Tamar Tak

Flow cytometers are highly sensitive scientific instruments that need to be in optimal condition to provide accurate and reliable data. To ensure consistent performance, cytometer performance is regularly assessed by measuring standardized reference particles during a quality control (QC) measurement. Cytometer manufacturers have developed dedicated modules in their operating software to monitor cytometer performance and award a pass or a fail for each QC to indicate if the instrument is performing to specifications. However, these are usually limited in their ease of use and data visualization options. For example, usually only a single parameter can be visualized and visualization over time is limited to 1 year or to the last baseline. Additionally, a shared interface to simultaneously monitor multiple cytometers from multiple manufacturers is not available. Furthermore, all instruments generate data that are not taken into account during QC, while these data can actually provide more insight in the performance of a flow cytometer and can allow early identification of problems. To facilitate more comprehensive analysis of instrument performance we have developed FlowCytoMonitoR, an interactive R‐shiny program that does not require coding experience and allows users to analyze QC data in full detail for multiple instruments. The program is currently compatible with instruments from three different vendors (BD Biosciences, Beckman Coulter, and Cytek Biosciences) and allows visualization of all QC metrics. FlowCytoMonitoR has proved invaluable to our Shared Resource Laboratory (SRL) in identifying and potentially predicting problems, thereby increasing flow cytometer uptime and service quality.

The kinetics of neutrophil development in human bone marrow remain largely unclear, despite the large implications for the mechanisms underlying host defense and inflammatory responses. Here, in situ DNA labeling was used to follow human neutrophil precursor maturation in bone marrow and blood. Human bone marrow samples were obtained by aspiration from healthy volunteers after short-term (6 hrs) labeling of dividing cells via oral intake of glucose containing the stable isotope deuterium. Mature and precursor populations were isolated at different times by fluorescence-activated cell sorting, and the deuterium incorporation in DNA was measured by GC/MS. Promyelocytes quickly incorporated deuterium in their DNA, whereas in mature neutrophils deuterium was only found from 6 days after intake. The enrichment curves of myelocytes, metamyelocytes, banded, and mature neutrophils mirrored that of the promyelocytes, shifted in time. This pattern indicated that neutrophil development in the bone marrow is well described with a linear conveyor belt model, i.e. following the "first-in/first-out" principle. In contrast to the current dogma, myelocytes did not divide, placing them in the postmitotic pool. Furthermore, the deuterium enrichment curves suggested that mature neutrophils constantly exchange between the bone marrow, blood, and tissue. Our unique labeling data of all stages of the neutrophil life cycle allowed us to estimate the lifespan of the mature neutrophil, deriving a value of at least 6 days. This study provides new insights into neutrophil development kinetics, informing the design and application of therapeutic strategies targeting granulopoiesis and the recruitment of mature neutrophils.

Accurate identification and quantification of eosinophils is critical for the diagnosis and monitoring of eosinophil-associated disorders. While flow cytometry remains a powerful tool for leukocyte characterization, conventional instruments equipped with 405 nm or 488 nm side scatter (SSC) detectors offer limited resolution for eosinophil discrimination overlap in scatter with neutrophils. Using a spectral flow cytometer equipped with six distinct SSC detectors, we report a novel, label-free approach for eosinophil detection leveraging high 808 nm near-infrared SSC (IRSSC) uniquely observed in human eosinophils. This optical signature is independent of antibody labeling, activation fixation, or permeabilization, and shows strong concordance with conventional CD66b/CD16 gating strategies (R = 0.997). Notably, the high 808 nm SSC is absent in murine eosinophils, suggesting a species-specific structural feature such as in human eosinophils. These findings establish IRSSC as a robust, reagent-free biomarker for eosinophil detection, with broad implications for both clinical diagnostics and translational immunology.

Runt-related transcription factor 1 (RUNX1) is a key regulator of hematopoietic differentiation. RUNX1 mutations in acute myeloid leukemia (AML) are associated with poor prognosis. One-third are frameshift mutations encoding an oncogenic protein with an elongated C-terminus translated in an alternative reading frame. Here, we investigated whether the alternative reading frame of oncogenic RUNX1 can be targeted by immunotherapy. We introduced a construct with a RUNX1 frameshift mutation into B-cell lines with common HLA class I alleles and identified 13 neopeptides by immunopeptidomics. To investigate whether these peptides are neoantigens, peptide-MHC tetramers were used to screen healthy individuals for RUNX1 neoantigen-specific CD8 T cells. T-cell clones were isolated against 5 neoantigens in 4 HLA alleles. Two neoantigens were recognized on an HLA-B*07:02-positive AML cell line with an endogenous RUNX1 frameshift mutation. The T-cell receptors (TCRs) of these clones were sequenced, and analyzed after transfer into CD8 T cells. One TCR induced effective killing of RUNX1-mutated AML cells in vitro and in immunodeficient mice. TCR-engineered T cells also killed patient-derived AML cells, including leukemic stem cells. In conclusion, we showed that RUNX1 frameshift mutations can be effectively targeted, demonstrating the potential relevance of TCR-based immunotherapy to treat patients with RUNX1-mutated AML.

