Biology subjects

Verdoes, M.

Publications and source records attributed to Verdoes, M..

9 recordsLinked to original sources

bioRxiv2025

Inhibition of SHP-1 /2 blocks antigen cross-presentation by human macrophages and dendritic cells

PD-1 immune checkpoint therapy aims to stimulate T-cell responses against cancer, but faces challenges due to resistance, rendering it ineffective for a significant subset of patients. Inhibitors of SHP-1 and SHP-2, widely expressed protein tyrosine phosphatases known for their pro-cancer and immunosuppressive properties, have attracted attention for their potential to enhance therapy efficacy and overcome resistance when combined with immune checkpoint PD-1 blockade. However, how SHP-1/2 inhibition affects antigen presenting cells is incompletely understood. In this study, we evaluated the effect of SHP-1/2 inhibition on antigen cross-presentation by human monocyte-derived macrophages and dendritic cells, using T cell reporter cell lines specific for epitopes derived from cancer antigens NY-ESO-1 and gp100. Our findings indicate that SHP-1/2 inhibitor NSC-87877 significantly reduces the cross-presentation efficiency of both antigens. Mechanistically, we show that SHP-1/2 inhibition blocks endo/lysosomal acidification and the activation of cathepsin proteases. The reduction of antigen cross-presentation upon SHP-1/2 inhibition potentially limits the effectiveness of the combination therapy with immune checkpoint inhibition.

immunology↗

bioRxiv2025

DC-SIGN Binding to the Surface Ig Oligomannose-type Glycans Promotes Follicular Lymphoma Cell Adhesion and Survival

The occupation of the surface immunoglobulin antigen-binding site by oligomannose-type glycans (sIg-Mann) is a tumor-specific post-translational modification of classic follicular lymphoma (FL). SIg-Mann switches binding from antigen to dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), known to be expressed on interfollicular macrophages and FL-associated follicular dendritic cells (FDCs). The interaction with DC-SIGN induces reorganization of sIg-Mann in wider and less dense clusters than anti-Ig, consistent with inefficient DC-SIGN-induced endocytosis and a low-level intracellular signaling. However, ligand-specific cell clusters form between sIg-Mann-expressing lymphoma and DC-SIGN-expressing cells, raising a need to understand the functional consequences of the interaction of DC-SIGN with sIg-Mann on primary FL cells. This engagement induces adhesion of FL cells to VCAM-1 via B-cell receptor proximal kinases and actin regulators in a fashion similar to anti-Ig, but without initiating apoptosis in vitro. Instead, antibody blockade of sIg-Mann contact with DC-SIGN expressed on FDC-derived YK6/SIGN cells inhibits adhesion and survival of primary FL cells in vitro. These data highlight that the specific interaction with DC-SIGN induces FL cell adhesion to VCAM-1, likely allowing FL cell retention in the lymph node, and survival of the FL cells. Adhesion and survival are inhibited by an anti-DC-SIGN blocking antibody, indicating a new early therapeutic approach against FL retention and survival in adaptive tumor tissue niches. Key pointsO_LIDC-SIGN promotes FL cell adhesion to VCAM-1 via B-cell receptor proximal kinases and actin regulators C_LIO_LIDC-SIGN interaction with sIg-Mann sustains the survival of FL cells C_LI

cancer biology↗

bioRxiv2024

Galectin-9 binding to HLA-DR in dendritic cells controls immune synapse formation and T cell proliferation

To initiate T cell-mediated immunity, dendritic cells (DCs) present antigens to specific T cells through the establishment of an immune synapse (IS). While the molecular mechanisms behind the formation of the IS on the T cell side are well understood, how IS components are organized at the DC membrane remain ill-defined. Galectins, a family of {beta}-galactoside binding proteins, modulate immune cell function via the establishment of specific glycan-dependent or independent interactions. Nonetheless, the molecular mechanisms that underlie galectin function are poorly described and very little is known regarding their contribution to DC-mediated T cell activation. Here, we demonstrate that intracellular galectin-9 (gal9) in DCs is required for T cell activation. Murine and human DCs lacking gal9 showed impaired induction of CD4+, but not CD8+, T cell proliferation, suggesting a conserved function for gal9 in modulating DC-T cell interactions. Live-cell imaging revealed that galectin-9-depleted DCs fail to establish stable ISs with T cells, resulting in reduced T cell activation and proliferation. Unbiased co-immunoprecipitation and mass spectrometry identified HLA-II as a gal9 binding partner in DCs, and we observed a marked reduction of HLA-II recruitment to the immune synapse in DCs lacking gal9. Conditional gal9 knockout in DCs led to enhanced tumor growth in vivo, compared to their wild-type (WT) counterparts, underscoring a role for gal9 in T cell-dependent anti-tumor immunity. Collectively, this study provides the first detailed account of gal9-mediated HLA-II organization at the synaptic site of DCs, revealing a novel mechanism by which galectins orchestrate immune receptor positioning from within the cytoplasm to enhance CD4+ T cell activation.

