Thomas Myles AshhurstThe University of Sydney · Sydney Cytometry Core Research Facility
Thomas Myles Ashhurst
Ph.D.
About
50
Publications
53,855
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
931
Citations
Citations since 2017
Introduction
Additional affiliations
January 2017 - present
May 2014 - June 2014
Education
March 2012 - December 2016
Publications
Publications (50)
Background and objectives
SARS-CoV-2 infection causes a spectrum of clinical disease presentation, ranging from asymptomatic to fatal. While neutralising antibody (NAb) responses correlate with protection against symptomatic and severe infection, the contribution of the T cell response to the resolution or progression of disease is still unclear. O...
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection causes severe coronavirus disease 2019 (COVID‐19) in a small proportion of infected individuals. The immune system plays an important role in the defence against SARS‐CoV‐2, but our understanding of the cellular immune parameters that contribute to severe COVID‐19 disease is inc...
Age can profoundly affect susceptibility to a broad range of human diseases. Children are more susceptible to some infectious diseases such as diphtheria and pertussis, while in others, such as COVID-19 and hepatitis A, they are more protected compared to adults. One explanation is that the composition of the immune system is a major contributing f...
Background: Patients affected by different types of autoimmune diseases, including common conditions such as Multiple Sclerosis (MS) and Rheumatoid Arthritis (RA), are often treated with immunosuppressants to suppress disease activity. It is not fully understood how the SARS-CoV-2 specific humoral and cellular immunity induced by infection and/or u...
Mapping the dynamics of immune cell populations over time or disease‐course is key to understanding immunopathogenesis and devising putative interventions. We present TrackSOM, a novel method for delineating cellular populations and tracking their development over a time‐ or disease‐course cytometry datasets. We demonstrate TrackSOM‐enabled elucida...
Respiratory tract infection with SARS-CoV-2 results in varying immunopathology underlying COVID-19. We examine cellular, humoral and cytokine responses covering 382 immune components in longitudinal blood and respiratory samples from hospitalized COVID-19 patients. SARS-CoV-2-specific IgM, IgG, IgA are detected in respiratory tract and blood, howev...
Background
Elevated production of the cytokines interleukin (IL)-6 or interferon (IFN)-α in the central nervous system (CNS) is implicated in the pathogenesis of neurological diseases such as neuromyelitis optica spectrum disorders or cerebral interferonopathies, respectively. Transgenic mice with CNS-targeted chronic production of IL-6 (GFAP-IL6)...
B cells play a major role in multiple sclerosis (MS), with many successful therapeutics capable of removing them from circulation. One such therapy, alemtuzumab, is thought to reset the immune system without the need for ongoing therapy in a proportion of patients. The exact cells contributing to disease pathogenesis and quiescence remain to be ide...
Background
Disease-specific studies have reported impaired humoral responses after SARS-CoV-2 vaccination in patients with immune-mediated inflammatory disorders treated with specific immunosuppressants. Disease-overarching studies, and data on recall responses and third vaccinations are scarce. Our primary objective was to investigate the effects...
High-dimensional cytometry experiments measuring 20–50 cellular markers have become routine in many laboratories. The increased complexity of these datasets requires added rigor during the experimental planning and the subsequent manual and computational data analysis to avoid artefacts and misinterpretation of results. Here we discuss pitfalls fre...
Although the respiratory tract is the primary site of SARS-CoV-2 infection and the ensuing immunopathology, respiratory immune responses are understudied and urgently needed to understand mechanisms underlying COVID-19 disease pathogenesis. We collected paired longitudinal blood and respiratory tract samples (endotracheal aspirate, sputum or pleura...
Although the respiratory tract is the primary site of SARS-CoV-2 infection and the ensuing immunopathology, respiratory immune responses are understudied and urgently needed to understand mechanisms underlying COVID-19 disease pathogenesis. We collected paired longitudinal blood and respiratory tract samples (endotracheal aspirate, sputum or pleura...
Background
Differentiating infiltrating myeloid cells from resident microglia in neuroinflammatory disease is challenging, because bone marrow-derived inflammatory monocytes infiltrating the inflamed brain adopt a ‘microglia-like’ phenotype. This precludes the accurate identification of either cell type without genetic manipulation, which is import...
Mapping the dynamics of immune cell populations over time or disease-course is key to understanding immunopathogenesis and devising putative interventions. We present TrackSOM, an algorithm which delineates cellular populations and tracks their development over a time- or disease-course of cytometry datasets. We demonstrate TrackSOM-enabled elucida...
Background:
Differentiating infiltrating myeloid cells from resident microglia in neuroinflammatory disease is challenging, because bone marrow-derived inflammatory monocytes infiltrating the inflamed brain adopt a ‘microglia-like’ phenotype. This precludes the accurate identification of either cell type without genetic manipulation, which is impor...
