Unlocking the secrets of Hofbauer cells in placental (patho-) physiology: Isolation and quality assessment in human term placenta.

INTRODUCTION: Hofbauer cells (HBCs) are macrophages of fetal origin that reside in the villous tissue. They are the only immune cells within healthy villi. While HBCs perform innate immune functions such as phagocytosis and antigen presentation, they are increasingly recognized for their diverse roles in placental physiology e.g. vascular functionality, tissue homeostasis, tolerance. Consequently, HBCs are of utmost interest in a variety of non-physiological placental conditions. ISOLATION: Villous tissue is collected freshly after delivery and finely minced. The resulting tissue is digested in a two-step process, using Trypsin/DNase to separate cytotrophoblasts and collagenase/DNase to penetrate deeper into the villous stroma, containing HBCs, and obtain a single cell suspension. After a density gradient centrifugation, the corresponding cell layer is collected and subjected to negative immune selection of HBCs, yielding unaffected cells that have not been activated during the isolation process. QUALITY CONTROL: In addition to a classical immunocytochemistry (ICC) approach including macrophage markers, and markers for potentially contaminating cell types (e.g. fibroblasts, muscle, mesenchymal cells), we have developed a multi-color flow cytometry (FC) panel. This panel assesses Hofbauer cell purity and polarization states more accurately and comprehensively than qualitative ICC, using percentage analysis of parent cells to estimate the expression levels of specific markers. DISCUSSION: The presented protocol allows us to isolate HBCs in significant numbers and high purity, even from placentae compromised by preeclampsia (PE) with limited placental volume. We have successfully developed and implemented this protocol to study healthy, diabetic and PE macrophages, aiding a better understanding of the underlying placental pathophysiology at the cellular level.

Efficient enzyme-free method to assess the development and maturation of the innate and adaptive immune systems in the mouse colon.

Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.

A comprehensive analysis of the immune system in healthy Vietnamese people.

Lifestyle, diet, socioeconomic status and genetics all contribute to heterogeneity in immune responses. Vietnam is plagued with a variety of health problems, but there are no available data on immune system values in the Vietnamese population. This study aimed to establish reference intervals for immune cell parameters specific to the healthy Vietnamese population by utilizing multi-color flow cytometry (MCFC). We provide a comprehensive analysis of total leukocyte count, quantitative and qualitative shifts within lymphocyte subsets, serum and cytokine and chemokine levels and functional attributes of key immune cells including B cells, T cells, natural killer (NK) cells and their respective subpopulations. By establishing these reference values for the Vietnamese population, these data contribute significantly to our understanding of the human immune system variations across diverse populations. These data will be of substantial comparative value and be instrumental in developing personalized medical approaches and optimizing diagnostic strategies for individuals based on their unique immune profiles.

Case Report: Balancing immune responses - multiple sclerosis disease exacerbation under BRAF/MEK treatment for malignant melanoma.

Background: Combination treatment with BRAF/MEK inhibitors favorably impact progression-free survival in malignant melanoma. However, it may cause paradoxical activation of the MAPK/ERK pathway in immune cells without BRAF mutation, which may lead to over activation of the immune system, especially in patients with pre-existing autoimmune conditions. In this case report, treatment of malignant melanoma with BRAF/MEK inhibitors was associated with radiological disease exacerbation of pre-existing multiple sclerosis (MS). Case presentation: A 47-year-old patient with pre-existing MS was diagnosed with malignant melanoma in June 2020. Anti-tumor treatment was initiated with a combination therapy of BRAF inhibitor dabrafenib and MEK inhibitor trametinib. In February 2022, the patient presented at our neurological clinic after routine MRI revealed exacerbation of radiological MS disease activity with ten new and gadolinium-enhancing lesions, and concomitant high levels of neurofilament light chain (NfL) in serum, a marker for axonal damage. In-depth analysis of immune cells in both peripheral blood and cerebrospinal fluid was performed by multi-color flow cytometry. After treatment with the B cell-depleting antibody ocrelizumab, MS disease stability was obtained and anti-tumor medication could be continued. Conclusions: Immunomodulatory treatment in cancer patients is highly effective from an oncological point of view, but may be associated with autoimmune side effects. This is of special importance in patients with pre-existing autoimmune diseases, as reflected by our case of MS disease reactivation under treatment with BRAF/MEK inhibitors. In our case, sequential modulation of immune cell subsets by B cell depletion, associated with marked shifts in B and T cell subsets, allowed for stabilization of disease and continuation of anti-tumor treatment.

