High-parameter Flow Cytometry Research Articles

Immune perturbations in human pancreas lymphatic tissues prior to and after type 1 diabetes onset.

Autoimmune destruction of pancreatic β cells results in type 1 diabetes (T1D), with pancreatic immune infiltrate representing a key feature in this process. Studies of human T1D immunobiology have predominantly focused on circulating immune cells in the blood, while mouse models suggest diabetogenic lymphocytes primarily reside in pancreas-draining lymph nodes (pLN). A comprehensive study of immune cells in human T1D was conducted using pancreas draining lymphatic tissues, including pLN and mesenteric lymph nodes, and the spleen from non-diabetic control, β cell autoantibody positive non-diabetic (AAb+), and T1D organ donors using complementary approaches of high parameter flow cytometry and CITEseq. Immune perturbations suggestive of a proinflammatory environment were specific for T1D pLN and AAb+ pLN. In addition, certain immune populations correlated with high T1D genetic risk independent of disease state. These datasets form an extensive resource for profiling human lymphatic tissue immune cells in the context of autoimmunity and T1D.

SARS-CoV-2 Vaccination Responses in Anti-CD20-Treated Progressive Multiple Sclerosis Patients Show Immunosenescence in Antigen-Specific B and T Cells.

Clinical, pathological, and imaging evidence in multiple sclerosis (MS) shows that inflammation starts early and progresses with age. B cells play a central role in this process, contributing to cytokine production, defective regulatory functions, and abnormal immunoglobulin production, even in the central nervous system. Anti-CD20 (aCD20) therapies, which deplete CD20+ B cells, are largely used in the treatment of both relapsing remitting (RR) and progressive (PR) forms of MS. Although effective against MS symptoms and lesions detectable by magnetic resonance imaging, aCD20 therapies can reduce the immune response to COVID-19 vaccination. By using high-parameter flow cytometry, we examined the antigen-specific (Ag+) immune response six months post-third COVID-19 mRNA vaccination in MS patients with RR and PR forms on aCD20 therapy. Despite lower Ag+ B cell responses and lower levels of anti-SARS-CoV2, both total and neutralizing antibodies, RR and PR patients developed strong Ag+ T cell responses. We observed similar percentages and numbers of Ag+ CD4+ T cells and a high proportion of Ag+ CD8+ T cells, with slight differences in T cell phenotype and functionality; this, however, suggested the presence of differences in immune responses driven by age and disease severity.

Interleukin-2 immunotherapy reveals human regulatory T cell subsets with distinct functional and tissue-homing characteristics

Due to its stimulatory potential for immunomodulatory CD4+ regulatory T (Treg) cells, low-dose interleukin-2 (IL-2) immunotherapy has gained considerable attention for the treatment of autoimmune diseases. In this investigator-initiated single-arm non-placebo-controlled phase-2 clinical trial of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE) patients, we generated a comprehensive atlas of invivo human immune responses to low-dose IL-2. We performed an in-depth study of circulating and cutaneous immune cells by imaging mass cytometry, high-parameter flow cytometry, transcriptomics, and targeted serum proteomics. Low-dose IL-2 stimulated various circulating immune cells, including Treg cells with a skin-homing phenotype that appeared in the skin of SLE patients in close interaction with endothelial cells. Analysis of surface proteins and transcriptomes revealed different IL-2-driven Treg cell activation programs, including gut-homing CD38+, skin-homing HLA-DR+, and highly proliferative inflammation-homing CD38+ HLA-DR+ Treg cells. Collectively, these data define the distinct human Treg cell subsets that are responsive to IL-2 immunotherapy.

Bacterial vaginosis-driven changes in vaginal T cell phenotypes and their implications for HIV susceptibility.

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome that is prevalent in reproductive-age women worldwide. Adverse outcomes associated with BV include an increased risk of sexually acquired Human Immunodeficiency Virus (HIV), yet the immunological mechanisms underlying this association are not well understood. To investigate BV driven changes to cervicovaginal tract (CVT) and circulating T cell phenotypes, participants with or without BV provided vaginal tract (VT) and ectocervical (CX) tissue biopsies and peripheral blood mononuclear cells (PBMC). Immunofluorescence analysis of genital mucosal tissues revealed a reduced density of CD3+CD4+CCR5+ cells in the VT lamina propria of individuals with compared to those without BV (median 243.8 cells/mm2 BV- vs 106.9 cells/mm2 BV+, p=0.043). High-parameter flow cytometry of VT biopsies revealed an increased frequency in individuals with compared to those without BV of dysfunctional CD39+ conventional CD4+ T cells (Tconv) (median frequency 15% BV- vs 30% BV+, padj=0.0331) and tissue-resident CD69+CD103+ Tconv (median frequency 24% BV- vs 38% BV+, padj=0.0061), previously reported to be implicated in HIV acquisition and replication. Our data suggests that BV elicits diverse and complex VT T cell alterations and expands on potential immunological mechanisms that may promote adverse outcomes including HIV susceptibility.

