Biology subjects

Wijfjes, Z.

Publications and source records attributed to Wijfjes, Z..

3 recordsLinked to original sources

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↗

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↗