Advances in Neuroscience With Flow Cytometry

NOW APPLICATIONS IN CYTOMETRY ADVANCES IN NEUROSCIENCE NEUROSCIENCEThis edition features impactful publications by researchers using high-dimensional flow cytometry with CyTOF® technology to better understand brain function and apply this knowledge to the prevention and treatment of neurological disease. CONTENT Promising therapeutic target for ALS The fatal neurological disease amyotrophic lateral sclerosis (ALS) might be close to becoming treatable. Research using CyTOF flow cytometry to investigate protein expression signatures on immune cells associated with ALS progression discovered that α5 integrin is predominantly expressed by microglia and endothelial cells in ALS patients. CyTOF panels that included antibodies for surface markers and intracellular cytokines were applied to microglial cells and sciatic nerve macrophages, enabling comprehensive characterization of these cell types. When we blocked its expression in mice, we were able to slow down the disease, said Bahareh Ajami, co-lead author on the research and Assistant Professor of Molecular Microbiology and Immunology and Behavioral Neuroscience in the Oregon Health & Science University School of Medicine. Blocking this protein could have therapeutic potential, leading to improved motor function, delayed disease progression and increased survival time. Read the news article covering the findings and find more details in the publication, Elevated α5 integrin expression on myeloid cells in motor areas in amyotrophic lateral sclerosis is a therapeutic target. WHAT’S INSIDE FEATURED ARTICLE ELUCIDATING BRAIN CELL INTERACTIONS AND ALTERATIONS DURING DYSFUNCTION OR DISEASE DECIPHERING CELL HETEROGENEITY AND NEUROINFLAMMATION IN NEUROLOGICAL DISORDERS MECHANISMS OF DISEASE IN BRAIN INJURY AND AUTOIMMUNITY PREDICTORS OF PROGRESSION BASED ON CHANGES IN IMMUNE CELL ACTIVITY THERAPEUTIC TARGETS OF GLIOBLASTOMA AND NEUROINFLAMMATION Advances in Neuroscience | Applications in Cytometry Now | 3 Deciphering cell heterogeneity and neuroinflammation in neurological disorders The brain is one of the most critical yet least understood parts of the human body. Its complexity lies in the organization and compartmentalization of billions of neurons, mixed among other cell types, to create a dynamic network of communication and functional pathways. Uncovering the makeup and interactions of so many cells is a difficult task, but current progress has already led to new ideas about how the brain functions in health and disease. With a better understanding of the purpose of each of these billions of densely packed cells, further insights can direct how we prevent, address and cure dysfunctions caused by disease and degeneration. Research approaches that cohesively analyze phenotypic and functional characteristics of cells across physiological and pathological states are essential when so many cell types act harmoniously for a given function. CyTOF flow cytometry enables this type of high-dimensional analysis, especially important for rare cell types and monitoring drug access and biodistribution, providing information on specific brain cells and biological pathways that are crucial to properly maintain cognitive activities. Here, we review impactful research focusing on understanding the central nervous system and its response to disease, and then applying that knowledge to predictors of progression and the development of novel approaches to treatment. CyTOF technology is widely used among neuroscience research as it provides reliable quantification regardless of cell input with higher sensitivity, increased detection capacity and little to no spectral overlap or background noise. Figure 1. The brain and its cell types Neuron Microglia Astrocyte Oligodendrocyte 4 | Applications in Cytometry Now | Advances in Neuroscience Mass cytometric analysis of the immune cell landscape after traumatic brain injury elucidates the role of complement and complement receptors in neurologic outcomes Toutonji, A. et al. Neuroinflammation following traumatic brain injury plays a role in secondary injury and influences the development of neurodegenerative pathologies. However, current interventions for brain injuries do not treat chronic inflammation. While the complement system is known to play a role in increasing inflammation during traumatic brain injury, ongoing activation can impact chronic neuroinflammation and exacerbate brain damage. Research from the Medical University of South Carolina investigated how