Setting the gold standard: Commentary on designing and optimizing high-parameter flow cytometry panels.

Setting the gold standard: Commentary on designing and optimizing high-parameter flow cytometry panels.

Abstract A021: The role of MGAT5 in sex-dependent regulation of CD8+ T cells in muscle-invasive bladder cancer

Abstract Biological sex significantly affects bladder cancer incidence and outcome. Although men have much greater incidence of bladder cancer than women, females tend to be diagnosed with more advanced tumors and have worse outcomes. The mechanisms driving these differences are likely multifactorial, with biological factors playing an important role, however, much of the biology underlying these differences is unknown. The immune system is a critical regulator of cancer biology, and immune features have been associated with bladder cancer grade, stage, progression, and response to therapies. Sex dramatically affects the immune system including anti-tumor immunity. However, how sex and immunity intersect to affect clinical outcomes in bladder cancer patients remains unclear. CD8+ T cells are a key part of anti-tumor immune responses and the importance of inhibitory receptors such as programmed cell death-1 (PD-1), is now well established as a mechanism restricting T cell responses in MIBC. However, there are a variety of other regulatory pathways that restrict their anti-tumor function, which have yet to be investigated. One such pathway involves alterations in protein glycosylation, which can affect T cell function at many levels. The activity of the glycosyltransferase N-acetylglucosaminyltransferase V (MGAT5), responsible for complex N-glycan branching, has been shown to regulate T cell function in autoimmunity and chronic viral infections, however, its role in regulating anti-tumor CD8+ T cell responses has yet to be explored. We assessed the activity of MGAT5 in circulating and tumor-infiltrating CD8+ T cells in mice using a panel of cell lines in a syngeneic orthotopic model of muscle-invasive bladder cancer (MIBC). We found that in all tumor models, MGAT5 activity was upregulated in tumor infiltrating CD8+ T cells compared with circulating cells. Furthermore, using high parameter flow cytometry, we assessed the activity of MGAT5 in CD8+ T cells from mice with bladder cancer induced by the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), and in patients with MIBC. In both mice and humans, we found that sex affects MGAT5 activity in memory CD8+ T cells, with females exhibiting decreased MGAT5 activity in these cells compared to males. The functional significance of altering MGAT5 activity in T cells was also assessed in vivo. To do this, chimeric mice were generated by mixed bone-marrow transplant resulting in mice with loss of Mgat5 in T cells. Bladder cancer was then induced using BBN, and morbidity (defined as either 10% weight loss in one week, 10% weight loss post-BBN, or humane endpoint), as a proxy for mortality, was assessed over time. In this model, male mice were protected from BBN-induced morbidity compared to female mice, and this protection was abrogated by homozygous or heterozygous loss of Mgat5 in T cells. Overall, our data suggest that sex-dependent regulation of MGAT5 activity in CD8+ T cells may be an important underlying factor affecting outcomes in bladder cancer. Citation Format: Morgan E. Roberts, Igor Moskalev, Peter C. Black. The role of MGAT5 in sex-dependent regulation of CD8+ T cells in muscle-invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A021.

Studying the cellular basis of small bowel enteropathy using high-parameter flow cytometry in mouse models of primary antibody deficiency.

