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The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells-especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell-cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1-2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHCP using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model., Competing Interests: A.C. is employee of Zellkraftwerk GmbH, which has developed and is selling Chipcytometry instrumentation and services.

The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure, and quantitate multiple fluorescent markers on a single cell makes the flow cytometer a useful tool for analyzing various aspects of cell phenotype and function. Here we describe a standardized protocol for surface and intracellular immune-phenotyping of murine lungs, beginning with the building of an optimal antibody panel and ending with data analysis and representation, including sample gating strategies for innate and adaptive immune responses.

Flow cytometry is a popular technique used for both clinical and research purposes. It involves laser-based technology to characterize cells based on size, shape, and complexity. Additionally, flow cytometers are equipped with the ability to take fluorescence measurements at multiple wavelengths. This capability makes the flow cytometer a practical resource in the utilization of fluorescently conjugated antibodies, fluorescent proteins, DNA binding dyes, viability dyes, and ion indicator dyes. As the technology advances, the number of parameters a flow cytometer can measure has increased tremendously, and now some has the capacity to analyze 30-50 or more parameters on a single cell. Here, we describe the basic principles involved in the mechanics and procedures of flow cytometry along with an insight into applications of flow cytometry techniques for biomedical and allergic disease research.

Background: Isolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The plasma cells are typically isolated by positive selection using plasma cell markers such as CD138. Here we have examined the effect of CD138 magnetic bead selection on the expression of other surface phenotypic markers on plasma cells., Materials and Methods: Bone marrow aspirates from 16 patients were split and prepared using 2 methods before staining for flow cytometric evaluation. The first method (whole bone marrow) used an ammonium-chloride-potassium lyse of whole bone marrow followed by 2 phosphate buffered saline washes. The second method used CD138-positive magnetic sorting technology (Stem Cell Technology). The cells were run on the FACSCanto flow cytometer after staining for CD38, CD45, CD56, activation markers CD71, CD69, CD154, adhesion markers CD49d, CD49e, CD11a, CD11b, and CD66, B cell markers CD19 and CD20, and for clonality., Results: There was a substantial loss in the expression of CD71, CD11b, CD11a, CD69, and CD49e on plasma cells following CD138-based sorting. Moreover, in 8 of the 16 cases, there was a nearly complete loss of the CD45-positive subset with a loss of discrimination between CD45-negative and CD45-positive plasma cell subsets in the remaining CD138-sorted preparations., Conclusions: The change in immunophenotype of the plasma cells on magnetic sorting should be kept in mind when isolating plasma cells using CD138-positive selection for analysis of plasma cells. The technique for characterizing plasma cells should be selected based on the study design to prevent loss of crucial and valuable information., (Copyright © 2020 Elsevier Inc. All rights reserved.)

B cells are primarily known for their capacity to differentiate into antibody-secreting cells (ASCs). ASCs are usually viewed as terminally differentiated cells sharing a unique phenotype. However, it lately became evident that ASCs exist in a variety of subsets differing by their lifespan, anatomic location, and immunological function. Thus, ASCs can exist as long-lived plasma cells (LLPC) that can persist for years in a nonproliferating state within particular niches in the bone marrow (BM), or as short-lived plasma cells (SLPC) that are primarily found in secondary lymphoid organs or inflamed tissues and wane upon the termination of the associated immune response. Another layer of ASC diversity was uncovered with the discovery of their capacity to produce various pro- or anti-inflammatory cytokines. Notably, a subset of natural regulatory plasma cells characterized by the distinctive expression of the inhibitory receptor lymphocyte activation gene (LAG)-3 and a unique capacity to produce interleukin (IL)-10 upon stimulation was recently identified. Here, we describe how to immunophenotypically characterize murine plasma cells as well as how to isolate them using cell sorting, with a special focus on these recently described natural regulatory plasma cells.

Immune infiltration in neuroblastoma (NBL) has been associated with clinical outcome. However, the diversity of distinct immune subpopulations that comprise immune infiltrates in NBL has not been examined. To this end, the present study investigated the immunological landscape of NBL tumors and its clinical significance. CIBERSORTx, an established RNA deconvolution algorithm, was used to impute immune cell proportions from 153 primary NBL tumors. Associations between immune proportions and overall/event-free survival were analyzed by Kaplan-Meier curves and evaluated using log-rank test. Of the 22 subpopulations imputed, M2 macrophages were the most abundant subtype in NBL tumors. Furthermore, monocytes, CD4+ naïve T cells, and CD4+ activated memory T cells were significantly associated with survival. Altogether, the findings suggest differences amongst certain immune cell subsets comprising NBL tumor infiltration and these differences may be important determinants of prognosis., (Copyright © 2020. Published by Elsevier Ltd.)

Myeloid-derived suppressor cells (MDSC) are immunosuppressive myeloid cells that accumulate in tumor sites and peripheral lymphoid organs such as the spleen. In murine cancer models, the spleen is a major reservoir for MDSC, representing an easily accessible tissue from which to isolate high numbers of these cell population for downstream applications. Here we describe an efficient method to phenotype as well as to isolate and assess the functionality of murine splenic MDSC.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of pathologically expanded myeloid cells with immunosuppressive activity. According to their phenotype, MDSC can be divided into three major subpopulations: early stage MDSC (e-MDSC), lacking myeloid lineage markers, monocytic MDSC (M-MDSC), and granulocytic MDSC (PMN-MDSC). Additionally, PMN-MDSC can be subdivided based on their activation and differentiation status, although it is not clear how this status contributes to immunosuppression and disease pathology. Here, we describe an immunophenotyping and gating strategy for the identification and isolation of MDSC subsets based on fluorescence-activated cell sorting. This method allows direct comparison of MDSC subsets in clinical settings.

