Advances In Flow Cytometry Research Articles

Distinct Immune Profiles of Exhausted Effector and Memory CD8+ T Cells in Individuals With Filarial Lymphedema

CD8+ T cells are crucial for the clearance of viral infections, and current research begins to highlight their importance in parasitic diseases too. In-depth research about characteristics of CD8+ T-cell subsets and exhaustion remains uncertain, especially during filariasis, a chronic helminth infection. Lymphatic filariasis, elicited by Wuchereria bancrofti, remains a serious health problem in endemic areas in Ghana, especially in those suffering from morbidity due to lymphedema (LE). In this observational study, the characteristics and profiles of CD8+ T cells were compared between asymptomatic Wuchereria bancrofti-infected individuals, uninfected endemic normals, and those with LE (grades 2–6). Focusing on exhausted memory (CD8+exmem: CD8+ T-betdimEomeshi) and effector (CD8+exeff: CD8+T-bethiEomesdim) CD8+ T-cell subsets, advanced flow cytometry revealed that LE individuals presented reduced frequencies of IFN-γ+CD8+exmem T cells expressing Tim-3 or LAG-3 which negatively correlated to the presence of LE. Moreover, the LE cohort further showed significantly higher frequencies of IL-10+CD8+exeff T cells expressing either Tim-3, LAG-3, CD39, KLRG-1, or PD-1, all associated markers of exhaustion, and that these frequencies positively correlated with the presence of LE. In summary, this study shows that distinct exhausted CD8+ T-cell subsets are prominent in individuals suffering from LE, suggesting that enhanced inflammation and constant immune activation might drive exhaustion of CD8+ T cells. Since T-cell exhaustion is known to be associated with insufficient control of persisting antigen, the data presented here reveals that these CD8+ T-cell exhaustion patterns in filarial LE should be taken into consideration for prevention and control management of LE.

Abstract 1856: Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry

Abstract Tumor-specific monoclonal antibodies (mAb) which induce NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) have proven to be successful against several types of cancer. Evaluating the potency of a new, tumor-specific mAb requires testing with multiple sources of donor cells since the degree of NK cell-mediated ADCC is known to be variable between patients. Thus, in addition to evaluating whether a new tumor-specific mAb can effectively induce NK cell-mediated ADCC, it is also important to characterize the donor NK cells utilized in order to provide insight into potential treatment efficacy. In the current study, we investigated the application of a novel, high throughput assay in assessing the ADCC potency of different anti-hCD20 mAbs against a Burkitt's lymphoma cell line using effector cells obtained from multiple donors. This multiplexed assay incorporates a cells and beads format to enable the simultaneous measurement of target cell killing, along with enumeration and phenotyping of the NK cells, and quantification of up to 8 secreted effector proteins and cytokines. The assay requires a sample volume of just 10 µl and can be performed in a single well of a 96 or 384 well plate using the iQue® platform for advanced flow cytometry. Data analysis and visualization was done using the integrated iQue Forecyt® software package. The results showed a mAb concentration-dependent increase in tumor cell killing, along with corresponding increases in secretion of Granzyme B and CD178 (Fas Ligand), and production of IFNγ, TNF, and RANTES. The degree of NK cell-mediated ADCC and effector protein secretion levels were dependent on both the anti-hCD20 mAb and donor cells used, with higher levels of ADCC and effector protein secretion observed using a non-fucosylated anti-hCD20 mAb as compared to a conventional, fucosylated anti-hCD20 mAb. This method enabled the rapid assessment of the potency of different mAbs in ADCC mediated by NK cells, and also effectively distinguished potential differences in mAb efficacy between donors. Citation Format: Julie Lovchik, Mark Carter. Donor and antibody diversity in NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) detected using an optimized multiplexed assay and advanced flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1856.

Identification of the predominant human NK cell effector subset mediating ADCC against HIV-infected targets coated with BNAbs or plasma from PLWH.