Primary immunodeficiencies are heritable defects in immune system function. Antibody deficiency is the most common form of primary immunodeficiency in humans, can be caused by abnormalities in both the development and activation of B cells, and may result from B-cell-intrinsic defects or defective responses by other cells relevant to humoral immunity. Inflammatory gastrointestinal complications are commonly observed in antibody-deficient patients, but the underlying immune mechanisms driving this are largely undefined. In this study, several mouse strains reflecting a spectrum of primary antibody deficiency (IgA-/-, Aicda-/-, CD19-/- and JH -/-) were used to generate a functional small-bowel-specific cellular atlas using a novel high-parameter flow cytometry approach that allows for the enumeration of 59 unique cell subsets. Using this cellular atlas, we generated a direct and quantifiable estimate of immune dysregulation. This estimate was then used to identify specific immune factors most predictive of the severity of inflammatory disease of the small bowel (small bowel enteropathy). Results from our experiments indicate that the severity of primary antibody deficiency positively correlates with the degree of immune dysregulation that can be expected to develop in an individual. In the SI of mice, immune dysregulation is primarily explained by defective homeostatic responses in T cell and invariant natural killer-like T (iNKT) cell subsets. These defects are strongly correlated with abnormalities in the balance between protein (MHCII-mediated) versus lipid (CD1d-mediated) antigen presentation by intestinal epithelial cells (IECs) and intestinal stem cells (ISCs), respectively. Multivariate statistical approaches can be used to obtain quantifiable estimates of immune dysregulation based on high-parameter flow cytometry readouts of immune function. Using one such estimate, we reveal a previously unrecognized tradeoff between iNKT cell activation and type 1 immunity that underlies disease in the small bowel. The balance between protein/lipid antigen presentation by ISCs may play a crucial role in regulating this balance and thereby suppressing inflammatory disease in the small bowel.

300 Intracranial Tumors Hijack the Meningeal Venolymphatic Network Through VEGFR-2 Signaling

INTRODUCTION: Glioblastoma and other intracranial neoplasms have been associated with breakdown of the blood brain barrier. Other protective CNS barriers have sparsely been studied in brain tumors. Here, we identify the ability of intracranial neoplasms to hijack the meningeal venolymphatic network, which is driven through VEGFR-2/VEGF-A signaling. METHODS: C57BL/6J mice were stereotactically injected with syngeneic SB28 (glioblastoma) cell lines. Mice were treated with either isotype control or a VEGFR-2 inhibitor. Two-photon microscopy was used to visualize vascular cytoarchitecture and dynamics in vivo. Meningeal whole mounts, brains and spleens were also collected and analyzed by confocal microscopy and high parameter flow cytometry. Human dura mater from glioblastoma patients underwent multiplex immunohistochemistry to confirm the translatability of our findings. RESULTS: Our imaging studies revealed that glioblastomas can generate an extensive de novo network of meningeal blood vessels and lymphatics both in mice and humans. Lymphatic vessels were often observed developing in parallel to the newly formed vasculature. Intravital two-photon microscopy further revealed that the peritumoral vascular bed in the meninges of mice was leaky, dysfunctional, and generated a hypoxic microenvironment. Importantly, inhibition of VEGFR signaling impeded formation of this faulty venolymphatic network, which slowed tumor growth, reduced peritumoral hypoxia, enhanced immune infiltration, and increased survival. CONCLUSIONS: Our results demonstrate that glioblastomas can invade the meninges and hijack the venolymphatic network through VEGFR-2 and PECAM-1 signaling. While VEGFR-2 is associated with angiogenesis, our data suggest that it may also have an important role in regulating the simultaneous development of de novo meningeal venous and lymphatic drainage during glioblastoma.

Development of a customizable mouse backbone spectral flow cytometry panel to delineate immune cell populations in normal and tumor tissues.

In vivo studies of cancer biology and assessment of therapeutic efficacy are critical to advancing cancer research and ultimately improving patient outcomes. Murine cancer models have proven to be an invaluable tool in pre-clinical studies. In this context, multi-parameter flow cytometry is a powerful method for elucidating the profile of immune cells within the tumor microenvironment and/or play a role in hematological diseases. However, designing an appropriate multi-parameter panel to comprehensively profile the increasing diversity of immune cells across different murine tissues can be extremely challenging. To address this issue, we designed a panel with 13 fixed markers that define the major immune populations -referred to as the backbone panel- that can be profiled in different tissues but with the option to incorporate up to seven additional fluorochromes, including any marker specific to the study in question. This backbone panel maintains its resolution across different spectral flow cytometers and organs, both hematopoietic and non-hematopoietic, as well as tumors with complex immune microenvironments. Having a robust backbone that can be easily customized with pre-validated drop-in fluorochromes saves time and resources and brings consistency and standardization, making it a versatile solution for immuno-oncology researchers. In addition, the approach presented here can serve as a guide to develop similar types of customizable backbone panels for different research questions requiring high-parameter flow cytometry panels.

