Biology subjects

Tolar, P.

Publications and source records attributed to Tolar, P..

10 recordsLinked to original sources

bioRxiv2026

Altered PI3K-PTEN balance promotes preferential killing of human IgE+ plasma cells by BCR crosslinking

Immunoglobulin E (IgE) drives allergic disease, yet what restrains the persistence of IgE production remains poorly understood. Mouse studies suggest that BCR-induced apoptosis limits the survival of IgE-producing plasma cells (PCs). Whether this mechanism applies to human IgE PCs is unclear. Using a human IgE class-switching system, we show that BCR crosslinking preferentially kills IgE PCs compared to IgG1+ PCs. However, this selective sensitivity is not explained by surface BCR levels or proximal BCR signaling as suggested in mice. Instead, elevated PTEN expression in IgE PCs constrains PI3K/Akt pro-survival signaling and lowers the apoptotic threshold by upregulating BIM, while JNK signaling sustains PTEN expression and amplifies their apoptotic sensitivity. CRISPR/Cas9 targeting of PTEN or BIM, or JNK inhibition protects IgE PCs from BCR-mediated killing. Therapeutic anti-IgE antibodies, including omalizumab and extracellular membrane-proximal domain (EMPD)-targeting antibodies, exploit this sensitivity to selectively eliminate IgE PCs and suppress IgE production, providing a mechanistic rationale for depleting IgE PCs in allergic disease. SummaryRamadani et al. identify a JNK/PTEN/BIM signaling axis that intrinsically limits human IgE plasma cell survival and drives their preferential sensitivity to BCR-induced apoptosis. This mechanism is distinct from that established in mice and has direct implications for anti-IgE therapeutic strategies.

immunology↗

bioRxiv2025

Fusion of Complement Fragment C3d Enhances Germinal Center Responses to HIV-1 Envelope Glycoproteins

Eliciting sustained germinal center (GC) responses is critical for the development of an effective HIV-1 vaccine, yet HIV-1 envelope glycoprotein (Env) immunogens often fail to elicit GC responses required for the maturation of cognate B cells that secrete broadly neutralizing antibodies (bNAbs). Effective antigen recognition is important for initial B cell priming, activation, and GC engagement. Since complement opsonization contributes to antigen recognition, we investigated whether C3d fusion could enhance the GC response of the stabilized HIV-1 Env immunogen based on a consensus sequence (ConM Env). Our results demonstrate that ConM Env-C3d induced potent HIV-specific B cell activation in vitro compared to ConM Env alone. We also observed that the C3d fusion enhanced antigen presentation by human tonsil-derived follicular dendritic cells (FDCs) to HIV-specific B cell lines. Moreover, mouse immunization studies combining ConM Env-C3d with the AddaS03 adjuvant revealed significantly enhanced early GC formation and prolonged antigen display and retention on FDCs for up to 56 days, highlighting improved antigen persistence within GCs. These immunological enhancements, including a more focused early antibody response, correlated with improved virus neutralization. Additionally, we observed sex-based differences in immune responses, with female mice showing stronger antibody responses and enhanced antigen retention compared to males. These findings suggest that C3d fusion can enhance GC engagement and improve the immunogenicity of HIV-1 vaccines.

immunology↗

bioRxiv2024

Mutations in the IgG B cell receptor associated with class-switched B cell lymphomas