Using the power of innate immunoprofiling to understand vaccine design, infection, and immunity.

In the field of immunology, a systems biology approach is crucial to understanding the immune response to infection and vaccination considering the complex interplay between genetic, epigenetic, and environmental factors. Significant progress has been made in understanding the innate immune response, including cell players and critical signaling pathways, but many questions remain unanswered, including how the innate immune response dictates host/pathogen responses and responses to vaccines. To complicate things further, it is becoming increasingly clear that the innate immune response is not a linear pathway but is formed from complex networks and interactions. To further our understanding of the crosstalk and complexities, systems-level analyses and expanded experimental technologies are now needed. In this review, we discuss the most recent immunoprofiling techniques and discuss systems approaches to studying the global innate immune landscape which will inform on the development of personalized medicine and innovative vaccine strategies.

Validation of a 12-color flow cytometry assay for acute myeloid leukemia minimal/measurable residual disease detection.

BACKGROUND: Acute myeloid leukemia (AML) minimal/measurable residual disease (MRD) by multicolor flow cytometry is a complex laboratory developed test (LDT), challenging for implementation. We share our experience in the validation of a 12-color AML MRD flow cytometry assay to meet stringent regulatory requirements. METHODS: We worked under the guidelines of the CLSI HL62 publication, illustrated the details of the validation process that was tailored to uniqueness of AML MRD, and tested its clinical validity in 61 patients. The "trueness" was determined by correlating with concurrent molecular genetic testing and follow-up bone marrow examinations. RESULTS: Under assay specificity, we shared the details of panel design, analysis, and criteria for interpretation and reporting. The assay accuracy was assessed by testing known positive and negative samples and correlating with molecular genetic testing and follow-up bone marrow examination. The limit of detection (LOD) and limit of quantification (LOQ) were validated to a level between 0.01% and 0.1%, varied from the leukemia-associated immunophenotypes (LAIP) and the numbers of events obtained for analysis. Assay linearity, precision and carry over studies all met acceptable criteria. In the clinical validity test, the concordance was 93%, specificity 98% and sensitivity 83%. The most challenging aspects of the assay were the discrimination of pre-leukemic cells (persistent clonal hematopoiesis) or underlying myelodysplastic clones from AML MRD with immunophenotypic switch or subclone selection. CONCLUSION: The validation met all criteria and obtained FDA IDE (investigational device exemption) approval. This study provides ample technical and professional details in setting up the AML MRD flow cytometry assay and illustrates through the example of the "fit for purpose" validation process. We also highlight the need for further characterization of abnormal blasts bearing the potential for AML relapse.

Validation of multiparametric panels for bovine mesenchymal stromal cell phenotyping.

Bovine mesenchymal stromal cells (MSCs) display important features that render them valuable for cell therapy and tissue engineering strategies, such as self-renewal, multi-lineage differentiation, as well as immunomodulatory properties. These cells are also promising candidates to produce cultured meat. For all these applications, it is imperative to unequivocally identify this cell population. The isolation and in vitro tri-lineage differentiation of bovine MSCs is already described, but data on their immunophenotypic characterization is not yet complete. The currently limited availability of monoclonal antibodies (mAbs) specific for bovine MSC markers strongly hampers this research. Following the minimal criteria defined for human MSCs, bovine MSCs should express CD73, CD90, and CD105 and lack expression of CD14 or CD11b, CD34, CD45, CD79α, or CD19, and MHC-II. Additional surface proteins which have been reported to be expressed include CD29, CD44, and CD106. In this study, we aimed to immunophenotype bovine adipose tissue (AT)-derived MSCs using multi-color flow cytometry. To this end, 13 commercial Abs were screened for recognizing bovine epitopes using the appropriate positive controls. Using flow cytometry and immunofluorescence microscopy, cross-reactivity was confirmed for CD34, CD73, CD79α, and CD90. Unfortunately, none of the evaluated CD105 and CD106 Abs cross-reacted with bovine cells. Subsequently, AT-derived bovine MSCs were characterized using multi-color flow cytometry based on their expression of nine markers. Bovine MSCs clearly expressed CD29 and CD44, and lacked expression of CD14, CD45, CD73, CD79α, and MHCII, while a variable expression was observed for CD34 and CD90. In addition, the mRNA transcription level of different markers was analyzed using reverse transcription quantitative polymerase chain reaction. Using these panels, bovine MSCs can be properly immunophenotyped which allows a better characterization of this heterogenous cell population.

