has remained elusive. EBER1 interacts with the ribosomal protein L22, which normally suppresses the expression of its paralog L22-like 1 (L22L1). Here we show that when L22 binds EBER1, it cannot suppress L22L1, resulting in L22L1 being expressed and incorporated into ribosomes. We further show that L22L1-containing ribosomes preferentially translate mRNAs involved in the oxidative phosphorylation pathway. Moreover, upregulation of L22L1 is indispensable for growth transformation and immortalization of resting B cells upon EBV infection. Taken together, our results suggest that the function of EBER1 is to modulate host gene expression at the translational level, thus bypassing the need for dysregulating host gene transcription.

Quantifying biomolecular organisation in membranes with brightness-transit statistics.

Cells crucially rely on the interactions of biomolecules at their plasma membrane to maintain homeostasis. Yet, a methodology to systematically quantify biomolecular organisation, measuring diffusion dynamics and oligomerisation, represents an unmet need. Here, we introduce the brightness-transit statistics (BTS) method based on fluorescence fluctuation spectroscopy and combine information from brightness and transit times to elucidate biomolecular diffusion and oligomerisation in both cell-free in vitro and in vitro systems incorporating living cells. We validate our approach in silico with computer simulations and experimentally using oligomerisation of EGFP tethered to supported lipid bilayers. We apply our pipeline to study the oligomerisation of CD40 ectodomain in vitro and endogenous CD40 on primary B cells. While we find a potential for CD40 to oligomerize in a concentration or ligand depended manner, we do not observe mobile oligomers on B cells. The BTS method combines sensitive analysis, quantification, and intuitive visualisation of dynamic biomolecular organisation.

Effect of Leishmania infantum infection on B cell lymphopoiesis and memory in the bone marrow and spleen.

Visceral leishmaniasis (VL) is characterized by an uncontrolled infection of internal organs such as the spleen, liver and bone marrow (BM) and can be lethal when left untreated. No effective vaccination is currently available for humans. The importance of B cells in infection and VL protective immunity has been controversial, with both detrimental and protective effects described. VL infection was found in this study to increase not only all analyzed B cell subsets in the spleen but also the B cell progenitors in the BM. The enhanced B lymphopoiesis aligns with the clinical manifestation of polyclonal hypergammaglobulinemia and the occurrence of autoantibodies. In line with earlier reports, flow cytometric and microscopic examination identified parasite attachment to B cells of the BM and spleen without internalization, and transformation of promastigotes into amastigote morphotypes. The interaction appears independent of IgM expression and is associated with an increased detection of activated lysosomes. Furthermore, the extracellularly attached amastigotes could be efficiently transferred to infect macrophages. The observed interaction underscores the potentially crucial role of B cells during VL infection. Additionally, using immunization against a fluorescent heterologous antigen, it was shown that the infection does not impair immune memory, which is reassuring for vaccination campaigns in VL endemic areas.

Control of germinal center B cell survival and IgE production by an adaptor molecule containing PH and SH2 domains, Aps/Sh2b2.

The germinal centers (GCs) are structure found within secondary lymphoid organs and are important for the antibody-producing response against foreign antigens. In GCs, antigen-specific B cells proliferate intensely, inducing immunoglobulin class switching. Recent studies have shown that GCs are also an important site for class switching to IgE, which is implicated in allergy. However, the mechanisms by which IgE production is regulated in GCs remain unclear. Here, we found impairment in IgE-specific production and a reduction of GC B cells after immunization in mice deficient in the Aps/Sh2b2 gene encoding the Lnk/Sh2b family adaptor protein Aps. GC B cells express higher levels of the Aps gene than non-GC B cells, and cell death of Aps-/- GC B cells is enhanced compared to wild-type GC B cells. An in vitro culture system with purified Aps-/- B cells induced the same level of IgE production and frequencies of IgE+ B cells as wild-type B cells. We found that Aps deficiency in B cells resulted in augmented depletion of IgE+ blasts by B cell receptor crosslinking with anti-CD79b antibodies compared to wild-type IgE+ cells. These results suggest that Aps regulates IgE production by controlling the survival of GC B cells and IgE+ plasma cells and may serve as a potential therapeutic target to control IgE production.

