Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting... Show moreIdiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting of a course of anti-thymocyte globulin (ATG) followed by long-term use of ciclosporin. However, the immune response that underlies the pathogenesis of AA remains poorly understood. In this study, we applied high-dimensional mass cytometry on bone marrow aspirates of AA patients pre-ATG, AA patients post-ATG and healthy donors to decipher which immune cells may be implicated in the pathogenesis of AA. We show that the bone marrow of AA patients features an immune cell composition distinct from healthy donors, with significant differences in the myeloid, B-cell, CD4+ and CD8+ T-cells lineages. Specifically, we discovered that AA pre-ATG is characterized by a disease-specific immune cell network with high frequencies of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells, CD45RA+CCR7+CD38+ CD8+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST strongly reduced the levels of CD16+ myeloid cells and showed a trend toward normalization of the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+EMRA CD8+ T-cells. Altogether, our study provides a unique overview of the immune landscape in bone marrow in AA at a single-cell level and proposes CCR6 as a potential new therapeutic target in AA. Show less
2947 Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer’s disease. The Norrin/FZD4... Show more2947 Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer’s disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/β-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier. Show less
Previous wireless motility capsule (WMC) studies demonstrated decreased small intestinal pH in people with CF (PwCF) however the data is lacking on the colonic pH profile. We re-analyzed... Show morePrevious wireless motility capsule (WMC) studies demonstrated decreased small intestinal pH in people with CF (PwCF) however the data is lacking on the colonic pH profile. We re-analyzed previously published WMC data to determine colonic pH/bicarbonate concentration and single cell RNA sequencing (sc-RNAseq) to examine the normal expression of acid-base transporters in the colon/rectum.CF patients showed significantly lower pH and bicarbonate concentration values, particularly in the distal rectosigmoid region. There was no difference in colonic motility parameters between CF and non-CF subjects. SLC26A3 is highly expressed bicarbonate transporter in the colon and rectum, more so than CFTR. While dysmotility can alter intraluminal pH, observed changes likely originate from alterations in intestinal ion transport rather than colonic dysmotility. SLC26A3 is abundantly expressed in the human colon and rectum and may be a therapeutic target for restoration of bicarbonate transport. These findings may help better understand the gastrointestinal symptoms in PwCF Show less
Guo, N.N.; Li, N.; Jia, L.; Jiang, Q.Y.; Schreurs, M.; Unen, V. van; ... ; Koning, F. 2023
T he intestine represents the largest immune compartment in the human body, yet its development and organisation during human foetal development is largely unknown. Here we show the immune subset... Show moreT he intestine represents the largest immune compartment in the human body, yet its development and organisation during human foetal development is largely unknown. Here we show the immune subset composition of this organ during development, by longitudinal spectral flow cytometry analysis of human foetal intestinal samples between 14 and 22 weeks of gestation. At 14 weeks, the foetal intestine is mainly populated by myeloid cells and three distinct CD3–CD7+ ILC, followed by rapid appearance of adaptive CD4+, CD8+ T and B cell subsets. Imaging mass cytometry identifies lymphoid follicles from week 16 onwards in a villus-like structure covered by epithelium and confirms the presence of Ki-67+ cells in situ within all CD3–CD7+ ILC, T, B and myeloid cell subsets. Foetal intestinal lymphoid subsets are capable of spontaneous proliferation in vitro. IL-7 mRNA is detected within both the lamina propria and the epithelium and IL-7 enhances proliferation of several subsets in vitro. Overall, these observations demonstrate the presence of immune subset-committed cells capable of local proliferation in the developing human foetal intestine, likely contributing to the development and growth of organized immune structures throughout most of the 2nd trimester, which might influence microbial colonization upon birth. Show less
Unen, V. van; Ouboter, L.F.; Li, N.; Schreurs, M.; Abdelaal, T.; Kooy-Winkelaar, Y.; ... ; Koning, F. 2022