The potential of immunotherapy strategies utilizing broadly neutralizing antibodies (BNAbs), such as 3BNC117 and 10-1074, to limit viral replication while also facilitating clearance of HIV infected cells has heightened interest in identifying the predominant NK effector subset(s) capable of mediating antibody dependent cellular cytotoxicity (ADCC). Utilizing advanced polychromatic flow cytometry, we identified that CD57 positive NK cells from ART-suppressed in People Living With HIV (PLWH) expressed significantly higher levels of the CD16 FcγR receptor, 2B4 ADCC coreceptor, and HLA-DR activation marker while NKG2C positive NK cells expressed significantly higher levels of the CD2 ADCC coreceptor (p<0.001, n = 32). Functionally, CD57 positive NK cells from ART-suppressed PLWH with either high or low NKG2C expansion exhibited significantly enhanced degranulation and IFN-γ production against heterologous gp120-coated ADCC targets coated with HIV reference plasma compared to CD57 negative NK cells (p = 0.0029, n = 11). CD57 positive NK cells from control donors lacking NKG2C expansion also exhibited significantly more degranulation and IFN-γ production at every timepoint tested against both heterologous ADCC targets (p = 0.019, n = 9) and HIV-1 infected autologous CD4+ primary T cells coated with BNAbs. Together, our data support CD57 positive and NKG2C positive NK cells as the predominant ADCC effector subsets capable of targeting HIV-infected CD4+ cells in the presence of 3BNC117 and 10-1074 immunotherapy.

Finding quality in complexity: how cellular therapeutics are shifting analytical paradigms for clinical supply and product manufacturing

Recent clinical successes of CAR-T cell approaches like Kymriah&#174;&#160;or Yescarta&#174;&#160;have promoted a boost of clinical trial applications for cellular therapeutics (CT). In terms of quality control, analytical strategies required to assess living cells represent a shift of paradigms compared to how chemical or recombinant biological drugs are typically addressed in a regulated GMP environment. This article elaborates on the corresponding CT-specific challenges and aims to provide standardized strategies for mitigation where available. Furthermore, a generic scheme of critical quality attributes is proposed, which takes into account largely different types of CT, including for example mesenchymal stem cells, CAR immune cells or stem-cell derived cardiomyocytes. Somewhat counterintuitively, rather few analytical core platforms (advanced GMP cell culture, flow cytometry, ddPCR, NGS and microscopy) should suffice to execute the vast majority of the corresponding analytical tasks throughout the product lifecycle. Obviously, internal mastery of these platforms should be achieved in order to ensure proper support of process development activities and product progression throughout clinical development, while other analytical tasks might be prone to be outsourced to contract research organizations.

14 Opening the Black Box of Fertility Prediction

Abstract Analysis of the U.S. swine herd shows variation in pregnancy rate is more attributable to male-factor subfertility than the dam. To date, a limited degree of correlations has been observed between conventional semen analysis parameters and actual fertility after standard quality cutoffs are met. Thus, a clear ability to predict male-factor fertility is lacking. Knowledge of what makes fertilization competent spermatozoa has been long sought after for centuries. It was only in the last half-century that we understood spermatozoa undergo a biological process after ejaculation to acquire the capacity to fertilize. Since then, work has been done to elucidate the molecular pathways involved in sperm capacitation. Recent technological advances in flow cytometry, namely image-based flow cytometry, allows for high-throughput, single-cell phenotyping. Single-cell phenotyping with biomarkers reflecting significant sperm capacitation events, mitochondrial status, cell health, and more, allows multi-million bioimage data sets to be easily attained. These datasets can then be analyzed utilizing machine and deep learning analytic methods and correlated with single sire field fertility data to open the black box of boar fertility prediction. Our findings establish a new paradigm in sperm function and pave the way for accurate fertility prediction in future precision agriculture applications. This work was supported by the National Institute of Food and Agriculture (NIFA), U.S. Department of Agriculture (USDA) award number 2019-67012-29714.

Research Techniques Made Simple: Experimental Methodology for Imaging Mass Cytometry

Technological advances in flow cytometry and the development of mass cytometry by time-of-flight (CyTOF) have led to progressive increases in the number of proteins and biochemical processes that can be simultaneously measured. The most recent development of these platforms, imaging mass cytometry (IMC), allows for the visualization of up to 40 unique cellular markers and also employs rare metal isotopes conjugated to antibodies. However, IMC also adds the important benefit of preserving two-dimensional (2D) tissue architecture; this is accomplished by staining in situ and direct tissue vaporization followed by generation of a 2D spectral reconstruction using CyTOF-captured events. We review the experimental methodology for IMC that enables high-resolution multilayer images depicting protein expression, cellular localization, and interaction in situ in dermatology research.

A High-Dimensional Window into the Micro-Environment of Triple Negative Breast Cancer.

