Midlands Innovation Flow Cytometry Conference 2024 resources

CD4+ and CD8+ type-2 T cells are enriched in sputum of patients with COPD eosinophilic exacerbations

Adam Wright¹, Hnin Wint Wint Aung¹, Tracy Thornton², Helen Evans², Akomeno Egbo², Sarah Edwards², Rebecca Dale¹, David J Cousins1, Neil Greening1,2#, Chris E Brightling1,2# (#co-last authors)

2University Hospitals of Leicester NHS Trust, Leicester, UK

Corresponding Author: Adam Wright (adam.wright@leicester.ac.uk) Research Fellow

Eosinophilic exacerbations account for a quarter of COPD exacerbations but the cellular mechanisms are poorly defined. Our objective was to determine the relationship between sputum type-2 polarised CD4⁺ and 8⁺ T cells with eosinophils in COPD.

We performed cytometry on spontaneous sputum samples from moderate-severe COPD patients; 30 during clinical stability and 25 during exacerbation. We measured viable CD4⁺ and CD8⁺ CRTH2⁺ CD161^– (conventional type-2) T cells and subsets co-expressing CD161 (TH2-A or CD161⁺ TC2, respectively) and reported as median [IQR]. Eosinophils were defined as viable, CD45⁺CD16^–CD193⁺CD294⁺, FcεR1a-. We partitioned patient samples into those without or with elevated sputum eosinophils (≥2% of total viable leukocytes).

At clinical stability, only sputum TH2% were significantly different between non-eosinophilic and eosinophilic sputum (n=12, 0.18% [0.09-0.37] vs n=18, 0.62% [0.33-1.8], p=0.008) and correlated with eosinophil % (n=30, r=0.45, p=0.013).  At exacerbation, sputum TH2% (n=17, 0.13% [0.05-0.22] vs n=8, 0.84% [0.3-2.2], p=0.006), TH2-A% (0.10% [0-0.2] vs 0.88% [0.5-1.3], p=0.006), TC2% (1.3% [0.5-3.4] vs 8.8 % [4.3-26], p=0.0002) and CD161⁺ TC2% (0% [0-0.3] vs 1.46% [0.4-3.3], p=0.004) were all significantly different between non-eosinophilic and eosinophilic sputum, respectively.  Sputum TH2% (r=0.51, p=0.0097), TH2-A% (r=0.60, p=0.0014) and TC2% (r=0.65, p=0.0005) positively correlated with sputum eosinophils at exacerbation (n=25). In eosinophilic sputum, CD8⁺ T cells were skewed towards a T2 profile to a greater extent than CD4⁺, both at stability (n=18, 3% [1.7-10.7] vs 0.6% [0.3-1.7], p=0.04) and at exacerbation (n=8, 8.8% [4-26] vs 0.84% [0.26-2.2], p=0.01), respectively. When utilising a common denominator (total CD3⁺), the % of TC2 and TH2 cells (of CD3⁺) were similar in eosinophilic sputum during clinical stability (1.0 [0.3-1.8] vs 0.45 [0.2-1] vs, p=0.21) but trended towards being significantly different at exacerbation (1.6 [0.9-3.3] vs 0.5 [0.14-1.6], p=0.018), respectively

Conclusion: Both CD4⁺ and CD8⁺ type-2 T cells are likely to contribute to eosinophilic exacerbations and warrant greater spatial and functional understanding.

CXCL12 inhibitor targets airway remodelling without modulating immune response in allergic asthma.

Rebecca E. Bignold & Jill R. Johnson

Aston University, Birmingham, UK

Corresponding Author: Rebecca Bignold (r.bignold1@aston.ac.uk). Research Fellow

Current allergic asthma treatments mainly target inflammation, with bronchodilators like salbutamol and medications which target inflammation. These include corticosteroids which reverse histone acetylation reducing the effect of inflammatory genes, leukotriene inhibitors that block the inflammatory effect of cysteinyl leukotrienes, as well as a handful of biologics that target specific inflammatory cytokines. Despite their efficacy in managing symptoms, they fail to address the underlying structural changes in asthma. LIT-927, a novel drug that disrupts the CXCL12/CXCR4 chemokine gradient, shows promise in preventing detrimental pericyte migration, a key factor in airway remodelling. However, its impact on immune cell infiltration requires further examination.