High‐dimensional cytometry represents an exciting new era of immunology research, enabling the discovery of new cells and prediction of patient responses to therapy. A plethora of analysis and visualisation tools and programmes are now available for both new and experienced users; however, the transition from low‐dimensional to high‐dimensional cyt...
As the size and complexity of high‐dimensional cytometry data continue to expand, comprehensive, scalable, and methodical computational analysis approaches are essential. Yet, contemporary clustering and dimensionality reduction tools alone are insufficient to analyze or reproduce analyses across large numbers of samples, batches, or experiments. M...
Cell counting is a critical step to determine the number of live leukocytes (white blood cells, WBC) and erythrocytes (red blood cells, RBC) per femur, and is accurately performed using a microscope and haemocytometer. This step allows enables the distribution of a specific number of cells per well in the staining plate. Some researchers prefer to...
There is an urgent need for novel vaccination strategies to combat respiratory pathogens. Mucosal vaccine delivery is an attractive option as it directly targets the site of infection; however, preclinical development has been hindered by a lack of suitable mucosal adjuvants and a limited understanding of their immune effects in the lung environmen...
SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill-defined. We analysed 85 SARS-CoV-2-infected individuals at acute and/or convalescent timepoints, up to 102 days post-symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18 and IL-10 and broad acti...
Motivation
Many ‘automated gating’ algorithms now exist to cluster cytometry and single cell sequencing data into discrete populations. Comparative algorithm evaluations on benchmark datasets rely either on a single performance metric, or a few metrics considered independently of one another. However, single metrics emphasise different aspects of c...
As the size and complexity of high-dimensional cytometry data continue to expand, comprehensive, scalable, and methodical computational analysis approaches are essential. Yet, contemporary clustering and dimensionality reduction tools alone are insufficient to analyze or reproduce analyses across large numbers of samples, batches, or experiments. M...
In conventional fluorescence cytometry, each fluorophore present in a panel is measured in a target detector, through the use of wide band‐pass optical filters. In contrast, spectral cytometry uses a large number of detectors with narrow band‐pass filters to measure a fluorophore's signal across the spectrum, creating a more detailed fluorescent si...
Objectives
Disease‐modifying therapies (DMTs) targeting B cells are amongst the most effective for preventing multiple sclerosis (MS) progression. IgG3 antibodies and their uncharacterised B‐cell clones are predicted to play a pathogenic role in MS. Identifying subsets of IgG3⁺ B cells involved in MS progression could improve diagnosis, could infor...
Signal transducers and activators of transcription (STAT) 1 is critical for cellular responses to type I interferons (IFN-Is), with the capacity to determine the outcome of viral infection. We previously showed that while wildtype (WT) mice develop mild disease and survive infection with lymphocytic choriomeningitis virus (LCMV), LCMV infection of...
Background:
Until the end of the twentieth century, Zika virus (ZIKV) was thought to cause a mostly mild, self-limiting disease in humans. However, as the geographic distribution of ZIKV has shifted, so too has its pathogenicity. Modern-day ZIKV infection is now known to cause encephalitis, acute disseminated encephalomyelitis, and Guillain-Barré...
Mass cytometry is a new high-throughput technology that is becoming a cornerstone in immunology and cell biology research. With technological advancement, the number of cellular characteristics cytometry can simultaneously quantify grows, making analysis increasingly computationally onerous. In this paper, we investigate the potential of dimensiona...
Despite success in treating hematological malignancies, T cells expressing chimeric antigen receptors (CARs) have shown poor efficacy in solid tumor indications. The failure to initiate and elicit a complete TCR response is arguably a primary underlying factor preventing CAR-T cell success in solid tumor indications. Here, we present a novel T cell...
Despite success in treating hematological malignancies, T cells expressing chimeric antigen receptors (CARs) have shown poor efficacy in solid tumor indications. The failure to initiate and elicit a complete TCR response is arguably a primary underlying factor preventing CAR-T cell success in solid tumor indications. Here, we present a novel T cell...
ORGANIZED by the International Society for Advancement of Cytometry (ISAC), the CYTO conference is one of the most important events for everyone interested in cytometry and quantitative single cell analysis. It is an annual assembly of cytometry experts as well as novices in this field—all hav- ing one common denominator—passion for cytometry. This...
Mass cytometry is a multi-parametric technique that offers insight into functional and biological systems at a single cell level (Tanner et al., Cancer Immunol Immunother 62:955–965, 2013). One of the major advantages of mass cytometry is the ability to measure multiple intracellular markers, including phosphorylated proteins that are part of major...
The hematopoietic system produces erythrocytes (red blood cells), leukocytes (white blood cells), and thrombocytes (platelets) throughout the life of an organism. Long-lived hematopoietic stem cells give rise to early progenitors with multi-lineage potential that progressively differentiate into lineage-specific progenitors. Following lineage commi...
In many scientific disciplines, the advent of new high-throughput technologies is giving rise to vast quantities of high-dimensional time-series data. A common requirement is to identify clusters of data-points with similar characteristics in this experimental data, and track their development over time. In this article we present ChronoClust, a no...
