Comparative transcriptomics reveals human-specific cortical features.

The cognitive abilities of humans are distinctive among primates, but their molecular and cellular substrates are poorly understood. We used comparative single-nucleus transcriptomics to analyze samples of the middle temporal gyrus (MTG) from adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets to understand human-specific features of the neocortex. Human, chimpanzee, and gorilla MTG showed highly similar cell-type composition and laminar organization as well as a large shift in proportions of deep-layer intratelencephalic-projecting neurons compared with macaque and marmoset MTG. Microglia, astrocytes, and oligodendrocytes had more-divergent expression across species compared with neurons or oligodendrocyte precursor cells, and neuronal expression diverged more rapidly on the human lineage. Only a few hundred genes showed human-specific patterning, suggesting that relatively few cellular and molecular changes distinctively define adult human cortical structure.

Reference-based cell type matching of in situ image-based spatial transcriptomics data on primary visual cortex of mouse brain.

With the advent of multiplex fluorescence in situ hybridization (FISH) and in situ RNA sequencing technologies, spatial transcriptomics analysis is advancing rapidly, providing spatial location and gene expression information about cells in tissue sections at single cell resolution. Cell type classification of these spatially-resolved cells can be inferred by matching the spatial transcriptomics data to reference atlases derived from single cell RNA-sequencing (scRNA-seq) in which cell types are defined by differences in their gene expression profiles. However, robust cell type matching of the spatially-resolved cells to reference scRNA-seq atlases is challenging due to the intrinsic differences in resolution between the spatial and scRNA-seq data. In this study, we systematically evaluated six computational algorithms for cell type matching across four image-based spatial transcriptomics experimental protocols (MERFISH, smFISH, BaristaSeq, and ExSeq) conducted on the same mouse primary visual cortex (VISp) brain region. We find that many cells are assigned as the same type by multiple cell type matching algorithms and are present in spatial patterns previously reported from scRNA-seq studies in VISp. Furthermore, by combining the results of individual matching strategies into consensus cell type assignments, we see even greater alignment with biological expectations. We present two ensemble meta-analysis strategies used in this study and share the consensus cell type matching results in the Cytosplore Viewer ( https://viewer.cytosplore.org ) for interactive visualization and data exploration. The consensus matching can also guide spatial data analysis using SSAM, allowing segmentation-free cell type assignment.

Fully Automated 3D Vestibular Schwannoma Segmentation with and without Gadolinium-based Contrast Material: A Multicenter, Multivendor Study.

Purpose: To develop automated vestibular schwannoma measurements on contrast-enhanced T1- and T2-weighted MRI scans. Materials and Methods: MRI data from 214 patients in 37 different centers were retrospectively analyzed between 2020 and 2021. Patients with hearing loss (134 positive for vestibular schwannoma [mean age ± SD, 54 years ± 12;64 men] and 80 negative for vestibular schwannoma) were randomly assigned to a training and validation set and to an independent test set. A convolutional neural network (CNN) was trained using fivefold cross-validation for two models (T1 and T2). Quantitative analysis, including Dice index, Hausdorff distance, surface-to-surface distance (S2S), and relative volume error, was used to compare the computer and the human delineations. An observer study was performed in which two experienced physicians evaluated both delineations. Results: The T1-weighted model showed state-of-the-art performance, with a mean S2S distance of less than 0.6 mm for the whole tumor and the intrameatal and extrameatal tumor parts. The whole tumor Dice index and Hausdorff distance were 0.92 and 2.1 mm in the independent test set, respectively. T2-weighted images had a mean S2S distance less than 0.6 mm for the whole tumor and the intrameatal and extrameatal tumor parts. The whole tumor Dice index and Hausdorff distance were 0.87 and 1.5 mm in the independent test set. The observer study indicated that the tool was similar to human delineations in 85%-92% of cases. Conclusion: The CNN model detected and delineated vestibular schwannomas accurately on contrast-enhanced T1- and T2-weighted MRI scans and distinguished the clinically relevant difference between intrameatal and extrameatal tumor parts.Keywords: MRI, Ear, Nose, and Throat, Skull Base, Segmentation, Convolutional Neural Network (CNN), Deep Learning Algorithms, Machine Learning Algorithms Supplemental material is available for this article. © RSNA, 2022.

Identification of a Disease-Associated Network of Intestinal Immune Cells in Treatment-Naive Inflammatory Bowel Disease.

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.

