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 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.@en

Motivation: High-dimensional mass cytometry (CyTOF) allows the simultaneous measurement of multiple cellular markers at single-cell level, providing a comprehensive view of cell compositions. However, the power of CyTOF to explore the full heterogeneity of a biological sample at the singlecell level is currently limited by the number of markers measured simultaneously on a single panel. Results: To extend the number of markers per cell, we propose an in silico method to integrate CyTOF datasets measured using multiple panels that share a set of markers. Additionally, we present an approach to select the most informative markers from an existing CyTOF dataset to be used as a shared marker set between panels. We demonstrate the feasibility of our methods by evaluating the quality of clustering and neighborhood preservation of the integrated dataset, on two public CyTOF datasets. We illustrate that by computationally extending the number of markers we can further untangle the heterogeneity of mass cytometry data, including rare cell-population detection.@en

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 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+ TCRγδ 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.@en

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 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.@en

The human fetal immune system must protect the infant against the sudden exposure to a large variety of pathogens upon birth. While it is known that the fetal immune system develops in sequential waves, relatively little is known about the composition of the innate and adaptive immune system in the tissues. Here, we applied high-dimensional mass cytometry to profile the immune system in human fetal liver, spleen, and intestine. With Hierarchical Stochastic Neighbor Embedding (HSNE) we distinguished 177 distinct immune cell clusters, including both previously identified and novel cell clusters. PCA analysis indicated substantial differences between the compositions of the immune system in the different organs. Through dual t-SNE we identified tissue-specific cell clusters, which were found both in the innate and adaptive compartment. To determine the spatial location of tissue-specific subsets we developed a 31-antibody panel to reveal both the immune compartment and surrounding stromal elements through analysis of snap-frozen tissue samples with imaging mass cytometry. Imaging mass cytometry reconstructed the tissue architecture and allowed both the characterization and determination of the location of the various immune cell clusters within the tissue context. Moreover, it further underpinned the distinctness of the immune system in the tissues. Thus, our results provide evidence for early compartmentalization of the adaptive and innate immune compartment in fetal spleen, liver, and intestine. Together, our data provide a unique and comprehensive overview of the composition and organization of the human fetal immune system in several tissues.@en

Mass cytometry by time-of-flight (CyTOF) is a valuable technology for high-dimensional analysis at the single cell level. Identification of different cell populations is an important task during the data analysis. Many clustering tools can perform this task, which is essential to identify “new” cell populations in explorative experiments. However, relying on clustering is laborious since it often involves manual annotation, which significantly limits the reproducibility of identifying cell-populations across different samples. The latter is particularly important in studies comparing different conditions, for example in cohort studies. Learning cell populations from an annotated set of cells solves these problems. However, currently available methods for automatic cell population identification are either complex, dependent on prior biological knowledge about the populations during the learning process, or can only identify canonical cell populations. We propose to use a linear discriminant analysis (LDA) classifier to automatically identify cell populations in CyTOF data. LDA outperforms two state-of-the-art algorithms on four benchmark datasets. Compared to more complex classifiers, LDA has substantial advantages with respect to the interpretable performance, reproducibility, and scalability to larger datasets with deeper annotations. We apply LDA to a dataset of ~3.5 million cells representing 57 cell populations in the Human Mucosal Immune System. LDA has high performance on abundant cell populations as well as the majority of rare cell populations, and provides accurate estimates of cell population frequencies. Further incorporating a rejection option, based on the estimated posterior probabilities, allows LDA to identify previously unknown (new) cell populations that were not encountered during training. Altogether, reproducible prediction of cell population compositions using LDA opens up possibilities to analyze large cohort studies based on CyTOF data.@en

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 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 TAI 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 TAI cells within the tumor micro-environment and improved tumor protection. Moreover, TAI 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 TAI cells can be targeted by combined immunotherapy leading to improved survival.@en

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 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+CD127-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 γδT cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδT cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδT 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.@en

During 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.@en

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 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-γ 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.@en

