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To better define the control of immune system regulation, we generated an atlas of microRNA (miRNA) expression from 63 mouse immune cell populations and connected these signatures with assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation followed by sequencing (ChIP-seq) and nascent RNA profiles to establish a map of miRNA promoter and enhancer usage in immune cells. miRNA complexity was relatively low, with >90% of the miRNA compartment of each population comprising

Several studies have revealed that the hyper-inflammatory response induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major cause of disease severity and death. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α and IL-1β in hospitalized patients with coronavirus disease 2019 (COVID-19) upon admission to the Mount Sinai Health System in New York. Patients (n = 1,484) were followed up to 41 d after admission (median, 8 d), and clinical information, laboratory test results and patient outcomes were collected. We found that high serum IL-6, IL-8 and TNF-α levels at the time of hospitalization were strong and independent predictors of patient survival (P

Improved identification of anti-tumor T cells is needed to advance cancer immunotherapies. CD39 expression is a promising surrogate of tumor-reactive CD8

Microglia are a heterogeneous population whose identity and function are dictated by signals from their microenvironment. Kana et al. show CSF-1 signaling is critical for cerebellar microglial transcriptional identity and homeostasis, and that altering the CSF-1–CSF-1R axis leads to motor and behavioral defects., Microglia, the brain resident macrophages, critically shape forebrain neuronal circuits. However, their precise function in the cerebellum is unknown. Here we show that human and mouse cerebellar microglia express a unique molecular program distinct from forebrain microglia. Cerebellar microglial identity was driven by the CSF-1R ligand CSF-1, independently of the alternate CSF-1R ligand, IL-34. Accordingly, CSF-1 depletion from Nestin+ cells led to severe depletion and transcriptional alterations of cerebellar microglia, while microglia in the forebrain remained intact. Strikingly, CSF-1 deficiency and alteration of cerebellar microglia were associated with reduced Purkinje cells, altered neuronal function, and defects in motor learning and social novelty interactions. These findings reveal a novel CSF-1–CSF-1R signaling-mediated mechanism that contributes to motor function and social behavior.

The COVID-19 pandemic caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to more than 100,000 deaths in the United States. Several studies have revealed that the hyper-inflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum IL-6, IL-8, TNF-α, and IL-1β in hospitalized COVID-19 patients upon admission to the Mount Sinai Health System in New York. Patients (n=1484) were followed up to 41 days (median 8 days) and clinical information, laboratory test results and patient outcomes were collected. In 244 patients, cytokine measurements were repeated over time, and effect of drugs could be assessed. Kaplan-Meier methods were used to compare survival by cytokine strata, followed by Cox regression models to evaluate the independent predictive value of baseline cytokines. We found that high serum IL-6, IL-8, and TNF-α levels at the time of hospitalization were strong and independent predictors of patient survival. Importantly, when adjusting for disease severity score, common laboratory inflammation markers, hypoxia and other vitals, demographics, and a range of comorbidities, IL-6 and TNF-α serum levels remained independent and significant predictors of disease severity and death. We propose that serum IL-6 and TNF-α levels should be considered in the management and treatment of COVID-19 patients to stratify prospective clinical trials, guide resource allocation and inform therapeutic options. We also propose that patients with high IL-6 and TNF-α levels should be assessed for combinatorial blockade of pathogenic inflammation in this disease.

Tissue-resident macrophages (TRMs) populate all tissues and play key roles in homeostasis, immunity and repair. TRMs express a molecular program that is mostly shaped by tissue cues. However, TRM identity and the mechanisms that maintain TRMs in tissues remain poorly understood. We recently found that serous-cavity TRMs (LPMs) are highly enriched in RXR transcripts and RXR-response elements. Here, we show that RXRs control mouse serous-macrophage identity by regulating chromatin accessibility and the transcriptional regulation of canonical macrophage genes. RXR deficiency impairs neonatal expansion of the LPM pool and reduces the survival of adult LPMs through excess lipid accumulation. We also find that peritoneal LPMs infiltrate early ovarian tumours and that RXR deletion diminishes LPM accumulation in tumours and strongly reduces ovarian tumour progression in mice. Our study reveals that RXR signalling controls the maintenance of the serous macrophage pool and that targeting peritoneal LPMs may improve ovarian cancer outcomes., Macrophages can differentiate to perform homeostatic tissue-specific functions. Here the authors show that RXR signalling is critical for large peritoneal macrophage (LPM) expansion during neonatal life and LPM lipid metabolism and survival during adult homeostasis, and that ovarian cancer growth relies on RXR-dependent LPMs.