A comprehensive multiparameter flow cytometry panel for immune profiling and functional studies of frozen tissue, bone marrow, and spleen.

Flow cytometry (FC) is a highly informative technology that can provide valuable information about immune phenotype monitoring and immune cell states. However, there is a paucity of comprehensive panels developed and validated for use on frozen samples. Here, we developed a 17-plex flow cytometry panel to detect subtypes, frequencies, and functions of different immune cells that can be leveraged to study the different cellular characteristics in different disease models, physiological, and pathological conditions. This panel identifies surface markers to characterize T cells (CD8+, CD4+), natural killer (NK) cells and their subtypes (immature, cytotoxic, exhausted, activated),natural killer T (NKT) cells, neutrophils, macrophages (M1 (pro-inflammatory) and M2 (anti-inflammatory)), monocytes and their subtypes (classical and non-classical), dendritic cells (DC) and their subtypes (DC1, DC2), and eosinophils. The panel was designed to include only surface markers to avoid the necessity for fixation and permeabilization steps. This panel was optimized using cryopreserved cells. Immunophenotyping of spleen and bone marrow using the proposed panel was efficient in correctly differentiating the immune cell subtypes in inflammatory model of ligature-induced periodontitis, in which we found increased percentage of NKT cells, activated and mature/cytotoxic NK cells in the bone marrow of affected mice. This panel enables in-depth immunophenotyping of murine immune cells in bone marrow, spleen, tumors, and other non-immune tissues of mice. It could be a tool for systematic analysis of immune cell profiling in inflammatory conditions, systemic diseases, and tumor microenvironments.

Immunophenotype and function of peripheral blood lymphocyte subsets in Chinese rhesus macaques / 中华微生物学和免疫学杂志

Objective:To provide data reference for using Chinese rhesus macaques as research model by studying the immunophenotype and function of peripheral blood lymphocytes in Chinese rhesus macaques.Methods:By optimizing antibody clones and fluorescent colors, the lymphocyte subset assay and T cell function assay panels were determined. Then the panels were used to analyze the proportion of T, B, NK and other cell subsets in peripheral blood mononuclear cells (PBMCs) in 15 healthy Chinese rhesus monkeys, and the ability of T cells to secrete cytokines after non-specific stimulation.Results:Two multi-color flow cytometry analytic panels were established. Panel 1 could simultaneously detect a variety of lymphocyte subsets, including cytotoxic T lymphocytes, follicular helper T cells, regulatory T cells, B cells and NK cells. Panel 2 could detect the functions of multiple T cell subsets and the expression of immune checkpoint moleculars. The mean percentages of T, B, NK, Tfh, Treg, CD16 + NK and CD56 + NK cells in PBMCs of the Chinese rhesus macaques were (75.32±7.73)%, (13.22±7.50)%, (0.88±0.48)%, (0.73±0.27)%, (0.75±0.43)%, (47.87±22.35)% and (10.69±12.41)%. After non-specific stimulation, the proportion of CD4 + T cells secreting IL-2 and TNF-α was higher than that of CD8 + T cells, and the proportion of CD8 + T cells secreting CD107a and IFN-γ was higher than that of CD4 + T cells, while the proportion of CD4 + and CD8 + T cells secreting IL-17A was low. Conclusions:This study established a multi-color flow detection scheme that could simultaneously detect multiple cellular surface molecules and cytokines at the single cell level and could accurately and comprehensively analyze the immune cell subsets, functions and the immune checkpoint molecules in peripheral blood of Chinese rhesus macaques, providing a new experimental method and basic data for the development of vaccines and drugs against infectious diseases.

OMIP 078: A 31-parameter panel for comprehensive immunophenotyping of multiple immune cells in human peripheral blood mononuclear cells.