Abstract 3974: Macronutrient composition dictates MASH-associated HCC immunosurveillance through microbiota alteration and metabolic adaptation

Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with an estimated incidence of over one million cases by 2025. The advances in vaccines against HBV and HCV have led to a decrease in the number of virus-related cases; however, the obesity epidemic has contributed to a drastic increase in metabolic dysfunction-associated steatohepatitis (MASH) related HCC cases (e.g., obesity and type 2 diabetes induced HCC). Considering this trend, we aim to understand how lifestyle choices, like food composition, in combination with alcohol consumption at a “social drinking level” impacts host metabolism, and subsequently the development of MASH-induced HCC. We hypothesize that the impact of dietary composition on dysbiosis and immune cells, particularly adaptive immune cell metabolic status, will drive the phenotype. We have developed a MASH-induced HCC murine model, with long-term dietary intervention including seven different customized diets: normal chow (NC), ketogenic, solid high fat diet (sHFD), or isocaloric liquid Lieber DeCarli Diets (LDC): high fat/low carb (HF), high fat/low carb with ethanol (HF-EtOH), low fat/high carb (LF), and low fat/high carb with ethanol (LF-EtOH). Mice began special diet in late adolescences and disease progression was monitored over the course of a year, where mice developed HCC. Our model allowed for a comprehensive understanding of MASH-induced HCC where disease initiation could be studied beyond known contributors like obesity or body mass index (BMI). The animal model was analyzed comprehensively with high-parameter flow cytometry (FC), 16S sequencing, bulk RNA-seq, scRNA/ATAC-seq, and WGS. Long-term diet intervention led to macronutrient specific microbiota dysregulation, alterations in systemic metabolism and suppression of adaptive immune response, consequently impacting the progression of HCC. Customized dietary interventions showed a specific phenotype regarding HCC development and progression with distinct mutational genotype and particular response to immunotherapy. T and B cell subpopulation deficiency had a significant impact on MASH-induced HCC development in a macronutrient composition dependent manner. In summary, each dietary intervention showed a distinct pattern and effect on dysbiosis, systemic metabolic reprograming, cellular transcriptomic and epigenetic alteration, and particularly immune cell effector function. Additionally, some compositions supported the immuno-escape mechanism of malignant cells through regulation of their MHC machinery. Collectively, these accumulated data construct an atlas for understanding the underlying molecular mechanisms contributing to lifestyle-induced HCC providing the knowledge needed to advance effective strategies for treatment and prevention. Citation Format: Nicolas T. Ryujin, Jian Huang, Albert Nguyen, Jared Edwards, Yuhe Cheng, Jessica Wen, Ashish Damania, Nadim Ajami, Spencer Rosario, Mark Long, Ludmil Alexandrov, Shabnam Shalapour. Macronutrient composition dictates MASH-associated HCC immunosurveillance through microbiota alteration and metabolic adaptation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3974.

Automated antibody dispensing to improve high-parameter flow cytometry throughput and analysis.

Over the past decade, the flow cytometry field has witnessed significant advancements in the number of fluorochromes that can be detected. This enables researchers to analyze more than 40 markers simultaneously on thousands of cells per second. However, with this increased complexity and multiplicity of markers, the manual dispensing of antibodies for flow cytometry experiments has become laborious, time-consuming, and prone to errors. An automated antibody dispensing system could provide a potential solution by enhancing the efficiency, and by improving data quality by faithfully dispensing the fluorochrome-conjugated antibodies and by enabling the easy addition of extra controls. In this study, a comprehensive comparison of different liquid handlers for dispensing fluorochrome-labeled antibodies was conducted for the preparation of flow cytometry stainings. The evaluation focused on key criteria including dispensing time, dead volume, and reliability of dispensing. After benchmarking, the I.DOT, a non-contact liquid handler, was selected and optimized in more detail. In the end, the I.DOT was able to prepare a 25-marker panel in 20 min, including the full stain, all FMOs and all single stain controls for cells and beads. Having all these controls improved the validation of the panel, visualization, and analysis of the data. Thus, automated antibody dispensing by dispensers such as the I.DOT reduces time and errors, enhances data quality, and can be easily integrated in an automated workflow to prepare samples for flow cytometry.

Epidermal barrier impairment predisposes for excessive growth of the allergy-associated yeast Malassezia on murine skin.