Background: Mass cytometry leverages inductively coupled mass spectrometry to perform high dimensional single cell analyses using antibodies tagged with rare earth isotopes that are considered to be largely absent in biological samples. We have recently noted an unusual exception to this rule while analyzing tissue samples from patients undergoing surgical resection for early stage lung cancer, and here we present a detailed cytometric characterization of cerium in a clinical patient sample. Methods: We performed a CyTOF analysis on cell suspensions derived from matched blood, tumor lesion, and non-involved lung tissue from an active smoker undergoing surgical resection for early stage lung adenocarcinoma. The samples were stained with a 31-parameter antibody panel to allow a detailed characterization of the cellular heterogeneity of the samples. The data were visualized using viSNE, major immune subsets were identified based on canonical marker expression patterns, and single cell cerium levels were evaluated across each of these defined subsets. Results: High dimensional immune cell mapping revealed that high levels of cerium were specifically associated with a phenotypically-distinct subset of lung macrophages that were most prevalent in non-involved lung tissue, whereas tumor associated macrophages showed relatively lower levels of cerium. We hypothesize that these findings reflect alveolar macrophage phagocytosis of inhaled cerium derived from cigarette flint lighters. Conclusions: These results demonstrate the first high-dimensional single cell characterization of environmental metal exposure associated with smoking, and offer a demonstration of the unique potential for applying mass cytometry to the field of environmental toxicology. This article is protected by copyright. All rights reserved.

The colony stimulating factor 1 receptor (CSF1R) functions as the major receptor for macrophage colony stimulating factor (CSF1) with crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by A disintegrin and metalloprotease 17 (ADAM17). Here we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2−/− mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2−/− BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2−/− Lin−SCA-1+c-Kit+ (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs.

Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses.

Macrophages are immune cells of haematopoietic origin that provide crucial innate immune defence and have tissue-specific functions in the regulation and maintenance of organ homeostasis. Recent studies of macrophage ontogeny, as well as transcriptional and epigenetic identity, have started to reveal the decisive role of the tissue stroma in the regulation of macrophage function. These findings suggest that most macrophages seed the tissues during embryonic development and functionally specialize in response to cytokines and metabolites that are released by the stroma and drive the expression of unique transcription factors. In this Review, we discuss how recent insights into macrophage ontogeny and macrophage–stroma interactions contribute to our understanding of the crosstalk that shapes macrophage function and the maintenance of organ integrity.

SummaryAlthough classified as hematopoietic cells, tissue-resident macrophages (MFs) arise from embryonic precursors that seed the tissues prior to birth to generate a self-renewing population, which is maintained independently of adult hematopoiesis. Here we reveal the identity of these embryonic precursors using an in utero MF-depletion strategy and fate-mapping of yolk sac (YS) and fetal liver (FL) hematopoiesis. We show that YS MFs are the main precursors of microglia, while most other MFs derive from fetal monocytes (MOs). Both YS MFs and fetal MOs arise from erythro-myeloid progenitors (EMPs) generated in the YS. In the YS, EMPs gave rise to MFs without monocytic intermediates, while EMP seeding the FL upon the establishment of blood circulation acquired c-Myb expression and gave rise to fetal MOs that then seeded embryonic tissues and differentiated into MFs. Thus, adult tissue-resident MFs established from hematopoietic stem cell-independent embryonic precursors arise from two distinct developmental programs.

e15644 Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths in men worldwide, and is the most rapidly rising cause of cancer mortality in the US. Patients with resectable disease experience high rates of locoregional recurrence, and there are few effective systemic treatment options available. PD-1 blockade was recently approved for 2nd line therapy, and there are promising response rates seen in the 1st-line setting. However, only 20% of patients achieve significant response, thus we must investigate further the tumor-immune microenvironment to understand how to better modulate the immune system in this highly immunosuppressive organ. Methods: We used mass cytometry (CyTOF) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to study protein and transcriptomics of untreated tumor and tumor adjacent tissue, on a single-cell level. Results: Paired CyTOF analysis of 10 tumor lesions/adjacent tissues identified a significant increase in frequency of CD4+ T cells and T regulatory cells, a significant decrease in NK cells and neutrophils, and no difference in CD8+ T cells, macrophages, or dendritic cells (DCs) in the tumor microenvironment compared to the adjacent liver tissue. We also analyzed four patients who received nivolumab and subsequently went on to have their liver tumors resected; two out of the four patients treated had near-complete necrosis of their tumors (radiographically and histologically). Of note, the tumors of the two patients with significant pathologic response contained a significantly higher number of CD103+ tissue resident CD8+ and CD4+ T cells, compared to adjacent tissue. Moreover, activated CD8+ T cells (TIGIT+, PD-1+, TIM3+, and/or CD38+) were significantly higher in the tumor of nivolumab responders, indicating a tumor-specific response. We will also present single cell transcriptomic analyses of T cell and myeloid populations in responders and non-responders. Interestingly, high levels of nivolumab were detected on PD-1+ T cells in the tumor in all 4 patients; we will discuss transcriptional differences between nivolumab-targeted T cells in responders and non-responder patients. Conclusions: Based on this preliminary data we will be opening a clinical trial of neoadjuvant PD-1 blockade in HCC (NCT# pending) in March 2019.