Simple SummaryTriple negative breast cancer (TNBC) shows a substantial level of genomic, cellular, and phenotypic heterogeneity. While genomic heterogeneity and subclonal diversity are prevalent in this subgroup of tumors, a growing body of evidence indicates that the disease course depends on the interaction between cancer cells and the tumor micro-environment (TME). The TME is not static and can change over time, owing to differences in cell numbers, phenotypes, and spatial relationships. Efforts to further elucidate the TME have been aided by a plethora of new technologies that study tumors in a high-dimensional manner. These high-dimensional technologies enable comprehensive analysis of cell phenotypes at the single cell level or the spatial relationships of tumor and immune cells. In this review, we discuss studies in TNBC that unravel specific spatial patterns of cells in the breast TME and single cell phenotypes.Providing effective personalized immunotherapy for triple negative breast cancer (TNBC) patients requires a detailed understanding of the composition of the tumor microenvironment. Both the tumor cell and non-tumor components of TNBC can exhibit tremendous heterogeneity in individual patients and change over time. Delineating cellular phenotypes and spatial topographies associated with distinct immunological states and the impact of chemotherapy will be necessary to optimally time immunotherapy. The clinical successes in immunotherapy have intensified research on the tumor microenvironment, aided by a plethora of high-dimensional technologies to define cellular phenotypes. These high-dimensional technologies include, but are not limited to, single cell RNA sequencing, spatial transcriptomics, T cell repertoire analyses, advanced flow cytometry, imaging mass cytometry, and their integration. In this review, we discuss the cellular phenotypes and spatial patterns of the lymphoid-, myeloid-, and stromal cells in the TNBC microenvironment and the potential value of mapping these features onto tumor cell genotypes.

Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical upbeta -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-upbeta -galactosidase activity using the fluorogenic substrate, C{_{12}}FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in upbeta -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p le 0.001 for the fluorogenic C{_{12}}FDG method, and r = 0.72, p le 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

PCR Technology to Identify Minimal Residual Disease.

Although new techniques (i.e., droplet digital-PCR, next-generation sequencing, advanced flow cytometry) are being developed, DNA-based allele-specific real-time quantitative (RQ)-PCR is still the gold standard for sensitive and accurate immunoglobulin/T cell receptor (IG/TR)-based minimal residual disease (MRD) monitoring, allowing the detection of up to 1 leukemic cell in 100,000 normal lymphoid cells. We herewith describe the standard PCR procedure which has been developed and standardized (with minor modification in single labs) through the last 20years of activity of the EuroMRD Consortium, a volunteer activity of expert laboratories that is continuously providing education, standardization, quality control rounds, and guidelines for interpretation of RQ-PCR data.

Chromosomal abnormality, laboratory techniques, tools and databases in molecular Cytogenetics.

Cytogenetics is concerned with the structure and number of chromosomes (Karyotyping) and their abnormalities not only in congenital but also in acquired genetic disorders. Chromosomal abnormalities can form when there is an error occurred in chromosome number and, or their structural changes. Such changes happen by itself or inductively by environmental agents like chemical reagents, radiation, etc. Cytogenetics techniques used to understand chromosomal disorders and their relationship to health and disease provide not only valuable clues about chromosome breakage and DNA repair mechanisms but also a more proper understanding of their relationship to cause various illnesses. In this sense, to evaluate chromosomal imbalance and rearrangement, alternative experimental methods have been expanded. Currently, Cytogenetics evolves into a multidimensional science that led to promoting both theoretically and technologically advanced molecular biology, flow cytometry, bioinformatics, and phylogeny. This study investigates the unique laboratory Cytogenetics methods, databases, algorithms, and software used molecular Cytogenetics to identify various chromosomal abnormalities.

Circulating histone signature of human lean metabolic-associated fatty liver disease (MAFLD)