Egg allergy is one of the most common food allergies in children, being the most important allergenic proteins found in the egg white (EW). Allergy to EW shows a complex phenotype that involves a multifaceted reaction that can only be assessed in vivo. Although other routes of sensitization have been described, oral exposure to food antigens is one of the most suitable in humans. In mice, oral administration of allergenic proteins results in the development of tolerance, and the use of adjuvants, such as cholera toxin (CT), is required to promote Th2-biased immune responses over tolerogenic responses. In this regard, among the mouse strains that readily display Th2 responses, Balb/c has been widely used. Here, we describe a frequently used protocol of oral EW sensitization by using CT as an adjuvant and we explain in detail the methods that we have developed to analyze the sensitizing and eliciting capacity of EW proteins including evaluation of signs, measurement of serum levels of specific immunoglobulins, mast cell degranulation, cytokine secretion profile of allergen-reactive T cells, phenotyping of mesenteric lymph node- and spleen-derived dendritic and T cells by flow cytometry, and quantification of intestinal gene expression.

High-dimensional cytometry is a powerful technique for deciphering the immunopathological factors common to multiple individuals. However, rational comparisons of multiple batches of experiments performed on different occasions or at different sites are challenging because of batch effects. In this study, we describe the integration of multibatch cytometry datasets (iMUBAC), a flexible, scalable, and robust computational framework for unsupervised cell-type identification across multiple batches of high-dimensional cytometry datasets, even without technical replicates. After overlaying cells from multiple healthy controls across batches, iMUBAC learns batch-specific cell-type classification boundaries and identifies aberrant immunophenotypes in patient samples from multiple batches in a unified manner. We illustrate unbiased and streamlined immunophenotyping using both public and in-house mass cytometry and spectral flow cytometry datasets. The method is available as the R package iMUBAC (https://github.com/casanova-lab/iMUBAC)., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Objective: Conventional karyotyping of multiple myeloma (MM) is hampered by the low mitotic index of plasma cells (PCs), and low proportion of PCs in some specimens may lead to false negative results in fluorescence in situ hybridisation (FISH) detection., Methods: Bone marrow cells were cultured in an ordinary medium for 24 h or in a medium containing 10 ng/mL IL-6 and 40 ng/mL GM-CSF for 6 d. Fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) was also conducted, combining CD138 fluorescent immunophenotype and FISH., Results: Under modified culture conditions, the successful rate of culture and abnormality detection rate during karyotype analysis increased to 86.4% and 40.9%, respectively. The abnormality detection rate of FICTION (89.5%) was significantly higher than that of FISH (60.0%). The genetic abnormality detection rate increased to 92.3% when FICTION and karyotyping were conducted under modified culture conditions., Conclusion: The established modified culture system could improve karyotyping quality in MM. Due to its obvious advantages compared with FISH, FICTION is recommended for detecting genetic abnormalities in MM., (Copyright © 2021 by the Association of Clinical Scientists, Inc.)

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r 2  > 0.95 for all cell types in PB and r 2  > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Transplantation is still the treatment of choice for organ failure; however, allograft induces inflammatory immune responses that require immunosuppressive treatment. The role of regulatory B cells (Bregs) in downregulating inflammation has been reported to be significant in several diseases including transplant rejection. Many reports have analyzed different B-cell subpopulations, including Bregs, in tolerant, stable, and rejecting transplant recipients as well as the influence of immunosuppressant on the frequencies and functions of the different B-cell subsets. In this chapter, the key techniques required to investigate human Breg frequencies and functions in transplant patients are discussed.

Myeloid-derived suppressor cells (MDSC) populate the peripheral blood and contribute to immune regulation in cancer. However, there is limited knowledge on the myeloid cell types with proinflammatory capacities that may serve as opponents of MDSC. In the circulation of cancer patients, a monocyte subpopulation was identified with a specific immunophenotype and transcriptomic signature. They were predominantly CD14 + CD33 hi CD16 -/+ HLA-DR +/hi cells that typically expressed CD66b. In accordance with the transcriptomics data, NALP3, LOX-1 and PAI-1 levels were also significantly upregulated. The CD66b + monocytes displayed high phagocytic activity, matrix adhesion and migration, and provided costimulation for T cell proliferation and IFN-γ secretion; thus, they did not suppress T cell responses. Irrespective of clinical stage, they were identified in various cancers. In conclusion, the CD66b + monocytes represent a novel myeloid subpopulation which is devoid of immune regulatory influences of cancer and displays enhanced proinflammatory capacities.

The tumor microenvironment (TME) contains noncancerous cells such as various types of immune cells and fibroblasts. Cancer cells direct these stromal cells to create a microenvironment favorable for tumor growth and intercellular interactions have a critical role in this process. In established tumors, interactions between CD137 and its ligand (CD137L) contribute to tumor immune evasion and tumor growth. Therefore, it is important to identify cells expressing CD137 and CD137L within tumors. In this chapter, we will introduce a simple, powerful method of analyzing CD137- and CD137L-expressing tumor cells using Fluorescence-activated cell sorting.

The pluripotency of human induced pluripotent stem cells (HiPSCs) cannot be tested strictly in a similar way as we can do for the mouse ones because of ethical restrictions. One common and initial approach to prove the pluripotency of an established human iPSC line is to demonstrate expression of a set of established surface and intracellular pluripotency markers. This chapter provides procedures of immunocytochemistry of the established HiPSC lines for a set of the signature intracellular pluripotency proteins, OCT4, SOX2, NANOG, and LIN28. We also describe cell phenotyping by flow cytometry for the five established human pluripotency surface markers, SSEA3, SSEA4, TRA-1-60, TRA-1-81, and TRA2-49 (ALP). Numbers of ALP + and TRA-1-60 + colonies are the most widely used parameters for evaluation of human iPSC reprogramming efficiency. Therefore, this chapter also provides detailed steps for substrate colorimetric reaction of the ALP activity, as well as the TRA-1-60 staining, of the iPSC colonies in the reprogramming population.

