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, Facility Tour & Conference Close
Circulating CD8+ effector memory cells differentiate immune checkpoint inhibitor-induced liver injury from other immune-mediated liver diseases
Stuart Astbury, Edmond Atallah, Jane I Grove, Amber G Bozward, Scott P Davies, Mark J Sheehan, Steven W Kumpf, Jessie Qian, Natalia M Krajewska, Grace E Wootton, Melanie R Lingaya, Davor Kresnik, Flavia Radulescu, Ankit Rao, Hester Franks, Lourdes Ruiz-Ortega, Mar Riveiro-Barciela, Shashi K Ramaiah, Thomas A Lanz, Changhua Ji, Poulam M Patel, Ye H Oo & Guruprasad P Aithal
Nottingham Digestive Diseases Centre, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham, UK
Checkpoint inhibitor-induced liver injury (ChILI) is an acute liver injury, occurring in cancer patients receiving immune checkpoint inhibitors (CPI). It shares clinical features with idiosyncratic drug-induced liver injury (DILI) and acute autoimmune hepatitis (AIH) with no biomarkers to differentiate between the three aetiologies. Using mass cytometry we have identified a circulating CD8+ effector memory cell subset, expressing high levels of CD38, HLA-DR and CXCR3. Analysis of this subset using single-cell RNAseq and flow cytometry revealed a significantly greater cytotoxic potential relative to remaining effector memory cells. Liver tissue bulk RNAseq and immune cell deconvolution showed a significant increase in resident CD8+ T-cells in ChILI compared to DILI and AIH, and a significant upregulation of genes related to CXCR chemokine receptor binding. Plasma cytokine profiles identified key inflammatory cytokines in ChILI, and T-cell dysregulation. We have shown that circulating CD8+ T-cells provide a potential biomarker to distinguish ChILI from DILI and AIH, and highlight different mechanistic pathways involved in liver damage between ChILI and other immune-mediated liver conditions.
Comparative in vitro efficacy of CD20xCD3 bispecific biosimilar constructs against diffuse large B cell lymphoma (DLBCL) cell lines with different levels of expression of CD20.
Joshua S Bray, Gethin R Thomas, Victoria M Smith, Sandrine Jayne, Martin J S Dyer, Harriet S Walter.
The Ernest and Helen Scott Haematological Research Institute, Leicester Cancer Research Centre, University of Leicester. @HaemResLeic
Multiple anti-CD20xCD3 bispecific antibodies (BsAb), that bind both CD20 on B cells and CD3 on T cells show unprecedented activity in relapsed/refractory Diffuse Large B Cell Lymphoma (DLBCL). Selection of BsAb may be key. Importantly, CD20 epitopes targeted vary, and glofitamab retains bivalent CD20 binding. Using biosimilar versions of glofitamab, epcoritamab, odronextamab and mosunetuzumab we compared their activity against a panel of DLBCL cell lines.
BsAb activity was assessed using a flow cytometry-based cytotoxicity assay. DLBCL cell lines with varying levels of CD20 expression: SU-DHL-10 (588400 mol/cell), UoL-RAD (148600 mol/cell), Karpas-1718 (52820 mol/cell) and UoL-AME (2333 mol/cell) were treated with 0.01-10,000pM BsAb biosimilar (Proteogenix) for 24 hours, using healthy volunteer peripheral blood mononuclear cells as effector cells. B cell depletion (BCD) was calculated and effector T and natural killer (NK) cell activity determined. Comparative data with biosimilar monospecific anti-CD20 antibodies were obtained. Absolute EC50 values were calculated.
All CD20xCD3 biosimilar constructs demonstrated dose-dependent killing against UoL-RAD and SU-DHL-10, with simultaneous activation and degranulation of T cells, but not NK cells. Glofitamab biosimilar demonstrated the highest activity against UoL-RAD and SU-DHL-10 (EC50 2pM and 59pM respectively). Comparative EC50 values for epcoritamab were 71pM and 537pM, for odronextamab 65pM and 129pM and mosunetuzumab 156pM and >10,000pM respectively. Despite low expression of CD20, UoL-AME was sensitive to glofitamab and odronextamab (EC50 802pM and 3248pM). Karpas-1718 was resistant to all biosimilar BsAb tested (EC50 >10,000pM). Monospecific antibodies demonstrated lower levels of B cell depletion and minimal T cell activation.