complement influences the posttraumatic brain injury immune cell landscape. Findings implicated several immune cell types in the pathogenesis of brain injury, highlighted the importance of CR4 and CR4+ microglia in chronic brain injury, and showed that complement C5aR1 signaling is involved in peripheral immune cell infiltration but does not alone significantly influence outcomes. Single-cell CyTOF flow cytometry was used as an unbiased and comprehensive approach to analyze 13 immune cell types, including peripheral and brain resident cells along with expression of various complement and phagocytic receptors, in injured brains having undergone CR2-Crry treatment, an inhibitor of complement C3 activation, at acute and subacute time points. Samples were barcoded, pooled and stained with a 33-antibody cocktail of lineage and functional markers. Mechanisms of disease Figure 2. CyTOF flow cytometry workflow, including immune cell isolation from the brains of seven experimental groups, cell staining and processing and data analysis (Toutonji, A. et al.) Advances in Neuroscience | Applications in Cytometry Now | 5 Single-cell mass cytometry on peripheral cells in myasthenia gravis identifies dysregulation of innate immune cells Verdier, J. et al. Myasthenia gravis (MG) is a neurological, antibody-mediated autoimmune disease known for disabling muscle weakness. Anti-acetylcholine receptor (AChR) autoantibodies are implicated in the disease, as is immune cell dysregulation involving CD4 T cells and B cells. Previous studies trying to characterize this dysregulation in MG patients have been limited by a lack of sufficiently high numbers of marker combinations to ensure unambiguous discrimination between cell populations expressing common markers and the inability to broaden the cell types studied. Researchers at Sorbonne University/INSERM performed an in-depth analysis of peripheral mononuclear blood cells (PBMC) using CyTOF flow cytometry to learn about the immune dysregulation underlying early-onset AChR+ MG. PBMC from 24 patients and 16 controls were stained with a 37-marker CyTOF panel, showing unexpected fluctuations in specific innate immune cell frequencies, such as lower percentages of monocyte subpopulations and higher percentages of type 2 innate lymphoid cells (ILC2) and CD27− γδ T cells in MG patients. CyTOF technology was chosen to gain improved depth and resolution of immune phenotyping over previous studies at the single-cell level using much higher numbers of markers at once, attempting to detect new immune cell dysregulation in early-onset MG patients. Figure 3. Landscape of immune cell subpopulations (Verdier, J. et al.) 6 | Applications in Cytometry Now | Advances in Neuroscience The landscape of PBMCs in AQP4-IgG seropositive NMOSD and MOGAD, assessed by high dimensional mass cytometry Yao, M. et al. Different inflammatory diseases affecting the central nervous system can present similar clinical manifestations and relapse behavior. Two of these conditions, neuromyelitis optica spectrum disorder (NMOSD) and MOG-IgG-associated disease (MOGAD), involve the optic nerves and the spinal cord. Early and accurate diagnosis of these distinct conditions is important to ensure correct and precise treatment. Researchers at Capital Medical University describe the circulating PBMC landscapes for these diseases and reveal separate signatures that can help guide treatment strategies and possible directions for precision therapy. CyTOF flow cytometry was used to phenotype samples from 27 AQP4-IgG seropositive NMOSD patients, 11 MOGAD patients and 15 healthy individuals, finding that mononuclear phagocytes and T, NK and B cells were all involved in pathogenesis and that a specific cell population could serve as a predictor for monitoring disease development or treatment responses. CyTOF technology was chosen to detail the pathogenesis of these diseases and map the phenotypes of circulating immune cell subsets in PBMC using a 36-marker panel that targeted intrinsic and cell surface markers. Predictors of progression Figure 4. Single-cell CyTOF flow cytometry profiling of PBMC in healthy controls (HC) or patients with AQP4-IgG seropositive NMOSD or MOGAD during acute phase reveals distinct cell subpopulations (Yao, M. et al.). Advances in Neuroscience | Applications in Cytometry Now | 7 Metabolic alterations of peripheral blood immune cells and heterogeneity of neutrophil in intracranial aneurysms patients Ya, X. et al. Another study out of Capital Medical University focused on better understanding