Chronic intestinal inflammation underlies inflammatory bowel disease (IBD). Previous studies indicated alterations in the cellular immune system; however, it has been challenging to interrogate the... Show moreChronic intestinal inflammation underlies inflammatory bowel disease (IBD). Previous studies indicated alterations in the cellular immune system; however, it has been challenging to interrogate the role of all immune cell subsets simultaneously. Therefore, we aimed to identify immune cell types associated with inflammation in IBD using high-dimensional mass cytometry. We analyzed 188 intestinal biopsies and paired blood samples of newly-diagnosed, treatment-naive patients (n=42) and controls (n=26) in two independent cohorts. We applied mass cytometry (36-antibody panel) to resolve single cells and analyzed the data with unbiased Hierarchical-SNE. In addition, imaging-mass cytometry (IMC) was performed to reveal the spatial distribution of the immune subsets in the tissue. We identified 44 distinct immune subsets. Correlation network analysis identified a network of inflammation-associated subsets, including HLA-DR(+)CD38(+) EM CD4(+) T cells, T regulatory-like cells, PD1(+) EM CD8(+) T cells, neutrophils, CD27(+) TCR gamma delta cells and NK cells. All disease-associated subsets were validated in a second cohort. This network was abundant in a subset of patients, independent of IBD subtype, severity or intestinal location. Putative disease-associated CD4(+) T cells were detectable in blood. Finally, imaging-mass cytometry revealed the spatial colocalization of neutrophils, memory CD4(+) T cells and myeloid cells in the inflamed intestine. Our study indicates that a cellular network of both innate and adaptive immune cells colocalizes in inflamed biopsies from a subset of patients. These results contribute to dissecting disease heterogeneity and may guide the development of targeted therapeutics in IBD. Show less
Li, J.; Zaslavsky, M.; Su, Y.P.; Guo, J.; Sikora, M.J.; Unen, V. van; ... ; Davis, M.M. 2022
Background Autoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more... Show moreBackground Autoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more severe disease and poor prognosis compared to ACPA-negative patients. However, the immune cell composition associated with antibody-positive/negative disease is incompletely defined. Mass cytometry (MC) is a high-dimensional technique offering new possibilities in the determination of the immune cell composition in rheumatic diseases. Here, we set up a broad phenotyping panel to study the immune cell profile of early untreated RA to investigate if specific immune cell subsets are associated with ACPA+ versus ACPA- RA. Methods Freshly obtained PBMCs of early, untreated RA patients (8 ACPA+ and 7 ACPA-) were analysed using a 36-marker MC panel, including markers related to various immune lineages. Data were processed using Cytosplore for dimensional reduction (HSNE) and clustering. Groups were compared using Cytofast. A second validation cohort of cryopreserved PBMCs obtained from early RA patients (27 ACPA+ and 20 ACPA-) was used to confirm MC data by flow cytometry (FC). FC data were processed and analysed using both an unsupervised analysis pipeline and through manual gating. Results MC indicated no differences when comparing major immune lineages (i.e. monocytes, T and B cells), but highlighted two innate subsets: CD62L(+) basophils (p = 0.33) and a subset of CD16(-) NK cells (p = 0.063). Although the NK cell subset did not replicate by FC, FC replication confirmed the difference in CD62L(+) basophil frequency when comparing ACPA+ to ACPA- patients (mean 0.32% vs. 0.13%; p = 0.01). Conclusions Although no differences in major lineages were found between early ACPA+ and ACPA- RA, this study identified the reduced presence of activated basophils in ACPA-negative disease as compared to ACPA-positive disease and thereby provides the first evidence for a connection between activated basophils and ACPA status. 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
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
Guo, N.N.; Unen, V. van; Ijsselsteijn, M.E.; Ouboter, L.F.; Meulen, A.E. van der; Lopes, S.M.C.D.; ... ; Li, N. 2020
Imaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass... Show moreImaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass cytometry. As such, it allows us to gain spatial information and antigen quantificationin situ, and can be applied to both snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue sections. Herein, we have developed and optimized the immunodetection conditions for a 34-antibody panel for use on human snap-frozen tissue sections. For this, we tested the performance of 80 antibodies. Moreover, we compared tissue drying times, fixation procedures and antibody incubation conditions. We observed that variations in the drying times of tissue sections had little impact on the quality of the images. Fixation with methanol for 5 min at -20 degrees C or 1% paraformaldehyde (PFA) for 5 min at room temperature followed by methanol for 5 min at -20 degrees C were superior to fixation with acetone or PFA only. Finally, we observed that antibody incubation overnight at 4 degrees C yielded more consistent results as compared to staining at room temperature for 5 h. Finally, we used the optimized method for staining of human fetal and adult intestinal tissue samples. We present the tissue architecture and spatial distribution of the stromal cells and immune cells in these samples visualizing blood vessels, the epithelium and lamina propria based on the expression of alpha-smooth muscle actin (alpha-SMA), E-Cadherin and Vimentin, while simultaneously revealing the colocalization of T cells, innate lymphoid cells (ILCs), and various myeloid cell subsets in the lamina propria of the human fetal intestine. We expect that this work can aid the scientific community who wish to improve IMC data quality. 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
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
Li, N.; Unen, V. van; Guo, N.N.; Abdelaal, T.; Somarakis, A.; Eggermont, J.; ... ; Koning, F. 2019