The skin barrier is vital for protection against environmental threats including insults caused by skin-resident microbes. Dysregulation of this barrier is a hallmark of atopic dermatitis (AD) and ichthyosis, with variable consequences for host immune control of colonizing commensals and opportunistic pathogens. While Malassezia is the most abundant commensal fungus of the skin, little is known about the host control of this fungus in inflammatory skin diseases. In this experimental study, MC903-treated mice were colonized with Malassezia spp. to assess the host-fungal interactions in atopic dermatitis. Additional murine models of AD and ichthyosis, including tape stripping, K5-Nrf2 overexpression and flaky tail mice, were employed to confirm and expand the findings. Skin fungal counts were enumerated. High parameter flow cytometry was used to characterize the antifungal response in the AD-like skin. Structural and functional alterations in the skin barrier were determined by histology and transcriptomics of bulk skin. Finally, differential expression of metabolic genes in Malassezia in atopic and control skin was quantified. Malassezia grows excessively in AD-like skin. Fungal overgrowth could, however, not be explained by the altered immune status of the atopic skin. Instead, we found that by upregulating key metabolic genes in the altered cutaneous niche, Malassezia acquired enhanced fitness to efficiently colonise the impaired skin barrier. This study provides evidence that structural and metabolic changes in the dysfunctional epidermal barrier environment provide increased accessibility and an altered lipid profile, to which the lipid-dependent yeast adapts for enhanced nutrient assimilation. Our findings reveal fundamental insights into the implication of the mycobiota in the pathogenesis of common skin barrier disorders.

A Noninvasive Method to Sample Immune Cells in the Lower Female Genital Tract Using Menstrual Discs.

T cells in the human female genital tract (FGT) are key mediators of susceptibility to and protection from infection, including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT, but current sampling methods require a healthcare provider and are expensive, limiting the ability to study these populations longitudinally. To address these challenges, we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are noninvasive, self-applied, and low in cost. To demonstrate reproducibility, we sampled the cervicovaginal fluid of healthy, reproductive-aged individuals using menstrual discs across 3 sequential days. Cervicovaginal fluid was processed for cervicovaginal cells, and high-parameter flow cytometry was used to characterize immune populations. We identified large numbers of live, CD45+ leukocytes, as well as distinct populations of T cells and B cells. Within the T cell compartment, activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches, including identification of both tissue-resident and migratory populations. In addition, the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies.

The complex heterogeneity of immune cell signatures across wasting tissues with C26 and 5-fluorouracil-induced cachexia.

Immune cell-driven pathways are linked to cancer cachexia. Tumor presence is associated with immune cell infiltration whereas cytotoxic chemotherapies reduce immune cell counts. Despite these paradoxical effects, both cancer and chemotherapy can cause cachexia; however, our understanding of immune responses in the cachexia condition with cancer and chemotherapy is largely unknown. We sought to advance our understanding of the immunology underlying cancer and cancer with chemotherapy-induced cachexia. CD2F1 mice were given 106 C26 cells, followed by five doses of 5-fluorouracil (5FU; 30 mg/kg LM, ip) or PBS. Indices of cachexia and tumor (TUM), skeletal muscle (SKM), and adipose tissue (AT) immune cell populations were examined using high-parameter flow cytometry. Although 5FU was able to stunt tumor growth, % body weight loss and muscle mass were not different between C26 and C26 + 5FU. C26 increased CD11b+Ly6g+ and CD11b+Ly6cInt inflammatory myeloid cells in SKM and AT; however, both populations were reduced with C26 + 5FU. tSNE analysis revealed 24 SKM macrophage subsets wherein 8 were changed with C26 or C26 + 5FU. C26 + 5FU increased SKM CD11b-CD11c+ dendritic cells, CD11b-NK1.1+ NK-cells, and CD11b-B220+ B-cells, and reduced Ly6cHiCX3CR1+CD206+CD163IntCD11c-MHCII- infiltrated macrophages and other CD11b+Ly6cHi myeloid cells compared with C26. Both C26 and C26 + 5FU had elevated CD11b+F480+CD206+MHCII- or more specifically Ly6cLoCX3CR1+CD206+CD163IntCD11c-MHCII- profibrotic macrophages. 5FU suppressed tumor growth and decreased SKM and AT inflammatory immune cells without protecting against cachexia suggesting that these cells are not required for wasting. However, profibrotic cells and muscle inflammatory/atrophic signaling appear consistent with cancer- and cancer with chemotherapy-induced wasting and remain potential therapeutic targets.NEW & NOTEWORTHY Despite being an immune-driven condition, our understanding of skeletal muscle and adipose tissue immune cells with cachexia is limited. Here, we identified immune cell populations in tumors, skeletal muscle, and adipose tissue in C26 tumor-bearing mice with/without 5-fluorouracil (5FU). C26 and C26 + 5FU had increased skeletal muscle profibrotic macrophages, but 5FU reduced inflammatory myeloid cells without sparing mass. Tumor presence and chemotherapy have contrasting effects on certain immune cells, which appeared not necessary for wasting.