Summary To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single-cell analysis of the tumor, non-involved lung, and blood cells, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor-infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single-cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies. Video Abstract

SummaryMacrophages are critical for innate immune defense and also control organ homeostasis in a tissue-specific manner. They provide a fitting model to study the impact of ontogeny and microenvironment on chromatin state and whether chromatin modifications contribute to macrophage identity. Here, we profile the dynamics of four histone modifications across seven tissue-resident macrophage populations. We identify 12,743 macrophage-specific enhancers and establish that tissue-resident macrophages have distinct enhancer landscapes beyond what can be explained by developmental origin. Combining our enhancer catalog with gene expression profiles and open chromatin regions, we show that a combination of tissue- and lineage-specific transcription factors form the regulatory networks controlling chromatin specification in tissue-resident macrophages. The environment is capable of shaping the chromatin landscape of transplanted bone marrow precursors, and even differentiated macrophages can be reprogramed when transferred into a new microenvironment. These results provide a comprehensive view of macrophage regulatory landscape and highlight the importance of the microenvironment, along with pioneer factors in orchestrating identity and plasticity.

Macrophages form a heterogeneous group of hematopoietic cells that reside in tissues, where they are required to maintain organ integrity. Tissue macrophages contribute to tissue formation, metabolism, homeostasis, and repair. They have a unique ability to sense and respond to tissue damage. They serve as the first line of defense during infection and help promote immune tolerance in the steady state. Although most tissue macrophages share a high phagocytic and degradative potential, they are heterogeneous in origin, as well as in homeostatic function and response to insults. Here, we will discuss recent developments in our understanding of the origin of tissue macrophages and their functional specialization in tissues. Cancer Immunol Res; 1(4); 201–9. ©2013 AACR.

Tumors form complex ecosystems that include normal epithelial cells, fibroblasts, blood and lymphatic vessels as well as structural components, and importantly, immune cells. The density, location, and organization of various tumor-infiltrating hematopoietic cells of myeloid and lymphoid origin can impact tumor growth, spreading, and clinical outcome of various cancers, including non-small cell lung cancer (NSCLC). Here, we propose to characterize the microenvironment of lung tumors using a unique set of matched fresh tissue single cell suspensions of tumor and normal lung, paraffin-embedded tissues, and peripheral blood plasma and cells obtained at time of surgery in the same patient. We aim to query mechanisms for local immunogenicity and immunosuppression of NSCLC, by defining interactions between tumor cells and various immune cell subsets in the local microenvironment, and by correlating these findings with presence of tumor-associated antigens (TAAs) and their capacity to elicit spontaneous local and systemic immune responses, as well as immune checkpoints known to impair immune responses. We applied multiplexed immunohistochemistry for formalin-fixed paraffin embedded cancer tissues (MICSSS method; n = 40), serological assays for immunogenicity of tumor antigens from plasma samples and supernatants of B cell cultures from the same patients (ELISA; n = 40), as well as mass cytometry on cell suspensions obtained from matched fresh specimens (CYTOF, n = 20) to (1) characterize the composition and organization of the immune microenvironment, (2) identify drivers of immunosuppression in NSCLC and (3) identify the expression profile of TAAs and B cell specificity in NSCLC. We found that TAAs, such as p53, NY-ESO-1, or ERG, were expressed by lung cancer cells in a subset of patients and that protein expression at the tumor site correlated with the antibody (Ab) response found in plasma of matched patients. While circulating plasma Ab titers did not appear to be correlated with the density of infiltrating B cells, potential associations with presence of tertiary-lymphoid structure, plasma cells (CD138+ cytokeratin− cells) or T follicular helper cells (CXCR5+ CD3+ CD8−) are currently being assessed. Association between molecular alterations and immune cell composition and location will also be discussed. We will also quantify expression of checkpoint molecules (PD-L1, VISTA and HHLA-2) on immune and tumor cells. The correlation between checkpoint inhibitor expression and immune cell densities and presence of immune responses will be presented, to assess whether immune cells contribute to the modulation of T cell effector functions. In conclusion, this study is the stepping-stone to a comprehensive understanding of the complex interactions between lung tumors and their immune microenvironment. By using novel multiplexed approaches, we intend to map the immune and antigenic landscape of human NSCLC, to identify mechanisms of early disease immunogenicity and immunosuppression, and to find new prognostic and predictive markers for the development of novel therapeutic strategies and immunotherapy trials in NSCLC. Citation Format: Romain Remark, Yonit Lavin, Adeeb Rahman, Camille Bigenwald, Soma Kobayashi, Christian Becker, Raja Flores, Sacha Gnjatic, Miriam Merad. Comprehensive multidimensional analysis of the immune contexture in non-small cell lung cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A092.

We developed a short questionnaire—Parental PTSD Questionnaire–(PPQ), designed to assess the presence of posttraumatic stress disorder (PTSD) symptoms in parents. Fifty-eight adult offspring of Holocaust survivors (23 men and 35 women) completed the questionnaire about a parent who was independently evaluated by a trained clinician using the Clinician Administered PTSD Scale (CAPS). Only 5.2% of the offspring reported, “not knowing” if their parent had experienced 10 or fewer symptoms, while 56.9% provided estimates for all 17 items. There were no significant differences between lifetime frequencies of the individual symptoms as endorsed on the PPQ compared to the CAPS when subjects with completed PPQs were compared with CAPS. Interrater reliability between offspring and clinician was highly significant for each of the items when evaluated separately so as to include data for subjects who endorsed not knowing if a certain symptom had been present. Further studies are warranted to examine the psychometric properties of this measure.