While myeloid-derived suppressor cells (MDSCs) in humans and mice have been intensively investigated, there is limited knowledge of these cells in nonhuman primates (NHPs). NHPs serve as critical models for late-stage testing of several biomedical inventions before proceeding with clinical trials and it is therefore important to fully understand their immune compartments and similarities with humans. Here, using antihuman cross-reactive antibodies, we provide flow cytometric analysis protocols for identification of MDSCs in the blood of rhesus macaques, one of the major NHP species as experimental models. Discrepancies and similarities between rhesus and human MDSCs are discussed.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population composed of mature and immature cells of myeloid origin that play a major role in tumor progression by inhibiting the antitumor immune responses mediated by T cells. In this chapter, we describe protocols for isolation, phenotypical and functional evaluation of MDSCs isolated from mouse tumors, with the aim at unifying and standardizing protocols set up by different laboratories.

Primordial germ cells (PGCs) are common ancestors of all germline cells. In mammals, PGCs emerge in early-stage embryos around the timing of gastrulation at or near epiblast, and specification of PGCs from their precursor cells involves multiple growth factors secreted by adjacent cells. Recent advancements in germline stem cell biology have made it possible to generate PGC-like cell culture models (PGCLCs for PGC-like cells) from human and mouse pluripotent stem cells by mimicking the embryonic growth factor environment in vitro. Here we describe a method of producing human PGCLCs from primed-pluripotency induced pluripotent stem cells (iPSCs) via temporal conversion to naive pluripotency followed by formation of embryoid bodies (EBs) using the spin-EB method.

The field of flow cytometry has witnessed rapid technological advancements in the last few decades. While the founding principles of fluorescent detection on cells (or particles) within a uniform fluid stream remains largely unchanged, the availability more sensitive cytometers with the ability to multiplex more and more florescent signals has resulted in very complex high-order assays. This results in the co-use of fluorophores with increased levels of emission overlap and/or spillover spreading than in years past and thus requires careful and well thought out planning for flow cytometry assay development. As an example, we present the development of a large 18-color (20 parameter) flow cytometry assay designed to take an in depth analysis of effector lymphocyte phenotypes, with careful attention to assay controls and panel design.

Wheat allergy is a pathological event involving immunocompetent cells against ingested wheat allergen and is clearly associated with transdermal sensitization. However, the molecular mechanisms involved in the disease etiology are not completely understood. A complex cellular and tissue network linking to food allergy makes it difficult to understand the molecular mechanism of allergenicity. Animal models are valuable tools to deduce basic principles of human disease without invasive intervention trials. A mouse model of wheat allergy has provided insights into effects of skin exposure to wheat protein; it is a plausible route of human sensitization for wheat anaphylaxis. Further investigation of this model will capture the essential occurrence and flow of events, bringing useful clues to develop effective treatment and control strategies against wheat allergy. Here, we describe a method for analyzing the expression of cell surface molecules in single cells isolated from lymphoid tissue with flow cytometry. Sensitization by wheat extracts significantly increases antigen-specific T cells in the spleen. Collecting information regarding the contribution of immune cells to allergic sensitization in the development of wheat allergy would be useful in preventing and treating food allergies.

Regulatory B cells (Bregs) have immunosuppressive capacity, primarily via the production of IL-10. IL-10 expression and immunosuppression have been described in a number of human B cell subsets, two of which include the CD19 + CD24 hi CD38 hi and CD19 + CD24 hi CD27 + populations. In this chapter, we describe how to identify and isolate these subsets from peripheral blood B cells via flow cytometry. We also explain how to expand Bregs in culture and how to identify them based on intracellular expression of IL-10.

Coronary artery disease remains the leading cause of death globally and is a major burden to every health system in the world. There have been significant improvements in risk modification, treatments, and mortality; however, our ability to detect asymptomatic disease for early intervention remains limited. Recent discoveries regarding the inflammatory nature of atherosclerosis have prompted investigation into new methods of diagnosis and treatment of coronary artery disease. This article reviews some of the highlights of the important developments in cardioimmunology and summarizes the clinical evidence linking the immune system and atherosclerosis. It provides an overview of the major serological biomarkers that have been associated with atherosclerosis, noting the limitations of these markers attributable to low specificity, and then contrasts these serological markers with the circulating immune cell subtypes that have been found to be altered in coronary artery disease. This review then outlines the technique of mass cytometry and its ability to provide high-dimensional single-cell data and explores how this high-resolution quantification of specific immune cell subpopulations may assist in the diagnosis of early atherosclerosis in combination with other complimentary techniques such as single-cell RNA sequencing. We propose that this improved specificity has the potential to transform the detection of coronary artery disease in its early phases, facilitating targeted preventative approaches in the precision medicine era.

Purpose: Molecular subtype classifications in glioblastoma may detect therapy sensitivities. IHC would potentially allow the identification of molecular subtypes in routine clinical practice., Experimental Design: Formalin-fixed, paraffin-embedded tumor samples of 124 uniformly treated, newly diagnosed patients with glioblastoma were submitted to RNA sequencing, IHC, and immune-phenotyping to identify differences in molecular subtypes associated with treatment sensitivities., Results: We detected high molecular and IHC overlapping of the The Cancer Genome Atlas (TCGA) mesenchymal subtype with instrinsic glioma subtypes (IGS) cluster 23 and of the TCGA classical subtype with IGS cluster 18. IHC patterns, gene fusion profiles, and immune-phenotypes varied across subtypes. IHC revealed that the TCGA classical subtype was identified by high expression of EGFR and low expression of PTEN, while the mesenchymal subtype was identified by low expression of SOX2 and high expression of two antibodies, SHC1 and TCIRG1, selected on the basis of RNA differential transcriptomic expression. The proneural subtype was identified by frequent positive IDH1 expression and high Olig2 and Ki67 expression. Immune-phenotyping showed that mesenchymal and IGS 23 tumors exhibited a higher positive effector cell score, a higher negative suppressor cell score, and lower levels of immune checkpoint molecules. The cell-type deconvolution analysis revealed that these tumors are highly enriched in M2 macrophages, resting memory CD4 + T cells, and activated dendritic cells, indicating that they may be ideal candidates for immunotherapy, especially with anti-M2 and/or dendritic cell vaccination., Conclusions: There is a subset of tumors, frequently classified as mesenchymal or IGS cluster 23, that may be identified with IHC and could well be optimal candidates for immunotherapy., (©2020 American Association for Cancer Research.)

