Search

The patient we present here had many clinical, morphological, and laboratory findings characteristic of acute leukemia. During the course of the disease, the diagnosis changed from acute leukemia to chronic small B-cell lymphoproliferative disease, a blastoid variant of mantle cell lymphoma, and finally to atypical plasma cell leukemia. Atypical plasma cell leukemia is a rare condition with aggressive biological behavior. Our patient relapsed a short time after achieving complete remission, in spite of aggressive therapy and autologous stem cell transplantation. During relapse, it was possible to morphologically identify malignant cells as being of plasma cell origin, although immature and atypical. Atypical plasma cell leukemia presents a diagnostic challenge as it may mimic other neoplasms both morphologically and clinically. It is also recognized that plasma cell neoplasm immunophenotype may not be entirely specific for its lineage where common diagnostic biomarkers are applied by immunohistochemistry or flow cytometry. Where this is the case, only focused investigation for plasma cell lineage will be more informative. This patient has unusual clinical presentation, a nondescript morphology of the circulating plasma cells, as well as an immunophenotype, detected by the initial panels used for flow cytometry and immunohistochemistry, that was not entirely specific for plasma cells. Such cases present a good reminder of the diagnostic complexity of atypical plasma cell leukemia and emphasize that plasma cell differentiation needs to be interrogated in cases where the initial work-up shows unusual results.

The use of flow cytometry in mice is constrained by several factors, including the limited availability of mouse-specific antibodies and the need to work with small volumes of peripheral blood. This is particularly challenging for longitudinal studies, as serial blood samples should not exceed 10% of the total blood volume in mice. To address this, we have developed two novel flow cytometry panels designed to extensively analyze immune cell populations in mice during longitudinal studies, using only 50 µL of peripheral blood per panel. Additionally, a third panel has been designed to conduct a more detailed analysis of cytotoxic and inhibitory markers at the end point. These panels have been validated on a lipopolysaccharide (LPS)-induced lung inflammation model. Two experiments were conducted to 1) validate the panels' sensitivity to immune challenges ( n =12) and 2) to assess intrinsic variability of measurements ( n =5). In both experiments, we collected 50 µL of peripheral blood for each cytometry panel from the maxillary venous sinus. All antibodies were titrated to identify the optimal concentration that maximized the signal from the positive population while minimizing the signal from the negative population. Samples were processed within 1 hour of collection using a MACSQuant Analyzer 16 cytometer. Our results demonstrate that these immunological panels are sensitive enough to detect changes in peripheral blood after LPS induction. Moreover, our findings help determine the sample size needed based on the immune population variability. In conclusion, the panels we have designed enable a comprehensive analysis of the murine immune system with a low blood volume requirement, enabling the measure of both absolute values and relative percentages effectively. This approach provides a robust platform for longitudinal studies in mice and can be used to uncover significant insights into immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Barco-Tejada, López-Esteban, Mulero, Pion, Correa-Rocha, Desco and Cussó.)

As the dimensionality, throughput and complexity of cytometry data increases, so does the demand for user-friendly, interactive analysis tools that leverage high-performance machine learning frameworks. Here we introduce FlowAtlas: an interactive web application that enables dimensionality reduction of cytometry data without down-sampling and that is compatible with datasets stained with non-identical panels. FlowAtlas bridges the user-friendly environment of FlowJo and computational tools in Julia developed by the scientific machine learning community, eliminating the need for coding and bioinformatics expertise. New population discovery and detection of rare populations in FlowAtlas is intuitive and rapid. We demonstrate the capabilities of FlowAtlas using a human multi-tissue, multi-donor immune cell dataset, highlighting key immunological findings. FlowAtlas is available at https://github.com/gszep/FlowAtlas.jl.git., Competing Interests: JJ reports receiving consulting fees and grant support from Sanofi Genzyme and Enhanc3DGenomics, and consulting fees from Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funders did not play a part in study design, data collection, decision to publish, or preparation of the manuscript. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Coppard, Szep, Georgieva, Howlett, Jarvis, Rainbow, Suchanek, Needham, Mousa, Menon, Feyertag, Mahbubani, Saeb-Parsy and Jones.)

Acute myeloid leukemia (AML) is a heterogenous blood cancer with a dismal prognosis. It emanates from leukemic stem cells (LSCs) arising from the genetic transformation of hematopoietic stem cells (HSCs). LSCs hold prognostic value, but their molecular and immunophenotypic heterogeneity poses challenges: there is no single marker for identifying all LSCs across AML samples. We hypothesized that imaging flow cytometry (IFC) paired with artificial intelligence-driven image analysis could visually distinguish LSCs from HSCs based solely on morphology. Initially, a seven-color IFC panel was employed to immunophenotypically identify LSCs and HSCs in bone marrow samples from five AML patients and ten healthy donors, respectively. Next, we developed convolutional neural network (CNN) models for HSC-LSC discrimination using brightfield (BF), side scatter (SSC), and DNA images. Classification using only BF images achieved 86.96% accuracy, indicating significant morphological differences. Accuracy increased to 93.42% when combining BF with DNA images, highlighting differences in nuclear morphology, although DNA images alone were inadequate for accurate HSC-LSC discrimination. Model development using SSC images revealed minor granularity differences. Performance metrics varied substantially between AML patients, indicating considerable morphologic variations among LSCs. Overall, we demonstrate proof-of-concept results for accurate CNN-based HSC-LSC differentiation, instigating the development of a novel technique within AML monitoring.

In recent years, the significance of detecting minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) has increased due to the availability of highly effective therapeutic agents. Flow cytometry provides notable cost-effectiveness and immediacy, with an expected sensitivity level of approximately 10 -4 . The critical aspect of MRD detection via flow cytometry lies in accurately defining the region containing tumor cells. However, a subset of CLL, known as CLL with atypical immunophenotype, exhibits a distinct cell surface marker expression pattern that can make MRD detection challenging, because these markers often resemble those of normal B cells. To enhance the sensitivity of MRD detection in such atypical cases of CLL, we have capitalized on the observation that cell surface immunoglobulin (sIg) light chains tend to be expressed at a higher level in this subtype. For every four two-dimensional plots of cell surface markers, we used a plot to evaluate the expression of sIg kappa/lambda light chains and identified regions where the kappa/lambda ratio of sIg light chains deviated from a designated threshold within the putative CLL cell region. Using this method, we could detect atypical CLL cells at a level of 10 -4 . We propose this method as an effective MRD assay., (© 2024. The Author(s).)

The mammalian immune system is a Janus-faced network of well-coordinated highly specialized cells and biomolecules [...].

Context.—: In 2018 the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee designed and implemented a new plasma cell neoplasia flow cytometry proficiency testing program-PCNEO-to allow clinical flow cytometry laboratories to monitor and assess their performance compared with a peer group., Objective.—: To report the results from the first 4 years of the PCNEO program., Design.—: Program participants were sent 2 sets of challenges per year, each including 1 wet challenge and 2 dry challenges, with associated clinical and laboratory findings. The wet challenges were composed of myeloma cell line specimens (with or without dilution in preserved whole blood) for flow cytometric analysis. The dry (paper) challenges were composed of clinical case summaries and images of flow cytometric test results from various flow cytometry laboratories of committee members., Results.—: A total of 116 to 145 laboratories from 17 countries enrolled in the proficiency testing program. For the wet challenges, almost all participants (97%-100%; cumulative, 98.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of undiluted myeloma cell lines. Slightly fewer participants (89.0%-97.4%; cumulative, 95.2%) correctly identified the presence of neoplastic plasma cell populations based on flow cytometric analysis of diluted myeloma cell lines (10% or 50% dilutions into peripheral blood) intended to better represent a typical clinical sample. There was generally agreement among 80% or more of participants for positive or negative staining for CD38, CD138, CD19, CD20, and surface and cytoplasmic κ and λ light chains. Similarly, 84% to 100% of participants were able to correctly identify the presence of neoplastic plasma cell populations in paper challenges, including the presence of small, neoplastic plasma cell populations (0.01%-5.0% clonal plasma cells) and the presence of nonneoplastic plasma cell populations (correctly identified by 91%-96% of participants)., Conclusions.—: Participant performance in the new proficiency testing program was excellent overall, with the vast majority of participants able to perform flow cytometric analysis and identify neoplastic plasma cell populations and to identify small plasma cell clones or expanded populations of reactive plasma cells in dry challenge flow cytometry results. This program will allow laboratories to verify the accuracy of their testing program and test interpretations for the assessment of patients suspected of having a plasma cell neoplasm., Competing Interests: All authors are current or past members of the College of American Pathologists Diagnostic Immunology and Flow Cytometry Committee, except for Bashleben, who is an employee of the College of American Pathologists., (© 2024 College of American Pathologists.)

