Biology subjects

Smits, E.

Publications and source records attributed to Smits, E..

8 recordsLinked to original sources

bioRxiv2026

Autologous biopsy-derived co-culture platform for interrogation of intestinal epithelial-T cell crosstalk

Interactions between intraepithelial lymphocytes (IELs) and the intestinal epithelium are central to mucosal homeostasis and disease. However, mechanistic in vitro studies describing their crosstalk in humans are limited by scarceness of primary material and insufficient knowledge about co-culture requirements. Here, we establish an autologous human duodenal IEL-organoid co-culture system encompassing expandable and bankable IEL and organoid protocols, with co-culture conditions that allow viability of both cell types. This system enables successive interrogation of lympho-epithelial interactions starting from minimal biopsy material. Under baseline conditions, CD45CD8CD103TCR{beta} IELs retain tissue-residency and effector features and induce an epithelial interferon response and chemokine production, without overt epithelial apoptosis. IL-15 and IL-21, essential cytokines involved in IEL-activation in intestinal enteropathies like celiac disease, increases granzyme B expression and interferon-{gamma} secretion but do not trigger epithelial cell death. However, enforcing IEL-epithelial contact using an anti-CD3-anti-Ep-CAM bispecific antibody induces epithelial apoptosis accompanied by increased tumor necrosis factor (TNF) and FAS-ligand (FASLG) secretion. These findings validate the platforms ability to resolve non-destructive and cytotoxic lympho-epithelial interaction and provide a tractable system for studying intestinal inflammation and immune-mediated epithelial cell death.

immunology↗

bioRxiv2025

HIF isoforms contribute distinctly during human NK cell activation

Hypoxia-inducible factors (HIFs) are critical transcriptional regulators that enable cellular adaptation to low oxygen environments. Natural killer (NK) cells are key effectors of innate immunity which frequently operate in hypoxic tissues during viral infection and tumor surveillance. In addition, HIF also play a role in NK cells beyond hypoxic adaptation. However, the distinct and overlapping roles of HIF-1 and HIF-2 in NK cell biology remain incompletely understood. In this study, we investigated the contribution of HIF-1 and HIF-2 to NK cell effector functioning in normoxia using pharmacological inhibition. When HIF-1 was inhibited, we observed a pronounced decline in cytotoxicity as well as IFN{gamma} and TNF production, but not granzyme B, accompanied by elevated mitochondrial reactive oxygen species. In contrast, HIF-2 did not alter these functions, nor did pan-HIF stabilization. These findings indicate a differential role of HIF-1 and HIF-2 in shaping NK cell responses in normoxia, at least during the acute activation phase as investigated here. Still, more mechanistic and contextual insights into the role of HIF isoforms in NK cells is warranted for future therapeutic applications.

immunology↗

bioRxiv2025

DRP1 depletion protects NK cells against hypoxia-induced dysfunction

Hypoxia within the tumor microenvironment poses a major barrier to the efficacy of NK cell-based immunotherapies for solid tumors. In this study, we investigated the influence of hypoxia on NK cell function and mitochondria. We found that hypoxia reduced NK cell cytotoxicity, mitochondrial content, and membrane potential, while increasing mtROS and inducing broad transcriptional changes in metabolic and stress response pathways. CAR engineering with CD70 and IL-15, while designed to enhance persistence and metabolic fitness, did not prevent hypoxia-induced impairment. Given the mitochondrial disruption, we then explored whether DRP1 ablation could mitigate hypoxia-induced dysfunction. Pharmacological inhibition of DRP1 restored mitochondrial content and cytotoxic function. To confirm the role of DRP1, we generated CRISPR-Cas9-mediated DRP1 KO NK cells, which preserved mitochondrial load and membrane potential under hypoxia. When armed with CD70-CAR-IL-15, DRP1KO cells retained cytotoxic activity under hypoxic conditions. These findings show that DRP1 inactivation can support NK cell function in hypoxic environments, and that metabolic engineering may enhance CAR NK cell efficacy in solid tumors. Graphical abstractNK cells become dysfunctional in hypoxic conditions, while DRP1KO NK cells retain their function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/661011v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@2c3a96org.highwire.dtl.DTLVardef@1ba7d20org.highwire.dtl.DTLVardef@6ceeecorg.highwire.dtl.DTLVardef@1b9e0ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Intestine-on-chip enhances nutrient and drug metabolism and maturation of iPSC-derived intestinal epithelial cells relative to organoids and Transwells