This 31-parameter panel was developed for simultaneously measuring multiple immune cell populations including T cells, B cells, natural killer cells, dendritic cells, monocytes, and hematopoietic progenitor cells in human peripheral blood mononuclear cells. This panel enables the capture of individual immune dynamics and assessments of single-cell changes in the immune system that are associated with aging and diseases. This panel includes markers to separate the differentiation status of each cell population and might be applicable to studies of infectious and autoimmune diseases, as patient samples are usually limited in volume and require an analysis system that provides a relatively large amount of information.

Analysis of Radiation-Induced Changes in Cell Cycle and DNA Damage of Murine Hematopoietic Stem Cells by Multi-Color Flow Cytometry.

Exposure of bone marrow to genotoxic stress such as ionizing radiation (IR) results in a rapid decline of peripheral blood cells and stimulates entry of the normally quiescent hematopoietic stem cells (HSCs) into the cell cycle to reconstitute the hematopoietic system. While several protocols have employed flow cytometry analysis of bone marrow cells to study changes in specific cell populations with respect to cell cycle proliferation and/or expression of γ-H2AX, a marker of DNA damage, these parameters were examined in separate panels. Here, we describe a flow cytometry-based method specifically designed to examine cell cycle distribution using Ki-67 and FXCycle violet in combination with γ-H2AX in HSCs and hematopoietic progenitor cells (HPCs) within the same sample. This method is very useful, particularly in studies involving genotoxic stresses such as IR, which substantially reduce the absolute numbers of HSCs and HPCs available for staining. Additionally, we describe several important considerations for the analysis of markers of HSCs in irradiated versus unirradiated samples. Examples include the use of fluorescence minus one (FMO) controls, the gating strategy for markers whose expression is typically impacted by IR such as Sca1, tips for staining of intracellular antigens like Ki67, and ensuring the detection of signal from at least 500 events in each gate to ensure robustness of the results. © 2021 Wiley Periodicals LLC. Basic Protocol: Immunostaining protocol for bone marrow mononuclear cells using a multi-fluorophore panel.

Immunophenotypic analysis of cerebrospinal fluid reveals concurrent development of ATL in the CNS of a HAM/TSP patient.

Both adult T-cell leukemia/lymphoma (ATL) and human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) can be induced by HTLV-1, but concurrent development has been rarely reported. We present the case of a 55-year-old female who developed cranial nerve symptoms after a 20-year history of HAM/TSP. Although multiple white matter lesions were observed on brain magnetic resonance imaging, no abnormalities were seen on a systemic computed tomography scan. Quantitative flow-cytometric analysis of cell populations in the cerebrospinal fluid (CSF) revealed that most of the infiltrating cells were not inflammatory cells, but HTLV-1-infected CD4+ CADM-1+ T-cells completely lacking CD7 expression. As stepwise downregulation of CD7 is correlated with disease progression from HTLV-1 carrier to aggressive ATL, the CSF cells were classified as aggressive ATL; these cells exhibited a more progressed phenotype than those in peripheral blood (PB). HAM/TSP disease activity was estimated to be low. From these and other examinations, we made a diagnosis of acute-type ATL, which unusually developed in the central nervous system at initial onset prior to systemic progression. In ATL cases with a challenging diagnosis, immunophenotypic characterization of CSF and PB is valuable for differential diagnosis and understanding disease status.

Methods for High-Dimensional Flow Cytometry Analysis of Human MAIT Cells in Tissues and Peripheral Blood.

Mucosal-associated invariant T (MAIT) cells can be found throughout the human body, in peripheral blood, at mucosal sites, and, among other organs, in the liver. As unconventional T cells, MAIT cells have the capacity to readily respond to bacterial infections and are also engaged during anti-viral responses. To thoroughly investigate the MAIT cell phenotype and function in such conditions, multi-color flow cytometry is an appropriate and powerful tool. Yet, the recent rapid technological development within this methodology, with generation of highly complex data, has increased the need for downstream dimensionality reducing methods to fully interpret obtained results. Among such methods, stochastic neighbor embedding (SNE) analysis stands out as it provides intuitive low-dimensional representations of complex data. Here, we describe techniques and workflow for high-dimensional state-of-the-art investigation and analysis of human MAIT cells from blood and peripheral tissues.

Quantification of Human MAIT Cell-Mediated Cellular Cytotoxicity and Antimicrobial Activity.