These experiments suggest that glofitamab biosimilar had superior efficacy against DLBCL cell lines compared to other CD20xCD3 biosimilars, perhaps due to bivalent CD20 binding. Interestingly, significant responses to glofitamab were observed in vitro for UoL-AME, despite low CD20 expression. The biological basis of inherent resistance to all CD20xCD3 BsAbs seen in Karpas-1718 requires further investigation.
Flow cytometry analysis of chloroplast shape changes after bacteria infection
Laura Civolani, Steven Servin, Murray Grant. University of Warwick
Biotic stress can lead to losses exceeding 25% of the crop yield. In order to avoid such crop loss, it is crucial to investigate all feasible approaches to limit pathogen infections. Chloroplast immunity is an emerging field, and increasing evidence highlights chloroplasts as a common pathogen virulence target. Using the model Arabidopsis thaliana – Pseudomonas syringae pv. tomato DC3000 (DC3000) pathosystem we demonstrated that recognition of pathogen-associated molecule pattern (PAMP) generates a burst of chloroplast derived reactive oxygen species (ROS). In response, pathogens produce and deliver proteins (effectors) to suppress ROS production. My project incorporates proteomics, genetics and cell biology to better understand how pathogens suppress chloroplast immunity.
By using Cytek ImageStream (ISX) Imaging Flow Cytometer, we are able to detect changes in the molecular and morphological structures of Arabidopsis thaliana (Col0) chloroplasts when infected with Pseudomonas syringae pv. tomato DC3000 at 18 hpi. This detected the reduction in length and diameter of the chloroplast, but not the reduction of intensity levels of chlorophyll.
After characterisation via imaging cytometry, we plan to use Fluorescent Activated Cell Sorting (FACS) to enrich samples of chloroplast for further proteomics analysis.
Optimising Synthetic Data Generation to Enhance the Accuracy and Reliability of Flow Cytometry-Based Haematopoietic Stem Cell Enumeration
Bradley Mason 1, Laura Justham 1, Stuart Scott 2, Liam Whitby 2 , Alison Whitby 2, Jon Petzing 1
1 Wolfson School of Mechanical, Electrical & Manufacturing Engineering, Loughborough University,
2 UK NEQAS for Leucocyte Immunophenotyping, Sheffield Teaching Hospital NHS Foundation Trust
Flow Cytometry (FC) enables the quantification of CD34+ haematopoietic stem cells, which are crucial for regenerative therapies in conditions like leukaemia. Despite its importance, validating the precision and accuracy of FC data remains a persistent challenge, particularly due to its inherently relative nature and the absence of definitive “ground truth” in biological samples. These challenges are compounded by variability introduced through operator practices, instrument inconsistencies, sample preparation methods, and diverse analysis protocols. Such factors often undermine confidence in the reliability of FC-based diagnostics.
This research addresses these challenges using synthetic datasets, which provide a stable and precise foundation for the analysis of clustering results. Synthetic data, unlike real biological samples, offers complete traceability and absolute accuracy by explicitly defining cluster memberships and enabling the generation of mathematically equivalent cluster replicants. The knowledge of all data point cluster allocations (exclusive to synthetic datasets) enables the ability to qualitatively and quantitatively analyse any method of clustering result output with certainty.
Central to this work is the optimisation of CluGen, a synthetic cluster data generator, to model haematological cell cluster characteristics with a high fidelity and accuracy. A key advancement in this study is the development of the ‘Rosetta Routine,’ a custom codebase that extracts statistical features from real data and converts them into computational parameters for CluGen. This ensures that the synthetic clusters closely mirror the properties of real-world datasets, tying together computational precision to biological complexity with the additional benefit of complete user controllability.