Background Aims: The CliniMACS Prodigy closed system is widely used for the manufacturing of chimeric antigen receptor T cells (CAR-T cells). Our study presents an extensive immunophenotypic and functional characterization and comparison of the properties of anti-CD19 CAR-T cell products obtained during long (11 days) and short (7 days) manufacturing cycles using the CliniMACS Prodigy system, as well as cell products manufactured from different donor sources of T lymphocytes: from patients, from patients who underwent HSCT, and from haploidentical donors. We also present the possibility of assessing the efficiency of transduction by an indirect method., Methods: Seventy-six CD19 CAR-T cell products were manufactured using the CliniMACS Prodigy automated system. Immunophenotypic properties, markers of cell activation and exhaustion, antitumor, anti-CD19 specific activity in vitro of the manufactured cell products were evaluated. As an indirect method for assessing the efficiency of transduction, we used the method of functional assessment of cytokine secretion and expression of the CD107a marker after incubation of CAR-T cells with tumor targets., Results: The CliniMACS Prodigy platform can produce a product of CD19 CAR-T cells with sufficient cell expansion (4.6 × 10 9 cells-median for long process [LP] and 1.6 × 10 9 -for short process [SP]), transduction efficiency (43.5%-median for LP and 41.0%-for SP), represented mainly by T central memory cell population, with low expression of exhaustion markers, and with high specific antitumor activity in vitro. We did not find significant differences in the properties of the products obtained during the 7- and 11-day manufacturing cycles, which is in favor of reducing the duration of production to 7 days, which may accelerate CAR-T therapy. We have shown that donor sources for CAR-T manufacturing do not significantly affect the composition and functional properties of the cell product., Conclusions: This study demonstrates the possibility of using the CliniMACS Prodigy system with a shortened 7-day production cycle to produce sufficient amount of functional CAR-T cells. CAR transduction efficiency can be measured indirectly via functional assays., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: The diagnosis of coeliac disease (CD) in adults is based on clinical, serological and histological criteria. The inappropriate performance of intestinal biopsies, non-specificity of mild histological lesions and initiation of a gluten-free diet (GFD) before biopsy may hamper the diagnosis. In these situations, determining the intraepithelial lymphogram of the duodenum by flow cytometry (IEL-FC) can be helpful., Objectives: To describe the clinical scenarios in which the IEL-FC is used and its impact on the diagnosis of CD., Methods: All adult patients with suspected CD at three tertiary centres for whom the duodenal histology and IEL-FC were available were identified. Catassi and Fasano's diagnostic criteria and changes to a CD diagnosis after the IEL-FCs were collected., Results: A total of 348 patients were included. The following indications for an IEL-FC formed part of the initial study for CD (38%): negative conventional work-up (32%), already on a GFD before duodenal biopsies (29%) and refractoriness to a GFD (2%). The IEL-FC facilitated a definitive diagnosis in 93% of patients with an uncertain diagnosis who had had a conventional work-up for CD or who were on a GFD before histology., Conclusions: The IEL-FC facilitates the confirmation or rejection of a diagnosis of CD in clinical scenarios in which a conventional work-up may be insufficient.

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein [M-protein], and skin changes) is a rare systemic disorder characterized by various symptoms caused by underlying plasma cell (PC) dyscrasia. Detection of monoclonal PCs is mandatory for the diagnosis of POEMS syndrome; however, the usefulness of EuroFlow-based next-generation flow cytometry (EuroFlow-NGF) in POEMS syndrome for detecting monoclonal PCs in bone marrow (BM) and the gating strategy suitable for flow cytometry study of POEMS syndrome remain unknown. We employed EuroFlow-NGF-based single-tube eight-color multiparameter flow cytometry (MM-flow) and established a new gating strategy (POEMS-flow) to detect the monoclonal PCs in POEMS syndrome, gating CD38 broadly from dim to bright and CD45 narrowly from negative to dim compared to MM-flow. MM-flow detected monoclonal PCs in 9/25 (36.0%) cases, including 2/2 immunofixation electrophoresis (IFE)-negative cases (100%). However, POEMS-flow detected monoclonal PCs in 18/25 cases (72.0%), including 2/2 IFE-negative cases (100%). POEMS-flow detected monoclonal PCs with immunophenotypes of CD19 - in 17/18 (94.4%). In six cases where post-treatment samples were available, the size of the clones was significantly reduced after the treatment (P = 0.031). POEMS-flow can enhance the identification rate of monoclonal PCs in POEMS syndrome and become a valuable tool for the diagnosis of POEMS syndrome., (© 2024. The Author(s).)

Patients with systemic autoimmune diseases, such as systemic lupus erythematosus, were at a higher risk for preeclampsia. The causal relationship between immunological inflammation and preeclampsia (PE) remains uncertain. We aimed to investigate the causal relationship between circulating immune inflammation and PE. Genetically predicted blood immune cells and circulating inflammatory proteins were identified using two genome-wide association studies (GWAS). We used a two-sample Mendelian randomization (MR) method to determine whether circulating immunological inflammation causes PE. Our findings indicated that ten immunophenotypes were identified to be significantly associated with PE risk: CD62L- Dendritic Cell Absolute Count, CD86+ myeloid Dendritic Cell %Dendritic Cell, CD62L- myeloid Dendritic Cell Absolute Count, CD86+ myeloid Dendritic Cell Absolute Count, CD62L- myeloid Dendritic Cell %Dendritic Cell, CD62L- CD86+ myeloid Dendritic Cell %Dendritic Cell, CD62L- CD86+ myeloid Dendritic Cell Absolute Count, CD16 on CD14+ CD16+ monocyte, HLA DR+ Natural Killer Absolute Count, and T cell Absolute Count. Ninety-one inflammation-related proteins had no statistically significant effect on PE following false discovery rate (FDR) correction. Certain proteins exhibited unadjusted low p-values that merited mention. These proteins include interleukin-10 (OR = 0.76, 95%CI = 0.63-0.93, p = .006), fibroblast growth factor 21 (OR = 1.23, 95%CI = 1.01-1.47, p = .035), and Caspase 8 (OR = 0.65, 95%CI = 0.50-0.85, p = .001). The ELISA analysis demonstrated elevated levels of FGF-21 and decreased levels of IL-10 and Caspase-8 in the plasma of patients with PE. These findings reveal that immunophenotypes and circulating inflammatory proteins may induce PE, confirming the importance of peripheral Immunity-Inflammation in PE. The discovery has the potential to lead to earlier detection and more effective treatment techniques., (© 2024 The Authors. The Journal of Clinical Hypertension published by Wiley Periodicals LLC.)