Local and systemic immune profiles of human pancreatic ductal adenocarcinoma revealed by single-cell mass cytometry.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in need of effective (immuno)therapeutic treatment strategies. For the optimal application and development of cancer immunotherapies, a comprehensive understanding of local and systemic immune profiles in patients with PDAC is required. Here, our goal was to decipher the interplay between local and systemic immune profiles in treatment-naïve patients with PDAC. METHODS: The immune composition of PDAC, matched non-malignant pancreatic tissue, regional lymph nodes, spleen, portal vein blood, and peripheral blood samples (collected before and after surgery) from 11 patients with PDAC was assessed by measuring 41 immune cell markers by single-cell mass cytometry. Furthermore, the activation potential of tumor-infiltrating lymphocytes as determined by their ability to produce cytokines was investigated by flow cytometry. In addition, the spatial localization of tumor-infiltrating innate lymphocytes in the tumor microenvironment was confirmed by multispectral immunofluorescence. RESULTS: We found that CD103+CD8+ T cells with cytotoxic potential are infrequent in the PDAC immune microenvironment and lack the expression of activation markers and checkpoint blockade molecule programmed cell death protein-1 (PD-1). In contrast, PDAC tissues showed a remarkable increased relative frequency of B cells and regulatory T cells as compared with non-malignant pancreatic tissues. Besides, a previously unappreciated innate lymphocyte cell (ILC) population (CD127-CD103+CD39+CD45RO+ ILC1-like) was discovered in PDAC tissues. Strikingly, the increased relative frequency of B cells and regulatory T cells in pancreatic cancer samples was reflected in matched portal vein blood samples but not in peripheral blood, suggesting a regional enrichment of immune cells that infiltrate the PDAC microenvironment. After surgery, decreased frequencies of myeloid dendritic cells were found in peripheral blood. CONCLUSIONS: Our work demonstrates an immunosuppressive landscape in PDAC tissues, generally deprived of cytotoxic T cells and enriched in regulatory T cells and B cells. The antitumor potential of ILC1-like cells in PDAC may be exploited in a therapeutic setting. Importantly, immune profiles detected in blood isolated from the portal vein reflected the immune cell composition of the PDAC microenvironment, suggesting that this anatomical location could be a source of tumor-associated immune cell subsets.

Deep Recursive Embedding for High-Dimensional Data.

Embedding high-dimensional data onto a low-dimensional manifold is of both theoretical and practical value. In this article, we propose to combine deep neural networks (DNN) with mathematics-guided embedding rules for high-dimensional data embedding. We introduce a generic deep embedding network (DEN) framework, which is able to learn a parametric mapping from high-dimensional space to low-dimensional space, guided by well-established objectives such as Kullback-Leibler (KL) divergence minimization. We further propose a recursive strategy, called deep recursive embedding (DRE), to make use of the latent data representations for boosted embedding performance. We exemplify the flexibility of DRE by different architectures and loss functions, and benchmarked our method against the two most popular embedding methods, namely, t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP). The proposed DRE method can map out-of-sample data and scale to extremely large datasets. Experiments on a range of public datasets demonstrated improved embedding performance in terms of local and global structure preservation, compared with other state-of-the-art embedding methods. Code is available at https://github.com/tao-aimi/DeepRecursiveEmbedding.

Stochastic neighbor embedding as a tool for visualizing the encoding capability of magnetic resonance fingerprinting dictionaries.

OBJECTIVE: To visualize the encoding capability of magnetic resonance fingerprinting (MRF) dictionaries. MATERIALS AND METHODS: High-dimensional MRF dictionaries were simulated and embedded into a lower-dimensional space using t-distributed stochastic neighbor embedding (t-SNE). The embeddings were visualized via colors as a surrogate for location in low-dimensional space. First, we illustrate this technique on three different MRF sequences. We then compare the resulting embeddings and the color-coded dictionary maps to these obtained with a singular value decomposition (SVD) dimensionality reduction technique. We validate the t-SNE approach with measures based on existing quantitative measures of encoding capability using the Euclidean distance. Finally, we use t-SNE to visualize MRF sequences resulting from an MRF sequence optimization algorithm. RESULTS: t-SNE was able to show clear differences between the color-coded dictionary maps of three MRF sequences. SVD showed smaller differences between different sequences. These findings were confirmed by quantitative measures of encoding. t-SNE was also able to visualize differences in encoding capability between subsequent iterations of an MRF sequence optimization algorithm. DISCUSSION: This visualization approach enables comparison of the encoding capability of different MRF sequences. This technique can be used as a confirmation tool in MRF sequence optimization.

Semi-automated background removal limits data loss and normalizes imaging mass cytometry data.

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 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.

Systems analysis and controlled malaria infection in Europeans and Africans elucidate naturally acquired immunity.

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.

Iron loading is a prominent feature of activated microglia in Alzheimer's disease patients.

Brain iron accumulation has been found to accelerate disease progression in amyloid-ß(Aß) 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 Aß-plaque infiltrating microglia. Finally, an increase of FTL+Iba1+-microglia was seen in patients with high Aß 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 Aß.

Visual cohort comparison for spatial single-cell omics-data.