Immature B cell homing shapes human lymphoid tissue structure and function.

Shortly after the emergence of newly formed human B cells from bone marrow as transitional cells, they diverge along two developmental pathways that can be distinguished by the level of IgM they express and migratory biases. Here, we propose that differential tissue homing of immature B cell subsets contributes to human lymphoid tissue structure and function.

B cell tolerance and autoimmunity: Lessons from repertoires.

Adaptive immune cell function is regulated by a highly diverse receptor recombined from variable germline-encoded segments that can recognize an almost unlimited array of epitopes. While this diversity enables the recognition of any pathogen, it also poses a risk of self-recognition, leading to autoimmunity. Many layers of regulation are present during both the generation and activation of B cells to prevent this phenomenon, although they are evidently imperfect. In recent years, our ability to analyze immune repertoires at scale has drastically increased, both through advances in sequencing and single-cell analyses. Here, we review the current knowledge on B cell repertoire analyses, focusing on their implication for autoimmunity. These studies demonstrate that a failure of tolerance occurs at multiple independent checkpoints in different autoimmune contexts, particularly during B cell maturation, plasmablast differentiation, and within germinal centers. These failures are marked by distinct repertoire features that may be used to identify disease- or patient-specific therapeutic approaches.

Atypical and non-classical CD45RBlo memory B cells are the majority of circulating SARS-CoV-2 specific B cells following mRNA vaccination or COVID-19.

Resting memory B cells can be divided into classical or atypical groups, but the heterogenous marker expression on activated memory B cells makes similar classification difficult. Here, by longitudinal analysis of mass cytometry and CITE-seq data from cohorts with COVID-19, bacterial sepsis, or BNT162b2 mRNA vaccine, we observe that resting B cell memory consist of classical CD45RB+ memory and CD45RBlo memory, of which the latter contains of two distinct groups of CD11c+ atypical and CD23+ non-classical memory cells. CD45RB levels remain stable in these cells after activation, thereby enabling the tracking of activated B cells and plasmablasts derived from either CD45RB+ or CD45RBlo memory B cells. Moreover, in both COVID-19 patients and mRNA vaccination, CD45RBlo B cells formed the majority of SARS-CoV2 specific memory B cells and correlated with serum antibodies, while CD45RB+ memory are activated by bacterial sepsis. Our results thus identify that stably expressed CD45RB levels can be exploited to trace resting memory B cells and their activated progeny, and suggest that atypical and non-classical CD45RBlo memory B cells contribute to SARS-CoV-2 infection and vaccination.

Comprehensive immune cell spectral library for large-scale human primary T, B, and NK cell proteomics.

Although proteomics is extensively used in immune research, there is currently no publicly accessible spectral assay library for the comprehensive proteome of immune cells. This study generated spectral assay libraries for five human immune cell lines and four primary immune cells: CD4 T, CD8 T, natural killer (NK) cells, and B cells. This was achieved by utilizing data-dependent acquisition (DDA) and employing fractionated samples from over 100 µg of proteins, which was applied to acquire the highest-quality MS/MS spectral data. In addition, Data-indedendent acquisition (DIA) was used to obtain sufficient data points for analyzing proteins from 10,000 primary CD4 T, CD8 T, NK, and B cells. The immune cell spectral assay library generated included 10,544 protein groups and 127,106 peptides. The proteomic profiles of 10,000 primary human immune cells obtained from 15 healthy volunteers analyzed using DIA revealed the highest heterogeneity of B cells among other immune cell types and the similarity between CD4 T and CD8 T cells. All data and spectral library are deposited in ProteomeXchange (PXD047742).

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus.

Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (encoded by IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-κB regulator IKKε. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a regulatory pathway involving rs2297550, Ikaros, and IKKε implicated by human genetics in risk for SLE.

Immune system dysregulation in the pathogenesis of non-alcoholic steatohepatitis: unveiling the critical role of T and B lymphocytes.

Non-alcoholic fatty liver disease (NAFLD), characterized by the excessive accumulation of fat within the cytoplasm of hepatocytes (exceeding 5% of liver weight) in individuals without significant alcohol consumption, has rapidly evolved into a pressing global health issue, affecting approximately 25% of the world population. This condition, closely associated with obesity, type 2 diabetes, and the metabolic syndrome, encompasses a spectrum of liver disorders ranging from simple steatosis without inflammation to non-alcoholic steatohepatitis (NASH) and cirrhotic liver disease. Recent research has illuminated the complex interplay between metabolic and immune responses in the pathogenesis of NASH, underscoring the critical role played by T and B lymphocytes. These immune cells not only contribute to necroinflammatory changes in hepatic lobules but may also drive the onset and progression of liver fibrosis. This narrative review aims to provide a comprehensive exploration of the effector mechanisms employed by T cells, B cells, and their respective subpopulations in the pathogenesis of NASH. Understanding the immunological complexity of NASH holds profound implications for the development of targeted immunotherapeutic strategies to combat this increasingly prevalent and burdensome metabolic liver disease.

Cell-specific gene networks and drivers in rheumatoid arthritis synovial tissues.

Rheumatoid arthritis (RA) is a common autoimmune and inflammatory disease characterized by inflammation and hyperplasia of the synovial tissues. RA pathogenesis involves multiple cell types, genes, transcription factors (TFs) and networks. Yet, little is known about the TFs, and key drivers and networks regulating cell function and disease at the synovial tissue level, which is the site of disease. In the present study, we used available RNA-seq databases generated from synovial tissues and developed a novel approach to elucidate cell type-specific regulatory networks on synovial tissue genes in RA. We leverage established computational methodologies to infer sample-specific gene regulatory networks and applied statistical methods to compare network properties across phenotypic groups (RA versus osteoarthritis). We developed computational approaches to rank TFs based on their contribution to the observed phenotypic differences between RA and controls across different cell types. We identified 18 (fibroblast-like synoviocyte), 16 (T cells), 19 (B cells) and 11 (monocyte) key regulators in RA synovial tissues. Interestingly, fibroblast-like synoviocyte (FLS) and B cells were driven by multiple independent co-regulatory TF clusters that included MITF, HLX, BACH1 (FLS) and KLF13, FOSB, FOSL1 (B cells). However, monocytes were collectively governed by a single cluster of TF drivers, responsible for the main phenotypic differences between RA and controls, which included RFX5, IRF9, CREB5. Among several cell subset and pathway changes, we also detected reduced presence of Natural killer T (NKT) cells and eosinophils in RA synovial tissues. Overall, our novel approach identified new and previously unsuspected Key driver genes (KDG), TF and networks and should help better understanding individual cell regulation and co-regulatory networks in RA pathogenesis, as well as potentially generate new targets for treatment.

Computational detection of antigen-specific B cell receptors following immunization.

B cell receptors (BCRs) play a crucial role in recognizing and fighting foreign antigens. High-throughput sequencing enables in-depth sampling of the BCRs repertoire after immunization. However, only a minor fraction of BCRs actively participate in any given infection. To what extent can we accurately identify antigen-specific sequences directly from BCRs repertoires? We present a computational method grounded on sequence similarity, aimed at identifying statistically significant responsive BCRs. This method leverages well-known characteristics of affinity maturation and expected diversity. We validate its effectiveness using longitudinally sampled human immune repertoire data following influenza vaccination and SARS-CoV-2 infections. We show that different lineages converge to the same responding Complementarity Determining Region 3, demonstrating convergent selection within an individual. The outcomes of this method hold promise for application in vaccine development, personalized medicine, and antibody-derived therapeutics.