Obesity‐induced chronic low‐grade inflammation, also known as metaflammation, results from alterations of the immune response in metabolic organs and contributes to the development of fatty liver diseases and type 2 diabetes. The diversity of tissue‐resident leukocytes involved in these metabolic dysfunctions warrants an in‐depth immunophenotyping in order to elucidate disease etiology. Here, we present a 30‐color, full spectrum flow cytometry panel, designed to (i) identify the major innate and adaptive immune cell subsets in murine liver and white adipose tissues and (ii) discriminate various tissue‐specific myeloid subsets known to contribute to the development of metabolic dysfunctions. This panel notably allows for distinguishing embryonically‐derived liver‐resident Kupffer cells from newly recruited monocyte‐derived macrophages and KCs. Furthermore, several adipose tissue macrophage (ATM) subsets, including perivascular macrophages, lipid‐associated macrophages, and pro‐inflammatory CD11c⁺ ATMs, can also be identified. Finally, the panel includes cell‐surface markers that have been associated with metabolic activation of different macrophage and dendritic cell subsets. Altogether, our spectral flow cytometry panel allows for an extensive immunophenotyping of murine metabolic tissues, with a particular focus on metabolically‐relevant myeloid cell subsets, and can easily be adjusted to include various new markers if needed.

Innate mononuclear phagocytic system (MPS) cells preserve mucosal immune homeostasis. We investigated their role at nasal mucosa following allergen challenge with house dust mite. We combined single-cell proteome and transcriptome profiling on nasal immune cells from nasal biopsies cells from 30 allergic rhinitis and 27 non-allergic subjects before and after repeated nasal allergen challenge. Biopsies of patients showed infiltrating inflammatory HLA-DRhi/CD14+ and CD16+ monocytes and proallergic transcriptional changes in resident CD1C+/CD1A+ conventional dendritic cells (cDC)2 following challenge. In contrast, non-allergic individuals displayed distinct innate MPS responses to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (MDSC: HLA-DRlow/CD14+ monocytes) and cDC2 expressing inhibitory/tolerogenic transcripts. These divergent patterns were confirmed in ex vivo stimulated MPS nasal biopsy cells. Thus, we identified not only MPS cell clusters involved in airway allergic inflammation but also highlight novel roles for non-inflammatory innate MPS responses by MDSC to allergens in non-allergic individuals. Future therapies should address MDSC activity as treatment for inflammatory airway diseases.

Summary: Tissue-macrophage populations are constituted by a mosaic of phenotypes, yet new methods are needed to link metabolic status to the range of phenotypes in vivo . We therefore designed a high-dimensional panel for spectral flow cytometry to investigate the heterogeneity of tissue macrophage metabolism at steady-state, and their metabolic adaptation in response to infection. Distinct metabolic profiles were observed between tissue macrophages from different peripheral organs, as well as within populations from a specific site. As such, our data show multiple metabolic states in macrophages corresponding to relative stages of maturity in both the peritoneal cavity and small intestine. Immune perturbation with helminth infection further showed that peritoneal macrophages acquire an overall highly metabolically active profile, whereas responding intestinal macrophages displayed minimal changes in their metabolic phenotype. Thus, we demonstrate that high-dimensional, flow-based analysis is an exciting method to interrogate the metabolic heterogeneity and dynamics of tissue-macrophage populations.

Neutrophils are the most abundant white blood cell, and they differentiate in homeostasis in the bone marrow from hematopoietic stem cells (HSCs) via multiple intermediate progenitor cells into mature cells that enter the circulation. Recent findings support a continuous model of differentiation in the bone marrow of heterogeneous HSCs and progenitor populations. Cell fate decisions at the levels of proliferation and differentiation are enforced through expression of lineage-determining transcription factors and their interactions, which are influenced by intrinsic (intracellular) and extrinsic (extracellular) mechanisms. Neutrophil homeostasis is subjected to positive-feedback loops, stemming from the gut microbiome, as well as negative-feedback loops resulting from the clearance of apoptotic neutrophils by mature macrophages. Finally, the cellular kinetics regarding the replenishing of the mature neutrophil pool is discussed in light of recent contradictory data.

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates^1,2,3,4,5,6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c⁺ natural killer (NK) cells and CD4⁺ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DR^low monocytes, CD38⁺PD1⁺CD4⁺ T effector (T_eff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127⁺ granulocytes and CD38⁺CD8⁺ tissue-resident memory T cells (T_RM). SARS-CoV-2-specific CD8⁺ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.