Comprehensive single-cell profiling of intratumoral T and B cells in gynecological malignancies:identifying targets to optimize immunotherapy

In this thesis, we described the preferential location, prognostic value and transcriptional profile of CD103+ T cells in endometrial and ovarian tumors. In our analyses, we found several potential therapeutic targets to enhance the (re)activation of CD103+ T cells in tumors. In addition, we found a clear link between CD103+ T cells, CXCL13 expression and B cells, leading to an extensive study of B cells and tertiary lymphoid structures in gynecological malignancies. The knowledge gained in this thesis can contribute to improving (response to) immunotherapy and also offers starting points for selecting patients with a high chance of responding to immunotherapy

Effect van bezettingsdichtheid op het gedrag van jonge vleeskuikens = Effect of stocking density on the behaviour of young broiler chickens

This report describes the effect of stocking density of young broiler chickens (until 21 days of age) on their behaviour

Tertiary lymphoid structures critical for prognosis in endometrial cancer patients

B-cells play a key role in cancer suppression, particularly when aggregated in tertiary lymphoid structures (TLS). Here, we investigate the role of B-cells and TLS in endometrial cancer (EC). Single cell RNA-sequencing of B-cells shows presence of naïve B-cells, cycling/germinal center B-cells and antibody-secreting cells. Differential gene expression analysis shows association of TLS with L1CAM overexpression. Immunohistochemistry and co-immunofluorescence show L1CAM expression in mature TLS, independent of L1CAM expression in the tumor. Using L1CAM as a marker, 378 of the 411 molecularly classified ECs from the PORTEC-3 biobank are evaluated, TLS are found in 19%. L1CAM expressing TLS are most common in mismatch-repair deficient (29/127, 23%) and polymerase-epsilon mutant EC (24/47, 51%). Multivariable Cox regression analysis shows strong favorable prognostic impact of TLS, independent of clinicopathological and molecular factors. Our data suggests a pivotal role of TLS in outcome of EC patients, and establishes L1CAM as a simple biomarker

Effect van bezettingsdichtheid op (de ontwikkeling van) het paargedrag en de technische resultaten bij vleeskuikenouderdieren = Effect of stocking density on (the development of) sexual behaviour and technical performance in broiler breeds

This report describes the effects of a reduced stocking density during rearing and/or production on mating behaviour and technical performance in broiler breeders

Transcriptional Activity and Stability of CD39+CD103+CD8+T Cells in Human High-Grade Endometrial Cancer

Tumor-infiltrating CD8+ T cells (TIL) are of the utmost importance in anti-tumor immunity. CD103 defines tumor-resident memory T cells (T-RM cells) associated with improved survival and response to immune checkpoint blockade (ICB) across human tumors. Co-expression of CD39 and CD103 marks tumor-specific T-RM with enhanced cytolytic potential, suggesting that CD39+CD103+ T-RM could be a suitable biomarker for immunotherapy. However, little is known about the transcriptional activity of T-RM cells in situ. We analyzed CD39+CD103+ T-RM cells sorted from human high-grade endometrial cancers (n = 3) using mRNA sequencing. Cells remained untreated or were incubated with PMA/ionomycin (activation), actinomycin D (a platinum-like chemotherapeutic that inhibits transcription), or a combination of the two. Resting CD39+CD103+ T-RM cells were transcriptionally active and expressed a characteristic T-RM signature. Activated CD39+CD103+ T-RM cells differentially expressed PLEK, TWNK, and FOS, and cytokine genes IFNG, TNF, IL2, CSF2 (GM-CSF), and IL21. Findings were confirmed using qPCR and cytokine production was validated by flow cytometry of cytotoxic TIL. We studied transcript stability and found that PMA-responsive genes and mitochondrial genes were particularly stable. In conclusion, CD39+CD103+ T-RM cells are transcriptionally active T-RM cells with a polyfunctional, reactivation-responsive repertoire. Secondly, we hypothesize that differential regulation of transcript stability potentiates rapid responses upon T-RM reactivation in tumors

Combined STING levels and CD103+ T cell infiltration have significant prognostic implications for patients with cervical cancer

Activation of STimulator of INterferon Genes (STING) is important for induction of anti-tumor immunity. A dysfunctional STING pathway is observed in multiple cancer types and associates with poor prognosis and inferior response to immunotherapy. However, the association between STING and prognosis in virally induced cancers such as HPV-positive cervical cancer remains unknown. Here, we investigated the prognostic value of STING protein levels in cervical cancer using tumor tissue microarrays of two patient groups, primarily treated with surgery (n = 251) or radio(chemo)therapy (n = 255). We also studied CD103, an integrin that marks tumor-reactive cytotoxic T cells that reside in tumor epithelium and that is reported to associate with improved prognosis. Notably, we found that a high level of STING protein was an independent prognostic factor for improved survival in both the surgery and radio(chemo)therapy group. High infiltration of CD103+ T cells was associated with improved survival in the radio(chemo)therapy group. The combination of STING levels and CD103+ T cell infiltration is strongly associated with improved prognosis. We conclude that combining the prognostic values of STING and CD103 may improve the risk stratification of cervical cancer patients, independent from established clinical prognostic parameters

Transcranial magnetic stunning of broilers: a preliminary trial to induce unconsciousness

This study was performed to identify whether non-focal transcranial magnetic stimulation (TMS) with an adapted coil for broilers has the potential for use as a non-invasive stunning method for broilers. Application of the TMS probe resulted in dominance of theta and delta waves and appearance of spikes in the electroencephalogram (EEG) after stimulation. Correlation dimension (CD) analyses of the EEG signals recorded prior to and following the application of TMS suggested that the birds might be unconscious for approximately 15 to 20 s assuming that a reduction in CD to 60% of the baseline value indicates unconsciousness. Other observations included loss of behavioural arousal or muscle tone (muscle flaccidity), and irregular heart rate after TMS. It can be suggested that TMS has the potential to be developed as a stunning method in the future. The technique, evaluated using small number of broilers in this study, requires further improvement and the use of a power supply optimised in future research. Transcranial magnetic stimulation of the brain has potential for application as a non-invasive stunning method for broilers, which could be acceptable to some religious groups opposed to the use of established or conventional stunning methods

Expression of CD39 Identifies Activated Intratumoral CD8+T Cells in Mismatch Repair Deficient Endometrial Cancer

Identification of human cancer-reactive CD8+ T cells is crucial for the stratification of patients for immunotherapy and determination of immune-therapeutic effects. To date, these T cells have been identified mainly based on cell surface expression of programmed cell death protein 1 (PD-1) or co-expression of CD103 and CD39. A small subset of CD103- CD39+ CD8+ T cells is also present in tumors, but little is known about these T cells. Here, we report that CD103- CD39+ CD8+ T cells from mismatch repair-deficient endometrial tumors are activated and characterized predominantly by expression of TNFRSF9. In vitro, transforming growth factor-beta (TGF-beta) drives the disappearance of this subset, likely through the conversion of CD103- CD39+ cells to a CD103+ phenotype. On the transcriptomic level, T cell activation and induction of CD39 was associated with a number of tissue residence and TGF-beta responsive transcription factors. Altogether, our data suggest CD39+ CD103- CD8+ tumor-infiltrating T cells are recently activated and likely rapidly differentiate towards tissue residence upon exposure to TGF-beta in the tumor micro-environment, explaining their relative paucity in human tumors