BackgroundAlthough metabolic associate fatty liver disease (MAFLD) is associated with obesity, it can also occur in lean patients. MAFLD is more aggressive in lean patients compared to obese patients, with a higher risk of mortality. Specific biomarkers to diagnose differentially lean or overweight MAFLD are missing. Histones and nucleosomes are released in the bloodstream upon cell death. Here, we propose a new, fast, imaging and epigenetics based approach to investigate the severity of steatosis in lean MAFLD patients.ResultsA total of 53 non-obese patients with histologically confirmed diagnosis of MAFLD were recruited. Twenty patients displayed steatosis grade 1 (0–33%), 24 patients with steatosis grade 2 (34–66%) and 9 patients with steatosis grade 3 (67–100%). The levels of circulating nucleosomes were assayed using enzyme-linked immunosorbent assay, while individual histones or histone dimers were assayed in serum samples by means of a new advanced flow cytometry ImageStream(X)-adapted method. Circulating nucleosome levels associated poorly with MAFLD in the absence of obesity. We implemented successfully a multi-channel flow methodology on ImageStream(X), to image single histone staining (H2A, H2B, H3, H4, macroH2A1.1 and macroH2A1.2). We report here a significant depletion of the levels of histone variants macroH2A1.1 and macroH2A1.2 in the serum of lean MAFLD patients, either individually or in complex with H2B.ConclusionsIn summary, we identified a new circulating histone signature able to discriminate the severity of steatosis in individuals with lean MAFLD, using a rapid and non-invasive ImageStream(X)-based imaging technology.

Abstract 311: Phenotyping tumor infiltrating lymphocytes (TILs) in vitro using 3D cell models and advanced flow cytometry

Abstract The importance of the immune system in cancer treatment is now recognized with a range of therapies utilizing its power to more effectively target autologous cancerous cells. Multiple immune cells actively infiltrate the tumor in response to chemokine signaling in an attempt to halt disease progression. Tumor infiltrating lymphocytes (TILs) elicit altered responses (anti- or pro-tumorigenic) dependent on the microenvironment and stage of disease. Individuals with high numbers of TILs are expected to be more responsive to neoadjuvant chemotherapy and have improved pathological complete response rates. Therefore the ability to study TIL response to immune and chemotherapies in vitro is ever more important. Here, we present an advanced 3D model of TILs, enabling the simultaneous quantification of phenotypes, cell type ratios and activation status of infiltrated and non-infiltrated cells. Single spheroids were formed in ultra-low attachment 96 well plates for 3 days from a variety of cell types, namely ovarian (SKOV3), breast (BT474) and lung (A549) cancer. PBMCs were added for a period of 24 h (pre-activated) or 40 h (in well activation) before non-infiltrated PBMCs were removed and the spheroids were dissociated. Analysis of subsets for both infiltrating and non-infiltrating T cells was performed using the T Cell Activation and Cytokine Profiling Kit (Sartorius) and the iQue3 Flow Cytometer (Sartorius). Tumor type-specific infiltration was observed, with A549 spheroids exhibiting 54% reduced infiltration of CD3+ TILs compared with spheroids formed of BT474. Despite this reduced infiltration, A549 exhibited a higher CD8:CD4 ratio of 1.4 ± 0.13 compared with 0.6 ± 0.04 for BT474. An altered ratio of CD8:CD4 T cells has been reported in multiple tumor types, with increasing CD4+ cells often recognized with poor outcome. Interestingly, infiltrating T cells were observed to have significantly increased activation markers (CD69, CD25 and HLA-DR) compared to their non-infiltrated counterparts. For example in BT474 spheroids, maximal CD69 expression of infiltrated CD3+ cells was reached with just 247 CD3/CD28 Dynabeads/well, compared with 20K/well for non-infiltrated CD3+ cells. T cell infiltration can be affected by stromal cells, which produce extracellular matrix components contributing to a stiffer ECM. This remodeling of the ECM affects the quantities and types of T cells that can infiltrate into a tumor. When including normal human dermal fibroblasts in BT474 spheroids at a 1:1 ratio, a 60% reduction of maximal infiltration of CD3+ cells was observed. Although infiltration declined, T cell activation levels were comparable to those observed in spheroids containing only BT474 cells. These data highlight the complexity of modelling TILs in vitro, with marked dependence on tumor cell type and the presence of stromal cells. Utilizing flow cytometry alongside advanced assays has the potential to offer translational models for the development of therapeutics modulating T cell infiltration. Citation Format: Donald Weldon, Lauren Kelsey, Kirsty McBain, Clare Szybut, Timothy Dale. Phenotyping tumor infiltrating lymphocytes (TILs) in vitro using 3D cell models and advanced flow cytometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 311.

