Biology subjects

Vandekerckhove, B.

Publications and source records attributed to Vandekerckhove, B..

8 recordsLinked to original sources

bioRxiv2026

A platform for high-throughput and ultrasensitive immunopeptidomics

Mass spectrometry (MS)-based immunopeptidomics is a powerful approach for untargeted discovery of peptides presented on major histocompatibility complex (MHC) molecules, which can guide the selection of vaccine antigens and immunotherapy targets. First-generation immunopeptidomics workflows require processing of hundreds of millions of cells using lengthy, manual procedures. More recent approaches focus on increasing either sensitivity or throughput, but rarely combine both aspects. Here, we describe a semi-automated immunopeptidomics platform that combines high sensitivity with high throughput by implementing highly optimized conditions for immunoprecipitation, elution and purification of MHC class I and II peptides on a 96-well positive-pressure device. Upon analysis of 25% of the eluate from 16 million cells, our workflow identified over 13,500 MHC I and 6,000 MHC II peptides on a timsTOF SCP mass spectrometer, operating in DDA-PASEF mode. Exploring the sensitivity limits of our platform, we identified over 1,000 MHC I peptides from as few as 20,000 JY cells. Validating the platforms performance for quantitative biological discovery, we report the identification of known and novel bacterial immunopeptides from U937 macrophages infected with Listeria monocytogenes or Bacillus Calmette-Guerin (BCG). Together, our optimized immunopeptidomics platform enables robust immunopeptide detection from lower-input samples in a high-throughput fashion, enabling its use for biological applications where sample amounts are limiting.

immunology↗

bioRxiv2025

Cell surface proteomics of patient-derived malignant rhabdoid tumor organoids identifies ROBO1 as potential CAR T cell target for pediatric solid tumors.

Malignant rhabdoid tumors are highly aggressive pediatric malignancies with limited treatment options and poor outcomes. To expand therapeutic options, we employed mass spectrometry-based cell surface proteomics on malignant rhabdoid tumor and patient-matched normal kidney organoids to identify potential CAR T cell targets. Integrating these findings with transcriptomics and protein expression data, we revealed ROBO1 as a promising target, showing strong and uniform expression across malignant rhabdoid and other pediatric tumors. ROBO1-targeted CAR T cells displayed potent anti-tumor activity in vitro, effectively eliminating tumor cells in co-cultures with organoids from malignant rhabdoid tumors, rhabdomyosarcoma, and neuroblastoma. In vivo, ROBO1 CAR T cells infiltrated tumors, induced potent tumor regression and significantly increased survival in malignant rhabdoid tumor-bearing mice. These findings establish ROBO1 as a compelling therapeutic target for CAR T cell therapy and offer a promising approach to address the critical need for effective treatments in high-risk pediatric solid tumors. Statement of significanceThis study identifies ROBO1 as a promising CAR T cell target for pediatric solid tumors. Using patient-derived tumor organoids and tissues, we demonstrate strong ROBO1 expression across several tumor entities and robust anti-tumor efficacy of ROBO1-targeted CAR T cells in vitro and in vivo, underscoring its potential for clinical translation.

cancer biology↗

bioRxiv2023

N-glycosylation engineering in chimeric antigen receptor T cells enhances anti-tumor activity

Chimeric antigen receptor (CAR) T cell therapy has had limited success in solid tumors, requiring novel enhancement strategies. Modifying the glycocalyx of CAR T cells is unexplored; we report on genome-editing of the MGAT5 gene to abolish human CAR T N-glycan poly-LacNAc modifications. This boosted tumor control in carcinoma and lymphoma models, for donors of whom the non-engineered CAR T cells largely failed in tumor control. More blood-circulating MGAT5 KO CD70 nanoCAR T cells were found, exhibiting potent tumor cell-killing activity ex vivo, while non-glycoengineered CAR T cells faltered. MGAT5 KO CD70 nanoCAR T cells also mediated durable anti-tumor immunity, improving control of secondary carcinoma challenge months later. Single-cell transcriptomics revealed increased mitotic activity and type I interferon signaling, indicating sustained intratumoral activation. The glyco-engineered cells had unaltered antigen sensitivity and dependence on T cell growth factors, preserving key safety features. MGAT5 KO is readily compatible with clinical manufacturing, representing a promising approach to enhance CAR T cell therapy.

immunology↗

bioRxiv2023

Knocking out CD70 rescues CD70-specific nanoCAR T cells from antigen induced exhaustion