Background Aims: The successful development of CD19-targeted chimeric antigen receptor (CAR) T-cell therapies has led to an exponential increase in the number of patients recieving treatment and the advancement of novel CAR T products. Therefore, there is a strong need to develop streamlined platforms that allow rapid, cost-effective, and accurate measurement of the key characteristics of CAR T cells during manufacturing (i.e., cell number, cell size, viability, and basic phenotype)., Methods: In this study, we compared the novel benchtop cell analyzer Moxi GO II (ORFLO Technologies), which enables simultaneous evaluation of all the aforementioned parameters, with current gold standards in the field: the Multisizer Coulter Counter (cell counter) and the BD LSRFortessa (flow cytometer)., Results: Our results demonstrated that the Moxi GO II can accurately measure cell number and cell size (i.e., cell volume) while simultaneously assessing simple two-color flow cytometry parameters, such as CAR T-cell viability and CD4 or CAR expression., Conclusions: These measurements are comparable with those of gold standard instruments, demonstrating that the Moxi GO II is a promising platform for quickly monitoring CAR T-cell growth and phenotype in research-grade and clinical samples., Competing Interests: Declaration of Competing Interest MR holds patents related to CAR T cells that are licensed to Novartis, Tmunity (Kite) and viTToria biotherapeutics. MR has served as a consultant for nanoString, BMS, GSK, GLG, GuidePoint, Sana, Bayer and AbClon. MR receives research funding from AbClon, Beckman Coulter, Oxford Nano Imaging, Curiox and viTToria biotherapeutics. MR is the scientific founder of viTToria biotherapeutics. The Ruella laboratory was provided a Moxi GO II device and supplies to perform this study. GD is an employee of ORFLO Technologies. All other authors declare no competing interests., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hemon, Ben-Guigui, Geier, Castillon, Paranthoen, Pers, Le Rochais and Uguen.)

Introduction: Exploring monocytes' roles within the tumor microenvironment is crucial for crafting targeted cancer treatments., Methods: This study unveils a novel methodology utilizing four 20-color flow cytometry panels for comprehensive peripheral immune system phenotyping, specifically targeting classical, intermediate, and non-classical monocyte subsets., Results: By applying advanced dimensionality reduction techniques like t-distributed stochastic neighbor embedding (tSNE) and FlowSom analysis, we performed an extensive profiling of monocytes, assessing 50 unique cell surface markers related to a wide range of immunological functions, including activation, differentiation, and immune checkpoint regulation., Discussion: This in-depth approach significantly refines the identification of monocyte subsets, directly supporting the development of personalized immunotherapies and enhancing diagnostic precision. Our pioneering panel for monocyte phenotyping marks a substantial leap in understanding monocyte biology, with profound implications for the accuracy of disease diagnostics and the success of checkpoint-inhibitor therapies. Key findings include revealing distinct marker expression patterns linked to tumor progression and providing new avenues for targeted therapeutic interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Xiao, Geng, Wang, Zeng, Lai, Gong and Chen.)

Introduction: B cells play a pivotal role in adaptive immunity which has been extensively characterised primarily via flow cytometry-based gating strategies. This study addresses the discrepancies between flow cytometry-defined B cell subsets and their high-confidence molecular signatures using single-cell multi-omics approaches., Methods: By analysing multi-omics single-cell data from healthy individuals and patients across diseases, we characterised the level and nature of cellular contamination within standard flow cytometric-based gating, resolved some of the ambiguities in the literature surrounding unconventional B cell subsets, and demonstrated the variable effects of flow cytometric-based gating cellular heterogeneity across diseases., Results: We showed that flow cytometric-defined B cell populations are heterogenous, and the composition varies significantly between disease states thus affecting the implications of functional studies performed on these populations. Importantly, this paper draws caution on findings about B cell selection and function of flow cytometric-sorted populations, and their roles in disease. As a solution, we developed a simple tool to identify additional markers that can be used to increase the purity of flow-cytometric gated immune cell populations based on multi-omics data ( AlliGateR ). Here, we demonstrate that additional non-linear CD20, CD21 and CD24 gating can increase the purity of both naïve and memory populations., Discussion: These findings underscore the need to reconsider B cell subset definitions within the literature and propose leveraging single-cell multi-omics data for refined characterisation. We show that single-cell multi-omics technologies represent a powerful tool to bridge the gap between surface marker-based annotations and the intricate molecular characteristics of B cell subsets., Competing Interests: RB-R is a co-founder of Alchemab Therapeutics Ltd and consultant for Alchemab Therapeutics Ltd, Roche, Enara Bio, UCB and GSK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pernes, Alsayah, Tucci and Bashford-Rogers.)

Purpose: Using a comprehensive flow cytometric panel, simultaneously obtained mid-luteal immunophenotypes from peripheral blood and endometrium were compared and values correlated. Is a peripheral blood evaluation of reproductive immunophenotype status meritorious relative to local endometrial evaluation to directly assess the peri-implantation environment?, Methods: Fifty-five patients had a mid-luteal biopsy to assess the local endometrial immunophenotype, while simultaneously providing a peripheral blood sample for analysis. Both samples were immediately assessed using a comprehensive multi-parameter panel, and lymphocyte subpopulations were described and compared., Results: Distinct lymphocyte proportions and percentage differences were noted across the two compartments, confirming the hypothesis that they are distinct environments. The ratio of CD4 + to CD8 + T cells were reversed between the two compartments, as were Th1 and Th2-type CD4 + T cell ratios. Despite these differences, some direct relationships were noted. Positive Pearson correlations were found between the levels of CD57 + expressing natural killer cells, CD3 + NK-T cells and CD4 + Th1 cells in both compartments., Conclusions: Flow cytometric evaluation provides a rapid and objective analysis of lymphocyte subpopulations. Endometrial biopsies have become the gold standard technique to assess the uterine immunophenotype in adverse reproductive outcome, but there may still a place for peripheral blood evaluation in this context. The findings demonstrate significant variations in cellular proportions across the two regions, but some positive correlations are present. Immunological assessment of these specific peripheral blood lymphocyte subtypes may provide insight into patients with potential alterations of the uterine immune environment, without the risks and inconveniences associated with an invasive procedure., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Quantitative or qualitative differences in immunity may drive clinical severity in COVID-19. Although longitudinal studies to record the course of immunological changes are ample, they do not necessarily predict clinical progression at the time of hospital admission. Here we show, by a machine learning approach using serum pro-inflammatory, anti-inflammatory and anti-viral cytokine and anti-SARS-CoV-2 antibody measurements as input data, that COVID-19 patients cluster into three distinct immune phenotype groups. These immune-types, determined by unsupervised hierarchical clustering that is agnostic to severity, predict clinical course. The identified immune-types do not associate with disease duration at hospital admittance, but rather reflect variations in the nature and kinetics of individual patient's immune response. Thus, our work provides an immune-type based scheme to stratify COVID-19 patients at hospital admittance into high and low risk clinical categories with distinct cytokine and antibody profiles that may guide personalized therapy., (© 2022. The Author(s).)

Background: The immune microenvironment of tumors provides information on prognosis and prediction. A prior validation of the immunoscore for breast cancer (IS BC ) was made on the basis of a systematic assessment of immune landscapes extrapolated from a large number of neoplastic transcripts. Our goal was to develop a non-invasive radiomics-based IS BC predictive factor., Methods: Immunocell fractions of 22 different categories were evaluated using CIBERSORT on the basis of a large, open breast cancer cohort derived from comprehensive information on gene expression. The IS BC was constructed using the LASSO Cox regression model derived from the Immunocell type scores, with 479 quantified features in the intratumoral and peritumoral regions as observed from DCE-MRI. A radiomics signature [radiomics ImmunoScore (RIS)] was developed for the prediction of IS BC using a random forest machine-learning algorithm, and we further evaluated its relationship with prognosis., Results: An IS BC consisting of seven different immune cells was established through the use of a LASSO model. Multivariate analyses showed that the IS BC was an independent risk factor in prognosis (HR=2.42, with a 95% CI of 1.49-3.93; P<0.01). A radiomic signature of 21 features of the IS BC was then exploited and validated (the areas under the curve [AUC] were 0.899 and 0.815). We uncovered statistical associations between the RIS signature with recurrence-free and overall survival rates (both P<0.05)., Conclusions: The RIS is a valuable instrument with which to assess the immunoscore, and offers important implications for the prognosis of breast cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Han, Cao, Wu and Liang.)