Abstract 3251: Assessment of NK cell-mediated killing and phenotypic analysis using advanced flow cytometry and an optimized multiplexed assay

Abstract In this study, a novel multiplexed assay was developed that simultaneously measures NK cell phenotype and function in relation to tumor target killing. Natural Killer (NK) cells are an essential part of the innate immune system, interface with the adaptive immune system, and play a critical role in tumor immune surveillance and anti-tumor responses. Although chimeric antigen receptor (CAR)-modified T cells have proven to be highly effective as an anti-cancer therapeutic, there are potential problems associated with their use, particularly the risk of inducing graft-versus-host disease (GVHD) resulting in aberrant cytokine release. In contrast to T cells, NK cells are potent cytotoxic effector cells that do not require MHC restriction and have a low risk of inducing GVHD. Recent advances in the development of CAR-modified NK cells have shown great promise for clinical utilization in cancer immunotherapy with capability for both direct tumor killing as well as for use in combination therapy with monoclonal antibodies to boost NK cell antibody-dependent cell-mediated cytotoxicity (ADCC). A critical process in the development of new NK cell-mediated therapeutics is the ability to expediently assay, screen, and analyze data for NK cell activation and tumor killing. To address this need, a novel multiplexed assay was developed that measures tumor target cell killing, target-dependent expression of activation markers (CD69 and CD25), and the quantitation of secreted granzyme B, IFNγ, and TNFα in a single well of a microtiter plate. To validate the assay, a direct NK cell-tumor cell killing model was used. Purified human NK cells were mixed with target cells at different ratios. Tumor cell killing, NK cell activation status, and quantification of secreted proteins were measured at 4 and 24 hours after co-culture. Assay data acquisition was performed on Intellicyt iQue3® platform for advanced flow cytometry. Data analysis and visualization was performed using the integrated ForeCyt® software package. Tumor cell killing along with granzyme B release, was evident by 4 hours following co-culture, and both were increased with the increase in effector to target ratio (E:T). Killing was further enhanced when NK cells were stimulated with IL-2 and IL-15. As expected, an increase in CD69 and CD25 expression was also observed following cytokine stimulation with further increase upon co-culture of NK cells with tumor cell targets, along with increased levels of secreted IFNγ and TNFα. Further validation of the assay using an ADCC model was also performed. These data show that a miniaturized, multiplexed NK cell killing assay, combined with high throughput flow cytometry, is a powerful tool to rapidly screen CAR-NK cell effector functions and other NK cell based therapeutics. Citation Format: Julie Lovchik, Sarita Chauhan, John O'Rourke. Assessment of NK cell-mediated killing and phenotypic analysis using advanced flow cytometry and an optimized multiplexed assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3251.

Impact of donor extracellular vesicle release on recipient cell "cross-dressing" following clinical liver and kidney transplantation.

In several murine models of transplantation, the "cross-dressing" of recipient antigen presenting cells (APCs) with intact donor major histocompatibility complex (MHC) derived from allograft-released small extracellular vesicles (sEVs) has been recently described as a key mechanism in eliciting and sustaining alloimmune responses. Investigation of these processes in clinical organ transplantation has, however, been hampered by the lack of sensitivity of conventional instruments and assays. We have employed advanced imaging flow cytometry (iFCM) to explore the kinetics of allograft sEV release and the extent to which donor sEVs might induce cross-dressing following liver and kidney transplantation. We report for the first time that recipient APC cross-dressing can be transiently detected in the circulation shortly after liver, but not kidney, transplantation in association with the release of HLA-bearing allograft-derived sEVs. In liver transplant recipients the majority of circulating cells exhibiting donor HLA are indeed cross-dressed cells and not passenger leukocytes. In keeping with experimental animal data, the downstream functional consequences of the transfer of circulating sEVs harvested from human transplant recipients varies depending on the type of transplant and time posttransplant. sEVs released shortly after liver, but not kidney, transplantation exhibit immunoinhibitory effects that could influence liver allograft immunogenicity.

Novel Markers of Angiogenesis in the Setting of Cognitive Impairment and Dementia.

Aberrant angiogenesis may play a role in the development of Alzheimer's disease and related dementia. To explore the relationship between angiogenesis activity and evidence of neurodegeneration among older adults. Cross-sectional study of 49 older adults clinically characterized as cognitively normal, mild cognitive impairment, or early Alzheimer's disease. In addition to neuroimaging, we completed assays on peripheral blood, including: vascular endothelial growth factor, tumor necrosis factor, fibroblast growth factor, and amyloid-β peptide 40. We used advanced polychromatic flow cytometry to phenotype circulating mononuclear cells to assess angiogenesis activity. Although we documented differences in cognitive performance, structural changes on neuroimaging, and burden of amyloid and tau on positron emission tomography, angiogenesis activity did not vary by group. Interestingly, VEGF levels were shown to be increased among subjects with mild cognitive impairment. In ANCOVA models controlling for age, sex, intracranial volume, and monocyte subpopulations, angiogenesis activity was correlated with increased white matter hyperintensities. We demonstrate a significant association between angiogenesis activity and cerebrovascular disease. To better understand the potential of angiogenesis as an intervention target, longitudinal studies are needed.

