Felix J. Hartmann

The Cytokine GM-CSF Drives the Inflammatory Signature of CCR2+ Monocytes and Licenses Autoimmunity

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage.

Multiple sclerosis-associated IL2RA polymorphism controls GM-CSF production in human T H cells

Genome-wide association studies implicate dysregulation of immune mechanisms in the pathogenesis of multiple sclerosis (MS). Particularly, polymorphisms in genes involved in T helper (TH) cell differentiation are associated with risk of developing MS. However, the underlying mechanism by which these risk alleles influence MS susceptibility has remained elusive. Initiation of neuroinflammation in animal models of MS has been shown to be dependent on TH cell-derived granulocyte-macrophage colony-stimulating factor (GM-CSF). We here report association of GM-CSF expression by human TH cells with MS disease severity. GM-CSF is strongly induced by interleukin 2 (IL-2). We show that an MS-associated polymorphism in the IL-2 receptor alpha (IL2RA) gene specifically increases the frequency of GM-CSF-producing TH cells. The IL2RA polymorphism regulates IL-2 responsiveness of naive TH cells and their propensity to develop into GM-CSF-producing memory TH cells. These findings mechanistically link an immunologically relevant genetic risk factor with a functional feature of TH cells in MS.

The end of gating? An introduction to automated analysis of high dimensional cytometry data.

Ever since its invention half a century ago, flow cytometry has been a major tool for single‐cell analysis, fueling advances in our understanding of a variety of complex cellular systems, in particular the immune system. The last decade has witnessed significant technical improvements in available cytometry platforms, such that more than 20 parameters can be analyzed on a single‐cell level by fluorescence‐based flow cytometry. The advent of mass cytometry has pushed this limit up to, currently, 50 parameters. However, traditional analysis approaches for the resulting high‐dimensional datasets, such as gating on bivariate dot plots, have proven to be inefficient. Although a variety of novel computational analysis approaches to interpret these datasets are already available, they have not yet made it into the mainstream and remain largely unknown to many immunologists. Therefore, this review aims at providing a practical overview of novel analysis techniques for high‐dimensional cytometry data including SPADE, t‐SNE, Wanderlust, Citrus, and PhenoGraph, and how these applications can be used advantageously not only for the most complex datasets, but also for standard 14‐parameter cytometry datasets.

High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy

Immune-checkpoint blockade has revolutionized cancer therapy. In particular, inhibition of programmed cell death protein 1 (PD-1) has been found to be effective for the treatment of metastatic melanoma and other cancers. Despite a dramatic increase in progression-free survival, a large proportion of patients do not show durable responses. Therefore, predictive biomarkers of a clinical response are urgently needed. Here we used high-dimensional single-cell mass cytometry and a bioinformatics pipeline for the in-depth characterization of the immune cell subsets in the peripheral blood of patients with stage IV melanoma before and after 12 weeks of anti-PD-1 immunotherapy. During therapy, we observed a clear response to immunotherapy in the T cell compartment. However, before commencing therapy, a strong predictor of progression-free and overall survival in response to anti-PD-1 immunotherapy was the frequency of CD14+CD16−HLA-DRhi monocytes. We confirmed this by conventional flow cytometry in an independent, blinded validation cohort, and we propose that the frequency of monocytes in PBMCs may serve in clinical decision support.

High-Dimensional Single-Cell Mapping of Central Nervous System Immune Cells Reveals Distinct Myeloid Subsets in Health, Aging, and Disease

Summary Individual reports suggest that the central nervous system (CNS) contains multiple immune cell types with diverse roles in tissue homeostasis, immune defense, and neurological diseases. It has been challenging to map leukocytes across the entire brain, and in particular in pathology, where phenotypic changes and influx of blood‐derived cells prevent a clear distinction between reactive leukocyte populations. Here, we applied high‐dimensional single‐cell mass and fluorescence cytometry, in parallel with genetic fate mapping systems, to identify, locate, and characterize multiple distinct immune populations within the mammalian CNS. Using this approach, we revealed that microglia, several subsets of border‐associated macrophages and dendritic cells coexist in the CNS at steady state and exhibit disease‐specific transformations in the immune microenvironment during aging and in models of Alzheimer’s disease and multiple sclerosis. Together, these data and the described framework provide a resource for the study of disease mechanisms, potential biomarkers, and therapeutic targets in CNS disease. Graphical Abstract Figure. No Caption available. HighlightsHigh‐dimensional cytometry reveals diverse immune cells in the steady‐state CNSCD38 and MHCII distinguish CNS border‐associated macrophage (BAM) subsetsA subset of microglia responds to aging and neurodegenerationAll microglia are homogenously affected in neuroinflammatory disease &NA; It has been challenging to map leukocytes in the brain, particularly during pathology. Mrdjen et al. combine high‐dimensional single‐cell cytometry with fate mapping to capture the immune landscape of the brain. They identify different subsets of myeloid cells and the phenotypic changes in CNS immune cells during aging and in models of Alzheimer’s disease and multiple sclerosis.