Background: Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The "inflammatory" nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies., Methods: Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients., Results: IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4 + T cells, whereas reductions in CD8 + T cells were more concentrated in memory subsets. Immature cytokine-producing CD56 bright NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56 dim cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4 + T cells and CD56 dim NK cells, and higher numbers of CD8 + effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20 + B cells in the tumor., Conclusions: Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC.

Numerous models are available for the preclinical study of sepsis, and they fall into one of three general categories: (1) administration of exogenous toxins (e.g., lipopolysaccharide, zymosan), (2) virulent bacterial or viral challenge, and (3) host barrier disruption, e.g., cecal ligation and puncture (CLP) or colon ascendens stent peritonitis (CASP). Of the murine models used to study the pathophysiology of sepsis, CLP combines tissue necrosis and polymicrobial sepsis secondary to autologous fecal leakage, as well as hemodynamic and biochemical responses similar to those seen in septic humans. Further, a transient numerical reduction of multiple immune cell types, followed by development of prolonged immunoparalysis, occurs in CLP-induced sepsis just as in humans. Use of the CLP model has led to a vast expansion in knowledge regarding the intricate physiological and cellular changes that occur during and after a septic event. This updated article details the steps necessary to perform this survival surgical technique, as well as some of the obstacles that may arise when evaluating the sepsis-induced changes within the immune system. It also provides representative monoclonal antibody (mAb) panels for multiparameter flow cytometric analysis of the murine immune system in the septic host. © 2020 Wiley Periodicals LLC. Basic Protocol: Cecal ligation and puncture in the mouse., (© 2020 Wiley Periodicals LLC.)

The aim of the present work was to evaluate counts and functional properties of PD-1 + and TIM-3 + T cells in peripheral blood (PB) and bone marrow (BM) of multiple myeloma (MM) patients following the induction therapy. Sixty patients were enrolled in the study, CD4 + and CD8 + T cells expressing PD-1 and TIM-3, intracellular production of IFNγ and intracellular expression of Granzyme B were assessed. Relative counts of the majority of circulating PD-1 + , TIM-3 + and PD-1 + TIM-3 + T cells were higher in MM patients with disease progression compared with individuals in remission. Frequencies of almost all evaluated PD-1 + and TIM-3 + T cell subsets were higher in BM samples compared with PB; circulating CD4 + PD-1 + , CD8 + PD-1 + , CD8 + TIM-3 + , CD8 + PD-1 + TIM-3 + T cells positively correlated with the same BM subsets. Circulating CD4 + T cells, expressing PD-1 and TIM-3 (including co-expressing subset), as well as CD8 + PD-1 + TIM-3 + T cells, and BM CD8 + PD-1 + T cells correlated with serum B2-M levels. Sufficient frequencies of GrB + and IFNγ + subsets in PD-1-expressing T cells indicated their retained functional properties. TIM-3-expressing T cells and double positive PD-1 + TIM-3 + populations showed diminished cytotoxic and cytokine-producing ability and therefore might be attributed to the exhausted compartment. To identify T cell exhaustion, it is necessary to evaluate T cells co-expressing PD-1, TIM-3 and other inhibitory signal molecules and to study their functional properties. Sustained functionality of PD-1-positive T cells may explain low efficacy and frequent immune-mediated adverse events during anti-PD-1 therapy in MM.

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

Background: Multiparameter flow cytometry (FC) is essential in the diagnostic work-up and classification of primary immunodeficiency (PIDs). The EuroFlow PID Orientation tube (PIDOT) allows identification of all main lymphocyte subpopulations in blood. To standardize data analysis, tools for Automated Gating and Identification (AG&I) of the informative cell populations, were developed by EuroFlow. Here, we evaluated the contribution of these innovative AG&I tools to the standardization of FC in the diagnostic work-up of PID, by comparing AG&I against expert-based (EuroFlow-standardized) Manual Gating (MG) strategy, and its impact on the reproducibility and clinical interpretation of results., Methods: FC data files from 44 patients (13 CVID, 12 PID, 19 non-PID) and 26 healthy donor (HD) blood samples stained with PIDOT were analyzed in parallel by MG and AG&I, using Infinicyt™ software (Cytognos). For comparison, percentage differences in absolute cell counts/µL were calculated for each lymphocyte subpopulation. Data files showing differences >20% were checked for their potential clinical relevance, based on age-matched percentile (p5-p95) reference ranges. In parallel, intra- and inter-observer reproducibility of MG vs AG&I were evaluated in a subset of 12 samples., Results: The AG&I approach was able to identify the vast majority of lymphoid events (>99%), associated with a significantly higher intra- and inter-observer reproducibility compared to MG. For most HD (83%) and patient (68%) samples, a high degree of agreement (<20% numerical differences in absolute cell counts/µL) was obtained between MG and the AG&I module. This translated into a minimal impact (<5% of observations) on the final clinical interpretation. In all except three samples, extended expert revision of the AG&I approach revealed no error. In the three remaining samples aberrant maturation and/or abnormal marker expression profiles were seen leading in all three cases to numerical alarms by AG&I., Conclusion: Altogether, our results indicate that replacement of MG by the AG&I module would be associated with a greater reproducibility and robustness of results in the diagnostic work-up of patients suspected of PID. However, expert revision of the results of AG&I of PIDOT data still remains necessary in samples with numerical alterations and aberrant B- and T-cell maturation and/or marker expression profiles., (Copyright © 2020 Linskens, Diks, Neirinck, Perez-Andres, De Maertelaere, Berkowska, Kerre, Hofmans, Orfao, van Dongen, Haerynck, Philippé and Bonroy.)