Future efforts aim to expand this approach to higher-dimensional FC datasets, with possible uses in the validation of clustering methods or as bespoke training scenarios to aid quality assurance programs. By enhancing the reliability of FC data analysis, this research has the potential to reduce diagnostic errors and improve patient outcomes, strengthening confidence in both research and clinical settings.
Multimodal Exploration of T Cell Fate Decision
Ilinca Patrascan, Matthias Merkenschlager
MRC Laboratory of Medical Sciences, Institute of Clinical Sciences, Imperial College London, London, United Kingdom
T cell fate decision can be described as a continuum of transcriptional states that are not always accurately captured by cell surface markers or steady-state transcript levels. RNA fluorescence in situ hybridisation enables the detection of nascent transcripts and the enumeration of mature transcripts. Here we harness the idea that proteins, mature transcripts, and primary transcripts turn over at different rates – slow for proteins, intermediate for mature (spliced) mRNAs and rapid for nascent pre-mRNA. With the help of imaging flow cytometry, we can track all these parameters at single-cell resolution, providing a powerful approach to view a cell’s present state in the context of its developmental trajectory. During T cell differentiation, bipotent progenitors commit to either the CD4+ helper or the CD8+ cytotoxic lineage. Pre-selection cells exhibit stable ratios of protein, mRNA and primary transcripts. Entry into the selection process triggers a ratio change where primary transcripts form the leading edge, mature RNA reflects recent and proteins more distant gene expression. Linear differentiation maintains coherent ratios that indicate lineage direction and speed, whereas incoherent ratios signal a change in differentiation trajectory. This distinction thus allows us to test current models of T cell fate decision, and to characterise the molecular basis of CD4 versus CD8 lineage differentiation.
Defining the impact of cigarette smoke extract in mice as a model on immune modulation and reproductive fitness
Yasmin Azizbayli1, Laura G. Bartlett2, Adam J. Watkins3 and Lucy C. Fairclough1
1School of Life Sciences, University of Nottingham, 2Institute of Life Sciences, University of Southampton, 3School of Medicine and Population Health, University of Sheffield.
Parental lifestyle factors, such as smoking around the time of conception, can significantly impact both parental health and the well-being of their offspring. This study aimed to define the effects of exposure to cigarette smoke extract (CSE) on inflammation and reproductive fitness in male and female mice.
Eight-week-old male and female C57BL/6J mice were intranasally administered either a 1% (light smoker), 3% (heavy smoker), or control (culture medium) CSE solution over a 12-week period. After 12 weeks, mice were euthanized for the collection of ovarian and testicular tissues for gene expression analysis. An in-vitro ovarian mouse model was also developed to assess immune cell composition within the ovary and testis by flow cytometry.
Histological analysis of ovarian and testicular tissue revealed a significant increase in the mean seminiferous tubule area in males exposed to the 3% CSE, while follicular count did not change in females exposed to both the 1% and 3% CSE, compared to controls. Transcriptomic analysis revealed upregulation of key inflammatory genes in the high-smoke group compared to controls, as well as alterations in genes related to male and female gamete production. Current in-vitro work has shown that the proportion of immune cells in the control mouse ovary ranged from 2-10%, with CD3+, CD11+, and Ly6G/C+ cells present.
We have shown that exposure to CSE in mice induces a local inflammatory response and affects testicular tissue morphology. In addition, we have optimised a flow panel for examining immune cell populations in reproductive organs. Future work will examine the effect of CSE on immune cell proportions in reproductive organs, as well as studying gamete quality and foetal reproductive health.
Darkness be my friend: When reduced laser power and clever chemistry can improve signal resolution
Steven Lim1, Sina Namjou1, Gavin Sooranna1, Muhammad Saeed1, Emma Russell1, Kerol Bartolovic1, Debipriya Das1, Ana Agua-Doce1, Mike Blundell2, Andrea Holme1, Philip Hobson1, Andy Riddell1
1 Francis Crick Institute, 2 Bio-Rad Laboratories
A key feature of any good fluorescent marker is how well a positive signal is resolved from the negative. This traditional understanding of a fluorochrome’s brightness meant our approaches to improving signal resolution involved using “brighter” dyes, increasing antibody concentration, or increasing input laser power. However, in instances where the target marker expression is already low, trying to obtain better separation can increase sample auto-fluorescence and quash any gains in resolution.