Unlocking Biomarkers for Cancer Immunotherapy: Immune Monitoring with High-Parameter Flow Cytometry

Unlocking Biomarkers for Cancer Immunotherapy: Immune Monitoring with High-Parameter Flow Cytometry

The ERK inhibitor LY3214996 augments anti-PD-1 immunotherapy in preclinical mouse models of BRAFV600E melanoma brain metastasis.

Immune checkpoint inhibitors (ICI) have revolutionized cancer treatment; however, only a subset of patients with brain metastasis (BM) respond to ICI. Activating mutations in the mitogen-activated protein kinase signaling pathway are frequent in BM. The objective of this study was to evaluate whether therapeutic inhibition of extracellular signal-regulated kinase (ERK) can improve the efficacy of ICI for BM. We used immunotypical mouse models of BM bearing dual extracranial/intracranial tumors to evaluate the efficacy of single-agent and dual-agent treatment with selective ERK inhibitor LY3214996 (LY321) and anti-programmed death receptor 1 (PD-1) antibody. We verified target inhibition and drug delivery, then investigated treatment effects on T-cell response and tumor-immune microenvironment using high-parameter flow cytometry, multiplex immunoassays, and T-cell receptor profiling. We found that dual treatment with LY321 and anti-PD-1 significantly improved overall survival in 2 BRAFV600E-mutant murine melanoma models but not in KRAS-mutant murine lung adenocarcinoma. We demonstrate that although LY321 has limited blood-brain barrier (BBB) permeability, combined LY321 and anti-PD-1 therapy increases tumor-infiltrating CD8+ effector T cells, broadens the T-cell receptor repertoire in the extracranial tumor, enriches T-cell clones shared by the periphery and brain, and reduces immunosuppressive cytokines and cell populations in tumors. Despite the limited BBB permeability of LY321, combined LY321 and anti-PD-1 treatment can improve intracranial disease control by amplifying extracranial immune responses, highlighting the role of extracranial tumors in driving intracranial response to treatment. Combined ERK and PD-1 inhibition is a promising therapeutic approach, worthy of further investigation for patients with melanoma BM.

Development of a Machine Learning Decision Support System for Clinical Flow Cytometry

Abstract High parameter flow cytometry is a highly utilized tool in both research and clinical settings for accurate immunophenotyping in immunology, oncology, and many other disciplines. With increasing dimensionality, manual analysis of data becomes more complex and time consuming. Over the past decade, several automated methods have been developed for cell population clustering and dimensionality reduction of high parameter flow cytometry data which have sped up and simplified the discovery of cell populations not observed by manual gating strategies. However, the input and output of such tools are stochastic in nature, thus making their results difficult to reuse with novel samples such as diagnostic patient samples. Several groups have recently attempted to utilize machine learning algorithms to analyze clinical flow cytometry data. These studies focused on training machine learning models with a goal of predicting a specific diagnosis for each sample. This approach can be limiting when the diagnostic features are not perfectly met to predict a specific diagnosis as well as the absence of the patient's clinical data. To our knowledge, no studies have utilized machine learning to identify and classify individual cells based on their raw fluorescence signature to various cellular populations, whether normal or abnormal, to aid a diagnostician to interpret their significance in a clinical context. In this study, we utilized a data set of ~240 million individual cells from ~9,500 flow cytometry files from 2,300 patients to train a machine learning model to predict cell population classifications as annotated by expert hematopathologists. These annotations were implemented into an end-to-end machine learning based decision support system allowing for rapid, automated analysis of high dimensional flow cytometry data with an output aiding the diagnostician in the generation of a diagnosis. A clinical validation highlighted the robustness of this methodology with 100% sensitivity and 94% specificity in the identification of abnormal B-cell populations on a cohort of 1,500 patients. This decision support system also identifies abnormal T-cell and myeloid cell populations with 100% sensitivity. Overall, the decision support system saves significant time as it removes the need for manual gating and analysis, and aids the diagnostician in identifying discrete cell populations and offering descriptive information about any abnormal populations identified.

