Biology subjects

Figdor, C. G.

Publications and source records attributed to Figdor, C. G..

10 recordsLinked to original sources

bioRxiv2025

Hiltonol and Protamine-RNA stimulation induce an immune-activating transcriptome profile in cDC1s

Ex-vivo stimulation of dendritic cells (DCs) is a critical step in DC-based cancer immunotherapies. In humans, conventional type 1 dendritic cells (cDC1s) are a rare myeloid dendritic cell (mDC) subset that express BDCA-3 (CD141). cDC1s promote CD8+ T cell cross-priming against tumor antigens and are therefore being explored for use in immunotherapy. We evaluated the impact of ex-vivo stimulation on human peripheral blood cDC1s. In contrast to routine evaluation, which focuses on pre-defined surface maturation markers or soluble factors released from the activated cells, we investigated the impact of stimulation on the transcriptome using both RNA-sequencing (RNA-seq) and microarrays. Specifically, we analyzed the mRNA of cDC1s upon activation with two clinical-grade adjuvants, Hiltonol (poly IC, a TLR3 ligand) and protamine-stabilized RNA (pRNA, a TLR7/8 ligand) compared to unstimulated controls. Both RNA-seq and microarray analysis showed profound and similar effects of both Hiltonol and pRNA on the transcriptome of cDC1s. A gene ontology (GO) analysis suggested that these changes were mainly related to activation and maturation pathways, including induction of type-I interferon (IFN) and interleukin (IL)-12 transcription, while pathways related to adverse effects or cell damage did not appear to be affected. Combination of both reagents did not appear to have a synergistic effect, as the transcriptome changes were similar to those induced by each stimulus alone. Together, our results indicate that both adjuvants have comparable effects on cDC1 maturation within an immunogenic short-term culture as performed in immunotherapy.

immunology↗

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↗

bioRxiv2021

A Segmentation-Free Machine Learning Architecture for Immune Landscape Phenotyping in Solid Tumors by Multichannel Imaging

Tissue specimens taken from primary tumors or metastases contain important information for diagnosis and treat-ment of cancer patients. Multiplex imaging allows in situ visualization of heterogeneous cell populations, such as immune cells, in tissue samples. Most image processing pipelines first segment cell boundaries and then measure marker expression to assign cell phenotypes. In dense tissue environments, this segmentation-first approach can be inaccurate due to segmentation errors or overlapping cells. Here we introduce the machine learning pipeline "ImmuNet" that identifies positions and phenotypes of cells without segmenting them. ImmuNet is easy to train: human annotators only need to click on an immune cell and score its expression of each marker. This approach al-lowed us to annotate 34,458 cells. We show that ImmuNet consistently outperforms a state-of-the-art segmentation-based pipeline for multiplex immunohistochemistry analysis across tissue types, cell types and tissue densities, achieving error rates below 5-10% on challenging detection and phenotyping tasks. We externally validate Im-muNet results by comparing them to flow cytometric measurements from the same tissue. In summary, ImmuNet is an effective, simpler alternative to segmentation-based approaches when only cell positions and phenotypes, but not their shapes, are required for downstream analyses. Thus, ImmuNet helps researchers to analyze multiplex tissue images more easily and accurately.

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↗

bioRxiv2020

Cancer cell elimination by cytotoxic T cell cooperation and additive damage

Cytotoxic T lymphocytes (CTL) eliminate tumor target cells in an antigen and cell-contact dependent manner. Lethal hit delivery occurs as a rapid and binary, "yes/no" process when immunogenicity is very high1-3, however in vivo CTL often fail to kill solid tumor cells during 1:1 conjugations4-6. Using long-term time-lapse microscopy in three distinct tumor cytotoxicity models and statistical modeling, we here show that migrating CTL transit between target cells and initiate apoptosis by a series of sublethal interactions ( additive cytotoxicity), while individual conjugations rarely induced apoptosis. Sublethal damage included perforin-dependent membrane pore formation, nuclear lamina rupture and DNA damage, and these events resolved within minutes to hours. In immunogenic B16F10 melanoma tumors in vivo, frequent serial engagements and sublethal hit delivery of CTL was largely confined to interstitial niches in the invasion front, resulting in eradication of invading tumor cells. Thus, additive cytotoxicity is a probabilistic process achieved by a series of CTL-target cell engagements and sublethal events. The need for additive "hits" has implications for the topographic mechanisms of elimination or immune evasion of tumor cells and microenvironmental regulation of CTL accumulation and cooperation by targeted therapy.

immunology↗

bioRxiv2019

Intracellular Galectin-9 controls dendritic cell function by maintaining plasma membrane rigidity

Extracellular Galectins constitute a novel mechanism of membrane protein organisation at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organisation and function. By combining functional, super-resolution and atomic force microscopy experiments to analyse membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure via modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organisation. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type lectin receptor-mediated pathogen uptake by human DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionary conserved.

cell biology↗

bioRxiv2019

The tumour microenvironment shapes dendritic cell plasticity in a human organotypic melanoma culture

The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light into multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We developed a novel human, organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multi-cellular tissue architecture. By co-culturing keratinocytes, fibroblasts and immune cells with melanoma cells, onto a de-cellularized dermis, we generated a reconstructed TME that closely recapitulates tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC we observed the tumour-driven conversion of cDC2s into CD14+ DCs, characterized by a an immunosuppressive phenotype. The OMC provides a valuable complement to current approaches to study the TME.

immunology↗

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↗

bioRxiv2017

C-type lectin-like receptor 2 (CLEC-2)-dependent DC migration is controlled by tetraspanin CD37

Cell migration is central to evoke a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2) expressed by DCs, with podoplanin expressed by lymph node stromal cells (LNSCs). However, the underlying molecular mechanisms by which CLEC-2 influences DC migration remain elusive. Here, we show that CLEC-2-dependent DC migration is tightly controlled by tetraspanin CD37, a membrane-organizing protein. Our findings demonstrate a specific molecular interaction between CLEC-2 and CD37. Myeloid cells lacking CD37 (Cd37-/-) expressed less CLEC-2 on their surface compared to wild-type cells, indicating that CD37 is required to stabilize membrane expression of CLEC-2. In addition, CLEC-2-expressing DCs lacking CD37 showed impaired adhesion, migration velocity and displacement on LNSCs. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs (CD11c{Delta}CLEC-2). Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights underlying CLEC-2-dependent DC migration.

cell biology↗