The house dust mite (HDM) model of allergic asthma was used in which male and female mice were dosed intranasally 5 times a week for 5 weeks (n= 6). After 3 weeks of HDM exposure, mice were treated with LIT-927 (300 nmol/kg) for 2 weeks. Lung sections were immunostained for fibrosis markers, while flow cytometry assessed immune cells and CXCR4 expression in lung tissue digests. Bronchoalveolar lavage (BAL) samples enumerated immune cells within the airway.

LIT-927 treatment effectively reduced allergic asthma symptoms and remodelling features such as smooth muscle thickening and extracellular matrix deposition. Flow cytometry indicated no significant changes in eosinophil, macrophage, or pericyte numbers, or their CXCR4 expression in lung tissue post-HDM or LIT-927 treatment. However, BAL analysis revealed increased eosinophils post-HDM, which was unchanged by LIT-927.

These findings suggest LIT-927’s positive effects are not driven by the modulation of the immune response, possibly due to its topical administration reducing off-target effects. Its commercialisation potential, especially in combination with existing anti-inflammatory drugs, is being explored. Further investigations, including lung function measurements and longer-term studies, are warranted to assess its efficacy in severe allergic asthma.

Defining the pathophysiological impact of cigarette smoke extract in mice as a model for Chronic Obstructive Pulmonary Disease (COPD)

Laura Bartlett¹, David Onion¹, Paddy Tighe¹, Adam Watkins², Lucy Fairclough¹

¹School of Life Sciences, University of Nottingham, ²School of Medicine, University of Nottingham

Corresponding Author: Laura Bartlett PhD Student (winner of best talk last year)

Chronic obstructive pulmonary disease (COPD) is a long-term and progressive respiratory condition most frequently caused by chronic cigarette smoking and characterised by marked pulmonary and systemic inflammation. Although it has a significant global burden in terms of its prevalence and impact on economic and healthcare resources, the immunopathogenic mechanisms driving COPD development and persistence are poorly understood. Previously, the majority of research on COPD has been conducted using murine models outside of the UK, due to ethical and welfare concerns that infringe upon UK legislations regarding the use of animals in science. To combat this, we have developed a novel 3Rs friendly murine model of COPD using a refined intranasal cigarette smoke extract (CSE) exposure method to investigate the role of pulmonary and systemic inflammation on COPD pathogenesis. Using a high-throughput 24 marker spectral flow cytometry panel, we have demonstrated that chronic exposure to CSE in mice results in a systemic immune profile reflective of that observed in human COPD patients. In addition, histological and RT-qPCR analysis demonstrates signs of significant airway remodelling, immune cell infiltration and activation and alterations to immune signalling pathways, thereby confirming the presence of a moderate COPD phenotype. This novel model system will provide researchers in the UK with the toolkit necessary to further characterise specific mechanisms of interest in COPD as well as identify potential markers for more effective treatment and therapeutic intervention.

Single Cell Sequencing of the Desert locust auditory organ

Christian Thomas., Benjamin Warren

Department of Neuroscience, Psychology & Behaviour, University of Leicester

Corresponding Author: Christian Thomas (clt54@leicester.ac.uk). Post-Doc

Auditory decline affects us all. As we get older it is inevitable that our auditory ability declines. Insects have long proven useful models for studying ageing and disease. In particular for auditory studies, the desert locust Schistocerca gregaria is emerging as a powerful insect model due to its easily accessible and large auditory organ the Muller’s organ. As with mammalian systems, we find that as this insect ages the electrophysiological output declines. Additionally, when exposed to an auditory attack we see declines in the electrophysiological output.

In mammalian systems we see a decline in the number of hair cells as auditory function declines. Unlike with mammalian systems, the desert locust does not a decrease in the number cells as auditory function declines. This raises the question, if age related hearing loss is conserved between insects and mammals but the mechanism of cell loss is not, what are the cellular mechanisms for insect auditory decline?