Combined Single Cell Flow Cytometry and Imaging Analyses Reveal Immunomodulatory Effects Exerted By Targeted Phospho-SYK Inhibitors with Elevated Sensitivity in NPM1 Mutated AML

Acute myeloid leukemia (AML) is driven by genetic lesions that can result in addiction to signaling pathways that lead to aberrant proliferation and survival. Several new targeted drugs are being assessed for activity in AML with some recently approved for the treatment of subgroups of patients with specific genetic features, such as mutations in FLT3, IDH1 or IDH2. Entospletinib (ENTO) and lanraplenib (LANRA) are inhibitors of spleen tyrosine kinase (SYK); the latter is a next-generation SYK inhibitor and is currently being evaluated in combination with gilteritinib (GILT) in patients with relapsed or refractory FLT3-mutated AML (NCT05028751). Although SYK is rarely mutated, it has been found to cooperate with FLT3, and is highly active especially in AML patients with mutated FLT3. In addition, SYK is important for immune cell signaling through antibody constant region Fc and B cell receptors. To examine the multi-modulatory effect of pSYK inhibitors in AML, we used combined high parameter flow cytometry (HP-FC) and single cell miniaturized imaging (sc-MI) approaches to profile the immunologic, mutation, and functional protein pathway targets of ENTO and LANRA. Two different AML patient cohorts were assessed by ex vivo treatment with ENTO, LANRA and combinations of LANRA (trametinib, gilteritinib (GILT), aPD-1) over a range of time points (2h, 4h, 3d and 9d) and readouts for cellular phenotype (e.g., differentiation state, immune cell populations) and functional readouts (pSYK, viability) for each cohort were analyzed by HP-FC and sc-MI and associated with available mutation information (bulk gene and single cell RNA sequencing). Differential analyses of data from flow cytometry (AML-HP-FC Cohort) and single cell imaging (AML-sc-MI Cohort) demonstrated: 1) sensitivity to ENTO and LANRA (³ 1uM) was higher in NPM1 and FLT3 mutated AML samples, as measured via cell viability and pSYK target inhibition; sensitivity was not drastically altered in the presence of other mutations (e.g. IDH1/2, MLL); 2) lower doses of ENTO and LANRA (<1uM) induced proliferation in specific cell types, including progenitor myeloid cells, and T and B lymphocytes, but induced more T cell proliferation in AML samples that have low baseline proliferation; analysis with next generation bulk gene and single cell RNA sequencing suggested downregulation of STAT1expression, downregulation of MYC- and mTOR-dependent pathways, and upregulation of IL-1 pathway genes; and 3) LANRA was more broadly effective at targeting pSYK inhibition than ENTO; however, both were effective in combinations (ENTO with trametinib and GILT, and LANRA with aPD-1). In sum, analysis using collective flow and single cell imaging provides corroboratory and complementary information revealing new roles for ENTO and LANRA as immune modulating agents supporting B and T cell proliferation at lower dosages, and with demonstrated greater effect in individuals with NPM1 and FLT3 mutations. These support studies investigating the use of pSYK inhibitors in combination with other therapies to reduce tumor burden, and as an improved line of treatment for AML. Acknowledgements: We are appreciative of the patients and families for their time and participation.

Splenic B Cell Lymphoma Innate Immune Cells: Detection of Red Versus White Pulp Lymphoma Differences in NK Cell Populations By High-Parameter Flow Cytometry