Immunoglobulin class-switching from IgM to IgG enhances B cell receptor (BCR) signalling1,2 and promotes germinal centre (GC) B cell responses to antigens3,4. In contrast, non-Hodgkin lymphomas derived from GC B cells typically avoid IgG BCR expression and retain the unswitched IgM BCR, suggesting that the IgG BCR may protect B cells from malignant transformation5,6. However, the mechanism of this phenomenon and its significance for the pathogenicity of IgG-expressing lymphomas remains unclear. Here, we report that IgG-positive follicular lymphoma (FL) and the related EZB subset of diffuse large B cell lymphoma (DLBCL) acquire mutations in the IgG heavy chain, disrupting its unique intracellular tail. Enforced class switching of IgM-expressing EZB DLBCL cell lines to IgG reduces BCR surface levels, signalling via phosphoinositide-3 kinase (PI3K), levels of MYC, cell proliferation and in vivo growth. Inhibiting GSK3, a target of BCR-PI3K signalling, or stimulating the BCR rescues IgG+ cell proliferation. In contrast, IgG tail-truncating mutations enhance BCR surface expression, intracellular signalling and competitive growth. These findings suggest that the expansion of IgG-switched GC-like B lymphoma cells is limited by low tonic PI3K activity of the wild-type IgG BCR, but a subset of these cancers acquires mutations of the IgG intracellular tail that reverse this effect, promoting the oncogenicity of their BCRs. The presence of IgG tail mutations underscores the importance of isotype-specific BCR signalling in the pathogenesis of FL and EZB DLBCL and can potentially inform therapeutic targeting with BCR signalling inhibitors or antibody-drug conjugates.

immunology↗

bioRxiv2023

Regulation of BCR-mediated Ca2+ mobilization by MIZ1-TIMBIM4 safeguards IgG1+ GC B cell positive selection

The transition from IgM to affinity-matured IgG antibodies is vital for effective humoral immunity. This is facilitated by germinal centers (GCs) through affinity maturation and preferential accumulation of IgG+ B cells over IgM+ B cells. However, it is not known whether the positive selection of the different immunoglobulin isotypes within GCs varies in its dependency on specific transcriptional mechanisms. Here, we identified IgG1+ GC B cell transcription factor dependency using CRISPR-Cas9 and conditional mouse genetics. We found that MIZ1 was specifically required for IgG1+ GC B cell survival during positive selection, whereas IgM+ GC B cells were largely independent. Mechanistically, MIZ1 induced TMBIM4, an ancestral anti-apoptotic protein that regulated inositol trisphosphate receptor mediated Ca2+ mobilization downstream of IgG1. The MIZ1-TMBIM4 axis prevented mitochondrial dysfunction-induced IgG1+ GC cell death caused by excessive Ca2+ accumulation. This study uncovers a unique immunoglobulin isotype-specific dependency, on a hitherto unidentified mechanism in GC positive selection.

immunology↗

bioRxiv2022

Structural basis for Fc receptor recognition of immunoglobulin M

FcR is the IgM-specific Fc receptor involved in the survival and activation of B cells. Using cryo-EM, we reveal eight binding sites for the human FcR Ig domain on the IgM pentamer, one of which overlaps with the receptor binding site for the transcytosis receptor pIgR, but a different mode of binding explains Ig isotype specificity. The complex explains engagement with polymeric serum IgM and the monomeric IgM B cell receptor.

molecular biology↗

bioRxiv2022

Long-term retention of antigens in germinal centres is controlled by the spatial organisation of the follicular dendritic cell network

Germinal centers (GCs) require sustained availability of antigens to promote antibody affinity maturation against pathogens and vaccines. A key source of antigens for GC B cells are immune complexes (ICs) displayed on follicular dendritic cells (FDCs). Here we show that FDC spatial organization regulates antigen dynamics in the GC. We show the existence of two light zone (LZ) FDC populations, which differ in the duration of antigen retention. While the entire light zone (LZ) FDC network captures ICs initially, only the central cells of the network function as a long-term antigen reservoir, where different antigens arriving from subsequent immunizations co-localize. Mechanistically, central FDCs constitutively express subtly higher CR2 membrane densities than peripheral FDCs, which strongly increases the IC retention half-life. Even though repeated immunizations gradually saturate central FDCs, B cell responses remain efficient because new antigens partially displace old ones. These results reveal the principles shaping antigen display on FDCs during the GC reaction.

