Biology subjects

ter Beest, M.

Publications and source records attributed to ter Beest, M..

5 recordsLinked to original sources

bioRxiv2026

T cells compete via reverse MHC class I signaling at the synapse with dendritic cells to secure Golgi recruitment for activation

Interleukin-12 (IL-12) is a key immunostimulatory cytokine produced by dendritic cells (DCs) upon infection that plays a central role in the activation and differentiation of cytotoxic T cells. Notably, IL-12 is secreted in a highly polarized manner from late endosomes or lysosomes at the contact interface between a DC and a T cell, known as the immunological synapse. However, the signaling mechanisms initiating this spatially restricted cytokine release remain poorly defined. Here, using artificial T cell mimics, beads coated with T cell surface ligands, we show that antigen recognition induces recruitment of the DCs microtubule organizing center (MTOC) and Golgi apparatus to the immunological synapse via reverse MHC class I signaling. Our results further indicate that the signaling pathways governing MTOC polarization are highly conserved in DCs, consistent with mechanisms described in other immune cell types. As newly synthesized IL-12 traffics through the Golgi, this intracellular reorientation facilitates localized release of IL-12 directly at the site of T cell engagement, likely enabling DCs to focus their stimulatory capacity on responsive T cells within the crowded environment of secondary lymphoid organs. Furthermore, by applying spatially patterned antibody arrays targeting MHC class I, we reveal that MTOC polarization is biased toward sites exhibiting the strongest reverse MHC class I signaling. This suggests that IL-12 is preferentially released at synapses formed with T cells displaying higher T cell receptor affinity, providing a mechanism for selection of the most potent T cell.

immunology↗

bioRxiv2025

DuoHexaBody-CD37 induces direct cytotoxic signaling in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a common aggressive form of Non-Hodgkin lymphoma. Tetraspanin CD37 is highly expressed on mature B cells and being studied as a therapeutic target for NHL, including DLBCL. DuoHexaBody-CD37 is a biparatopic antibody with an E430G hexamerization-enhancing mutation targeting two non-overlapping CD37 epitopes shown to promote complement-dependent cytotoxicity. However, the impact of DuoHexaBody-CD37 on direct cytotoxic signaling has not yet been studied. Here we demonstrate that DuoHexaBody-CD37 induces direct cytotoxicity in DLBCL-derived tumor cell lines independent of the subtype. DuoHexaBody-CD37 induced significant CD37 clustering and was retained at the cell surface in contrast to rituximab, which was internalized. Unbiased screening identified the modulation of 26 (phospho)proteins upon DuoHexaBodyCD37 treatment of primary B cells or DLBCL cells. Whereas DLBCL cells predominantly upregulated p-SHP1(Y564) upon DuoHexaBody-CD37 treatment, primary B cells showed significantly increased p-AKT(S473) and MAPK signaling which is linked to cell survival. Studies using CD37-mutants identified the N-terminus to be involved in DuoHexaBody-CD37-induced signaling. Finally, DuoHexaBody-CD37 treatment inhibited cytokine pro-survival signaling in DLBCL cells. These findings provide novel insights into the signaling functions of CD37 upon DuoHexaBody-CD37 treatment, and open up opportunities for developing CD37-targeted immunotherapy in combination with small molecule inhibitors to maximize tumor cell death.

cancer biology↗

bioRxiv2023

Trafficking Machinery is Rapidly Primed to Facilitate Polarised IL-6 Secretion in Dendritic Cells

The mounting of an adaptive immune response is critical for removing pathogens from the body and generating immunological memory. Central to this process are myeloid cells, which sense pathogens through a variety of cell surface receptors, engulf and destroy pathogens and become activated. Activation is essential for the release of cytokines as well as the cell-surface presentation of pathogen-derived-antigens. Activation-induced cytokine release by myeloid cells requires a complex series of molecular events to facilitate cytokine expression. However, although the transcriptional machinery regulating cytokine expression is well defined, it is becoming increasingly clear that trafficking machinery has to be re-programmed through post-translational modifications to dynamically regulate cytokine secretory events. We demonstrate through quantitative total internal-resonance fluorescence (TIRF) microscopy that short-term stimulation with the pathogenic stimulus lipopolysaccharide (LPS) is sufficient to up-regulate IL-6 secretion rates in human blood monocyte-derived dendritic cells and that this secretion is asymmetric and thus polarised. Using bioinformatics analysis of our phosphoproteomic data, we demonstrate that LPS stimulation of monocyte-derived dendritic cells rapidly reprograms SNARE-associated membrane trafficking machinery, through phosphorylation/dephosphorylation events. Finally, we link this enhanced rate of secretion to the phosphorylation of the SNARE protein VAMP3 at serine 44 (48 in mice), by showing that this phosphorylation drives the release of VAMP3 by its chaperone WDFY2 and the complexing of VAMP3 with STX4 at the plasma membrane.

cell biology↗

bioRxiv2022

Galectin-9 interacts with Vamp-3 to regulate cytokine secretion in dendritic cells

Intracellular vesicle transport is essential for cellular homeostasis and is partially mediated by SNARE proteins. Endosomal trafficking to the plasma membrane ensures cytokine secretion in dendritic cells (DCs) and the initiation of immune responses. Despite its critical importance, the specific molecular agents that regulate DC cytokine secretion are poorly characterised. Galectin-9, a {beta}-galactoside-binding protein, has emerged as a novel cellular modulator although its exact intracellular roles in regulating (immune) cell homeostasis and vesicle transport are virtually unknown. We investigated galectin-9 function in primary human DCs and report that galectin-9 is essential for intracellular cytokine trafficking to the cell surface. Galectin-9-depleted DCs accumulate cytokine-containing vesicles in the Golgi complex that eventually undergo lysosomal degradation. We observed galectin-9 to molecularly interact with Vamp-3 using immunoprecipitation-mass-spectrometry and identified galectin-9 was required for rerouting Vamp-3-containing endosomes upon DC activation as the underlying mechanism. Overall, this study identifies galectin-9 as a necessary mechanistic component for intracellular trafficking. This may impact our general understanding of vesicle transport and shed new light into the multiple roles galectins play in governing cell function.

cell biology↗

bioRxiv2021

Fluorescence lifetime imaging of pH along the secretory pathway

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein (GFP), pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. We therefore applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block ER-Golgi trafficking. Finally, we followed the pH changes of newly-synthesized molecules of the inflammatory cytokine tumor necrosis factor (TNF)- while they were in transit from the endoplasmic reticulum via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution, and can be used to assess organellar pH in disease models.

cell biology↗