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 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.

SCHNEL: scalable clustering of high dimensional single-cell data.

MOTIVATION: Single cell data measures multiple cellular markers at the single-cell level for thousands to millions of cells. Identification of distinct cell populations is a key step for further biological understanding, usually performed by clustering this data. Dimensionality reduction based clustering tools are either not scalable to large datasets containing millions of cells, or not fully automated requiring an initial manual estimation of the number of clusters. Graph clustering tools provide automated and reliable clustering for single cell data, but suffer heavily from scalability to large datasets. RESULTS: We developed SCHNEL, a scalable, reliable and automated clustering tool for high-dimensional single-cell data. SCHNEL transforms large high-dimensional data to a hierarchy of datasets containing subsets of data points following the original data manifold. The novel approach of SCHNEL combines this hierarchical representation of the data with graph clustering, making graph clustering scalable to millions of cells. Using seven different cytometry datasets, SCHNEL outperformed three popular clustering tools for cytometry data, and was able to produce meaningful clustering results for datasets of 3.5 and 17.2 million cells within workable time frames. In addition, we show that SCHNEL is a general clustering tool by applying it to single-cell RNA sequencing data, as well as a popular machine learning benchmark dataset MNIST. AVAILABILITY AND IMPLEMENTATION: Implementation is available on GitHub (https://github.com/biovault/SCHNELpy). All datasets used in this study are publicly available. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcriptomic signatures of brain regional vulnerability to Parkinson's disease.

The molecular mechanisms underlying caudal-to-rostral progression of Lewy body pathology in Parkinson's disease remain poorly understood. Here, we identified transcriptomic signatures across brain regions involved in Braak Lewy body stages in non-neurological adults from the Allen Human Brain Atlas. Among the genes that are indicative of regional vulnerability, we found known genetic risk factors for Parkinson's disease: SCARB2, ELOVL7, SH3GL2, SNCA, BAP1, and ZNF184. Results were confirmed in two datasets of non-neurological subjects, while in two datasets of Parkinson's disease patients we found altered expression patterns. Co-expression analysis across vulnerable regions identified a module enriched for genes associated with dopamine synthesis and microglia, and another module related to the immune system, blood-oxygen transport, and endothelial cells. Both were highly expressed in regions involved in the preclinical stages of the disease. Finally, alterations in genes underlying these region-specific functions may contribute to the selective regional vulnerability in Parkinson's disease brains.

Eleven grand challenges in single-cell data science.

The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.

Helminth infections drive heterogeneity in human type 2 and regulatory cells.

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.

GPGPU Linear Complexity t-SNE Optimization.

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. 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.

High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity.

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

Deep Learning for Quantitative Cardiac MRI.

OBJECTIVE. The recent advancement of deep learning techniques has profoundly impacted research on quantitative cardiac MRI analysis. The purpose of this article is to introduce the concept of deep learning, review its current applications on quantitative cardiac MRI, and discuss its limitations and challenges. CONCLUSION. Deep learning has shown state-of-the-art performance on quantitative analysis of multiple cardiac MRI sequences and holds great promise for future use in clinical practice and scientific research.

Automatic coronary artery plaque thickness comparison between baseline and follow-up CCTA images.

PURPOSE: Currently, coronary plaque changes are manually compared between a baseline and follow-up coronary computed tomography angiography (CCTA) images for long-term coronary plaque development investigation. We propose an automatic method to measure the plaque thickness change over time. METHODS: We model the lumen and vessel wall for both the baseline coronary artery tree (CAT-BL) and follow-up coronary artery tree (CAT-FU) as smooth three-dimensional (3D) surfaces using a subdivision fitting scheme with the same coarse meshes by which the correspondence among these surface points is generated. Specifically, a rigid point set registration is used to transform the coarse mesh from the CAT-FU to CAT-BL. The plaque thickness and the thickness difference is calculated as the distance between corresponding surface points. To evaluate the registration accuracy, the average distance between manually defined markers on clinical scans is calculated. Artificial CAT-BL and CAT-FU pairs were created to simulate the plaque decrease and increase over time. RESULTS: For 116 pairs of markers from nine clinical scans, the average marker distance after registration was 0.95 ± 0.98 mm (two times the voxel size). On the 10 artificial pairs of datasets, the proposed method successfully located the plaque changes. The average of the calculated plaque thickness difference is the same as the corresponding created value (standard deviation ± 0.1 mm). CONCLUSIONS: The proposed method automatically calculates local coronary plaque thickness differences over time and can be used for 3D visualization of plaque differences. The analysis and reporting of coronary plaque progression and regression will benefit from an automatic plaque thickness comparison.

Multidimensional analyses of proinsulin peptide-specific regulatory T cells induced by tolerogenic dendritic cells.

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.