Biology subjects

Wienke, J.

Publications and source records attributed to Wienke, J..

9 recordsLinked to original sources

bioRxiv2026

Inhibitory Nectin-2/TIGIT interactions between CD163+ macrophages and CD8+ T cells in spatially defined immune niches associate with poor outcomes in neuroblastoma

Background: While immunotherapies have transformed outcomes in several adult cancers, more than half of pediatric patients with high-risk neuroblastoma fail to achieve sustained responses, resulting in poor outcomes. Therefore, understanding the phenotypic and spatial organization of immune cells within the neuroblastoma tumor microenvironment is essential to identify mechanisms driving immunotherapy failure. Methods: To identify spatial determinants of the neuroblastoma immune landscape that may contribute to treatment failure, we dissected immunoregulatory mechanisms at single-cell and protein levels in neuroblastoma samples from 28 patients, using imaging mass cytometry (IMC, n=14) and high-dimensional spectral flow cytometry (n=18). Results: We characterized four distinct cellular neighborhoods, ranging from tumor-dominant to stromal and immune niches. Macrophages were broadly distributed across these niches. In contrast, T and B cells were largely confined to immune niches, forming immature tertiary lymphoid structure-like aggregates within the tumor and stromal cores. T cells displayed a CD69+ tissue-resident memory phenotype, with an overall reduced cytotoxic profile (in CD8+ T cells) and increased expression of inhibitory receptors (predominantly in CD4+ T cells), indicative of dysfunctional features. Intriguingly, T cell functional states differed between spatial niches, with a TCF7+/Ki-67+ self-renewing profile within immune niches, and increased granzyme B expression in CD8+ T cells in tumor cores. Interaction analysis pinpointed CD163+ macrophages as the dominant immunosuppressive interaction partner of T cells, particularly within the tumor edge and immune cores. Analysis of inhibitory ligand-receptor interactions identified the TIGIT/PVR/Nectin-2 axis as the main engaged axis between CD163+ macrophages and T cells. Nectin-2 expression on macrophages was particularly high within immune cores, and elevated in macrophages with direct T cell contact, suggesting a high probability of in situ engagement. Consistent with this immunosuppressive role, both CD163 and Nectin-2 expression on macrophages correlated with poor patient survival, substantiating the clinical relevance of inhibitory macrophage-T cell interactions. Conclusions: This study reveals T cell-excluded tumor niches and targetable inhibitory interactions between CD163+ macrophages and T cells. These insights provide a rationale for combinatorial immunotherapeutic strategies that both enhance T cell infiltration into tumor-rich areas and disrupt inhibitory signaling to improve immunotherapy efficacy in neuroblastoma.

cancer biology↗

bioRxiv2026

A microscopy-based readout to assess tumour-specific viability in neuroblastoma co-cultures and short-term cultured patient samples

High-risk neuroblastoma patients face poor survival despite intensive treatment. Drug testing using patient-derived models can support therapy prioritization for precision medicine and drug development. Models incorporating tumour microenvironmental components, such as co-cultures and short-term cultured patient samples containing substantial non-malignant cell fractions, could better recapitulate microenvironment-dependent drug responses. However, conventional viability assays measure the combined signal from all viable cells in a well and therefore cannot determine tumour-specific drug responses. Here, we establish a microscopy-based readout to quantify cell-type-specific viability in two complementary settings: neuroblastoma-PBMC co-cultures and freshly dissociated patient tumour samples. In the co-cultures, PBMCs were pre-labelled with a cell-tracking dye, and Calcein staining was used to independently quantify the viability of tumour cells and PBMCs in the same well. The Calcein-based viability readout correlated strongly with conventional CellTiter-Glo measurements and was compatible with automated high-throughput drug screening. The imaging workflow enabled identification of compounds with differential efficacy in co-culture versus monoculture and distinguished tumour-specific effects from PBMC toxicity. The microscopy-based viability readout was further adapted to short-term cultured patient samples. Neuroblastoma tumour cells were distinguished from the non-malignant cells using a combination of tumour-specific surface markers NCAM, L1CAM and B7H3. This enabled determination of tumour fractions and measurement of tumour-specific drug responses. Tumour fractions varied substantially between patient samples, highlighting the importance of tumour-specific viability measurements. Together, the microscopy-based viability readout for co-cultures and patient samples enables scalable assessment of tumour-specific drug responses.