Helminth infections induce strong type 2 and regulatory responses, but the degree of heterogeneity of such cells is not well characterized. Using mass cytometry, we profiled these cells in Europeans and Indonesians not exposed to helminths and in Indonesians residing in rural areas infected with soil-transmitted helminths. To assign immune alteration to helminth infection, the profiling was performed before and 1 year after deworming. Very distinct signatures were found in Europeans and Indonesians, showing expanded frequencies of T helper 2 cells, particularly CD161+ cells and ILC2s in helminth-infected Indonesians, which was confirmed functionally through analysis of cytokine-producing cells. Besides ILC2s and CD4+ T cells, CD8+ T cells and γδ T cells in Indonesians produced type 2 cytokines. Regulatory T cells were also expanded in Indonesians, but only those expressing CTLA-4, and some coexpressed CD38, HLA-DR, ICOS, or CD161. CD11c+ B cells were found to be the main IL-10 producers among B cells in Indonesians, a subset that was almost absent in Europeans. A number of the distinct immune profiles were driven by helminths as the profiles reverted after clearance of helminth infections. Moreover, Indonesians with no helminth infections residing in an urban area showed immune profiles that resembled Europeans rather than rural Indonesians, which excludes a major role for ethnicity. Detailed insight into the human type 2 and regulatory networks could provide opportunities to target these cells for more precise interventions.@en

Helminth infections induce strong type 2 and regulatory responses, but the degree of heterogeneity of such cells is not well characterized. Using mass cytometry, we profiled these cells in Europeans and Indonesians not exposed to helminths and in Indonesians residing in rural areas infected with soil-transmitted helminths. To assign immune alteration to helminth infection, the profiling was performed before and 1 year after deworming. Very distinct signatures were found in Europeans and Indonesians, showing expanded frequencies of T helper 2 cells, particularly CD161+ cells and ILC2s in helminth-infected Indonesians, which was confirmed functionally through analysis of cytokine-producing cells. Besides ILC2s and CD4+ T cells, CD8+ T cells and γδ T cells in Indonesians produced type 2 cytokines. Regulatory T cells were also expanded in Indonesians, but only those expressing CTLA-4, and some coexpressed CD38, HLA-DR, ICOS, or CD161. CD11c+ B cells were found to be the main IL-10 producers among B cells in Indonesians, a subset that was almost absent in Europeans. A number of the distinct immune profiles were driven by helminths as the profiles reverted after clearance of helminth infections. Moreover, Indonesians with no helminth infections residing in an urban area showed immune profiles that resembled Europeans rather than rural Indonesians, which excludes a major role for ethnicity. Detailed insight into the human type 2 and regulatory networks could provide opportunities to target these cells for more precise interventions.@en

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.@en

Purpose: The tumor immune microenvironment determines clinical outcome. Whether the original tissue in which a primary tumor develops influences this microenvironment is not well understood. Experimental Design: We applied high-dimensional single-cell mass cytometry [Cytometry by Time-Of-Flight (CyTOF)] analysis and functional studies to analyze immune cell populations in human papillomavirus (HPV)–induced primary tumors of the cervix (cervical carcinoma) and oropharynx (oropharyngeal squamous cell carcinoma, OPSCC). Results: Despite the same etiology of these tumors, the composition and functionality of their lymphocytic infiltrate substantially differed. Cervical carcinoma displayed a 3-fold lower CD4:CD8 ratio and contained more activated CD8þCD103þCD161þ effector T cells and less CD4þCD161þ effector memory T cells than OPSCC. CD161þ effector cells produced the highest cytokine levels among tumor-specific T cells. Differences in CD4þ T-cell infiltration between cervical carcinoma and OPSCC were reflected in the detection rate of intratumoral HPV-specific CD4þ T cells and in their impact on OPSCC and cervical carcinoma survival. The peripheral blood mononuclear cell composition of these patients, however, was similar. Conclusions: The tissue of origin significantly affects the overall shape of the immune infiltrate in primary tumors.@en