In vitro evaluation of liposomes containing bio-enhancers for the oral delivery of macromolecules

The aim of this work was to develop a new type of liposomes containing bio-enhancers for oral delivery of hydrophilic macromolecules. The study focused on EPC/cholesterol-based formulations combined with TPGS 1000 and 400, cholylsarcosine (CS), cetylpyridinium chloride (CpCl) and stearylamine (SA) covering a broad range of different types of enhancers. Most of the tested liposomal formulations and enhancers showed neither influence on cell viability in the Alamar Blue® assay nor an increase in lactate dehydrogenase LDH release. But, at a concentration of 1 mM, CpCl exhibited a strong toxicity after 2 h, TPGS 1000 reduced the cell viability at the same concentration after 8h significantly. Only one liposomal formulation with 25% CpCl led to a decrease in viability to 60.0% after 8h at a total lipid concentration of 5 mM. In the Caco-2 Transwell® model, one formulation with 5% TPGS 400 improved the permeation of FITC-dextran 70 kDa 3.34 ± 0.62-fold, one with 10% CpCl 3.69 ± 0.67 and one with 10% CS and 2.5% SA 3.41 ± 0.51-fold without influencing the TER. Liposomes with 10% SA or 25% CpCl increased the permeation of FITC-dextran 29.02 ± 5.84, respectively 39.28 ± 2.10-fold, but led also to a strong reduction in the TER. Especially, the three formulations which enhanced the permeation of FITC-dextran around 3.5-fold without showing any cell toxicity or decrease in TER should be safe and effective candidates for the development of an oral delivery system for hydrophilic macromolecules.

High-dimensional single-cell analysis reveals the immune signature of narcolepsy

Hartmann et al. show that, in narcolepsy, T cells exhibit a proinflammatory signature characterized by increased production of TNF, IL-2, and B cell–supporting cytokines.

HLA Class II tetramers reveal tissue-specific regulatory T cells that suppress T-cell responses in breast carcinoma patients.

Regulatory T cells (Tregs) play an important role in controlling antitumor T-cell responses and hence represent a considerable obstacle for cancer immunotherapy. The abundance of specific Treg populations in cancer patients has been poorly analyzed so far. Here, we demonstrate that in breast cancer patients, Tregs often control spontaneous effector memory T-cell responses against mammaglobin, a common breast tissue-associated antigen that is overexpressed by breast carcinoma. Using functional assays, we identified a HLA-DRB1*04:01- and HLA-DRB1*07:01-restricted epitope of mammaglobin (mam34–48) that was frequently recognized by Tregs isolated from breast cancer patients. Using mam34–48-labeled HLA Class II tetramers, we quantified mammaglobin-specific Tregs and CD4+ conventional T (Tcon) cells in breast carcinoma patients as well as in healthy individuals. Both mammaglobin-specific Tregs and Tcon cells were expanded in breast cancer patients, each constituting approximately 0.2% of their respective cell subpopulations. Conversely, mammaglobin-specific Tregs and CD4+ Tcon cells were rare in healthy individuals (0.07%). Thus, we provide here for the first time evidence supporting the expansion of breast tissue-specific Tregs and CD4+ Tcon cells in breast cancer patients. In addition, we substantiate the potential implications of breast tissue-specific Tregs in the suppression of antitumor immune responses in breast cancer patients. The HLA Class II tetramers used in this study may constitute a valuable tool to elucidate the role of antigen-specific Tregs in breast cancer immunity and to monitor breast cancer-specific CD4+ T cells.

CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets

High dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high throughput interrogation and characterization of cell populations.Here, we present an R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signaling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g. multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g. plots of aggregated signals).

High Dimensional Cytometry of Central Nervous System Leukocytes During Neuroinflammation

Autoimmune diseases like multiple sclerosis (MS) develop from the activation and complex interactions of a wide network of immune cells, which penetrate the central nervous system (CNS) and cause tissue damage and neurological deficits. Experimental autoimmune encephalomyelitis (EAE) is a model used to study various aspects of MS, including the infiltration of autoaggressive T cells and pathogenic, inflammatory myeloid cells into the CNS. Various signature landscapes of immune cell infiltrates have proven useful in shedding light on the causes of specific EAE symptoms in transgenic mice. However, single cell analysis of these infiltrates has thus far been limited in conventional fluorescent flow cytometry methods by 14-16 parameter staining panels. With the advent of mass cytometry and metal-tagged antibodies, a staining panel of 35-45 parameters is now possible. With the aid of dimensionality reducing and clustering algorithms to visualize and analyze this high dimensional data, this allows for a more comprehensive picture of the different cell populations in an inflamed CNS, at a single cell resolution level. Here, we describe the induction of active EAE in C56BL/6 mice and, in particular, the staining of microglia and CNS invading immune cells for mass cytometry with subsequent data visualization and analysis.