intracranial aneurysms (IAs), a severe cerebrovascular disease that can potentially lead to subarachnoid hemorrhage. While it is known that peripheral immune cells are involved in the formation and progression of IAs, little is understood about how metabolic or heterogeneity changes impact these cells. The team used CyTOF flow cytometry to profile the single-cell atlas of PBMC and polymorphonuclear cells (PMNs) in 72 patients with IAs, also looking at any shifts in key enzyme expression levels to identify metabolic changes. Findings enabled the generation of a predictive model based on CyTOF data, which could assist in diagnosis and monitoring of IA progression. CyTOF technology was chosen for its high-dimensional single-cell analysis capabilities, allowing for simultaneous comprehensive characterization of cell populations based on numerous parameters and enabling a direct comparative analysis of the metabolic states of all immune cell subtypes without the need for presorting. Figure 5. Study workflow (Ya, X. et al.) 8 | Applications in Cytometry Now | Advances in Neuroscience The immunological landscape of peripheral blood in glioblastoma patients and immunological consequences of age and dexamethasone treatment Dusoswa, S.A. et al. Glioblastomas have a poor prognosis, and current treatments can be undercut by administration of steroids that lessen symptoms or by aging patients with diminishing response. Since glioblastomas are known to suppress the immune system both locally and systemically, a comprehensive understanding of glioblastoma-specific weaknesses in the immune system would support the development of effective immunotherapies. Researchers at Amsterdam UMC compared the peripheral immune composition of glioblastoma patients against other tumor types using CyTOF flow cytometry. Examining the effects of disease pathology, dexamethasone treatment, tumor volume, sex and age on immune composition, the team found glioblastoma profiles contain fewer naive CD4+ T cells but higher percentages of mature NK cells. Steroid treatment, age and tumor volume influenced T and B cell frequencies, demonstrating associations between intracranial pathology and peripheral immune cell status and providing insight into mechanisms that impact treatments. CyTOF flow cytometry captures the phenotypic heterogeneity of PBMC samples in patients with intracranial malignancies. The ability to analyze the heterogeneity of immune fingerprints among glioblastoma patients indicates a role for high-dimensional CyTOF analysis as a tool for monitoring treatment effects and predicting response to treatment. Therapeutic targets Figure 6. Study design using CyTOF flow cytometry to characterize PBMC from patients undergoing intracranial surgery (Dusoswa, S.A. et al.) Advances in Neuroscience | Applications in Cytometry Now | 9 LCP1 knockdown in monocyte-derived macrophages: mitigating ischemic brain injury and shaping immune cell signaling and metabolism Wang, Y. et al. Strokes cause devastating impacts extending beyond mortality to disability, with treatments that are effective only within a narrow window of patients. Exploring new targets could help develop novel therapeutic strategies that could assist a greater number of people. Since ischemic stroke can trigger a complex immune response that involves neuroinflammation and severe immunosuppression, a collaboration led by researchers at Peking University Third Hospital investigated the role of lymphocyte cytosolic protein 1 (LCP1), which facilitates cell migration and immune responses but when silenced can reduce ischemic brain damage. The team used CyTOF flow cytometry to evaluate the impact of LCP1 on ischemic brain injury and immune cell signaling and metabolism. A deeper understanding of LCP1 in the context of ischemic stroke could provide valuable insights into potential therapeutic strategies targeting LCP1 and metabolic pathways aimed at attenuating neuroinflammation and lymphopenia. Figure 7. Study design overview (Wang, Y. et al.) 10 | Applications in Cytometry Now | Advances in Neuroscience Extended interval dosing of ocrelizumab modifies the repopulation of B cells without altering the clinical efficacy in multiple sclerosis Rodriguez-Mogeda, C. et al. Because B cells are known to play a crucial role in the pathophysiology of multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system, they are the target of multiple therapies. However, the depletion of B cells can affect a patient’s ability to respond to respiratory infections, such as