Blood malignancies provide unique opportunities for longitudinal tracking of disease evolution following therapeutic bottlenecks and for the monitoring of changes in anti-tumor immunity. The expanding development of multi-modal single-cell sequencing technologies affords newer platforms to elucidate the mechanisms underlying these processes at unprecedented resolution. Furthermore, the identification of molecular events that can serve as in-vivo barcodes now facilitate the tracking of the trajectories of malignant and of immune cell populations over time within primary human samples, as these permit unambiguous identification of the clonal lineage of cell populations within heterogeneous phenotypes. Here, we provide an overview of the potential for chromosomal copy number changes, somatic nuclear and mitochondrial DNA mutations, single nucleotide polymorphisms, and T and B cell receptor sequences to serve as personal natural barcodes and review technical implementations in single-cell analysis workflows. Applications of these methodologies include the study of acquired therapeutic resistance and the dissection of donor- and host cellular interactions in the context of allogeneic hematopoietic stem cell transplantation., Competing Interests: CW holds equity in BioNTech,Inc; and receives research support from Pharmacyclics. SG undertakes consultancy for Novalgen Limited and has received speakers fees and honoraria from Abbvie, Janssen and AstraZeneca. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Penter, Gohil and Wu.)

Mesenchymal stem/stromal cells or medicinal signaling cells (MSC)-based therapy holds promise as a beneficial strategy for treating knee OA (osteoarthritis), but there is no standardized protocols nor mechanistic understanding. In order to gain a better insight into the human MSC from adipose tissue applied for autologous OA treatment, we performed extensive comparative immunophenotyping of the stromal vascular fraction from lipoaspirate or microfragmented lipoaspirates by polychromatic flow cytometry and investigated the cellular components considered responsible for cartilage regeneration. We found an enrichment of the regenerative cellular niche of the clinically applied microfragmented stromal vascular fraction. Sex-related differences were observed in the MSC marker expression and the ratio of the progenitor cells from fresh lipoaspirate, which, in female patients, contained a higher expression of CD90 on the three progenitor cell types including pericytes, a higher expression of CD105 and CD146 on CD31 high CD34 high endothelial progenitors as well as of CD73 on supra-adventitialadipose stromal cells. Some of these MSC-expression differences were present after microfragmentation and indicated a differential phenotype pattern of the applied MSC mixture in female and male patients. Our results provide a better insight into the heterogeneity of the adipose MSC subpopulations serving as OA therapeutics, with an emphasis on interesting differences between women and men.

Detecting the presence of prostate cancer (PCa) and distinguishing low- or intermediate-risk disease from high-risk disease early, and without the need for potentially unnecessary invasive biopsies remains a significant clinical challenge. The aim of this study is to determine whether the T and B cell phenotypic features which we have previously identified as being able to distinguish between benign prostate disease and PCa in asymptomatic men having Prostate-Specific Antigen (PSA) levels < 20 ng/ml can also be used to detect the presence and clinical risk of PCa in a larger cohort of patients whose PSA levels ranged between 3 and 2617 ng/ml. The peripheral blood of 130 asymptomatic men having elevated Prostate-Specific Antigen (PSA) levels was immune profiled using multiparametric whole blood flow cytometry. Of these men, 42 were subsequently diagnosed as having benign prostate disease and 88 as having PCa on biopsy-based evidence. We built a bidirectional Long Short-Term Memory Deep Neural Network (biLSTM) model for detecting the presence of PCa in men which combined the previously-identified phenotypic features (CD8 + CD45RA - CD27 - CD28 - ( CD8 + Effector Memory cells ), CD4 + CD45RA - CD27 - CD28 - ( CD4 + Effector Memory cells ), CD4 + CD45RA + CD27 - CD28 - ( CD4 + Terminally Differentiated Effector Memory Cells re-expressing CD45RA ), CD3 - CD19 + ( B cells ), CD3 + CD56 + CD8 + CD4 + ( NKT cells ) with Age. The performance of the PCa presence 'detection' model was: Acc: 86.79 ( ± 0.10), Sensitivity: 82.78% (± 0.15); Specificity: 95.83% (± 0.11) on the test set (test set that was not used during training and validation); AUC: 89.31% (± 0.07), ORP-FPR: 7.50% (± 0.20), ORP-TPR: 84.44% (± 0.14). A second biLSTM 'risk' model combined the immunophenotypic features with PSA to predict whether a patient with PCa has high-risk disease (defined by the D'Amico Risk Classification) achieved the following: Acc: 94.90% (± 6.29), Sensitivity: 92% (± 21.39); Specificity: 96.11 (± 0.00); AUC: 94.06% (± 10.69), ORP-FPR: 3.89% (± 0.00), ORP-TPR: 92% (± 21.39). The ORP-FPR for predicting the presence of PCa when combining FC+PSA was lower than that of PSA alone. This study demonstrates that AI approaches based on peripheral blood phenotyping profiles can distinguish between benign prostate disease and PCa and predict clinical risk in asymptomatic men having elevated PSA levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cosma, McArdle, Foulds, Hood, Reeder, Johnson, Khan and Pockley.)

CD8+ tumor-infiltrating T cells can be regarded as one of the most relevant predictive biomarkers in immune-oncology. Highly infiltrated tumors, referred to as inflamed (clinically "hot"), show the most favorable response to immune checkpoint inhibitors in contrast to tumors with a scarce immune infiltrate called immune desert or excluded (clinically "cold"). Nevertheless, quantitative and reproducible methods examining their prevalence within tumors are lacking. We therefore established a computational diagnostic algorithm to quantitatively measure spatial densities of tumor-infiltrating CD8+ T cells by digital pathology within the three known tumor compartments as recommended by the International Immuno-Oncology Biomarker Working Group in 116 prospective metastatic melanomas of the Swiss Tumor Profiler cohort. Workflow robustness was confirmed in 33 samples of an independent retrospective validation cohort. The introduction of the intratumoral tumor center compartment proved to be most relevant for establishing an immune diagnosis in metastatic disease, independent of metastatic site. Cut-off values for reproducible classification were defined and successfully assigned densities into the respective immune diagnostic category in the validation cohort with high sensitivity, specificity, and precision. We provide a robust diagnostic algorithm based on intratumoral and stromal CD8+ T-cell densities in the tumor center compartment that translates spatial densities of tumor-infiltrating CD8+ T cells into the clinically relevant immune diagnostic categories "inflamed", "excluded", and "desert". The consideration of the intratumoral tumor center compartment allows immune phenotyping in the clinically highly relevant setting of metastatic lesions, even if the invasive margin compartment is not captured in biopsy material., (© 2021. The Author(s).)

The complex interactions among different immune cells have important functions in the development of malignant pleural effusion (MPE). Here we perform single-cell RNA sequencing on 62,382 cells from MPE patients induced by non-small cell lung cancer to describe the composition, lineage, and functional states of infiltrating immune cells in MPE. Immune cells in MPE display a number of transcriptional signatures enriched for regulatory T cells, B cells, macrophages, and dendritic cells compared to corresponding counterparts in blood. Helper T, cytotoxic T, regulatory T, and T follicular helper cells express multiple immune checkpoints or costimulatory molecules. Cell-cell interaction analysis identifies regulatory B cells with more interactions with CD4 + T cells compared to CD8 + T cells. Macrophages are transcriptionally heterogeneous and conform to M2 polarization characteristics. In addition, immune cells in MPE show the general up-regulation of glycolytic pathways associated with the hypoxic microenvironment. These findings show a detailed atlas of immune cells in human MPE and enhance the understanding of potential diagnostic and therapeutic targets in advanced non-small cell lung cancer., (© 2021. The Author(s).)

