Microglia have been identified as key players in Alzheimer's disease pathogenesis, and other neurodegenerative diseases. Iba1, and more specifically TMEM119 and P2RY12 are gaining ground as... Show moreMicroglia have been identified as key players in Alzheimer's disease pathogenesis, and other neurodegenerative diseases. Iba1, and more specifically TMEM119 and P2RY12 are gaining ground as presumedly more specific microglia markers, but comprehensive characterization of the expression of these three markers individually as well as combined is currently missing. Here we used a multispectral immunofluorescence dataset, in which over seventy thousand microglia from both aged controls and Alzheimer patients have been analysed for expression of Iba1, TMEM119 and P2RY12 on a single-cell level. For all markers, we studied the overlap and differences in expression patterns and the effect of proximity to beta-amyloid plaques. We found no difference in absolute microglia numbers between control and Alzheimer subjects, but the prevalence of specific combinations of markers (phenotypes) differed greatly. In controls, the majority of microglia expressed all three markers. In Alzheimer patients, a significant loss of TMEM119(+)-phenotypes was observed, independent of the presence of beta-amyloid plaques in its proximity. Contrary, phenotypes showing loss of P2RY12, but consistent Iba1 expression were increasingly prevalent around beta-amyloid plaques. No morphological features were conclusively associated with loss or gain of any of the markers or any of the identified phenotypes. All in all, none of the three markers were expressed by all microglia, nor can be wholly regarded as a pan-or homeostatic marker, and preferential phenotypes were observed depending on the surrounding pathological or homeostatic environment. This work could help select and interpret microglia markers in previous and future studies. Show less
Abdulrahman, Z.; Santegoets, S.J.; Sturm, G.; Charoentong, P.; Ijsselsteijn, M.E.; Somarakis, A.; ... ; Burg, S.H. van der 2022
Background The composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre... Show moreBackground The composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre-existing tumor-specific T cells. Here, the blueprint of the TIME associated with such an ongoing tumor-specific T-cell response was dissected in a unique prospective oropharyngeal squamous cell carcinoma (OPSCC) cohort, in which tumor-specific tumor-infiltrating T cells were detected (immune responsiveness (IR+)) or not (lack of immune responsiveness (IR-)). Methods A comprehensive multimodal, high-dimensional strategy was applied to dissect the TIME of treatment-naive IR+ and IR- OPSCC tissue, including bulk RNA sequencing (NanoString), imaging mass cytometry (Hyperion) for phenotyping and spatial interaction analyses of immune cells, and combined single-cell gene expression profiling and T-cell receptor (TCR) sequencing (single-cell RNA sequencing (scRNAseq)) to characterize the transcriptional states of clonally expanded tumor-infiltrating T cells. Results IR+ patients had an excellent survival during >10 years follow-up. The tumors of IR+ patients expressed higher levels of genes strongly related to interferon gamma signaling, T-cell activation, TCR signaling, and mononuclear cell differentiation, as well as genes involved in several immune signaling pathways, than IR- patients. The top differently overexpressed genes included CXCL12 and LTB, involved in ectopic lymphoid structure development. Moreover, scRNAseq not only revealed that CD4(+) T cells were the main producers of LTB but also identified a subset of clonally expanded CD8(+) T cells, dominantly present in IR+ tumors, which secreted the T cell and dendritic cell (DC) attracting chemokine CCL4. Indeed, immune cell infiltration in IR+ tumors is stronger, highly coordinated, and has a distinct spatial phenotypical signature characterized by intratumoral microaggregates of CD8(+)CD103(+) and CD4(+) T cells with DCs. In contrast, the IR- TIME comprised spatial interactions between lymphocytes and various immunosuppressive myeloid cell populations. The impact of these chemokines on local immunity and clinical outcome was confirmed in an independent The Cancer Genome Atlas OPSCC cohort. Conclusion The production of lymphoid cell attracting and organizing chemokines by tumor-specific T cells in IR+ tumors constitutes a positive feedback loop to sustain the formation of the DC-T-cell microaggregates and identifies patients with excellent survival after standard therapy. Show less