The human intestinal epithelial barrier is shaped by various biological and biomechanical influences such as growth factor gradients and the flow of intestinal contents. Exposure to these cues in vitro impacts the cell type composition and function of adult stem cell (ASC)-derived intestinal epithelial cells, but their effect on human induced pluripotent stem cell (hiPSC)-derived cells is largely unexplored. Here, we characterize and compare the cellular composition and gene expression profiles of hiPSC-derived intestinal epithelial cells exposed to various medium compositions and cultured as organoids, in Transwell and in microfluidic intestine-on-chip systems. We demonstrate that inhibition and activation of the WNT, BMP, NOTCH and MAPK pathways regulates the presence of dividing, absorptive and secretory epithelial lineages within these systems, as has been described for ASC-based systems. Upon differentiation, intestinal epithelial organoids and monolayers in Transwell systems expressed genes involved in important intestinal functions, including digestive enzymes, nutrient transporters and members of the Cytochrome P450 family implicated in drug metabolism. However, the dynamic microenvironment of the intestine-on-chip system induced the strongest upregulation of these genes, with an expression profile that suggests a more mature developmental state. Overall, these results underscore the value of hiPSC-derived intestinal epithelial cells for modeling important functions of the human intestinal epithelial barrier and facilitates the selection of relevant culture conditions for specific applications.

cell biology↗

bioRxiv2023

Analysis of Wilms' tumor protein 1 specific TCR repertoire in AML patients uncovers higher diversity in patients in remission than in relapsed

The Wilms tumor protein 1 (WT1) is a well-known and prioritized tumor-associated antigen expressed in numerous solid and blood tumors. Its abundance and immunogenicity have led to the development of different WT1-specific immune therapies. The driving player in these therapies, the WT1-specific T-cell receptor (TCR) repertoire, has received much less attention. Importantly, T cells with high affinity against the WT1 self-antigen are normally eliminated after negative selection in the thymus and are thus rare in peripheral blood. Here, we developed computational models for the robust and fast identification of WT1-specific TCRs from TCR repertoire data. To this end, WT137-45 (WT1-37) and WT1126-134 (WT1-126)-specific T cells were isolated from WT1 peptide-stimulated blood of healthy individuals. The TCR repertoire from these WT1-specific T cells was sequenced and used to train a pattern recognition model for the identification of WT1-specific TCR patterns for the WT1-37 or WT1-126 epitopes. The resulting computational models were applied on an independent published dataset from acute myeloid leukemia (AML) patients, treated with hematopoietic stem cell transplantation, to track WT1-specific TCRs in silico. Several WT1-specific TCRs were found in AML patients. Subsequent clustering analysis of all repertoires indicated the presence of more diverse TCR patterns within the WT1-specific TCR repertoires of AML patients in complete remission in contrast to relapsing patients. We demonstrate the possibility of tracking WT1-37 and WT1-126-specific TCRs directly from TCR repertoire data using computational methods, eliminating the need for additional blood samples and experiments for the two studied WT1 epitopes.