Introduction Splenic B-cell lymphomas, (SLs, total incidence ~1:100,000/year) comprise four distinct indolent B-cell non-Hodgkin lymphomas. Three predominantly involve the red pulp of the spleen (classical hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv) and splenic diffuse red pulp small B-cell lymphoma (SDRPL)) and the fourth predominantly involves the white pulp (splenic marginal zone lymphoma (SMZL)). HCL is usually diagnosed by detection of an activation mutation in BRAF and responds well to treatment. We focused our study on the remaining three diseases, which have overlapping clinical presentations with only subtle differences in their morphology and phenotypes, but have considerable differences in prognosis, response to treatment and outcome. The white pulp lymphoma SMZL generally has good treatment responses and longer survival, whereas the red pulp SLs, HCLv and SDRPL are less responsive to treatments with shorter survival. Cells of the tumor microenvironment (TME) may have distinct characteristics between these three diseases. We hypothesized that TME innate immune cells from red pulp SLs (HCLv, and SDRPL) would be similar to each other and distinct from the white pulp SL, SMZL.In this study, we report that natural killer (NK) cells, which are important innate immune cells critical for immune surveillance, vary in proportions among red and white pulp SLs. Methods We studied splenectomy specimens from patients with SMZL (n=8), HCLv (n=8), SDRPL (n=7), and controls (healthy, post-trauma, n=8). Cryo-preserved bulk splenic suspension cell isolates from fresh (<4 hour) surgical specimens and intact formalin-fixed paraffin embedded (FFPE) tissue sections were used. Human specimen collection and usage was conducted with written informed consent after approval of the Institutional Research Subjects Review Board. FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells were analyzed by high-parameter fluorescence flow cytometry using a Cytek Aurora spectral flow cytometer with a 25-color myeloid and a 28-color NK/B-cell panel. Results IHC characterization showed an extensive network of innate immune cells as reflected by CD163 + macrophages. Using high-parameter flow cytometry, we focused on innate immune cells in the non-B- non-T-cell (CD3 -CD19 -) population. The average proportions of this population were: NK cells 25-36%, monocyte/macrophages 20-34%, dendritic cells 5-11%, and granulocytes 2%. Among the patient groups, the median percentage of NK cells was greater for controls (34%) and SMZL (35%) as compared to red pulp lymphomas (HCLv (28%), SDRPL (24%)). Next, we examined NK subtypes: 1) cytotoxic NK cells (CD56 +CD16 +), 2) cytokine-secreting weakly cytotoxic NK cells (CD56 +CD16 -), and 3) undefined NK cells (CD56 -CD16 +) as a median percentage of total NK cells. The major subtype for control and SMZL was CD56 +CD16 - with values of 58 and 44%, respectively. This subtype comprises only 38%-40% of red pulp SLs. Proportionally the CD56 +CD16 + subtype was the largest for red pulp SLs at 52-57% as compared to 30% for control and 38% for SMZL. The remaining CD56 -CD16 + NK cell subtype was generally higher among red pulp SLs (HCLv (9%), SDRPL (12%)) as compared to control (6%) and SMZL (4%) patients. The increase in cytotoxic CD56 +CD16 + subtype among red pulp SLs was confirmed by measuring the percentage of granzymeB +perforin + cells among mature cytotoxic NK cells (CD56 +CD16 +/ CD57 +NKG2a -). SLs had higher proportions of this NK phenotype (78-86% compared to control 69%) and red pulp SLs had the most granzymeB +perforin + proportions with 82 and 86% for HCLv and SDRPL, respectively. Discussion In summary, spleens from patients with SMZL had similar percentages of total NK cells to controls, with lower percentages in red pulp SLs. All SLs had a higher percentage of cytotoxic NK cells than control, with the red pulp SLs having the most. These data suggest that monoclonal antibody treatments targeting SL antigen, such as anti-CD20, may be effective by activating cytotoxic NK cell to target lymphoma cells by antibody-dependent cellular cytotoxicity. Thus, uncovering the characteristics of the TME may aid the development of non-invasive diagnostic procedures and/or precision therapies.

MODL-01. INTRACRANIAL TUMORS HIJACK THE MENINGEAL VENOLYMPHATIC NETWORK THROUGH VEGFR-2 SIGNALING

Abstract INTRODUCTION Glioblastoma and other intracranial neoplasms have been associated with breakdown of the blood brain barrier. Other protective CNS barriers have sparsely been studied in brain tumors. Here, we identify the ability of intracranial neoplasms to hijack the meningeal venolymphatic network, which is driven through VEGFR-2/VEGF-A signaling. METHODS C57BL/6J mice were stereotactically injected with syngeneic SB28 (glioblastoma) cell lines. Mice were treated with either isotype control or a VEGFR-2 inhibitor. Two-photon microscopy was used to visualize vascular cytoarchitecture and dynamics in vivo. Meningeal whole mounts, brains and spleens were also collected and analyzed by confocal microscopy and high parameter flow cytometry. Similar experiments were conducted with a PECAM-1 inhibitor to assess the role of endothelial cell crosstalk in venolymphatic expansion. Human dura mater from glioblastoma patients underwent multiplex immunohistochemistry to confirm the translatability of our findings. RESULTS Our imaging studies revealed that glioblastomas can generate an extensive de novo network of meningeal blood vessels and lymphatics both in mice and humans. Lymphatic vessels were often observed developing in parallel to the newly formed vasculature. Intravital two-photon microscopy further revealed that the peritumoral vascular bed in the meninges of mice was leaky, dysfunctional, and generated a hypoxic microenvironment. Importantly, inhibition of VEGFR signaling impeded formation of this faulty venolymphatic network, which slowed tumor growth, reduced peritumoral hypoxia, enhanced immune infiltration, and increased survival. PECAM-1 (CD31) inhibition had a similar effect on the tumor microenvironment, suggesting that endothelial cell crosstalk is required for glioblastomas to optimally invade the meninges. CONCLUSION Our results demonstrate that glioblastomas can invade the meninges and hijack the venolymphatic network through VEGFR-2 and PECAM-1 signaling. While VEGFR-2 is associated with angiogenesis, our data suggest that it may also have an important role in regulating the simultaneous development of de novo meningeal venous and lymphatic drainage during glioblastoma.