The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals(1). Here, using high-throughput transcriptomic and epigenomic profiling of... Show moreThe primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals(1). Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations. Show less
Imaging mass cytometry (IMC) allows the detection of multiple antigens (approximately 40 markers) combined with spatial information, making it a unique tool for the evaluation of complex biological... Show moreImaging mass cytometry (IMC) allows the detection of multiple antigens (approximately 40 markers) combined with spatial information, making it a unique tool for the evaluation of complex biological systems. Due to its widespread availability and retained tissue morphology, formalin-fixed, paraffin-embedded (FFPE) tissues are often a material of choice for IMC studies. However, antibody performance and signal to noise ratios can differ considerably between FFPE tissues as a consequence of variations in tissue processing, including fixation. In contrast to batch effects caused by differences in the immunodetection procedure, variations in tissue processing are difficult to control. We investigated the effect of immunodetection-related signal intensity fluctuations on IMC analysis and phenotype identification, in a cohort of 12 colorectal cancer tissues. Furthermore, we explored different normalization strategies and propose a workflow to normalize IMC data by semi-automated background removal, using publicly available tools. This workflow can be directly applied to previously acquired datasets and considerably improves the quality of IMC data, thereby supporting the analysis and comparison of multiple samples. Show less
Jong, S.E. de; Unen, V. van; Manurung, M.D.; Stam, K.A.; Goeman, J.J.; Jochems, S.P.; ... ; Yazdanbakhsh, M. 2021
Controlled human infections provide opportunities to study the interaction between the immune system and malaria parasites, which is essential for vaccine development. Here, we compared immune... Show moreControlled human infections provide opportunities to study the interaction between the immune system and malaria parasites, which is essential for vaccine development. Here, we compared immune signatures of malaria-naive Europeans and of Africans with lifelong malaria exposure using mass cytometry, RNA sequencing and data integration, before and 5 and 11 days after venous inoculation with Plasmodium falciparum sporozoites. We observed differences in immune cell populations, antigen-specific responses and gene expression profiles between Europeans and Africans and among Africans with differing degrees of immunity. Before inoculation, an activated/differentiated state of both innate and adaptive cells, including elevated CD161(+)CD4(+) T cells and interferon-gamma production, predicted Africans capable of controlling parasitemia. After inoculation, the rapidity of the transcriptional response and clusters of CD4(+) T cells, plasmacytoid dendritic cells and innate T cells were among the features distinguishing Africans capable of controlling parasitemia from susceptible individuals. These findings can guide the development of a vaccine effective in malaria-endemic regions.Malaria immunity can be acquired through natural infection, but the correlates of protection are still being determined. Yazdanbakhsh and colleagues combine experimental infection of volunteers with Plasmodium falciparum with systems analysis to throw light on the nature of protective immune responses. Show less
Kenkhuis, B.; Somarakis, A.; Haan, L. de; Dzyubachyk, O.; IJsselsteijn, M.E.; Miranda, N.F.C.C. de; ... ; Weerd, L. van der 2021
Brain iron accumulation has been found to accelerate disease progression in amyloid-beta(A beta) positive Alzheimer patients, though the mechanism is still unknown. Microglia have been identified... Show moreBrain iron accumulation has been found to accelerate disease progression in amyloid-beta(A beta) positive Alzheimer patients, though the mechanism is still unknown. Microglia have been identified as key players in the disease pathogenesis, and are highly reactive cells responding to aberrations such as increased iron levels. Therefore, using histological methods, multispectral immunofluorescence and an automated in-house developed microglia segmentation and analysis pipeline, we studied the occurrence of iron-accumulating microglia and the effect