Plasmablasts represent a specialized class of antibody-secreting effector B cells that transiently appear in blood circulation following infection or vaccination. The expansion of these cells generally tends to be massive in patients with systemic infections such as dengue or Ebola that cause hemorrhagic fever. To gain a detailed understanding of human plasmablast responses beyond antibody expression, here, we performed immunophenotyping and RNA sequencing (RNA-seq) analysis of the plasmablasts from dengue febrile children in India. We found that plasmablasts expressed several adhesion molecules and chemokines or chemokine receptors that are involved in endothelial interactions or homing to inflamed tissues, including skin, mucosa, and intestine, and upregulated the expression of several cytokine genes that are involved in leukocyte extravasation and angiogenesis. These plasmablasts also upregulated the expression of receptors for several B-cell prosurvival cytokines that are known to be induced robustly in systemic viral infections such as dengue, some of which generally tend to be relatively higher in patients manifesting hemorrhage and/or shock than in patients with mild febrile infection. These findings improve our understanding of human plasmablast responses during the acute febrile phase of systemic dengue infection. IMPORTANCE Dengue is globally spreading, with over 100 million clinical cases annually, with symptoms ranging from mild self-limiting febrile illness to more severe and sometimes life-threatening dengue hemorrhagic fever or shock, especially among children. The pathophysiology of dengue is complex and remains poorly understood despite many advances indicating a key role for antibody-dependent enhancement of infection. While serum antibodies have been extensively studied, the characteristics of the early cellular factories responsible for antibody production, i.e., plasmablasts, are only beginning to emerge. This study provides a comprehensive understanding of the transcriptional profiles of human plasmablasts from dengue patients.

Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections ( n = 22), compared to those that had recovered from other mild respiratory infections ( n = 11). Individuals who had experienced mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1-3 months after symptom onset, and changes in phenotype and function of circulating T-cells that were not apparent in individuals 6-9 months post-symptom onset. Markers of monocyte activation, and expression of adherence and chemokine receptors indicative of altered migratory capacity, were also higher at 1-3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6-9 months post-infection. Perhaps most surprisingly, significantly more T-cells could be activated by polyclonal stimulation in individuals who had recently experienced a mild SARS-CoV-2, infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for at least three months after mild or asymptomatic SARS-CoV-2 infections.

Tacrolimus (FK506) and rapamycin (RAPA) are widely used to maintain long-term immunosuppression after organ transplantation. However, the impact of accumulative drug administration on the recipients' immune systems remains unclear. We investigated the impact of 3-year FK506 or RAPA treatment after renal transplantation on the human immune systems. A discovery cohort of 30 patients was first recruited, and we discovered two distinctive T lineage suppressive regulatory patterns induced by chronic treatment of FK506 and RAPA. The increased percentage of senescent CD8 + CD57 + T lineages and less responsive T cell receptor (TCR) pathway in the FK506 group indicate better graft acceptance. Meanwhile, percentages of regulatory T cells (Tregs) and expression of CTLA-4 were both up to two-fold higher in the RAPA group, suggesting the inconsistent reactivation potential of the FK506 and RAPA groups when an anti-tumour or anti-infection immune response is concerned. Additionally, up-regulation of phosphorylated signaling proteins in T lineages after in vitro CD3/CD28 stimulation suggested more sensitive TCR-signaling pathways reserved in the RAPA group. An independent validation cohort of 100 renal transplantation patients was further investigated for the hypothesis that long-term RAPA administration mitigates the development of tumours and infections during long-term intake of immunosuppressants. Our results indicate that RAPA administration indeed results in less clinical oncogenesis and infection. The deep phenotyping of T-cell lineages, as educated by the long-term treatment of different immunosuppressants, provides new evidence for personalized precision medicine after renal transplantations., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

This protocol provides instructions to improve flow cytometry analysis of marrow/peripheral blood cells by avoiding erythrolytic solutions, density gradients, and washing steps. We describe two basic approaches for identifying cell surface antigens with minimal sample perturbation, which have been successfully used to identify healthy and pathologically rare cells. The greatest advantage of these approaches is that they minimize the unwanted effect caused by sample preparation, allowing for improved study of live cells at the point of analysis. For complete details on the use and execution of this protocol, please refer to Petriz et al. (2018)., Competing Interests: M.D.W. and J.A.B. are employees of Thermo Fisher Scientific, which is in the business of selling flow cytometers and flow cytometry reagents. The rest of the authors declare no competing interests., (© 2021 The Author(s).)

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Identification of stage-specific erythroid cells is critical for studies of normal and disordered human erythropoiesis. While immunophenotypic strategies have previously been developed to identify cells at each stage of terminal erythroid differentiation, erythroid progenitors are currently defined very broadly. Refined strategies to identify and characterize BFU-E and CFU-E subsets are critically needed. To address this unmet need, a flow cytometry-based technique was developed that combines the established surface markers CD34 and CD36 with CD117, CD71, and CD105. This combination allowed for the separation of erythroid progenitor cells into four discrete populations along a continuum of progressive maturation, with increasing cell size and decreasing nuclear/cytoplasmic ratio, proliferative capacity and stem cell factor responsiveness. This strategy was validated in uncultured, primary erythroid cells isolated from bone marrow of healthy individuals. Functional colony assays of these progenitor populations revealed enrichment of BFU-E only in the earliest population, transitioning to cells yielding BFU-E and CFU-E, then CFU-E only. Utilizing CD34/CD105 and GPA/CD105 profiles, all four progenitor stages and all five stages of terminal erythroid differentiation could be identified. Applying this immunophenotyping strategy to primary bone marrow cells from patients with myelodysplastic syndrome, identified defects in erythroid progenitors and in terminal erythroid differentiation. This novel immunophenotyping technique will be a valuable tool for studies of normal and perturbed human erythropoiesis. It will allow for the discovery of stage-specific molecular and functional insights into normal erythropoiesis as well as for identification and characterization of stage-specific defects in inherited and acquired disorders of erythropoiesis., (© 2021 Wiley Periodicals LLC.)

Background and Aim: This study analyzed peripheral blood lymphocyte subsets to determine their role in the etiopathogenesis of IGM., Materials and Methods: This study includes 51 pathologically proven IGM patients (active disease: 26 and in remission: 25) and 28 healthy volunteers. The analyses of lymphocyte subsets were performed by flow cytometric immunophenotyping., Results: The percentage of T helper lymphocyte of all IGM patients were lower than control groups (p = 0.001). Absolute cytotoxic T lymphocyte count (p = 0.03), both percentage (p = 0.035) and absolute count (p = 0.002) of the natural killer cells, and both percentage (p = 0.038) and absolute count (p = 0.008) of natural killer T cells, were higher than the control group. The T helper lymphocyte percentage of the patients with active disease was lower than the control group (p = 0.0003). The absolute cytotoxic T lymphocyte (p = 0.029) and natural killer T cells (p = 0.012) of the patients with active disease were higher than the control group., Conclusion: Idiopathic granulomatous mastitis is defined as a localized form of granulomatous disorders. However, the observed changes in T cells, NK, and NKT cells suggest that there is systemic immune dysregulation in patients with IGM., (This work is licensed under a Creative Commons Attribution 4.0 International License.)