Cancer Biology↗

bioRxiv2025

Proteomic profiling of the neuroblastoma secretome identifies extracellular vesicles as drivers of T cell suppression

Immunosuppressive tumor microenvironments in solid tumors hamper the efficacy of immunotherapies, including CAR T-cell therapy. To identify immunosuppressive secreted factors in high-risk neuroblastoma, a childhood solid tumor with a 5-year overall survival of below 60%, we mapped secreted factors of 16 genetically diverse patient-derived neuroblastoma tumoroids by LC-MS. The secretomes suppressed T cell activation, proliferation and cytotoxicity to varying extents, suggesting that this panel constituted a valuable discovery foundation to identify clinically relevant immunosuppressive factors. 29 proteins were significantly enriched in highly suppressive secretomes, of which Neuropeptide Y (NPY) had the highest correlation with functionally-determined immunosuppression. No direct or indirect effect of NPY on (CAR) T cell activation could be validated. Instead, NPY proved to be a biomarker for extracellular vesicle (EV) secretion. Finally, we demonstrated that neuroblastoma-derived EVs potently suppress T cell activation. Altogether, this study provides an atlas of the neuroblastoma secretome and reveals immunosuppressive effects of EVs on T cells. These insights contribute to the understanding of the immunosuppressive neuroblastoma TME.

cancer biology↗

bioRxiv2025

The immune landscape of pediatric Brain and Solid Tumors: A systematic review and integration of immunohistochemistry and single-cell RNA sequencing data

BackgroundImmunotherapies achieved remarkable success in adult cancers, yet their efficacy in pediatric brain and solid tumors remains limited. Insights into the unique immune landscape of pediatric tumors are crucial to improve immunotherapies for pediatric patients. MethodsWe performed a systematic search for studies reporting immunohistochemistry (IHC), single-cell- or single-nucleus RNA-sequencing (sc/snSeq) data on the immune landscape of pediatric tumors. For IHC studies, data on macrophages, T cells, T helper cells, regulatory T cells, NK cells and B cells were extracted. For sc/snSeq studies, cell cluster counts were extracted. 47 IHC studies and 26 sc/snSeq studies were included in the analysis. ResultsOur integrative in-depth analysis of 73 studies covered 35 unique pediatric tumor types with 17 tumor types analyzed by IHC, 4 by sc/snSeq, and 14 by both techniques. Regardless of variability in analysis methods, both IHC and sc/snSeq showed that peripheral nerve tumors and soft tissue sarcomas had relatively immune-infiltrated, T cell-rich tumor microenvironments (TME). Brain tumors exhibited a macrophage/microglia-rich, NK cell- infiltrated and T cell-depleted TME. Sc/snSeq data confirmed these observations, showing a macrophage/microglia-rich brain TME. Compared to adult tumors, (CD8+) T cell infiltration and macrophage infiltration was low for all pediatric tumor types. Integrated IHC and sc/snSeq data were visualized in interactive heatmaps, publicly available on R2 as a comprehensive atlas [https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2_targetmap_v1; map Immune_landscape_mm2(by patient)_v4]. ConclusionWe provide a comprehensive, integrated overview of the immune landscape of pediatric brain and solid tumors. These insights can aid the development and selection of immunotherapeutic strategies for specific pediatric cancers, tailored to their unique immune characteristics.

immunology↗

bioRxiv2024

Blocking MIF secretion enhances CAR T-cell efficacy against neuroblastoma

While chimeric antigen receptor (CAR) T-cell therapies are showing highly promising first results in neuroblastoma, immunosuppressive tumor microenvironments (TME) limit T cell persistence and durable clinical efficacy. To improve CAR T-cell efficacy further, we applied a multi-omics approach including single-cell RNA sequencing and proteomics, which identified 13 targetable immunosuppressive factors in neuroblastoma. Of these, macrophage migration inhibitory factor (MIF) and midkine (MDK) were validated across multiple published RNA datasets. Moreover, they were secreted in high abundance by neuroblastoma tumoroids. Functional validation experiments revealed MIF as a potent inhibitor of CAR T-cells, in vitro and in vivo. Degradation of MIF by PROTAC technology significantly enhanced CAR T-cell activation targeting GPC2 and B7-H3, providing a potential intervention against MIF. By defining the immunosuppressive effects of neuroblastomas TME on CAR T-cell efficacy, particularly the pivotal role of MIF, we provide a therapeutic strategy for improving adoptive cell therapies for this pediatric malignancy.

cancer biology↗

bioRxiv2024

Functional avidity of anti-B7H3 CAR-T constructs predicts antigen density thresholds for triggering effector function