The mucosa-associated invariant T (MAIT) cells represent the most abundant population of antimicrobial T cells in humans. When encountering cells infected with riboflavin-producing bacteria, this innate-like T cell population rapidly release a plethora of pro-inflammatory cytokines, mediates antimicrobial activity, and kill infected cells. Here, we describe methodological approaches and protocols to measure their cytotoxicity and antimicrobial effector function using multi-color flow cytometry-based and standard microbiological techniques. We provide specific guidance on protocols and describe potential pitfalls for each of the presented methodologies. Finally, we discuss potential applications and current limitations of our approaches to the study of human MAIT cell antimicrobial properties.

Detection of Immune Cell Subsets of Peripheral Blood in Hepatocellular Carcinoma before Liver Transplantation / 中山大学学报(医学科学版)

[Objective]To investigate the characteristics of immunophenotypes in hepatocellular carcinoma(HCC) before liver transplantation.[Methods]The immunophenotypes of T-,B- cells,monocytes,dendritic cells(DC)and NK-cells in peripheral blood from 6 HCC patient who were ready to have liver transplantation and 6 healthy volunteers were analyzed by multicolor flow cytometry.[Results]In the patients,the proportions of CD4+PD-1+T cells,Treg cell (CD4+CD25+CD39+T cells),CD19+B cells,Plasmablasts(CD27highCD38highIgD-IgM-),classical monocytes(CD14high CD16-)and mature NK-cells(CD3-CD56high)were all higher than those in the healthy controls(all P<0.05).However, marginal zone B cell(CD27+IgD+),Non-switched B cells(CD27+CD38dimIgM+),intermediate monocytes(CD14high CD16+)and immature NK-cells(CD3-CD56+)were lower than those in the healthy controls(all P<0.05). And there wasn't any obvious difference in quantity being observed among other cell types.[Conclusion]There was difference in the immunophenotypes of immune cells in peripheral blood between HCC patients before liver transplantation and healthy people.And this finding exerts important effects on monitoring the immune status of the patients after liver transplantation and guiding the administrations of immunosuppressors.

Enhancing acute myeloid leukemia therapy - monitoring response using residual disease testing as a guide to therapeutic decision-making.

INTRODUCTION: Current standards for monitoring the response of acute myeloid leukemia (AML) are based on morphologic assessments of the bone marrow and recovery of peripheral blood counts. A growing experience is being developed to enhance the detection of small amounts of AML, or minimal residual disease (MRD). Areas covered: Available techniques include multi-color flow cytometry (MFC) of leukemia associated immunophenotypes (LAIP), quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) for detecting fusion and mutated genes (RUNX1-RUNX1T1, CBFB-MYH11, and NPM1), overexpression of genes such as WT1, and next generation sequencing (NGS) for MRD. Expert commentary: While MRD monitoring is standard of care in some leukemia subsets such as acute promyelocytic leukemia, this approach for the broader AML population does not universally predict outcomes as some patients may experience relapse in the setting of undetectable leukemia while others show no obvious disease progression despite MRD positivity. However, there are instances where MRD can identify patients at increased risk for relapse that may change recommended therapy. Currently, prospective investigations to define clinically relevant MRD thresholds are ongoing. Risk-adapted trials are needed to best define the use of MRD in the follow up of AML patients after initial induction therapy.

Multiparameter flow cytometry: advances in high resolution analysis.

Over the past 40 years, flow cytometry has emerged as a leading, application-rich technology that supports high-resolution characterization of individual cells which function in complex cellular networks such as the immune system. This brief overview highlights advances in multiparameter flow cytometric technologies and reagent applications for characterization and functional analysis of cells modulating the immune network. These advances significantly support high-throughput and high-content analyses and enable an integrated understanding of the cellular and molecular interactions that underlie complex biological systems.

Multiparameter Flow Cytometry: Advances in High Resolution Analysis

Over the past 40 years, flow cytometry has emerged as a leading, application-rich technology that supports high-resolution characterization of individual cells which function in complex cellular networks such as the immune system. This brief overview highlights advances in multiparameter flow cytometric technologies and reagent applications for characterization and functional analysis of cells modulating the immune network. These advances significantly support high-throughput and high-content analyses and enable an integrated understanding of the cellular and molecular interactions that underlie complex biological systems.