Midlands Innovation Flow Cytometry Conference 2024 resources

9:00 Registration and Coffee

9:30 Welcome

9:45 Session 1

10:45 Coffee Break & Cell Sorter Demonstrations

11:15 Session 2

12:30 Lunch, Posters, Exhibition & Cell Sorter Demonstrations

13:30 Session 3

14:45 Coffee Break

15:00 Keynote Speaker

15:45 Concluding Remarks & Prizes

16:00 Reception & Conference Close

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+CD16CD193+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. Ezema1,2,3,4, Carmen Hierro-Iglesias1, Kingsley. O. Omeje3, Roslyn Bill5, Alan Goddard5, Alfred Fernandez-Castane1,4*

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



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.

For FACS sake: Exploring embryonic neurogenesis in Drosophila melanogaster using FACS-enriched single-cell RNA sequencing

Clifton Lewis1, David Onion2, Nicola Croxall2, Charalambos Kyriacou1, Ezio Rosato1, Roberto Feuda1


1 Department of Genetics and Genome Biology, University of Leicester, University Rd, Leicester LE1 7RH, United Kingdom

2 Queen’s Medical Centre, University of Nottingham, Derby Rd, Lenton, Nottingham, NG7 2UH, United Kingdom

Corresponding author: Clifton Lewis,  cl486@leicester.ac.uk PhD Student

The complexity in morphology and function of the nervous system is defined through development by the type, number, and positioning of neurons. In order to build such complex systems, the temporal and spatial coordination is paramount and is coordinated by an elaborate, yet tightly controlled regulatory system to define specific cell fates. Despite extensive characterisation of the Drosophila nervous system, there are gaps in our understanding of the underlying genetics and molecular control of embryonic neurogenesis. Resolving the specification and differentiation molecular program is a challenge due to complexity and rapidity of cell type evolution, with a fully-formed embryonic nervous system formed within the first 24 hours of development. However, the simplicity in cell number in Drosophila, as well as the predefined set of tissue- and cell-type specific markers, makes it ideal for characterising different neuronal subtypes and tracking their developmental trajectories. We utilised single-cell RNA sequencing, across overlapping consecutive time-points, to provide a high-resolution view of the expression dynamics as well as the underlying regulatory programs that are involved in defining different neuronal subtypes. To account for the rapid changes in cell types, we use a fixation-based approach to temporally stage the embryos across developmental time. However, as embryogenesis involves the formation of all the various organ systems, a targeted approach using FACS was used to enrich specifically for the neuronal lineage. This approach reduces the cost as well allowing for a deeper dissection of the cell lineage of interest and should help us resolve the subtypes in finer details. Using this FACS-enriched scRNA-seq method allows for high-resolution, time-resolved view of Drosophila embryonic neurogenesis, from early progenitors to differentiated neurons. This will provide the in-depth resolution to resolve cell type evolution in neurogenesis along a developmental trajectory by regulatory dynamics.

Building Confidence in Fully Synthetic High-Dimensional Cluster Generation solutions to model Flow Cytometry Data for Haematopoietic Stem Cell Analysis


Bradley Mason1, Laura Justham1, Liam Whitby2, Jon Petzing1


1Wolfson School of Mechanical, Electrical & Manufacturing Engineering

Loughborough University, Loughborough, UK

2UK NEQAS-LI, Sheffield, UK

Corresponding author: Bradley Mason (b.mason6@lboro.ac.uk) PhD Student



Flow cytometry grapples with notable challenges in both the metrological and biological domains. Like any measurement system, uncertainty in flow cytometry data, often stems from its relativistic nature and the absence of a definitive ‘ground truth’, further compounded by diverse sources of random variability (e.g. operator, instrument, preparation, data analysis). These uncertainties can cause a lack of confidence in flow cytometry data, with potential diagnosis impact for applications such as diagnostics and therapeutics in medicine. Automated unsupervised clustering methods enable populations recognition, often without requiring the user to define a known cellular collation. However, the types of available clustering algorithms are constantly increasing, necessitating the development of robust methods capable of drawing performance comparisons and providing traceability of data processing.

This research aims to address some of these challenges by further developing synthetic data generation to help manage and mitigate operator-induced variability. The work primarily focuses on the synthetic generation of multi-cluster high-dimensional Haematopoietic CD34+ stem cell blood sample datasets. The synthetic data utilises probabilistic terms derived from previously ethically cleared and anonymised patient data and agnostic in nature, safeguarding individual privacy. Cluster algorithm generated synthetic benchmarking datasets can be used to quantify and test how effective these differing clustering algorithms are compared to one another, in addition to providing EQA based bespoke training aids for operators.

The novelty lies in the creation of highly realistic synthetic high-dimensional haematological flow cytometric datasets. The study aims to demonstrate real-world validation of high-dimensional synthetic datasets as suitable surrogates for traditional live cell flow cytometry datasets. Synthetic data provides analytically superior data models compared to the current ‘gold-standard’ methodologies by granting absolute accuracy, traceability, and reproducibility in the data analysis. This research holds promise in enhancing the reliability of flow cytometry data analysis, offering tangible benefits to clinical users, patients, and the broader flow cytometry community.