CD70 is an attractive target for chimeric antigen receptor (CAR) T cell therapy as treatment for both solid and liquid malignancies. However, functionality of CD70-specific CARs is only modest. Here, we optimized a CD70-specific VHH based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo by using a diffuse large B cell lymphoma (DLBCL) patient-derived xenograft (PDX) model. Whereas the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. Knocking out CD70 expression by the nanoCAR T cells resulted in dramatically enhanced functionality in the PDX model, suggesting that endogenous CD70 interaction with the nanoCAR induces exhaustion. Through single-cell transcriptomics, we obtained evidence that CD70KO CD70-specific nanoCAR T cells are protected from antigen induced exhaustion. Our data shows that CARs targeted to endogenous T cell antigens, negatively affect CAR T cell functionality by inducing an exhausted state which can be overcome by knocking out the specific target, in this case CD70.

immunology↗

bioRxiv2022

Immunopeptidomics-based design of highly effective mRNA vaccine formulations against Listeria monocytogenes

Listeria monocytogenes is a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine against Listeria is available. Attenuated Listeria strains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screened for Listeria epitopes presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detected 68 Listeria epitopes from 42 different bacterial proteins, including several known antigens. Peptide epitopes presented on different cell lines were often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations resulted in specific CD8+ T-cell responses and high levels of protection in vaccination challenge experiments in mice. Our results pave the way for the development of a clinical mRNA vaccine against Listeria and aid to improve attenuated Listeria vaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.

immunology↗

bioRxiv2022

Single-cell profiling identifies a spectrum of human unconventional intraepithelial T lineage cells

In the human thymus, a CD10+ PD-1+ TCR{beta}+ differentiation pathway diverges from the conventional single positive T cell lineages at the early double positive stage. These cells are phenotypically and functionally similar to murine unconventional intraepithelial lymphocyte (uIEL) precursors. Here, the progeny of the human uIEL lineage was identified in antigen-inexperienced blood. The uIELs in thymus and peripheral blood share a transcriptomic profile, characterized by hallmark transcription factors (i.e. ZNF683 and IKZF2), and polyclonal TCR repertoire with autoreactive features, exhibiting a bias towards early TCR alpha chain rearrangements. Single-cell RNA sequencing confirmed a common developmental trajectory between the thymic and peripheral uIELs, and clearly delineated this unconventional lineage in peripheral blood. This population is phenotypically defined as CD3+ TCR{beta}+ CD4- CCR7- CD26-. It contains CD10+ recent thymic emigrants, Helios+ KIR+ CD8+ Tregs and CD8+ T cells. Thus, the uIEL lineage represents a well-defined but heterogeneous, unconventional TCR{beta}+ lineage mostly confined in human within the CD8 single positive T cells. SummaryBilliet et al. identify the postthymic progeny of the intraepithelial lymphocyte precursors in human based on shared characteristics of the T cell receptor repertoire and the transcriptome. This lineage represents a well-defined but heterogeneous, unconventional TCR{beta}+ lineage mostly confined within the CD8 single positive T cells.

immunology↗

bioRxiv2022

The transcription factor RUNX2 drives the generation of human NK cells and promotes tissue residency

NK cells are innate lymphocytes that eliminate virus-infected and cancer cells by cytotoxicity and cytokine secretion. In addition to circulating NK cells, distinct tissue-resident NK subsets have been identified in various organs. Although transcription factors regulating NK cell development and function have been extensively studied in mice, the role of RUNX2 in these processes has not been investigated, neither in mice nor in human. Here, by manipulating RUNX2 expression with either knockdown or overexpression in human hematopoietic stem cell-based NK cell differentiation cultures, combined with transcriptomic and ChIP-sequencing analyses, we established that RUNX2 drives the generation of NK cells, possibly through induction of IL-2R{beta} expression in NK progenitor cells. Importantly, RUNX2 promotes tissue residency in human NK cells. Our findings have the potential to improve existing NK cell-based cancer therapies and can impact research fields beyond NK cell biology, since tissue-resident subsets have also been described in other lymphocyte subpopulations.

immunology↗

bioRxiv2020

Single cell profiling of immature human postnatal thymocytes resolves the complexity of intra-thymic lineage differentiation and thymus seeding precursors.

During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone marrow derived hematopoietic progenitors that migrate through the bloodstream. In human, the nature of these thymus immigrants has remained unclear. Here, we employ single-cell RNA sequencing on approximately 70.000 CD34+ thymocytes to unravel the heterogeneity of the human immature postnatal thymocytes. Integration of bone marrow and peripheral blood precursors datasets identifies several putative thymus seeding precursors that display heterogeneity for currently used surface markers as revealed by CITEseq. Besides T cell precursors, we discover branches of intrathymic developing dendritic cells with predominantly plasmacytoid DCs. Trough trajectory inference, we delineate the transcriptional dynamics underlying early human T-lineage development from which we predict transcription factor modules that drive stage-specific steps of human T cell development. Thus, our work resolves the heterogeneity of thymus seeding precursors in human and reveals the molecular mechanisms that drive their in vivo cell fate.

immunology↗