immunology↗

bioRxiv2022

Digital holography-based 3D particle localisation for single molecule tweezer techniques

We present a three-dimensional imaging technique for fast tracking of microscopic objects in a fluid environment. Our technique couples digital holographic microscopy with three-dimensional localisation via parabolic masking. Compared with existing approaches, our method reconstructs 3D volumes from single-plane images, which greatly simplifies image acquisition, reduces the demand on microscope hardware, and facilitates tracking higher densities of microscopic particles while maintaining similar levels of precision. We demonstrate utility of this method in magnetic tweezer experiments, opening their use to multiplexed single-molecule force spectroscopy assays. We propose that our technique will also be useful in other applications that involve the tracking of microscopic objects in three dimensions. SIGNIFICANCETracking objects in 3D is a common task in biology, but typically requires the acquisition of image stacks, which is limited by speed, the depth of field of microscope objectives and by the presence of other objects that obscure the illumination. Here we develop HoloMiP (Holographic Microscopy with Parabolic masking), which uses digital holography to reconstruct the three-dimensional images from a single plane allowing tracking of light-scattering objects in 3D. HoloMiP outperforms existing methods in precision, speed, simplicity and tolerance to crowding. We show that it is particularly suitable for fast, multiplexed magnetic tweezer experiments, opening new avenues to high-throughput force spectroscopy.

biophysics↗

bioRxiv2021

Genome-wide screens identify calcium signaling as a key regulator of IgE+ plasma celldifferentiation and survival

IgE antibodies protect against toxins and parasites, however, they also mediate allergic reactions. In contrast to other antibody isotypes, B cells switched to IgE respond transiently and do not give rise to long-lived plasma cells (PCs) or memory B cells. Although the intrinsic differences of IgE+ B cells have been linked to signaling by the IgE-B cell receptor (BCR), the molecular pathways controlling their behavior remain poorly understood. Here we employ whole-genome CRISPR screening to identify genes regulating IgE+ B cell proliferation, survival and differentiation into PCs. We show that IgE+ B cells are selectively suppressed by the IgE-BCR signaling to intracellular calcium, which inhibits PC differentiation and limits their lifespan after differentiation. Consequently, manipulation of calcium signaling in vivo enhances IgE+ PC responses. Insights from this pathway shed new light on the self-limiting character of IgE responses and open new avenues to eliminate IgE+ PCs in allergy.

immunology↗

bioRxiv2020

Endophilin A2 regulates B cell protein trafficking and humoral responses

Antigen-specific B cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B cell endosomal trafficking pathways and their specific roles in B cell responses have not been systematically investigated. Here we report high-throughput identification of genes regulating B cell receptor (BCR)-mediated antigen internalization using genome-wide functional screens. We show that antigen internalization depends both on clathrin-coated pits and on clathrin-independent endocytosis mediated by endophilin A2. Although endophilin A2 is dispensable for presentation of the endocytosed antigen, it is required for metabolic support of germinal center (GC) B cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2 deficient mice show selective defects in GC B cell responses and production of high-affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin-independent intracellular trafficking in GC B cell clonal expansion and antibody responses. HIGHLIGHTSO_LIGenome-wide CRISPR screens comprehensively identify genes regulating antigen uptake in B cells C_LIO_LIB cell receptor-mediated antigen internalization is mediated by both epsin1- dependent clathrin-coated pits and a novel fast endophilin A2-mediated endocytosis. C_LIO_LIEndophilin A2 is required for peripheral B cell development, antigen-specific germinal center responses and high-affinity IgG production. C_LIO_LIEndophilin A2 is broadly essential for B cell intracellular trafficking pathways providing metabolic support of germinal center B cell proliferation, in part through regulation of iron uptake. C_LI

immunology↗

bioRxiv2019

B cells extract antigens using Arp2/3-generated actin foci interspersed with linear filaments

Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in B cell synapses forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction.

immunology↗