Development of novel FRET sensors to decode T cell signalling dynamics using flow cytometry


Authors: Emma Davey, John James

Affiliations: Warwick Medical School, University of Warwick

Corresponding author Emma Davey (emma.davey@warwick.ac.uk) PhD Student

Our immune system relies on T cells making appropriate and robust decisions to protect our healthy cells whilst destroying pathogens or cancerous cells. To achieve this behaviour, extracellular cues are converted into information through biochemical reactions within the cell. These individual reactions, such as phosphorylation driven by kinases, integrate to form a complex signalling network that drives the overall functional outcome. However, current methods for probing this network lack the necessary resolution to further our understanding of these decision-making processes. To address this limitation, we use spectral flow cytometry to probe key parts of the T cell signalling network using ratiometric FRET sensors. By combining a new, very efficient fluorophore combination for FRET and an Erk kinase biosensor originally designed for microscopy, we have developed a new approach for measuring Erk activity within T cells using flow cytometry. Temporal analysis of the FRET ratio showed a digital increase in Erk activity once the cells were activated. This exciting novel approach can easily be extended by multiplexing with other biosensors, leveraging the power of spectral detection, to simultaneously measure other key signalling pathways. Future work to incorporate optogenetic receptors to also control the cellular input will drive new insights into how signalling dynamics are decoded by the T cell signalling network.

OX40 agonism boosts CD4+ T cell responses to anti-PD-L1 blockade through increasing T cell receptor signal strength and duration


Emma K Jennings, Lozan Sheriff, David Bending (University of Birmingham)


Corresponding author Emma Jennings (Emma.Jennings@warwick.ac.uk) PhD Student at the time of work post-doc now


Background: Immunotherapy, such as immune checkpoint modulation (ICM), has proved promising in recent years. Immunotherapeutic blockade of the PD-1:PD-L1 pathway strengthens T cell receptor (TCR) signalling, leading to higher expression of the costimulatory receptor OX40 on CD4+ T cells. We hypothesised that agonising OX40 in combination with blockade of the PD-L1 pathway would therefore represent a rational ICM combination, which would have superior ability to re-activate CD4+ T cells.

Methods: We employed the Tg4 Nr4a3-Tocky accelerated adaptive tolerance model to induce recalibrated activation thresholds in CD4+ T cells in vivo. Using a novel screening culture system, recalibrated CD4+ T cells were evaluated for their ability to re-activate TCR signalling and relative TCR signal strengths in response to different combinations of ICM. Promising combinations were then chosen to test the ability to sustain CD4+ T cell activation in vivo and T cells were analysed by flow and spectral cytometry.

Results: Screenings of different combinations identified that OX40 but not ICOS agonism in combination with PD-L1 blockade resulted in enhanced TCR signal strength and cytokine secretion, compared with either PD-L1, OX40 or ICOS-targeting monotherapies. In vivo experiments on adaptively tolerised T cells revealed that anti-PD-L1+anti-OX40 combination therapy resulted in an additive increase in the proportions of T cells able to respond to restimulation in vivo compared to monotherapies. Combination ICM also drove increases in markers of sustained TCR signalling (OX40, CD25, GITR, ICOS) which was linked to a longer TCR signal duration as evidenced by higher levels of Nr4a3-Timer Red proteins in combination-treated CD4+ T cells.

Conclusions: Anti-PD-L1 and agonistic OX40 ICM combination therapy augment T cell activation, both in vitro and in vivo through modulating the strength and duration of TCR signalling in CD4+ T cells.

Keywords: Immunotherapy, OX40, PD-L1, Checkpoint Modulation

Novel 355nm (and Lower) Excitable and Tuneable Emission (Blue through Red) Fluorophores for Utility in Flow Cytometry


Sareena Sund,a Sunil Claire,a Parvez Iqbal,a Michael Butlin, a Julianna Malicay,aKarolis Virzbickasa,b and Jon A. Preecea,b,*


a ChromaTwist Ltd, Lab PD206, Science Centre, University of Wolverhampton Science Park, Glaisher Drive, Wolverhampton, WV10 9RU, UK.

bSchool of Chemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.


The chemical modularity of ChromaTwist’s fluorescent dye technology has allowed 50+ UV excitable fluorescent dyes to be created that emit across the visible spectrum (395-635 nm, with potential for reaching into IR emission).1, 2 ChromaTwist dyes can achieve red emission without the need for tandem polymer dye based approaches and their drawbacks for end-users. We have previously demonstrated that the ChromaTwist antibody (CD4 & CD8) conjugates have utility in both multicolour and spectral flow cytometry (BD Bioscience Fortessa X-20, Cytek Aurora, Sony Biotechnology ID7000, ThermoFisher Bigfoot).3

Multicolour Flow Cytometry: The chemical tunability of the ChromaTwist dyes means that we are able to match the fluorescent emission outputs to the 6 visible flow cytometry filters and ultimately the 2 IR filters, and bring to the multicolour market a portfolio of UV excitable molecular based dyes.

Spectral Flow Cytometry: The chemical tunability of the ChromaTwist dyes means that we will be able to go far beyond the multicolour 8 dye range and be able to present a portfolio of 50+ UV excitable dyes (320 nm and/or 355 nm), each with a unique spectral fingerprint.  Ultimately ChromaTwist could offer 50+ ChromaTwist-dye antibody conjugates which are all excited by 320 and/or 355nm UV laser.

Reported here is the characterisation of the blue and the green ChromaTwist (CT) Dye CD4 (SK3) conjugates, CT450●CD4 CT525●CD4 respectively. Flow cytometry was performed on Compensation beads (Invitrogen™ UltraComp eBeads™) and CYTO-TROL control cells (Beckman Coulter). Duplex staining of CT Dye CD4 conjugates on compensation beads was achieved with CoraLite® Plus 750 Anti-Human CD4 (OKT4) (Proteintech), demonstrating the use of ChromaTwist dye antibody conjugates in multicolour panels.