Background: The Matutes score (MS) was proposed to differentiate chronic lymphocytic leukemia (CLL) from other B-cell non-Hodgkin lymphomas (B-NHLs). However, ambiguous immunophenotypes are common and remain a diagnostic challenge. Therefore, we evaluated the diagnostic benefit of measuring CD200 and CD43 expression together with the standard MS antigens., Methods: 138 lymphoma patient samples and a validation cohort of 138 additive samples were classified according to the standard MS and further assigned with one or two additional points, for high CD200 and/or CD43 expression levels. The "classical" MS and the "Matutes score-extended" (MS-e) were categorized as high (4-5/6-7), intermediate (2-3/4-5), and low (0-1/0-3). Samples were reclassified into the MS-e with focus on ambiguous cases with an intermediate "classical" MS., Results: A total of 35 of 138 (25.4%) patient samples were assigned to the intermediate MS group and confirmed by histopathological reports as CLL (14/40.0%) and B-NHLs other than CLL (21/60%). MS-e analysis identified 13 of 14 (92.9%) of CLL cases (MS-e 4-5) and 18/21 (85.7%) non-CLL cases (MS-e ≤ 3) correctly. Overall, the sensitivity of the CLL diagnosis was significantly increased by application of MS-e compared to the "classical" MS (98.8% vs. 82.7%; p = 0.0009), while specificity of both methods was almost equal (94.7% vs. 98.3%; p = 0.4795). Of note, sole measurement of CD43 and CD200 on B-cells sufficiently differentiated CLL from non-CLL with a test accuracy superior to the "classical" MS (F1 score 96.2 vs. 93.6)., Conclusion: CD200 and CD43 have a high informative value in diagnostic immunophenotyping and facilitate the separation of CLL from other B-NHLs particularly in ambiguous cases., (© 2020 International Clinical Cytometry Society.)

Diabetic foot ulcers are a major health care concern with limited effective therapies. Mesenchymal stem cell (MSC)-based therapies are promising treatment options due to their beneficial effects of immunomodulation, angiogenesis, and other paracrine effects. We investigated whether a bioengineered scaffold device containing hypoxia-preconditioned, allogeneic human MSCs combined with the beta-adrenergic antagonist timolol could improve impaired wound healing in diabetic mice. Different iterations were tested to optimize the primary wound outcome, which was percent of wound epithelialization. MSC preconditioned in 1 μM timolol at 1% oxygen (hypoxia) seeded at a density of 2.5 × 10 5  cells/cm 2 on Integra Matrix Wound Scaffold (MSC/T/H/S) applied to wounds and combined with daily topical timolol applications at 2.9 mM resulted in optimal wound epithelialization 65.6% (24.9% ± 13.0% with MSC/T/H/S vs 41.2% ± 20.1%, in control). Systemic absorption of timolol was below the HPLC limit of quantification, suggesting that with the 7-day treatment, accumulative steady-state timolol concentration is minimal. In the early inflammation stage of healing, the MSC/T/H/S treatment increased CCL2 expression, lowered the pro-inflammatory cytokines IL-1B and IL6 levels, decreased neutrophils by 44.8%, and shifted the macrophage ratio of M2/M1 to 1.9 in the wound, demonstrating an anti-inflammatory benefit. Importantly, expression of the endothelial marker CD31 was increased by 2.5-fold with this treatment. Overall, the combination device successfully improved wound healing and reduced the wound inflammatory response in the diabetic mouse model, suggesting that it could be translated to a therapy for patients with diabetic chronic wounds., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Introduction: There are no established criteria in selecting pleural effusion (PE) specimens for flow cytometric analysis (FCA). FCA on effusion specimens may be ordered by a clinician or a cytopathologist. In an effort to improve lab test utilization, this retrospective study aims to identify characteristics of PE specimens on which the addition of FCA has high diagnostic yield., Materials and Methods: We identified consecutive cases of PE cytology specimens on which FCA was performed over a 5-year period (2014-2019). Patient demographic data and history, FCA diagnosis, cytologic diagnosis, cellular quantity and composition, and peripheral blood cell counts were collected. Chi-square, Mann-Whitney U, and t tests were used when appropriate with a significance level of P < 0.05., Results: We identified 164 FCA cases corresponding to 142 patients (age: 19-90 years; male:female 2:1). The majority of cases had no abnormality by cytologic examination, whereas others were obviously malignant due to non-hematologic malignancy. Most (119 of 164, 73%) had negative immunophenotypic studies by FCA. Forty-five of 164 (27%) FCA cases were positive for a monoclonal myeloid or lymphoid population. Clinicopathologic features associated with positive FCA results included a history of hematologic malignancy, peripheral blood lymphocytes of ≥20%, the presence of a monomorphic lymphoid population, large atypical cells, and mitoses., Conclusions: This study identifies features that are associated with positive FCA in PE cytology specimens. Using these features by cytopathologists to order FCA on PE specimens as a reflex test would significantly reduce unnecessary testing and improve FCA utilization., (Copyright © 2020 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Background: Immune response against cancer has prognostic impact but its role in gastric cancer is poorly known. The aim of the study was to assess the prognostic significance of immune cell score (CD3+, CD8+), tumour immune escape (PD-L1, PD-1) and immune tolerance (Clever-1)., Methods: After exclusion of Epstein-Barr virus positive (n = 4) and microsatellite instable (n = 6) tumours, the study included 122 patients with GC undergoing D2 gastrectomy. CD3+ and CD8+ based ICS, PD-L1, PD-1 and Clever-1 expressions were evaluated. Differences in survival were examined using Cox regression adjusted for confounders. The primary outcome was 5-year survival., Results: The 5-year overall survival rate was 43.4%. High ICS was associated with improved overall survival (adjusted HR 0.48 (95% CI 0.26-0.87)) compared to low ICS. In the high ICS group, patients with PD-L1 expression (5-year survival 69.2 vs. 53.1%, p = 0.317), high PD-1 (5-year survival 70.6 vs. 55.3% p = 0.312) and high Clever-1 (5-year survival 72.0% vs. 45.5% (p = 0.070) had poor prognosis., Conclusions: High ICS was associated with improved survival. In the high ICS group, patients with high PD-L1, PD-1 and Clever-1 had poor prognosis highlighting the importance of immune escape and immune tolerance in GC.