on its activation state in human Alzheimer brains. We identified a subset of microglia with increased expression of the iron storage protein ferritin light chain (FTL), together with increased Iba1 expression, decreased TMEM119 and P2RY12 expression. This activated microglia subset represented iron-accumulating microglia and appeared morphologically dystrophic. Multispectral immunofluorescence allowed for spatial analysis of FTL(+)Iba1(+)-microglia, which were found to be the predominant A beta-plaque infiltrating microglia. Finally, an increase of FTL(+)Iba1(+)-microglia was seen in patients with high A beta load and Tau load. These findings suggest iron to be taken up by microglia and to influence the functional phenotype of these cells, especially in conjunction with A beta. Show less
Spatially-resolved omics-data enable researchers to precisely distinguish cell types in tissue and explore their spatial interactions, enabling deep understanding of tissue functionality. To... Show moreSpatially-resolved omics-data enable researchers to precisely distinguish cell types in tissue and explore their spatial interactions, enabling deep understanding of tissue functionality. To understand what causes or deteriorates a disease and identify related biomarkers, clinical researchers regularly perform large-scale cohort studies, requiring the comparison of such data at cellular level. In such studies, with little a-priori knowledge of what to expect in the data, explorative data analysis is a necessity. Here, we present an interactive visual analysis workflow for the comparison of cohorts of spatially-resolved omics-data. Our workflow allows the comparative analysis of two cohorts based on multiple levels-of-detail, from simple abundance of contained cell types over complex co-localization patterns to individual comparison of complete tissue images. As a result, the workflow enables the identification of cohort-differentiating features, as well as outlier samples at any stage of the workflow. During the development of the workflow, we continuously consulted with domain experts. To show the effectiveness of the workflow, we conducted multiple case studies with domain experts from different application areas and with different data modalities. Show less
Somarakis, A.; Unen, V. van; Koning, F.; Lelieveldt, B.; Hollt, T. 2021
Tissue functionality is determined by the characteristics of tissue-resident cells and their interactions within their microenvironment. Imaging Mass Cytometry offers the opportunity to distinguish... Show moreTissue functionality is determined by the characteristics of tissue-resident cells and their interactions within their microenvironment. Imaging Mass Cytometry offers the opportunity to distinguish cell types with high precision and link them to their spatial location in intact tissues at sub-cellular resolution. This technology produces large amounts of spatially-resolved high-dimensional data, which constitutes a serious challenge for the data analysis. We present an interactive visual analysis workflow for the end-to-end analysis of Imaging Mass Cytometry data that was developed in close collaboration with domain expert partners. We implemented the presented workflow in an interactive visual analysis tool; ImaCytE. Our workflow is designed to allow the user to discriminate cell types according to their protein expression profiles and analyze their cellular microenvironments, aiding in the formulation or verification of hypotheses on tissue architecture and function. Finally, we show the effectiveness of our workflow and ImaCytE through a case study performed by a collaborating specialist. Show less
Zwan, A. van der; Unen, V. van; Beyrend, G.; Laban, S.; Keur, C. van der; Kapsenberg, H.J.M.; ... ; Heidt, S. 2020
During healthy pregnancy, a balanced microenvironment at the maternal-fetal interface with coordinated interaction between various immune cells is necessary to maintain immunological tolerance.... Show moreDuring healthy pregnancy, a balanced microenvironment at the maternal-fetal interface with coordinated interaction between various immune cells is necessary to maintain immunological tolerance. While specific decidual immune cell subsets have been investigated, a system-wide unbiased approach is lacking. Here, mass cytometry was applied for data-driven, in-depth immune profiling of the total leukocyte population isolated from first, second, and third trimester decidua, as well as maternal peripheral blood at time