bioinformatics↗

bioRxiv2023

Uncovering the hidden threat: single-organoid analysis reveals clinically relevant treatment-resistant and invasive subclones in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases, characterized by a treatment-resistant and invasive nature. In-line with these inherent aggressive characteristics, only a subset of patients show a clinical response to the standard of care therapies, thereby highlighting the need for a more personalized treatment approach. In this study, we comprehensively unraveled the intra-patient response heterogeneity and intrinsic aggressive nature of PDAC on bulk and single-organoid resolution. We leveraged a fully characterized PDAC organoid panel (N=8) and matched our artificial intelligence-driven, live-cell organoid image analysis with retrospective clinical patient response. In-line with the clinical outcomes, we identified patient-specific sensitivities to the standard of care therapies (gemcitabine-paclitaxel and FOLFIRINOX) using a growth rate-based and normalized drug response metric. Moreover, the single-organoid analysis was able to detect resistant as well as invasive PDAC organoid clones, which was orchestrates on a patient, therapy, drug, concentration and time-specific level. Furthermore, our in vitro organoid analysis indicated a strong correlation with the matched patient progression-free survival (PFS) compared to the current, conventional drug response readouts. This work not only provides valuable insights on the response complexity in PDAC, but it also highlights the potential applications (extendable to other tumor types) and clinical translatability of our approach in drug discovery and the emerging era of personalized medicine.

cancer biology↗

bioRxiv2021

OrBITS: A High-throughput, time-lapse, and label-free drug screening platform for patient-derived 3D organoids

BackgroundPatient-derived organoids are invaluable for fundamental and translational cancer research and holds great promise for personalized medicine. However, the shortage of available analysis methods, which are often single-time point, severely impede the potential and routine use of organoids for basic research, clinical practise, and pharmaceutical and industrial applications. MethodsHere, we developed a high-throughput compatible and automated live-cell image analysis software that allows for kinetic monitoring of organoids, named Organoid Brightfield Identification-based Therapy Screening (OrBITS), by combining computer vision with a convolutional network machine learning approach. The OrBITS deep learning analysis approach was validated against current standard assays for kinetic imaging and automated analysis of organoids. A drug screen of standard-of-care lung and pancreatic cancer treatments was also performed with the OrBITS platform and compared to the gold standard, CellTiter-Glo 3D assay. Finally, the optimal parameters and drug response metrics were identified to improve patient stratification. ResultsOrBITS allowed for the detection and tracking of organoids in routine extracellular matrix domes, advanced Gri3D(R)-96 well plates, and high-throughput 384-well microplates, solely based on brightfield imaging. The obtained organoid Count, Mean Area, and Total Area had a strong correlation with the nuclear staining, Hoechst, following pairwise comparison over a broad range of sizes. By incorporating a fluorescent cell death marker, intra-well normalization for organoid death could be achieved, which was tested with a 10-point titration of cisplatin and validated against the current gold standard ATP-assay, CellTiter-Glo 3D. Using this approach with OrBITS, screening of chemotherapeutics and targeted therapies revealed further insight into the mechanistic action of the drugs, a feature not achievable with the CellTiter-Glo 3D assay. Finally, we advise the use of the growth rate-based normalised drug response metric to improve accuracy and consistency of organoid drug response quantification. ConclusionsOur findings validate that OrBITS, as a scalable, automated live-cell image analysis software, would facilitate the use of patient-derived organoids for drug development and therapy screening. The developed wet-lab workflow and software also has broad application potential, from providing a launching point for further brightfield-based assay development to be used for fundamental research, to guiding clinical decisions for personalized medicine.

cancer biology↗

bioRxiv2020

Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon vaccination

Antigen recognition through the T cell receptor (TCR) {beta} heterodimer is one of the primary determinants of the adaptive immune response. Vaccines activate naive T cells with high specificity to expand and differentiate into memory T cells. However, antigen-specific memory CD4 T cells exist in unexposed antigen-naive hosts. In this study, we use high-throughput sequencing of memory CD4 TCR{beta} repertoire and machine learning to show that individuals with preexisting vaccine-reactive memory CD4 T cell clonotypes elicited earlier and higher antibody titers and mounted a more robust CD4 T cell response to hepatitis B vaccine. In addition, integration of TCR{beta} sequence patterns into a hepatitis B vaccine specific model can predict which individuals will have an early and more vigorous vaccine-elicited immunity. Thus, the presence of preexisting memory T clonotypes has a significant impact on immunity and can be used to predict immune responses to vaccination.

immunology↗