Development of a cells and bead-based, advanced flow cytometry assay for exhausted T cell quantitation

Abstract T cells can become exhausted due to persistent antigen exposure during chronic infections and cancer. Exhausted T cells exhibit decreased effector functions and express inhibitory receptors (IRs). The exhausted phenotype may be protective in the context of chronic infection, however, it allows tumors to avoid immune surveillance and immune cell mediated killing. Recent therapies block the IRs to reinvigorate exhausted T cells. A miniaturized, multiplexed flow cytometry assay was developed to measure expression of IRs on T cells while simultaneously measuring viability, proliferation, and cytokine secretion in microtiter plates. Data was acquired on the Intellicyt® iQue platform and analyzed using Forecyt®. Templates were prepared for ease of data analysis, to auto-generate gating, standard curves and sample quantitation and rapid, actionable results. For proof of concept studies, human PBMCs, purified CD3+, or in vitro exhausted purified CD3+ cells were treated with anti-CD3/CD28 and cells were analyzed at 1, 2, and 3 days post-stimulation. Non-exhausted cells showed a concentration and time dependent increase in IR expression. Persistent, high levels of the IR markers PD-1, LAG-3 and TIM-3 were measured in exhausted cells. Stimulated exhausted T cells secreted stable and moderate levels of IFN-γ (&amp;lt;1650 pg/ml) and TNF (&amp;lt;750 pg/ml) over time whereas non-exhausted cells showed time and concentration dependent increases in IFN-γ (13,319 pg/ml) and TNF (3,711 pg/ml) upon stimulation. The ability of this assay to examine T cell response allows screening for molecules that reverse T cell exhaustion, screening of modified cells such as CAR-Ts or selection of clones that exhibit low levels of IRs and show robust response to stimulation.

Pitfalls in the characterization of circulating and tissue-resident human γδ T cells.

Dissection of the role and function of human γδ T cells and their heterogeneous subsets in cancer, inflammation, and auto-immune diseases is a growing and dynamic research field of increasing interest to the scientific community. Therefore, harmonization and standardization of techniques for the characterization of peripheral and tissue-resident γδ T cells is crucial to facilitate comparability between published and emerging research. The application of commercially available reagents to classify γδ T cells, in particular the combination of multiple Abs, is not always trouble-free, posing major demands on researchers entering this field. Occasionally, even entire γδ T cell subsets may remain undetected when certain Abs are combined in flow cytometric analysis with multicolor Ab panels, or might be lost during cell isolation procedures. Here, based on the recent literature and our own experience, we provide an overview of methods commonly employed for the phenotypic and functional characterization of human γδ T cells including advanced polychromatic flow cytometry, mass cytometry, immunohistochemistry, and magnetic cell isolation. We highlight potential pitfalls and discuss how to circumvent these obstacles.

Minimal Residual Disease Negativity Does Not Overcome Poor Prognosis in High-Risk Multiple Myeloma: A Single-Center Retrospective Study

BackgroundMinimal residual disease (MRD) is a standard measurement for response assessment in multiple myeloma (MM). Despite new treatments, high-risk MM patients continue to have poor prognosis. We evaluated the effect of MRD negativity in high-risk versus standard-risk patients. Patients and MethodsWe retrospectively evaluated all consecutive MM patients who underwent routine MRD testing by 1-tube 8-color advanced flow cytometry with 2,000,000 events and sensitivity level 10−5 at our center from 2015 to 2018 after initial therapy. Kaplan-Meier and log-rank test were used to assess survival estimates and differences between study groups. ResultsOne hundred thirty-six patients with MRD testing after initial therapy or autologous stem-cell transplantation were identified. At a median follow-up of 14 months (range, 1-36 months), progression-free survival and overall survival were significantly worse in high-risk versus standard-risk patients. During the study period, 50% of high-risk group had experienced disease progression (relapse and/or death) versus 20% in the standard-risk group (P = .0006). No patients with standard-risk died, but 4 (14%) in the high-risk group did (P = .0007). Regardless of MRD status, high-risk patients had statistically significant worse progression-free survival than standard-risk patients. At median follow-up, those with disease 10% standard-risk/MRD negative; 20% standard-risk/MRD positive; 40% high-risk/MRD negative; and 45% high-risk/MRD positive had either experienced relapse or died (P = .0041). MRD status did not significantly affect overall survival in either group (P = .0914); however, longer follow-up is needed to assess survival. ConclusionGenetic abnormalities remain a powerful prognostic indicator for MM, regardless of MRD status. For newly diagnosed MM patients treated with novel triple-drug initial therapy and frontline autologous stem-cell transplantation, MRD-negative status did not mitigate the poor-prognosis outcomes of high-risk MM patients.