The efficiency of immunotherapy using monoclonal antibodies that inhibit immune checkpoints has been proven in many clinical studies and well documented by numerous registration approaches. To date, PD-L1 expression on tumor and immune cells, tumor mutation burden (TMB), and microsatellite instability (MSI) are the only validated predictive factors used for the qualification of cancer patients for immunotherapy. However, they are not the ideal predictive factors. No response to immunotherapy could be observed in patients with high PD-L1 expression, TMB, or MSI. On the other hand, the effectiveness of this treatment method also may occur in patients without PD-L1 expression or with low TMB and with microsatellite stability. When considering the best predictive factor, we should remember that the effectiveness of immunotherapy relies on an overly complex process depending on many factors. To specifically stimulate lymphocytes, not only should their activity in the tumor microenvironment be unlocked, but above all, they should recognize tumor antigens. The proper functioning of the anticancer immune system requires the proper interaction of many elements of the specific and non-specific responses. For these reasons, a multi-parameter analysis of the immune system at its different activity levels is considered a very future-oriented predictive marker. Such complex immunological analysis is performed using modern molecular biology techniques. Based on the gene expression studies, we can determine the content of individual immune cells within the tumor, its stroma, and beyond. This includes all cell types from active memory cytotoxic T cells, M1 macrophages, to exhausted T cells, regulatory T cells, and M2 macrophages. In this article, we summarize the possibilities of using an immune system analysis to predict immunotherapy efficacy in cancer patients. Moreover, we present the advantages and disadvantages of immunoprofiling as well as a proposed future direction for this new method of immune system analysis in cancer patients who receive immunotherapy.

Background: Mutations along PSTPIP1 gene are associated to two specific conditions, PAPA syndrome and PAMI syndrome, both autoinflammatory disorders associated to disturbances in cytoskeleton formation. Immunological aspects of PAMI syndrome has not yet been reported neither the clinical impact on therapeutical decisions., Methods: Clinical data of patients records were retrospectively accessed. Genomic DNA were extracted and sequenced following standard procedures. Peripheral lymphocytes were quantified in T, B e FOXP3 phenotypes., Results: We describe two related patients with PAMI syndrome harboring the usual E250K mutation. Anti-IL1 therapy could partially control the disease in the index patient. A broad spectrum of immunological effects as well as an aberrant expression of FOXP3 could be observed., Conclusions: Here we report two related brazilian patients with PAMI syndromes harboring the E250K mutation in PSTPIP1, their immunological aspects and the therapeutical response to canakinumab., (© 2021. The Author(s).)

The ectocervix is part of the lower female reproductive tract (FRT), which is susceptible to sexually transmitted infections (STIs). Comprehensive knowledge of the phenotypes and T cell receptor (TCR) repertoire of tissue-resident memory T cells (TRMs) in the human FRT is lacking. We took single-cell RNA-Seq approaches to simultaneously define gene expression and TCR clonotypes of the human ectocervix. There were significantly more CD8+ than CD4+ T cells. Unsupervised clustering and trajectory analysis identified distinct populations of CD8+ T cells with IFNGhiGZMBloCD69hiCD103lo or IFNGloGZMBhiCD69medCD103hi phenotypes. Little overlap was seen between their TCR repertoires. Immunofluorescence staining showed that CD103+CD8+ TRMs were preferentially localized in the epithelium, whereas CD69+CD8+ TRMs were distributed evenly in the epithelium and stroma. Ex vivo assays indicated that up to 14% of cervical CD8+ TRM clonotypes were HSV-2 reactive in HSV-2-seropositive persons, reflecting physiologically relevant localization. Our studies identified subgroups of CD8+ TRMs in the human ectocervix that exhibited distinct expression of antiviral defense and tissue residency markers, anatomic locations, and TCR repertoires that target anatomically relevant viral antigens. Optimization of the location, number, and function of FRT TRMs is an important approach for improving host defenses to STIs.

Background: Considering the clinical and genetic characteristics, acute lymphoblastic leukemia (ALL) is a rather heterogeneous hematological neoplasm for which current standard diagnostics require various analyses encompassing morphology, immunophenotyping, cytogenetics, and molecular analysis of gene fusions and mutations. Hence, it would be desirable to rely on a technique and an analytical workflow that allows the simultaneous analysis and identification of all the genetic alterations in a single approach. Moreover, based on the results with standard methods, a significant amount of patients have no established abnormalities and hence, cannot further be stratified., Methods: We performed WTS and WGS in 279 acute lymphoblastic leukemia (ALL) patients (B-cell: n = 211; T-cell: n = 68) to assess the accuracy of WTS, to detect relevant genetic markers, and to classify ALL patients., Results: DNA and RNA-based genotyping was used to ensure correct WTS-WGS pairing. Gene expression analysis reliably assigned samples to the B Cell Precursor (BCP)-ALL or the T-ALL group. Subclassification of BCP-ALL samples was done progressively, assessing first the presence of chromosomal rearrangements by the means of fusion detection. Compared to the standard methods, 97% of the recurrent risk-stratifying fusions could be identified by WTS, assigning 76 samples to their respective entities. Additionally, read-through fusions (indicative of CDKN2A and RB1 gene deletions) were recurrently detected in the cohort along with 57 putative novel fusions, with yet untouched diagnostic potentials. Next, copy number variations were inferred from WTS data to identify relevant ploidy groups, classifying an additional of 31 samples. Lastly, gene expression profiling detected a BCR-ABL1-like signature in 27% of the remaining samples., Conclusion: As a single assay, WTS allowed a precise genetic classification for the majority of BCP-ALL patients, and is superior to conventional methods in the cases which lack entity defining genetic abnormalities., (© 2021. The Author(s).)

Chronic graft-versus-host disease (cGVHD) is a frequent complication of allogeneic haematopoietic stem cell transplantation. Low density neutrophils (LDNs) in autoimmunity, which shares disease features with cGVHD, are proinflammatory, whereas those in cancer and sepsis suppress T cell immunity. Mature LDNs can be distinguished from immature LDNs on the basis of expression of CD10 and suppressive neutrophils can be identified using lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression. The functionality of LDNs in cGVHD has not been specifically investigated. Here, we have determined the relative contribution of immature and mature neutrophils to LDNs in cGVHD and assessed whether these were suppressive or potentially proinflammatory. Peripheral blood LDNs and normal density neutrophils (NDNs) from 30 cGVHD patients and NDNs from 10 healthy controls (HCs) were immunophenotyped by flow cytometry. The ability of LDNs and NDNs to influence T cell proliferation and cytokine production in co-cultures was quantified. To further characterize LDNs, their propensity to undergo constitutive apoptosis and differentiate ex vivo was assessed. LDNs were elevated in cGVHD versus HCs, heterogeneous in phenotype, with a predominance of immature CD10 - cells in most patients, but some mature CD10 + LOX-1 + LDNs were also detected. LDNs enhanced autologous T cell proliferation, interleukin (IL)-6 and interferon (IFN)-γ production. LDN, but not NDN, CD10 expression was inversely correlated with LOX-1, which correlated with IL-6 production. LDNs resisted apoptosis and differentiated into antigen-presenting/neutrophil-hybrid-like cells, which co-expressed major histocompatibility complex (MHC) class II HLA-DR and immuno-inhibitory programmed cell death ligand 1 (PD-L1), but did not suppress T cell proliferation. These data suggest LDNs in cGVHD are predominantly immature, proinflammatory and may have pathogenic potential., (© 2021 British Society for Immunology.)