immunology↗

bioRxiv2024

Head-to-head comparison of nuclear imaging approaches to quantify tumor CD8+ T-cell infiltration

Many immunotherapies focus on (re)invigorating CD8+ T cell anti-cancer responses and different nuclear imaging techniques have been developed to measure CD8+ T cell distributions. In vivo labeling approaches using radiotracers primarily show CD8+ T cell distributions, while ex vivo labeled CD8+ T cells can show CD8+ T cell migration patterns, homing, and tumor infiltration. Currently, a comprehensive head-to-head comparison of in vivo and ex-vivo cell labeling with respect to their tumor and normal tissue targeting properties and correlation to the presence of CD8+ T cells is lacking, yet essential for correct interpretation of clinical CD8+ imaging applications. Therefore, we performed a head-to-head comparison of three different CD8+ T cell imaging approaches: 1) 89Zr-labeled DFO-conjugated Fc-silent anti-CD8 antibody ([89Zr]Zr-anti-CD8-IgG2asilent), 2) ex vivo 89Zr-oxine labeled ovalbumin-specific CD8+ T cells ([89Zr]Zr-OT-I cells), and 3) 18F-labeled IL2 ([18F]AlF-RESCA-IL2). MethodsB16F10/OVA tumor-bearing C57BL/6 mice (n=10/group) received intravenously one of the three radiopharmaceuticals. PET/CT images were acquired starting 72 h ([89Zr]Zr-anti-CD8-IgG2asilent), 24 and 48 h ([89Zr]Zr-OT-I cells), and 10 min ([18F]AlF-RESCA-IL2) post injection. Subsequently, ex vivo biodistribution analysis of the radiopharmaceuticals was performed followed by flow cytometric analysis to evaluate the number of intratumoral CD8+ T cells. Additionally, the intratumoral radiolabel distributions was assessed by autoradiography and immunohistochemistry (IHC) on tumor slices. Results[89Zr]Zr-anti-CD8-IgG2asilent, [89Zr]Zr-OT-I cells, and [18F]AlF-RESCA-IL2 showed uptake in CD8-rich tissues, with preferential targeting to the spleen. Biodistribution analysis showed tumor uptake above blood level for all radiopharmaceuticals, except [18F]AlF-RESCA-IL2. For all three approaches, the uptake in the tumor-draining lymph node was significantly higher compared with the contralateral axial lymph node, suggesting that all approaches allow evaluation of immune responses involving CD8+ T cells. Tumor uptake of [89Zr]Zr-anti-CD8-IgG2asilent (R2=0.65, p<0.01) and [89Zr]Zr-OT-I cells (R2=0.74, p<0.01) correlated to the number of intratumoral CD8+ T cells (flow cytometry). The intratumoral distribution pattern of the radiosignal was different for ex vivo and in vivo radiolabeling techniques. The short half-life of 18F precluded autoradiography assessment of [18F]AlF-RESCA-IL2. ConclusionWe show that [89Zr]Zr-anti-CD8-IgG2asilent and [89Zr]Zr-OT-I cells PET/CT imaging can be used to evaluate intratumoral CD8+ T cells, even though their normal tissues and intratumoral distribution patterns are significantly different. Based on their characteristics, [89Zr]Zr-anti-CD8-IgG2asilent might be most useful to immunophenotyping the TME, while the ex vivo cell labeling approach visualizes CD8+ T cell migrations patterns and the permissiveness of tumors for invasion, whereas [18F]AlF-RESCA-IL2 allows for rapid recurrent imaging and might prove useful for tracking rapid changes in CD8+ T cell distributions. In conclusion, our head-to-head comparison of the three prototype CD8+ T cell labeling approaches provides new insights which can aid in correct interpretation of clinical CD8 imaging and may guide in the selection of the optimal imaging approach for the research question of interest.

immunology↗

bioRxiv2023

Cathepsin B abundance, activity and microglial localisation in Alzheimer's disease-Down syndrome and early onset Alzheimer's disease; the role of elevated cystatin B