Intact Viral Particle Counts Measured by Flow Virometry Provide Insight into the Infectivity and Genome Packaging Efficiency of Moloney Murine Leukemia Virus

Murine leukemia viruses (MLVs) have long been used as a research model to further our understanding of retroviruses. These simple gammaretroviruses have been studied extensively in various facets of science for nearly half a century, yet we have surprisingly little quantitative information about some of the basic features of these viral particles. These include parameters such as the genome packaging efficiency and the number of particles required for a productive infection. The reason for this knowledge gap relies primarily on the technical challenge of accurately measuring intact viral particles from infected cell supernatants. Virus-infected cells are well known to release soluble viral proteins, defective viruses, and extracellular vesicles (EVs) harboring viral proteins that may mimic viruses, all of which can skew virus titer quantifications. Flow virometry, also known as nanoscale flow cytometry or simply small-particle flow cytometry, is an emerging analytical method enabling high-throughput single-virus phenotypic characterizations. By utilizing the viral envelope glycoprotein (Env) and monodisperse light scattering characteristics as discerning parameters of intact virus particles, here, we analyzed the basic properties of Moloney MLV (M-MLV). We show that <24% of the total p30 capsid protein measured in infected cell supernatants is associated with intact viruses. We calculate that about one in five M-MLV particles contains a viral RNA genome pair and that individual intact particle infectivity is about 0.4%. These findings provide new insights into the characteristics of an extensively studied prototypical retrovirus while highlighting the benefits of flow virometry for the field of virology.IMPORTANCE Gammaretroviruses, or, more specifically, murine leukemia viruses (MLVs), have been a longstanding model for studying retroviruses. Although being extensively analyzed and dissected for decades, several facets of MLV biology are still poorly understood. One of the primary challenges has been enumerating total intact virus particles in a sample. While several analytical methods can precisely measure virus protein amounts, MLVs are known to induce the secretion of soluble and vesicle-associated viral proteins that can skew these measurements. With recent technological advances in flow cytometry, it is now possible to analyze viruses down to 90 nm in diameter with an approach called flow virometry. The technique has the added benefit of being able to discriminate viruses from extracellular vesicles and free viral proteins in order to confidently provide an intact viral particle count. Here, we used flow virometry to provide new insights into the basic characteristics of Moloney MLV.

Current Projection Methods-Induced Biases at Subgroup Detection for Machine-Learning Based Data-Analysis of Biomedical Data.

Advances in flow cytometry enable the acquisition of large and high-dimensional data sets per patient. Novel computational techniques allow the visualization of structures in these data and, finally, the identification of relevant subgroups. Correct data visualizations and projections from the high-dimensional space to the visualization plane require the correct representation of the structures in the data. This work shows that frequently used techniques are unreliable in this respect. One of the most important methods for data projection in this area is the t-distributed stochastic neighbor embedding (t-SNE). We analyzed its performance on artificial and real biomedical data sets. t-SNE introduced a cluster structure for homogeneously distributed data that did not contain any subgroup structure. In other data sets, t-SNE occasionally suggested the wrong number of subgroups or projected data points belonging to different subgroups, as if belonging to the same subgroup. As an alternative approach, emergent self-organizing maps (ESOM) were used in combination with U-matrix methods. This approach allowed the correct identification of homogeneous data while in sets containing distance or density-based subgroups structures; the number of subgroups and data point assignments were correctly displayed. The results highlight possible pitfalls in the use of a currently widely applied algorithmic technique for the detection of subgroups in high dimensional cytometric data and suggest a robust alternative.