In this work, we report on two novel monoclonal antibodies, specific for porcine CD9. CD9 is a tetraspanin that is expressed on a wide variety of cells. We phenotyped porcine immune cell subsets and found that CD9 was expressed on all monocytes as well as a subset of B cells. CD9 was variably expressed on T cells, with CD4 T cells containing the highest frequency of CD9 + cells. CD9 expression positively correlated with the frequency of central memory CD4 T cells in ex vivo PBMC. Therefore, we proceeded to explore CD9 as a marker of T cell function. Here we observed that CD9 was expressed on the vast majority of long-lived influenza A virus-specific effector cells that retained the capacity for cytokine production in response to in vitro recall antigen. Therefore, the new antibodies enable the detection of a cell surface molecule with functional relevance to T cells. Considering the importance of CD9 in membrane remodelling across many cell types, they will also benefit the wider field of swine biomedical research., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Ageing dramatically affects number and function of both innate and adaptive arms of immune system, particularly T cell subsets, contributing to reduced vaccination efficacy, decreased resistance to infections and increased prevalence of cancer in older people. In the present paper, we analysed the age-related changes in the absolute number of lymphocytes in 214 Sicilian subjects, and in the percentages of T and natural killer (NK) cells in a subcohort of donors. We compared these results with the immunophenotype of the oldest living Italian supercentenarian (aged 111 years). The results were also sorted by gender. The correlation between number/percentage of cells and age in all individuals. and separately in males and females, was examined using a simple linear regression analysis. We did not record the increase in the rate of inversion of the CD4/CD8 ratio, frequently reported as being associated with ageing in literature. Our observation was the direct consequence of a flat average trend of CD4 + and CD8 + T cell percentages in ageing donors, even when gender differences were included. Our results also suggest that CD4 + and CD8 + subsets are not affected equally by age comparing females with males, and we speculated that gender may affect the response to cytomegalovirus (CMV) infection. The supercentenarian showed a unique immunophenotypic signature regarding the relative percentages of her T cell subsets, with CD4 + and CD8 + T cell percentages and CD4 + naive T cell values in line with those recorded for the octogenarian subjects. This suggests that the supercentenarian has a naive 'younger' T cell profile comparable to that of a >80-year-old female., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

IFN-γ-driven responses to malaria have been shown to modulate the development and function of T follicular helper (TFH) cells and memory B cells (MBCs), with conflicting evidence of their involvement in the induction of antibody responses required to achieve clinical immunity and their association with disease outcomes. Using high-dimensional single-cell mass cytometry, we identified distinct populations of TH1-polarized CD4+ T cells and MBCs expressing the TH1-defining transcription factor T-bet, associated with either increased or reduced risk of Plasmodium vivax (P. vivax) malaria, demonstrating that inflammatory responses to malaria are not universally detrimental for infection. Furthermore, we found that, whereas class-switched but not IgM+ MBCs were associated with a reduced risk of symptomatic malaria, populations of TH1 cells with a stem central memory phenotype, TH17 cells, and T regulatory cells were associated with protection from asymptomatic infection, suggesting that activation of cell-mediated immunity might also be required to control persistent P. vivax infection with low parasite burden.

Background: The current desirable endpoint of treatment against chronic hepatitis B virus infection (cHBV) is to achieve a functional cure, which is defined as HBsAg loss (sAg-L) with or without anti-HBs seroconversion. However, the immunological features that are associated with functional cure have not been studied in detail., Methods: 172 cHBV patients (67 HBeAg+ and 105 HBeAg-), including 141 HBsAg retained (sAg-R) patients (115 chronic hepatitis and 26 asymptomatic carriers), 31 sAg-L patients, and 24 healthy individuals (vaccinated but not infected with HBV) were examined for their T cell phenotypic profile and HBV-specific T cell responses by flow cytometry. 18 cHBV patients with low serum HBsAg levels were also longitudinally followed for their T cell phenotypic profile and HBV-specific T cell responses up to 60 weeks., Findings: sAg-L patients showed distinct CD4 + and CD8 + T cell phenotype fingerprints compared to those of sAg-R patients, as mainly indicated by the upregulation of HLA-DR on both CD4 + and CD8 + T cells, and a potent HBcAg-specific CD8 + T cell response. The changes in the T cell phenotype in cHBV patients were even more profound during rapid HBsAg decrease and was associated with interferon α treatment. The expression of HLA-DR (r = 0·3269, p = 0·0037), CD95 (r = 0·2796, p = 0·0151), CD40L (r = 0·2747, p = 0·0156), CTLA-4 (r = 0·2786, p = 0·0148), TIM-3 (r = 0·3082, p = 0·0068), CD107a (r = 0·3597, p = 0·0013) on CD4 + T cells, and HLA-DR (r = 0·3542, p = 0·0016), CD69 (r = 0·2507, p = 0·0279), CD107a (r = 0·2875, p = 0·0112) on CD8 + T cells were positively correlated with the rate of HBsAg decrease. The expression of HLA-DR (r = 0·2846, p = 0·0009) and CD95 (r = 0·2442, p = 0·0049) on CD8 + T cells were positively correlated with the magnitude of the HBcAg-specific T cell responses in cHBV patients. Importantly, CTLA-4, CD95 and CD107a expression on CD4 + T cells, as well as HLA-DR and TIM-3 expression on CD8 + T cells in combination with HBsAg quantification were identified as potential predictive factors for sAg-L within 48 weeks in cHBV patients., Interpretation: The onset of HBsAg decrease and subsequent loss in cHBV patients on treatment is associated with significant alterations of both CD4 + and CD8 + T cell phenotypes. Characterization of the T cell phenotype in cHBV patients may present predicative value for sAg-L., Funding: National Natural Science Foundation of China, National Scientific and Technological Major Project of China, Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, "Double-First Class" Project for the International Cooperation Center on Infection and Immunity, HUST., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The skin lesion erythema migrans (EM) is an initial sign of the Ixodes tick-transmitted Borreliella spirochetal infection known as Lyme disease. T cells and innate immune cells have previously been shown to predominate the EM lesion and promote the reaction. Despite the established importance of B cells and antibodies in preventing infection, the role of B cells in the skin immune response to Borreliella is unknown. Here, we used single-cell RNA-Seq in conjunction with B cell receptor (BCR) sequencing to immunophenotype EM lesions and their associated B cells and BCR repertoires. We found that B cells were more abundant in EM in comparison with autologous uninvolved skin; many were clonally expanded and had circulating relatives. EM-associated B cells upregulated the expression of MHC class II genes and exhibited preferential IgM isotype usage. A subset also exhibited low levels of somatic hypermutation despite a gene expression profile consistent with memory B cells. Our study demonstrates that single-cell gene expression with paired BCR sequencing can be used to interrogate the sparse B cell populations in human skin and reveals that B cells in the skin infection site in early Lyme disease expressed a phenotype consistent with local antigen presentation and antibody production.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. MS is characterized by infiltrations of leukocytes such as T and B lymphocytes and macrophages. Macrophages have been identified as major effectors of inflammation and demyelination in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the activation and heterogeneity of macrophages in MS has been poorly investigated. Thus, in this study, we evaluated M1 and M2 macrophages immunophenotype from EAE and control mice by analyzing over 30 surface and intracellular markers through polychromatic flow cytometry, qRT-PCR, and ELISA assay. We showed that M1 macrophages possessed a higher proinflammatory profile in EAE compared to control mice, since they expressed higher levels of activation/co-stimulatory markers (iNOS, CD40, and CD80) and cytokines/chemokines (IL-6, IL-12, CCL2, and CXCL10), whereas M2 lost their M2-like phenotype by showing a decreased expression of their signature markers CD206 and CCL22, as well as a concomitant upregulation of several M1 makers. Furthermore, immunization of M1 and M2 macrophages with MOG35-55 led to a significant hyperactivation of M1 and a concomitant shift of anti-inflammatory M2 to pro-inflammatory M1 macrophages. Overall, we provide evidence for a phenotypic alteration of M1/M2 balance during MS, which can be of crucial importance not only for a better understanding of the immunopathology of this neurodegenerative disease but also to potentially develop new macrophage-centered therapeutic strategies.