To investigate this, we first want to characterize the cells of the locust auditory organ the Muller’s organ to better understand the conservation of auditory cell type. To do this we performed single cell sequencing on the cell types in the locust ear. By dissociating nuclei from the auditory organ, and sorting of dissociated debris using the cell sorting facility at the University of Nottingham we were able to index individual cell nuclei and perform single cell sequencing. We now have annotated cell clusters and propose their function.

Investigating the Impact of Fluorescence Activated Cell Sorting on the Metabolome of Glioblastoma Tumoursphere model

Une Kontrimaite, Ruman Rahman, Dong-Hyun Kim, Stuart Smith

School of Medicine, University of Nottingham

Corresponding Author: Une Kontrimaite (mzxuk1@exmail.nottingham.ac.uk). PhD Student

Fluorescence-activated cell sorting (FACS) is an essential technique in cancer research, facilitating the isolation of specific cell populations within tissues. Metabolic profiling of distinct cellular populations enhances our understanding of unique functions and contributions to tumorigenesis. This study aimed to elucidate the effects of FACS on the metabolic profiles of cells derived from glioblastoma tumourspheres, which serve as a model for aggressive brain cancer, compared with control human cortical astrocytes. We prepared a singlecell suspension from dissociated tumourspheres and employed CellTrace Violet labelling for astrocytes and eGFP for intracellular marking of tumoursphere cells. Post-FACS, we analysed the metabolomic profiles of both live cells and those fixed with 4% paraformaldehyde (PFA).

Our results revealed alterations in the cellular metabolome attributable to the mechanical stresses encountered during the sorting process. The research underscores the necessity to consider FACS-induced metabolic changes when designing experiments and interpreting data. Our findings stress the importance of optimising cell processing and developing strategies to mitigate the impact of sorting on cellular metabolism.

Blood Leukocyte Characterisation in Eosinophilic Granulomatosis with Polyangiitis (EGPA)

Dale R1, Naveed S 2, Thornton T 3, Turner H 3, Evans H 3, Porter K 3, Charalambou A 3, Bourne M 3, Siddiqui S 4, Amrani Y 1 and Wright A 1

1 – Department of Respiratory Sciences, University of Leicester, Leicester

2 – Department of Respiratory Medicine, Glenfield Hospital, University Hospitals of Leicester, Leicester

3 – NIHR Respiratory Biomedical Research Unit, Glenfield Hospital, University Hospitals of Leicester NHS Trust, Leicester

4 – National Heart and Lung Institute, Imperial College, London

Corresponding author: Rebecca Dale rbd8@leicester.ac.uk PhD Student

Rationale: Peripheral blood leukocyte characteristics are poorly understood in Eosinophilic Granulomatosis with Polyangiitis (EGPA), a rare disease whereby Severe Eosinophilic Asthmatic (SEA) patients develop blood and tissue eosinophilia, and other comorbidities.

Objectives: To measure whole blood leukocyte counts from EGPA patients, specifically granulocytes (eosinophils, neutrophils, basophils), Type 2-polarised Innate Lymphoid cells (ILC2s) and T helper/cytotoxic cell subsets (Type 1/2/2, CD161+/17/22).

Methods: Lithium-heparin blood was collected from stable EGPA (n=9), SEA (n=8) and healthy volunteers (n=10). Leukocyte populations were measured using a 12-colour panel on the Attune NxT acoustic cytometer and reported /100uL blood. EGPA and SEA patients were on continued benralizumab, mepolizumab and/or oral corticosteroid at blood collection.

ILC subsets (ILC1/ILC2/ILC3) were measured in PBMCs (n=5/group), using a 10-colour panel. Absolute counts are expressed /2×106 PBMCs, and proportions expressed as % of total ILCs. Maturity status was measured using CD45RO+/CD45RA+ expressed as % of each ILC subset.

Results: Type 2-polarised granulocyte (eosinophils, basophils) and lymphocyte (Th/Tc2, Th/Tc2 CD161+) counts were similar across groups. However, ILC2s were significantly lower in EGPA compared to health (p=0.0457). Th17 and B cell counts were significantly lower in EGPA than health (p=0.0136; p=0.0495 respectively), whereas neutrophils, Tc17 and Th/Tc22 cells were similar. There were no disease specific differences between EGPA and SEA.