HIV type 1 is associated with pulmonary dysfunction that is exacerbated by cigarette smoke. Alveolar macrophages (AM) are the most prominent immune cell in the alveolar space. These cells play an important role in clearing inhaled pathogens and regulating the inflammatory environment; however, how HIV infection impacts AM phenotype and function is not well understood, in part because of their autofluorescence and the absence of well-defined surface markers. The main aim of this study was to evaluate the impact of HIV infection on human AM and to compare the effect of smoking on their phenotype and function. Time-of-flight mass cytometry and RNA sequencing were used to characterize macrophages from human bronchoalveolar lavage of HIV-infected and -uninfected smokers and nonsmokers. We found that the frequency of CD163 + anti-inflammatory AM was decreased, whereas CD163 - CCR7 + proinflammatory AM were increased in HIV infection. HIV-mediated proinflammatory polarization was associated with increased levels of inflammatory cytokines and macrophage activation. Conversely, smoking heightened the inflammatory response evident by change in the expression of CXCR4 and TLR4. Altogether, these findings suggest that HIV infection, along with cigarette smoke, favors a proinflammatory macrophage phenotype associated with enhanced expression of inflammatory molecules. Further, this study highlights time-of-flight mass cytometry as a reliable method for immunophenotyping the highly autofluorescent cells present in the bronchoalveolar lavage of cigarette smokers., (Copyright © 2020 by The American Association of Immunologists, Inc.)

SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients reveals a dramatic increase in the number of immature neutrophils. This increase strongly correlates with disease severity and is associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts is observed for CD8 T-cells and VD2 γδ T-cells, which both exhibit increased differentiation and activation. ROC analysis reveals that the count ratio of immature neutrophils to VD2 (or CD8) T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.

The antitumor activity of NK cells in patients with chronic myeloid leukemia (CML) is inhibited by the leukemia microenvironment. Recent studies have identified that the expression of TIGIT, CD57, and KLRG1 is related to the function, maturation, and antitumor capabilities of NK cells. However, the characteristics of the expression of these genes in the peripheral blood (PB) and bone marrow (BM) from patients with CML remain unknown. In this study, we used multicolor flow cytometry to assay the quantity and phenotypic changes of NK cells in PB and BM from de novo CML (DN-CML) and CML patients acquiring molecular response (MR-CML). We found that the expression of TIGIT, which inhibits NK cell function, is increased on CD56 + and CD56 dim NK cells in DN-CML PB compared with those in healthy individuals (HIs), and it is restored to normal in patients who achieve MR. We also found that the expression of CD57 on NK cells was approximately the same level in PB and BM from DN-CML patients, while decreased CD57 expression was found on CD56 + and CD56 dim NK cells in HI BM compared with PB. Additionally, those two subsets were significantly increased in DN-CML BM compared to HI BM. The expression of CD57 correlates with replicative senescence and maturity for human NK cells; therefore, the increase in TIGIT on PB NK cells together with an increase in CD57 on BM NK cells may explain the subdued NK cell antileukemia capacity and proliferative ability in DN-CML patients. These results indicate that reversing the immune suppression of PB NK cells by blocking TIGIT while improving the proliferation of BM NK cells via targeting CD57 may be more effective in removing tumor cells., Competing Interests: The authors declare that they have no conflict of interests., (Copyright © 2020 Danlin Yao et al.)

Bone marrow fibrosis (BMF) is a rare complication in acute leukemia. In pediatrics, it predominantly occurs in acute megakaryoblastic leukemia (AMKL) and especially in patients with trisomy 21, called myeloid leukemia in Down syndrome (ML-DS). Defects in mesenchymal stromal cells (MSC) and cytokines specifically released by the myeloid blasts are thought to be the main drivers of fibrosis in the bone marrow niche (BMN). To model the BMN of pediatric patients with AMKL in mice, we first established MSCs from pediatric patients with AMKL ( n = 5) and ML-DS ( n = 9). Healthy donor control MSCs ( n = 6) were generated from unaffected children and adolescents ≤18 years of age. Steady-state analyses of the MSCs revealed that patient-derived MSCs exhibited decreased adipogenic differentiation potential and enrichment of proliferation-associated genes. Importantly, TGFB1 exposure in vitro promoted early profibrotic changes in all three MSC entities. To study BMF induction for longer periods of time, we created an in vivo humanized artificial BMN subcutaneously in immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice, using a mixture of MSCs, human umbilical vein endothelial cell, and Matrigel. Injection of AMKL blasts as producers of TGFB1 into this BMN after 8 weeks induced fibrosis grade I/II in a dose-dependent fashion over a time period of 4 weeks. Thus, our study developed a humanized mouse model that will be instrumental to specifically examine leukemogenesis and therapeutic targets for AMKL blasts in future. IMPLICATIONS: TGFB1 supports fibrosis induction in a pediatric AMKL model generated with patient-derived MSCs. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/10/1603/F1.large.jpg., (©2020 American Association for Cancer Research.)

Background: Anaplastic thyroid carcinoma (ATC) is nearly always fatal. Large studies on ATC are exceedingly rare. We aimed to study the clinical, genotypic, and histologic characteristics of ATC in the largest retrospective cohort of ATC to date. Methods: Three hundred sixty patients with ATC from two tertiary centers were studied. Molecular testing was performed in 126 cases including 107 using next-generation sequencing. Results: The median patients' age was 68 years. Differentiated thyroid carcinoma (DTC) was present in 208 cases (58%), the most common being papillary carcinoma ( n  = 150). The 1-, 2-, 3-, and 5-year overall survival (OS) was 36%, 17%, 13%, and 11%, respectively. On univariate analysis, age, resectability, chemotherapy, radiotherapy, margin status, encapsulation, gross residual disease, gross extrathyroidal extension, percentage, and size of ATC in the primary tumor predicted OS ( p  < 0.05). Age, resectability, chemotherapy, and gross residual disease were independent prognostic factors in the entire cohort, while gross residual disease was the only independent predictor of OS in patients who had resection of their tumor. BRAF , RAS , TERT promoter, TP53 , PIK3CA , E1F1AX , and PTEN mutations were detected in 45%, 24%, 75%, 63%, 18%, 14%, and 14% of ATC, respectively. Concomitant BRAF/RAS and TERT mutations were associated with worse outcome than mutation in only one of the genes. BRAF- mutated and RAS -mutated ATCs had similar frequency of nodal and distant metastasis. Twelve cases were pure squamous cell carcinoma, 60% of which carried BRAF V600E mutation and showed a similar OS to other ATCs. Conclusions: (i) Gross residual disease remains the most crucial indicator of outcome in ATC. (ii) Encapsulation, margin status, percentage, and size of ATC in the primary were prognostically relevant. (iii) Pure thyroid squamous cell carcinoma may be considered as ATC given a BRAF V600E genotype and similar outcome. (iv) In contrast to DTC, BRAF -mutated and RAS -mutated ATCs have similar metastatic spread. (v) Concomitant mutations of BRAF or RAS with TERT confer a worse prognosis.