Cathepsin B is a cysteine protease that is implicated in multiple aspects of Alzheimers disease pathogenesis. The endogenous inhibitor of this enzyme, cystatin B (CSTB) is encoded on chromosome 21. Thus, individuals who have Down syndrome, a genetic condition caused by having an additional copy of chromosome 21, have an extra copy of an endogenous inhibitor of the enzyme. Individuals who have Down syndrome are also at significantly increased risk of developing early-onset Alzheimers disease (EOAD). The impact of the additional copy of cystatin B (CSTB) on Alzheimers disease development in people who have Down syndrome is not well understood. Here we compared the biology of cathepsin B and cystatin B (CSTB) in individuals who had Down syndrome and Alzheimers disease, with disomic individuals who had Alzheimers disease or were ageing healthily. We find that the activity of cathepsin B enzyme is decreased in the brain of people who had Down syndrome and Alzheimers disease compared with disomic individuals who had Alzheimers disease. This change occurs independently of an alteration in the abundance of the mature enzyme or the number of cathepsin B+ cells. We find that the abundance of cystatin B (CSTB) is significantly increased in the brains of individuals who have Down syndrome and Alzheimers disease compared to disomic individuals both with and without Alzheimers disease and we go on to investigate how this impacts enzyme activity in mouse and human cellular preclinical models of Down syndrome.

neuroscience↗

bioRxiv2022

Immunofilaments Provide a Nanoscale Platform for In Vivo T Cell Expansion and Cancer Immunotherapy

Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, the precarious ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. Here, we propose a radically different approach aiming at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), consisting of a soluble semiflexible polyisocyanopeptide polymer backbone that presents peptide-loaded major histocompatibility complexes and co-stimulatory molecules in a multivalent fashion. We demonstrate that IFs readily activate and expand antigen-specific T cells in a manner highly similar to natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach lymphoid organs including spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display remarkable anti-tumor efficacy resulting in inhibition of melanoma metastases formation and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful new type of aAPC that provide a modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.

immunology↗

bioRxiv2021

Efficient targeting of NY-ESO-1 tumor antigen to human cDC1s by lymphotactin results in cross-presentation and antigen-specific T cell expansion

Type 1 conventional dendritic cells (cDC1s) are characterized by their ability to induce potent CD8+ T cell responses. In efforts to generate novel vaccination strategies, notably against cancer, human cDC1s emerge as an ideal target to deliver antigens. cDC1s uniquely express XCR1, a seven transmembrane G protein-coupled receptor (GPCR). Due to its restricted expression and endocytic nature, XCR1 represents an attractive receptor to mediate antigen-delivery to human cDC1s. To explore tumor antigen delivery to human cDC1s, we used an engineered version of XCR1-binding lymphotactin (XCL1), XCL1(CC3). Site-specific sortase-mediated transpeptidation was performed to conjugate XCL1(CC3) to an analog of the HLA-A*02:01 epitope of the cancer testis antigen New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1). While poor epitope solubility prevented isolation of stable XCL1-antigen conjugates, incorporation of a single polyethylene glycol (PEG) chain upstream of the epitope-containing peptide enabled generation of soluble XCL1(CC3)-antigen fusion constructs. Binding and chemotactic characteristics of the XCL1-antigen conjugate, as well as its ability to induce antigen-specific CD8+ T cell activation by cDC1s, was assessed. PEGylated XCL1(CC3)-antigen conjugates retained binding to XCR1, and induced cDC1 chemoattraction in vitro. The model epitope was efficiently cross-presented by human cDC1s to activate NY-ESO-1-specific CD8+ T cells. Importantly, vaccine activity was increased by targeting XCR1 at the surface of cDC1s. Our results present a novel strategy for the generation of targeted vaccines fused to insoluble antigens. Moreover, our data emphasize the potential of targeting XCR1 at the surface of primary human cDC1s to induce potent CD8+ T cell responses.

immunology↗

bioRxiv2020

Dual site-specific chemoenzymatic antibody fragment conjugation using CRISPR-based hybridoma engineering

I.Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theragnostics, and antibody-drug conjugates (ADC). Antibody functionalization is classically achieved by coupling payloads onto lysine or cysteine residues. However, such stochastic strategies typically lead to heterogenous products, bearing a varying number of payloads. This affects bioconjugate efficacy and stability, as well as its in vivo biodistribution, and therapeutic index, while potentially obstructing the binding sites and leading to off-target toxicity. In addition, therapeutic and theragnostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labelling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogenous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab fragments bearing two distinct site-specific labelling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab molecule (DTFab), which can be easily isolated in high yields. To demonstrate feasibility, we functionalized the DTFab with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theragnostics, and next-generation ADCs.

biochemistry↗

bioRxiv2019

Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas which secrete antibodies in the preferred format, species and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab fragments, isotype switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemo-enzymatic modification. We believe this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research. One Sentence SummaryWe demonstrate a universal CRISPR/HDR based platform for rapid genetic engineering of hybridomas to obtain functionally diverse antibody isotype panels in the species and format of choice.

bioengineering↗