Total ILCs were lower in EGPA, with a trend for decreased ILC2s in EGPA compared to health, in concordance with blood staining (p=0.0710). A greater proportion of ILC2s expressed CD45RO in EGPA versus health (p=0.0385). In contrast, fewer ILC2s expressed CD45RA in EGPA versus SEA and health (p=0.0027; p=0.002 respectively).

Conclusion: T2-polarised lymphocytes circulate at similar levels between health, SEA and EGPA. ILC2s in EGPA are decreased and show evidence of increased maturation compared to controls, suggesting disease specific differences. Whether functional differences for these cells exist between diseases remains to be defined.

Analysis of Tumour-Astrocyte Crosstalk in 3D using a Human Brain Specific Extracellular Matrix Hydrogel.

Harry Porter¹, Phoebe McCrorie¹, Mohammed Diksin¹, Cara Moloney¹, Richard Grundy¹, Ruman Rahman¹

1. Children’s Brain Tumour Research Centre, School of Medicine, University of Nottingham.

Corresponding Author: Harry Porter (mzxhp2@exmail.nottingham.ac.uk) PhD Student

Atypical Teratoid Rhabdoid Tumours (ATRT) are a rare and aggressive paediatric brain tumour, conferring a poor survival rate. Other malignant brain tumour types can hijack astrocytes in the healthy brain to promote tumour growth, but this has not been previously investigated in ATRT. Our lab has developed an innovative model system to recapitulate tumour-astrocyte interaction in vitro by co-culturing patient derived cell lines with astrocytes in a decellularized human brain extracellular matrix hydrogel. This system has been extensively characterized as a platform for spheroid culture. However, with new models comes new challenges and a requirement for novel methods of analysis. Namely, when conducting assays on co-cultures it is difficult to conclude whether readout is generated from one cell line or another. To address this, we have developed a suite of techniques supporting the use of this model based on fluorescently labelling live cells. Firstly, a high throughput imaging and analysis technique was developed, profiling the expansion of monoculture and coculture spheroids over a 7-day period. As we aim to accurately isolate ATRT and astrocyte populations from co-culture spheroids, we have developed a bespoke methodology for harvesting cells from the hydrogel and preparing single-cell solutions for flow cytometry. Using fluorescence activated cell sorting we have successfully purified co-culture tumour cells and astrocytes from the model system. These distinct cell populations will be compared to monoculture counterparts using transcriptomics to elucidate how ATRT cells crosstalk with astrocytes. Candidate therapeutics which could inhibit tumour promoting signalling from astrocytes will be identified and trialled by adapting this co-culture model as drug screen. Furthermore, the success of our staining and flow cytometry to date indicates that this method could be combined with a viability stain to obtain viability readouts from tumour cells and astrocytes independently to further validate the action of future molecular targeted therapies.

Flow cytometry as a tool to study the physiology of the recombinant yeast, Pichia pastoris: Optimising lipase enzyme expression

Lewis Yandle¹, Benjamin. O. Ezema^1,2,3,4, Carmen Hierro-Iglesias¹, Kingsley. O. Omeje³, Roslyn Bill⁵, Alan Goddard⁵, Alfred Fernandez-Castane^1,4*

¹Energy and Bioproducts Research Institute, Aston University, Birmingham, B4 7ET, UK, ²The Biochemistry Unit, Department of Science Laboratory Technology, University of Nigeria, Nsukka, Nigeria, ³Department of Biochemistry, University of Nigeria, Nsukka, ⁴Aston Institute of Materials Research, Aston University, Birmingham, B4 7ET, UK, , ⁵College of Health and Life Sciences, Birmingham, B4 7ET, UK

Corresponding author: Lewis Yandle: lyand18@aston.ac.uk PhD Student

Flow cytometry (FCM) provides insights into physiological responses, enabling effective optimisation of protein expression in recombinant systems. FCM enables the monitoring single-cell physiology of microbes, such as the yeast Pichia pastoris, in real-time, offering more nuanced insights over traditional methods (e.g., plate counting). Light scatter measures relative cell size and complexity; and combined with fluorescent dyes such as propidium iodide (PI) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (BOX), allows the quantitative determination of cell viability via membrane permeability and polarisation, respectively.