Background: Although immune checkpoint blockade is effective for several malignancies, a substantial number of patients remain refractory to treatment. The future of immunotherapy will be a personalized approach adapted to each patient's cancer-immune interactions in the tumor microenvironment (TME) to prevent suppression of antitumor immune responses. To demonstrate the feasibility of this kind of approach, we developed combination therapy for a preclinical model guided by deep immunophenotyping of the TME., Methods: Gastric cancer cell lines YTN2 and YTN16 were subcutaneously inoculated into C57BL/6 mice. YTN2 spontaneously regresses, while YTN16 grows progressively. Bulk RNA-Seq, single-cell RNA-Seq (scRNA-Seq) and flow cytometry were performed to investigate the immunological differences in the TME of these tumors., Results: Bulk RNA-Seq demonstrated that YTN16 tumor cells produced CCL20 and that CD8 + T cell responses were impaired in these tumors relative to YTN2. We have developed a vertical flow array chip (VFAC) for targeted scRNA-Seq to identify unique subtypes of T cells by employing a panel of genes reflecting T cell phenotypes and functions. CD8 + T cell dysfunction (cytotoxicity, proliferation and the recruitment of interleukin-17 (IL-17)-producing cells into YTN16 tumors) was identified by targeted scRNA-Seq. The presence of IL-17-producing T cells in YTN16 tumors was confirmed by flow cytometry, which also revealed neutrophil infiltration. IL-17 blockade suppressed YTN16 tumor growth, while tumors were rejected by the combination of anti-IL-17 and anti-PD-1 (Programmed cell death protein 1) mAb treatment. Reduced neutrophil activation and enhanced expansion of neoantigen-specific CD8 + T cells were observed in tumors of the mice receiving the combination therapy., Conclusions: Deep phenotyping of YTN16 tumors identified a sequence of events on the axis CCL20->IL-17-producing cells->IL-17-neutrophil-angiogenesis->suppression of neoantigen-specific CD8 + T cells which was responsible for the lack of tumor rejection. IL-17 blockade together with anti-PD-1 mAb therapy eradicated these YTN16 tumors. Thus, the deep immunological phenotyping can guide immunotherapy for the tailored treatment of each individual patient's tumor., Competing Interests: Competing interests: KK reports grants from TAKARA BIO, grants from MSD, outside the submitted work. The Department of Immunotherapeutics, University of Tokyo Hospital, is endowed by TAKARA BIO., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: There are no immunophenotypic guidelines for the investigation of MYC-rearranged lymphomas. We aimed to identify simple immunophenotypic features that would help to differentiate between MYC-rearranged lymphomas and guide cytogenetic analysis., Methods: We reviewed diagnostic samples from patients diagnosed with Burkitt lymphoma (BL), double-hit lymphoma (DHL), MYC-rearranged diffuse large B-cell lymphoma (MYC-DLBCL), and standard (non-MYC-rearranged) DLBCL over the last decade in our Institution. Using flow cytometry (with antibodies CD20, CD10, CD38, bcl-2, Ki-67, FMC-7, CD43, CD27, CD79b, CD23, and CD22) we determined antigen% expression and median-fluorescence intensity ratios (MFIR). The forward scatter (FS) and side scatter (SS) characteristics of tumor B-cells were compared with normal T-cells (B/T ratios) for patients with MYC-rearranged lymphomas., Results: We identified 51 patients of whom 14 had BL, 10 had DHL (6 MYC+/BCL2+; 4 MYC+/BCL6+), 8 MYC-DLBCL, and 19 standard DLBCL. The significant differences (p <.05) were: higher CD38% in BL than standard DLBCL; higher CD10% in BL and DHL versus MYC-DLBCL and standard DLBCL; higher CD10MFIR in BL than MYC-DLBCL and standard DLBCL; higher Ki-67% in BL than DHL and MYC-DLBCL; higher bcl-2% in DHL than BL; higher FMC-7% in BL than MYC-DLBCL and standard DLBCL; and lower SS (B/T) ratio in DHL than MYC-DLBCL., Conclusions: The combination of CD38% > 90, CD10% > 80, CD10MFIR > 10, bcl-2% < 30, and Ki-67% > 70 was characteristic of BL. "Deviation" from these cut-offs should raise suspicion for DHL and, therefore, BCL2 and/or BCL6 FISH is required. We also found that a diagnosis of DHL rather than of MYC-DLBCL was significantly associated with CD10% > 60, Ki-67% > 50, and SS (B/T) <1.5., (© 2020 International Clinical Cytometry Society.)

Background: The role of CD200 in the differential diagnosis of chronic lymphocytic leukemia (CLL) and classical mantle cell lymphoma (MCL) is well established. Its role in the differential diagnosis of CLL and other lymphoproliferative disorders (LPD) is less clear, in particular its positive predictive value (PPV)., Materials and Methods: We conducted a systematic review of the use of CD200 in the differential diagnosis of CLL, MCL, and other predominantly leukemic, typically CD103-negative LPD. With the results, we then derived a curve to determine the PPV based on the prevalence of the disorders included in the differential diagnosis., Results: Of 43 publications screened, 27 were included in the systematic review (5,764 patients). The median CD200 positivity rate in all studies and the percentage of CD200-positive (pooled) patients was 100% and 95% (3,061/3,208) in CLL, 4 and 8% (86/1112) in MCL and 56 and 62% (425/689) in other LPD., Conclusion: CD200 is suboptimal for the differential diagnosis of CLL and disorders other than nodal MCL., (© 2019 International Clinical Cytometry Society.)