of delivery. The maternal-fetal interface showed a unique composition of immune cells, different from peripheral blood, with significant differences between early and term pregnancy samples. Profiling revealed substantial heterogeneity in the decidual lymphoid and myeloid cell lineages that shape gestational-specific immune networks and putative differentiation trajectories over time during gestation. Uncovering the overall complexity at the maternal-fetal interface throughout pregnancy resulted in a human atlas that may serve as a foundation upon which comprehension of the immune microenvironment and alterations thereof in pregnancy complications can be built. Show less
Vries, N.L. de; Unen, V. van; Ijsselsteijn, M.E.; Abdelaal, T.; Breggen, R. van der; Sarasqueta, A.F.; ... ; Miranda, N.F.C.C. de 2020
Objective A comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune... Show moreObjective A comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC.Design Thirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence.Results We discovered that a previously unappreciated innate lymphocyte population (Lin(-)CD7+(C)D127(-)CD56(+)CD45RO(+)) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103(+)CD69(+)) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and gamma delta T cells with highly similar activated (HLA-DR(+)CD38(+)PD(-)1(+)) phenotypes. Remarkably, activated gamma delta T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and gamma delta T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes.Conclusion This work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting. Show less
Induction of antigen-specific regulatory T cells (Tregs) in vivo is the holy grail of current immune-regulating therapies in autoimmune diseases, such as type 1 diabetes. Tolerogenic dendritic... Show moreInduction of antigen-specific regulatory T cells (Tregs) in vivo is the holy grail of current immune-regulating therapies in autoimmune diseases, such as type 1 diabetes. Tolerogenic dendritic cells (tolDCs) generated from monocytes by a combined treatment with vitamin D and dexamethasone (marked by CD52(hi) and CD86(lo) expression) induce antigen-specific Tregs. We evaluated the phenotypes of these Tregs using high-dimensional mass cytometry to identify a surface-based T cell signature of tolerogenic modulation. Naive CD4(+) T cells were stimulated with tolDCs or mature inflammatory DCs pulsed with proinsulin peptide, after which the suppressive capacity, cytokine production and phenotype of stimulated T cells were analysed. TolDCs induced suppressive T cell lines that were dominated by a naive phenotype (CD45RA(+)CCR7(+)). These naive T cells, however, did not show suppressive capacity, but were arrested in their naive status. T cell cultures stimulated by tolDC further contained memory-like (CD45RA(-)CCR7(-)) T cells expressing regulatory markers Lag-3, CD161 and ICOS. T cells expressing CD25(lo) or CD25(hi) were most prominent and suppressed CD4(+) proliferation, while CD25(hi) Tregs also effectively supressed effector CD8(+) T cells.We conclude that tolDCs induce antigen-specific Tregs with various phenotypes. This extends our earlier findings pointing to a functionally diverse pool of antigen-induced and specific Tregs and provides the basis for immune-monitoring in clinical trials with tolDC. Show less
Induction of antigen-specific regulatory T cells (Tregs) in vivo is the holy grail of current immune-regulating therapies in autoimmune diseases, such as type 1 diabetes. Tolerogenic dendritic... Show moreInduction of antigen-specific regulatory T cells (Tregs) in vivo is the holy grail of current immune-regulating therapies in autoimmune diseases, such as type 1 diabetes. Tolerogenic dendritic cells (tolDCs) generated from monocytes by a combined treatment with vitamin D and dexamethasone (marked by CD52hi and CD86lo expression) induce antigen-specific Tregs. We evaluated the phenotypes of these Tregs using high-dimensional mass cytometry to identify a surface-based T cell signature of tolerogenic modulation. Naïve CD4+ T cells were stimulated with tolDCs or mature inflammatory DCs pulsed with proinsulin peptide, after which the suppressive capacity, cytokine production and phenotype of stimulated T cells were analysed. TolDCs induced suppressive T cell lines that were dominated by a naïve phenotype (CD45RA+CCR7+). These naïve T cells, however, did not show suppressive capacity, but were arrested in their naïve status. T cell cultures stimulated by tolDC further contained memory-like (CD45RA-CCR7-) T cells expressing regulatory markers Lag-3, CD161 and ICOS. T cells expressing CD25lo or CD25hi were most prominent and suppressed CD4+ proliferation, while CD25hi Tregs also effectively supressed effector CD8+ T cells.We conclude that tolDCs induce antigen-specific Tregs with various phenotypes. This extends our earlier findings pointing to a functionally diverse pool of antigen-induced and specific Tregs and provides the basis for immune-monitoring in clinical trials with tolDC. Show less