This book details a compilation of up-to-date and cutting-edge protocols in mass cytometry.. Chapters guide readers through setting up a facility, panel design and reagent preparation, sample preparation, specific applications, and data analysis. Written in the highly successful Methods in Molecular Biology series format, chapters include introduct...
Anti-CD4 or anti-CD8α Ab-mediated depletion strategies are widely used to determine the role of T cell subsets. However, surface expression of CD4 and CD8α is not limited to T cells and occurs on other leukocyte populations as well. Using both unbiased t-distributed stochastic neighbor embedding of flow cytometry data and conventional gating strate...
Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus, responsible for varicella upon primary infection and herpes zoster following reactivation from latency. To establish lifelong infection, VZV employs strategies to evade and manipulate the immune system to its advantage in disseminating virus. As innate lymphocytes, natural killer...
Cell-free VARIVAX VZV infects human peripheral blood NK cells.
Healthy human donor PBMCs were inoculated with VARIVAX vaccine (MOI 0.001) for 2 days then analysed for infection by flow cytometry. Shown are example flow cytometry plots of live NK cells (CD3–CD56+) examining surface VZV gE:gI expression (A) or Fluorescence Minus One (FMO) control for...
Transmission of infection from VZV cultured NK cells stripped with citrate buffer.
(A) NK cells (CD3–CD56+) were FACS sorted from healthy human donor PBMCs following VZV infection for 1 day. Isolated NK cells were subsequently washed with citrate buffer and PBS before being added to ARPE-19 epithelial cell monolayers. After 4 days in culture, monol...
VZV-GFP infects human peripheral blood NK cells, CD3+CD56+ lymphocytes, and T cells.
Healthy human donor PBMCs were inoculated with ARPE-19 epithelial cells mock infected or infected with VZV expressing GFP (VZV-GFP) for 2 days then analysed for infection by flow cytometry. (A) Representative flow cytometry plots of live T cells (CD3+CD56–), CD3+CD...
Increase in viral genome copies in VZV cultured NK cells over time.
Healthy human donor PBMCs were infected with VZV for 4 hours and then NK cells (CD3–CD56+) were isolated by FACS sorting. A sample of isolated NK cells were harvested immediately following sorting, while remaining NK cells were further cultured at 37°C and harvested at the specifie...
The immune system consists of a complex network of cells, all expressing a wide range of surface and/or intracellular proteins. Using flow cytometry, these cells can be analyzed by labeling with fluorophore-conjugated antibodies. The recent expansion of fluorescence flow cytometry technology, in conjunction with the ever-expanding understanding of...
Effective CD8⁺ T cell responses play an important role in determining the course of a viral infection. Overwhelming antigen exposure can result in suboptimal CD8⁺ T cell responses, leading to chronic infection. This altered CD8⁺ T cell differentiation state, termed exhaustion, is characterized by reduced effector function, upregulation of inhibitor...
IRF8 (interferon-regulatory factor-8) plays a critical role in regulating myeloid cell differentiation. However, the role of this transcription factor in the development of Ly6C+ inflammatory monocytes and their migration to the infected brain has not been examined. We have previously shown that West Nile virus (WNV) infection of wild-type (WT) mic...
Over the last three decades it has become increasingly clear that monocytes, originally thought to have fixed, stereotypic responses to foreign stimuli, mediate exquisitely balanced protective and pathogenic roles in disease and immunity. This balance is crucial in core functional organs, such as the central nervous system (CNS), where minor change...
Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegrada...
Mosquito-borne flaviviruses are a major current and emerging threat, affecting millions of people worldwide. Global climate change, combined with increasing proximity of humans to animals and mosquito vectors by expansion into natural habitats, coupled with the increase in international travel, have resulted in significant spread and concomitant in...
Infiltration of Ly6Chi monocytes from the blood is a hallmark of viral encephalitis. In mice with lethal encephalitis caused by West Nile virus (WNV), an emerging neurotropic flavivirus, inhibition of Ly6Chi monocyte trafficking into the brain by anti-very late antigen (VLA)-4 integrin antibody blockade at the time of first weight loss and leukocyt...
Projects
Project (1)
Spectre is an R package that enables comprehensive end-to-end integration and analysis of high-dimensional cytometry data from different batches or experiments. Spectre streamlines the analytical stages of raw data pre-processing, batch alignment, data integration, clustering, dimensionality reduction, visualisation and population labelling, as well as quantitative and statistical analysis. To manage large cytometry datasets, Spectre was built on the data.table framework – this simple table-like structure allows for fast and easy processing of large datasets in R. Critically, the design of Spectre allows for a simple, clear, and modular design of analysis workflows, that can be utilised by data and laboratory scientists. For more information, check out our Github page (https://github.com/ImmuneDynamics/spectre) or preprint (https://www.biorxiv.org/content/10.1101/2020.10.22.349563v1).