Chimeric Antigen receptor T cell (CAR-T) treatments for solid cancers have been compromised by limited expansion and survival in the tumor microenvironment following interaction with antigen-expressing target cells. Using B7H3 as a model antigen with broad clinical applicability, we evaluated the relationship between the antibody/antigen affinity of three clinical candidate binders and the three following functional characteristics: functional avidity, prolonged cytotoxicity in tumoroid re-stimulation assays, and in vivo anti-tumoral responses. BEHAV3D video-microscopy assessed distinct CAR-T cell behaviors at single cell resolution. T cell exhaustion did not dictate effector function. Rather, we demonstrated a threshold avidity of CAR-T / tumor cell interaction, characterized by longer cumulative CD8+ CAR-T / tumor target interaction times, and required for adequate CAR-T cell expansion to result in sustained tumor control upon re-challenge. These results provide new insights into design of CAR-T cells for antigen-dim cell targeting, and avoidance of antigen-dim tumor relapse.

bioengineering↗

bioRxiv2022

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastomas vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies.

cancer biology↗

bioRxiv2021

Human placental bed transcriptomic profiling reveals inflammatory activation of endothelial cells in preeclampsia

RationaleFunctional characteristics of endothelial cells (ECs) within the human placental bed are unknown and may provide insight into the adaptive biology of ECs in disorders of vascular remodelling like preeclampsia. ObjectiveTo determine transcriptional profiles of human placental bed ECs and systemic biomarker profiles in women with normal pregnancy, and women with preeclampsia, a condition characterized by extensive EC dysfunction, poor development of spiral arteries underlying the placenta and long-term susceptibility to atherosclerosis and hypertension. Methods & resultsWe obtained biopsy samples from the uterine placental bed, of five women with preeclampsia with fetal growth restriction (FGR) due to impaired spiral artery development and four controls undergoing Caesarean section. CD31+CD146+ ECs were isolated and sorted by flow cytometry for RNA-sequencing using CEL-Seq2 protocol. Data were analyzed by unsupervised clustering, gene set enrichment (GSEA) and pathway analysis. 67 circulating biomarkers of EC function and inflammation were measured in 20 women with preeclampsia with FGR and 20 controls by multiplex immunoassay. Transcriptional profiling showed various differentially expressed genes (FDR<0.05) in placental bed ECs of preeclampsia patients, with enhanced activity of pathways associated with vasoconstriction, platelet activation and innate immunity. GSEA was suggestive of a VEGF- and PlGF deprived state of preeclampsia-derived ECs. Moreover, the transcriptomic profile was similar to that of human umbilical vein endothelial cells (HUVECs) treated with plasma from preeclampsia patients, pointing towards a central role for circulating factors in EC dysfunction. Unsupervised clustering of subjects by EC-related circulating factors identified distinct profiles for healthy pregnancy and preeclampsia, in particular for those women with low platelets and elevated liver enzymes, which was predominantly driven by sFLT-1, endoglin, PlGF, leptin, SAA-1 and sICAM-1. ConclusionsWe revealed inflammatory activation of EC and a key role for systemic factors in EC dysfunction in women with preeclampsia associated with impaired spiral artery development.

immunology↗

bioRxiv2019

Human regulatory T cells at the maternal-fetal interface show functional site-specific adaptation with tumor-infiltrating-like features

ObjectivesRegulatory T cells (Tregs) are crucial for maintaining immune tolerance against the semi-allogeneic fetus during pregnancy. Since their functional profile at the human maternal-fetal interface is still elusive, we investigated the transcriptional profile and functional adaptation of human uterine Tregs (uTregs) during pregnancy.\n\nMethodsBlood and uterine biopsies from the placental bed (=maternal-fetal interface) and incision site (=control), were obtained from women with uneventful pregnancies undergoing primary Caesarean section. Tregs and CD4+ non-Tregs (Tconv) were isolated for transcriptomic profiling by Cel-Seq2. Results were validated on protein and single cell level by flow cytometry.\n\nResultsPlacental bed uterine Tregs (uTregs) showed elevated expression of Treg signature markers compared to blood Tregs, including FOXP3, CTLA4 and TIGIT. The uTreg transcriptional profile was indicative of late-stage effector Treg differentiation and chronic activation with high expression of immune checkpoints GITR, TNFR2, OX-40, 4-1BB, genes associated with suppressive capacity (CTLA4, HAVCR2, IL10, IL2RA, LAYN, PDCD1), activation (HLA-DR, LRRC32), and transcription factors MAF, PRDM1, BATF, and VDR. uTregs mirrored uTconv Th1 polarization, and characteristics indicating tissue-residency, including high CD69, CCR1, and CXCR6. The particular transcriptional signature of placental bed uTregs overlapped strongly with the specialized profile of human tumor-infiltrating Tregs, and, remarkably, was more pronounced at the placental bed than uterine control site.\n\nConclusionuTregs at the maternal-fetal interface acquire a highly differentiated effector Treg profile similar to tumor-infiltrating Tregs, which is locally enriched compared to a distant uterine site. This introduces the novel concept of site-specific transcriptional adaptation of human Tregs within one organ.

immunology↗