Ruiter, K. de; Jochems, S.P.; Tahapary, D.L.; Stam, K.A.; Konig, M.; Unen, V. van; ... ; Yazdanbakhsh, M. 2020
In recent years the t-distributed Stochastic Neighbor Embedding (t-SNE) algorithm has become one of the most used and insightful techniques for exploratory data analysis of high-dimensional data.... Show moreIn recent years the t-distributed Stochastic Neighbor Embedding (t-SNE) algorithm has become one of the most used and insightful techniques for exploratory data analysis of high-dimensional data. It reveals clusters of high-dimensional data points at different scales while only requiring minimal tuning of its parameters. However, the computational complexity of the algorithm limits its application to relatively small datasets. To address this problem, several evolutions of t-SNE have been developed in recent years, mainly focusing on the scalability of the similarity computations between data points. However, these contributions are insufficient to achieve interactive rates when visualizing the evolution of the t-SNE embedding for large datasets. In this work, we present a novel approach to the minimization of the t-SNE objective function that heavily relies on graphics hardware and has linear computational complexity. Our technique decreases the computational cost of running t-SNE on datasets by orders of magnitude and retains or improves on the accuracy of past approximated techniques. We propose to approximate the repulsive forces between data points by splatting kernel textures for each data point. This approximation allows us to reformulate the t-SNE minimization problem as a series of tensor operations that can be efficiently executed on the graphics card. An efficient implementation of our technique is integrated and available for use in the widely used Google TensorFlow.js, and an open-source C++ library. Show less
Background The clinical benefit of immunotherapeutic approaches against cancer has been well established although complete responses are only observed in a minority of patients. Combination... Show moreBackground The clinical benefit of immunotherapeutic approaches against cancer has been well established although complete responses are only observed in a minority of patients. Combination immunotherapy offers an attractive avenue to develop more effective cancer therapies by improving the efficacy and duration of the tumor-specific T-cell response. Here, we aimed at deciphering the mechanisms governing the response to PD-1/PD-L1 checkpoint blockade to support the rational design of combination immunotherapy. Methods Mice bearing subcutaneous MC-38 tumors were treated with blocking PD-L1 antibodies. To establish high-dimensional immune signatures of immunotherapy-specific responses, the tumor microenvironment was analyzed by CyTOF mass cytometry using 38 cellular markers. Findings were further examined and validated by flow cytometry and by functional in vivo experiments. Immune profiling was extended to the tumor microenvironment of colorectal cancer patients. Results PD-L1 blockade induced selectively the expansion of tumor-infiltrating CD4(+) and CD8(+) T-cell subsets, co-expressing both activating (ICOS) and inhibitory (LAG-3, PD-1) molecules. By therapeutically co-targeting these molecules on the T-AI cell subsets in vivo by agonistic and antagonist antibodies, we were able to enhance PD-L1 blockade therapy as evidenced by an increased number of T-AI cells within the tumor micro-environment and improved tumor protection. Moreover, T-AI cells were also found in the tumor-microenvironment of colorectal cancer patients. Conclusions This study shows the presence of T cell subsets in the tumor micro-environment expressing both activating and inhibitory receptors. These T-AI cells can be targeted by combined immunotherapy leading to improved survival. Show less
Beyrend, G.; Gracht, E. van der; Yilmaz, A.; Duikeren, S. van; Camps, M.; Hollt, T.; ... ; Ossendorp, F. 2019