Aspergillus flavus lipase (AFL) has applications in various industries, from pharmaceuticals to biofuels. However, low native yields and carcinogenic aflatoxins produced by A.flavus limit industrial application. The AFL gene was recently isolated and expressed in P.pastoris, aiming to scale-up expression to industry-relevant yields. Evaluating physiology is imperative for understanding stress responses, its influence on enzyme expression, and facilitating yield optimisation.

Here, we evaluated the efficacy of two promoter systems namely, methanol inducible (AOX1) and constitutive (GAP), for the expression of AFL in P.pastoris under varied culture conditions. FCM revealed discrepancies between optimal conditions for maximum enzyme activity versus cell growth and viability. Intriguingly, conditions favouring maximum enzyme activity related to those optimal for cell growth of the native organism, rather than the recombinant host. Notably, AFL expressed with the GAP promoter yielded 2.37-fold higher maximum lipase activity than when expressed with the AOX1 promoter, revealing key differences between constitutive and inducible expression systems for efficient protein production.

This research advances bioprocess development by harnessing FCM to study cell physiology, enabling effective promoter selection and culture condition optimisation. This approach will pave the way for enhanced enzyme yield and stability, contributing to sustainable, cost-effective industrial applications.

Embracing Failure: Insights Gained from Seminal Vesicle Exosome Research Setbacks

Vipul Batra¹, David Onion², David Boocock³, Clare Coveney³, Nadine Holmes⁴, Victoria Wright⁴, and Adam J. Watkins¹

1Lifespan and Population Health, School of Medicine, University of Nottingham, United Kingdom

2School of Life Sciences, University of Nottingham, Nottingham, United Kingdom

3John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, United Kingdom

4DeepSeq, University of Nottingham, United Kingdom

Corresponding author: Vipul Batra (mszvb2@exmail.nottingham.ac.uk) Post-Doc

Abstract

Seminal plasma exosomes play various roles in reproductive biology, including modulating sperm function, influencing the immune response in the female reproductive tract, facilitating intercellular communication, and potentially affecting embryo development. The seminal vesicle glands produce approximately 90% of the ejaculate volume in mice underscoring their substantial contribution to the semen composition. We aimed to investigate whether the previously observed alterations in the transcriptomic and proteomic profiles of the C57BL6 mice sperm and seminal plasma upon feeding sub-optimal diets are mediated by the seminal vesicle exosomes (svExos) cargo. The svExos were enriched from the mice’s seminal vesicle fluid and were characterized by Nano-particle Tracking Assay, Imaging Flow cytometry (ImageStream), and Transmission Electron Microscopy. Subsequently, total RNA and protein were isolated from these exosomes using QIAGEN miRNeasy technology and RIPA lysis buffer (and acetone precipitation), respectively and subject to RNA-Seq and LC-MS/MS. The svExos measured between 80-150 nm in size with a concentration range of 108-109 particles/cm³ and had a typical cup-shaped round morphology. The svExos exhibited positive staining for internal extracellular vesicle (EV) components after labelling with an EV-specific, high-quantum efficiency dye (ExoFlow-ONE EV Labeling Kit). Analysis of RNA content via the high-sensitivity RNA ScreenTape assay failed to detect any RNA, while the zSWATH DIA data (analyzed using DIA-NN) revealed the absence of any substantial proteomic cargo within these exosomes.

These data indicate that svExos (which are neither epididymosomes nor prostasomes) may have a unique cargo composition (metabolites, lipids or information-rich glycans) distinct from other known types of exosomes, as they lacked RNA and proteomic cargo. Alternatively, the generation of svExos could be a component of the normal cellular homeostasis process (e.g. cellular disposal mechanism in senescent cells), or it is plausible that the non-cargo molecules of svExos (e.g. membrane proteins) play a role in communication with sperm and the female reproductive tract. Nonetheless, further studies are warranted to clarify the functional significance of svExos in male reproductive physiology and offspring health.