The aim of this study is to investigate the gene expression module of tumor-infiltrating CD4 + T cells and its potential roles in modulating immune cell functions in triple-negative breast cancer. Differentially expressed genes were identified by comparison of the expression profile in CD4 + T cells isolated from tumor tissues and peripheral blood of TNBC patients respectively. The differential expression analysis was conducted using R, and then the functional and pathway enrichment of the DEGs were analyzed using GSEA, followed by integrated regulatory network construction and genetic analysis of tumor-infiltrating immune cells based on a scientific deconvolution algorithm. As a result, abundant Treg and exhausted lymphocytes were detected, accompanied by largely decreased of effector/memory and cytotoxic T cells. Immune-related gene correlation analysis showed that the extent of follicular helper T cells gene expression signatures were inversely associated with those of CD4 +  naive T cells and CD4 +  memory resting T cells, but positively correlated with that of CD4 +  memory activated T cells. In addition, we found five core genes including IFNG, CTLA4, FAS, CXCR6, and JUN were significantly over expressed in CD4 +  TILs which may contribute to exhaustion of lymphocytes and participate in biological processes associated with regulation of chemotaxis. Study provides a comprehensive understanding of the roles of DEGs associated with the chemotactic and exhausted immunophenotypes of CD4 +  TILs that are a valuable resource from which future investigation may be carried out to better understand the mechanisms that promote TNBC progression.

Background: Kawasaki disease (KD) is an acute, self-limited febrile illness of unknown cause. Intravenous immunoglobulin (IVIG)-resistance are related to greater risk for permanent cardiac complications. We aimed to determine the correlation between monocytes and the phenotype of KD in relation to IVIG responsiveness in children., Materials and Methods: The study cohort included 62 patients who were diagnosed with KD, 20 non febrile healthy controls (NFC), and 15 other febrile controls (OFC). In all enrolled patients, blood was taken at least 4 times and laboratory tests were performed. In addition, subtypes of monocytes were characterized via flow cytometry., Results: The numbers of intermediate monocytes were significantly lower in IVIG-resistant group compared to IVIG-responsive group before IVIG infusion (p < 0.0001). After infusion, intermediate monocytes decreased in the responsive group, while a trend of increase was observed in the resistant group. Only intermediate monocytes were significant in logistic regression with adjusted OR of 0.001 and p value of 0.03., Conclusions: CD14 + CD16 + intermediate monocyte may play an important role in IVIG responsiveness among KD children. Low starting levels of intermediate monocytes, followed by a dramatic increase post-IVIG infusion during acute phase of KD are associated with IVIG-resistance. Functional studies on intermediate monocyte may help to reveal the pathophysiology.

In this study, the minimal residual disease (MRD) levels in patients with multiple myeloma (MM) were assessed by comparing the new 8-color single-tube multiparameter flow cytometry method (DuraClone), which reduces the cost of antibodies and labor burden of laboratories, with the EuroFlow next-generation flow (NGF) method. A total of 96 samples derived from 69 patients with MM were assessed to determine the total cell acquisition number (tCAN), percentages of total and normal plasma cells (PCs), and MRD levels using two methods. We found that the tCAN was significantly higher with EuroFlow-NGF than with DuraClone (median 8.6 × 10 6 vs. 5.7 × 10 6 ; p < 0.0001). In addition, a significant correlation in the MRD levels between the two methods was noted (r = 0.92, p < 0.0001). However, in the qualitative analysis, 5.2% (5/96) of the samples showed discrepancies in the MRD levels. In conclusion, the DuraClone is a good option to evaluate MRD in multiple myeloma but it should be used with caution.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient's immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process., Competing Interests: TM, MB, and MK are employees of Beckman Coulter Life Sciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Blache, Weiss, Boldt, Kapinsky, Blaudszun, Quaiser, Pohl, Miloud, Burgaud, Vucinic, Platzbecker, Sack, Fricke and Koehl.)

The tumor microenvironment (TME) plays a crucial role in cancer progression and recent evidence has clarified its clinical significance in predicting outcomes and efficacy. However, there are no studies on the systematic analysis of TME characteristics in bladder cancer. In this study, we comprehensively evaluated the TME invasion pattern of bladder cancer in 1,889 patients, defined three different TME phenotypes, and found that different subtypes were associated with the clinical prognosis and pathological characteristics of bladder cancer. We further explored the signaling pathways, cancer-immunity cycle, copy number, and somatic mutation differences among the different subtypes and used the principal component analysis algorithm to calculate the immune cell (IC) score, a tool for comprehensive evaluation of TME. Univariate and multivariate Cox regression analyses showed that ICscore is a reliable and independent prognostic biomarker. In addition, the use of anti-programmed death-ligand (PD-L1) treatment cohort, receiver operating characteristic (ROC) curve, Tumor Immune Dysfunction and Exclusion (TIDE), Subnetwork Mappings in Alignment of Pathways (SubMAP), and other algorithms confirmed that ICscore is a reliable prognostic biomarker for immune checkpoint inhibitor response. Patients with higher ICscore showed a significant therapeutic advantage in immunotherapy. In conclusion, this study improves our understanding of the characteristics of TME infiltration in bladder cancer and provides guidance for more effective personalized immunotherapy strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2021 Chen, Chen, He, Cheng, Zhu, Xiao, Lan, Wang and Cao.)

Peripheral blood involvement by cutaneous T-cell lymphoma is typically assessed by flow cytometry and plays a critical role in diagnosis, classification, and prognosis. Simplified strategies to detect tumor cells (Sezary cells) fail to exclude reactive subsets, whereas tumor-specific abnormalities are subtle and inconsistently present. We implemented a flow cytometric strategy to detect clonal Sezary cells based on the monotypic expression of one of two mutually exclusive TCR constant β chains, TRBC1 and TRBC2. Analysis of CD4 + T-cell subsets and TCR variable β classes from healthy donors showed polytypic TRBC1 staining. Clonal Sezary cells were identified by TRBC1 staining in 56 of 111 (50%) samples from patients with cutaneous T-cell lymphoma, accounting for 7-18,155 cells/μl and including 13 cases (23%) lacking tumor-specific immunophenotypic abnormalities. CD4 + T-cell subsets from 86 patients without T-cell lymphoma showed polytypic TRBC1 staining, except for five patients (6%) with minute T-cell clones of uncertain significance accounting for 53-136 cells/μl. Assessment of TRBC1 expression within a comprehensive single-tube flow cytometry assay effectively overcomes interpretative uncertainties in the identification of Sezary cells without the need for a separate T-cell clonality assay., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Gender differences in individual immune responses to external stimuli have been elucidated in many invertebrates. However, it is unclear if gender differences do exist in the Hong Kong oyster Crassostrea hongkongensis , one of the most valuable marine species cultivated along the coast of South China. To clarify this, we stimulated post-spawning adult C. hongkongensis with Vibrio harveyi and lipopolysaccharide (LPS). Gender-based differences in some essential functional parameters of hemocytes were studied via flow cytometry. Obvious gender-, subpopulation-, and immune-specific alterations were found in the hemocyte immune parameters of C. hongkongensis . Three hemocyte subpopulations were identified: granulocytes, semi-granulocytes, and agranulocytes. Granulocytes, the chief phagocytes and major producers of esterase, reactive oxygen species, and nitric oxide, were the main immunocompetent hemocytes. Immune parameter alterations were notable in the accumulation of granulocyte esterase activities, lysosomal masses, nitric oxide levels, and granulocyte numbers in male oysters. These results suggest that post-spawning-phase male oysters possess a more powerful immune response than females. Gender and subpopulation differences in bivalve immune parameters should be considered in the future analysis of immune parameters when studying the impact of pathogenic or environmental factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lu, Shi, Yao, Bai, Jiang and Ye.)

Autoimmune disease development depends on multiple factors, including genetic and environmental. Abnormalities such as sialylation levels and/or quality have been recently highlighted. The adjunction of sialic acid at the terminal end of glycoproteins and glycolipids is essential for distinguishing between self and non-self-antigens and the control of pro- or anti-inflammatory immune reactions. In autoimmunity, hyposialylation is responsible for chronic inflammation, the anarchic activation of the immune system and organ lesions. A detailed characterization of this mechanism is a key element for improving the understanding of these diseases and the development of innovative therapies. This review focuses on the impact of sialylation in autoimmunity in order to determine future treatments based on the regulation of hyposialylation.