Background and Aims: Intrahepatic cholangiocarcinoma (ICC) is a severe malignant tumor in which the standard therapies are mostly ineffective. The biological significance of the desmoplastic tumor microenvironment (TME) of ICC has been stressed but was insufficiently taken into account in the search for classifications of ICC adapted to clinical trial design. We investigated the heterogeneous tumor stroma composition and built a TME-based classification of ICC tumors that detects potentially targetable ICC subtypes., Approach and Results: We established the bulk gene expression profiles of 78 ICCs. Epithelial and stromal compartments of 23 ICCs were laser microdissected. We quantified 14 gene expression signatures of the TME and those of 3 functional indicators (liver activity, inflammation, immune resistance). The cell population abundances were quantified using the microenvironment cell population-counter package and compared with immunohistochemistry. We performed an unsupervised TME-based classification of 198 ICCs (training set) and 368 ICCs (validation set). We determined immune response and signaling features of the different immune subtypes by functional annotations. We showed that a set of 198 ICCs could be classified into 4 TME-based subtypes related to distinct immune escape mechanisms and patient outcomes. The validity of these immune subtypes was confirmed over an independent set of 368 ICCs and by immunohistochemical analysis of 64 ICC tissue samples. About 45% of ICCs displayed an immune desert phenotype. The other subtypes differed in nature (lymphoid, myeloid, mesenchymal) and abundance of tumor-infiltrating cells. The inflamed subtype (11%) presented a massive T lymphocyte infiltration, an activation of inflammatory and immune checkpoint pathways, and was associated with the longest patient survival., Conclusion: We showed the existence of an inflamed ICC subtype, which is potentially treatable with checkpoint blockade immunotherapy., (© 2020 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Our understanding of programmed cell death 1 (PD-1) biology is limited due to technical difficulties in establishing reproducible, yet simple, in vitro assays to study PD-1 signaling in primary human T cells. The protocols in this article were refined to test the consequences of PD-1 ligation on short-term T cell signaling, long-term T cell function, and the structural consequences of PD-1 ligation with PD-1 ligands. Basic Protocol 1 addresses the need for a robust and reproducible short-term assay to examine the signaling cascade triggered by PD-1. We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr 142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Protocol 4 we outline a tetramer-based method useful to interrogate the quality of PD-1/PD-L interactions. These protocols can be easily adapted for mouse studies and other inhibitory receptors. They provide a valuable resource to investigate PD-1 signaling in T cells and the functional consequences of various PD-1-based therapeutics on T cell responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: PD-1 crosslinking assay to determine CD3ζ phosphorylation in primary human T cells Basic Protocol 2: Plate-based ligand binding assay to study PD-1 function in human T cells Support Protocol 1: T cell proliferation assay in the presence of PD-1 ligation Basic Protocol 3: In vitro APC/T cell co-culture system to evaluate therapeutic interventions targeting the PD-1/PD-L1 axis Support Protocol 2: Microscopy-based approach to evaluate the consequences of PD-1 ligation on immune synapse formation Basic Protocol 4: Tetramer-based approach to study PD-1/PD-L1 interactions., (© 2020 Wiley Periodicals LLC.)

Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90 + SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90 + podoplanin + CD105 + CD146 + CD271 + VCAM-1 + ICAM-1 + α-SMA + ) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90 + CD34 + CD105 + CD271 + ) represents a novel population of CD34 + SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90 + CD146 + CD36 + NG2 - pericytes in the walls of high endothelial venules and other small vessels, and CD90 + CD146 + NG2 + CD36 - pericytes in the walls of larger vessels. Distinguishing between these CD90 + SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes., (©2020 American Association for Cancer Research.)

Background & Aims: Studies are needed to determine the mechanisms of mucosal dysregulation in patients with inflammatory bowel diseases (IBDs) and differences in inflammatory responses of patients with ulcerative colitis (UC) vs Crohn's disease (CD). We used mass cytometry (CyTOF) to characterize and compare immune cell populations in the mucosa and blood from patients with IBD and without IBD (controls) at single-cell resolution., Methods: We performed CyTOF analysis of colonic mucosa samples (n = 87) and peripheral blood mononuclear cells (n = 85) from patients with active or inactive UC or CD and controls. We also performed single-cell RNA sequencing, flow cytometry, and RNA in situ hybridization analyses to validate key findings. We used random forest modeling to identify differences in signatures across subject groups., Results: Compared with controls, colonic mucosa samples from patients with IBD had increased abundances of HLA-DR+CD38+ T cells, including T-regulatory cells that produce inflammatory cytokines; CXCR3+ plasmablasts; and IL1B+ macrophages and monocytes. Colonic mucosa samples from patients with UC were characterized by expansion of IL17A+ CD161+ effector memory T cells and IL17A+ T-regulatory cells; expansion of HLA-DR+CD56+ granulocytes; and reductions in type 3 innate lymphoid cells. Mucosal samples from patients with active CD were characterized by IL1B+HLA-DR+CD38+ T cells, IL1B+TNF+IFNG + naïve B cells, IL1B+ dendritic cells (DCs), and IL1B+ plasmacytoid DCs. Peripheral blood mononuclear cells from patients with active CD differed from those of active UC in that the peripheral blood mononuclear cells from patients with CD had increased IL1B+ T-regulatory cells, IL1B+ DCs and IL1B+ plasmacytoid DCs, IL1B+ monocytes, and fewer group 1 innate lymphoid cells. Random forest modeling differentiated active UC from active CD in colonic mucosa and blood samples; top discriminating features included many of the cellular populations identified above., Conclusions: We used single-cell technologies to identify immune cell populations specific to mucosa and blood samples from patients with active or inactive CD and UC and controls. This information might be used to develop therapies that target specific cell populations in patients with different types of IBD., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)