COVID-19. Researchers from Amsterdam UMC tested the hypothesis that an extended interval of B cell-depleting therapy could help patients better respond to and recover from secondary illness by enabling repopulation of specific B cell subsets without affecting initial treatment effectiveness. The team characterized the peripheral immune landscape of MS patients after standard or extended interval dosing of ocrelizumab using high-dimensional single-cell CyTOF flow cytometry. Data detailed B cell type and the timing of repopulation and correlated the frequencies and changes in B cell subsets after both dosing schemes to clinical symptoms, relapse and progression, showing no effect on clinical efficacy with the extended dosing. CyTOF technology enabled flexible sample collection, batch staining, freezing and storage during this longitudinal study. Samples were collected, frozen and stored, then thawed and stained in pooled aliquots, followed by freezing and storage until acquisition. This versatility in sample processing is unique to CyTOF flow cytometry. Figure 8. Study design and timeline for immune cell phenotyping (Rodriguez-Mogeda, C. et al.) Advances in Neuroscience | Applications in Cytometry Now | 11 Elevated α5 integrin expression on myeloid cells in motor areas in amyotrophic lateral sclerosis is a therapeutic target Chiot, A. et al. In a collaborative effort led by researchers at Oregon Health & Science University, a therapeutic target for ALS, a fatal disease leading to progressive paralysis with no cure to date, was identified and tested. Single-cell CyTOF flow cytometry analysis of ALS mouse and ALS postmortem tissues showed that α5 integrin, expressed by microglial cells and macrophages, is highly upregulated in the spinal cord and peripheral nerves during ALS. The heterogeneity of microglia and the need for single-cell studies have made it difficult to decipher molecular mechanisms involved in inflammation-induced ALS pathology. High-parameter CyTOF flow cytometry has bridged this gap by enabling identification of α5 integrin expression on microglia and peripheral nerve macrophages. Results showed a significant increase of α5 integrin on microglia in ALS spinal cords compared with normal controls and other neurodegenerative disorders. The team further demonstrated that targeting α5 integrin with anti-α5 integrin antibody extended survival and improved motor performance in superoxide dismutase-1 G93A mice. Figure 9. Microglial cells and sciatic nerve macrophages express α5 integrin in ALS mice and display a highly inflammatory profile (Chiot, A. et al.) 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Any other trademarks are the sole property of their respective owners. ©2024 Standard BioTools Inc. (f.k.a. Fluidigm Corporation). All rights reserved. FLDM-01351 Rev 01 References 1. Chiot, A. et al. “Elevated α5 integrin expression on myeloid cells in motor areas in amyotrophic lateral sclerosis is a therapeutic target.” Proceedings of the National Academy of Sciences of the United States of America 120 (2023): e2306731120. 2. Dusoswa, S.A. et al. “The immunological landscape of peripheral blood in glioblastoma patients and immunological consequences of age and dexamethasone treatment.” Frontiers in Immunology 15 (2024): 1343484. 3. Rodriguez-Mogeda, C. et al. “Extended interval dosing of ocrelizumab modifies the repopulation of B cells without altering the clinical efficacy in multiple sclerosis.” Journal of Neuroinflammation 20 (2023): 215. 4. Toutonji, A. et al. “Mass cytometric analysis of the immune cell landscape after traumatic brain injury elucidates the role of complement and complement receptors in neurologic outcomes.” Acta Neuropathologica Communications 11 (2023): 92. 5. Verdier, J. et al. “Single-cell mass cytometry on peripheral cells in myasthenia gravis identifies dysregulation of innate immune cells.” Frontiers in Immunology 14 (2023): 1083218. 6. Wang, Y. et al. “LCP1 knockdown in monocyte-derived macrophages: mitigating ischemic brain injury and shaping immune cell signaling and metabolism.” Theranostics 14 (2024): 159–175. 7. Ya, X. et al. “Metabolic alterations of peripheral blood immune cells and heterogeneity of neutrophil in intracranial aneurysms patients.” Clinical and Translational Medicine 14 (2024): e1572. 8. Yao, M. et al. “The landscape of PBMCs in AQP4-IgG seropositive NMOSD and MOGAD, assessed by high dimensional mass cytometry.” CNS Neuroscience & Therapeutics 30 (2024): e14608. 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