In recent years, novel fluorochromes have become commercially available with unique dye chemistry that offer an alternative solution to improving signal-to-noise. Bio-Rad’s semi-conducting polymer quantum dots, marketed as StarBright dyes, have impressive chemical and spectral properties which may be useful in any cytometrist’s toolkit.
Using stain index to evaluate signal resolution, a human T-cell line (SUP-T1) was stained with anti-CD4 StarBright Violet 515 and analysed on a conventional (Bio-Rad ZE5) and spectral analyser (Sony ID7000). Initial analysis of the fluorochrome-stained sample showed clearly unique emission spectra, but when considering the photo-physical properties of the dye, we found that reducing laser power also decreases the contribution of autofluorescence thereby improving signal-to-noise. Provisional data suggests that the StarBright dyes display isotropic emission, hinting at its emission structure.
By gaining a better understanding of the photo-physics of these novel fluorochromes, we are able to show that they saturate at low power. When used in noisy tissue such as lung cells, low laser power dramatically reduces the auto-fluorescent contribution of the cells while saturating the StarBright dye. The result is clearer separation of the target population from the negative. This provides an alternative approach in sorting highly auto-fluorescent cells.
A guide to choosing and using antibodies for flow cytometry
The Only Good Antibodies community is a non-profit organisation led from the University of Leicester with the mission to make best practices in antibody choice and use more feasible, easy and rewarded. As part of this mission, it hosts the UK YCharOS lab, performing knockout characterisation of commercial antibodies from 12 antibody manufacturers in flow cytometry and other applications. Drs Biddle and Virk will present the lessons learnt from large scale antibody characterisation and how this might impact the flow cytometry community. There will be a discussion around how to perform antibody validation for your specific experimental needs, within resource and time constraints of most labs. There will also be an opportunity to discuss collaborative approaches to identifying the most suitable antibodies for experiments and how to promote good practices in antibody choice and use.
Antibody validation for flow cytometry
Workshop organised and supported by the OGA community at the Midlands Innovation Flow Cytometry meeting
Workshop leaders: Harvinder Virk, Michael Biddle.
Background
Antibody validation methods are performed to confirm (or refute) that the antibody selectively detects the target of interest in your assay and sample-of-interest. Validation approaches broadly map onto the five pillars of antibody validation (see: Uhlen et al., 2016). In this workshop, we will describe the approaches that can be used to determine the selectivity of an antibody for flow cytometry experiments. This will include a discussion of the Human Leucocyte Differentiation Antigens (HCDM) workshop approach, which focuses on validating antibodies against markers of human blood leukocyte populations, as well as a pragmatic approach to determining the selectivity of antibodies for other sample types and for non-surface (intracellular) antigens.
Unfortunately, antibodies frequently do not work as assumed (Ayoubi et al., 2023). Additionally, their performance depends on the specific protocol used and the abundance of the epitope in the sample, relative to cross-reactive antigens. That is, an antibody shown to perform well in one set of conditions may not perform as well in other conditions. For these reasons, it is essential to test the selectivity of your antibody. But testing is not one-size-fits-all. The approaches used will be different depending on the sample and target type.
To start the validation process, one must first confirm that a candidate antibody can detect the antigen of interest in the given protocol. This requires use of one of the five pillars of antibody validation, and it is recommended to screen multiple candidate antibodies when possible. If cost is prohibitive, consider an approach that prioritises more reproducible/renewable reagents, such as recombinant or hybridoma-derived monoclonal antibodies. It is also important to seek out supportive data on candidate antibodies from the manufacturer and/or in the published literature. Note that the number of citations doesn’t always indicate the presence of supportive data. To be supportive, the data must show that the antigen being detected in the intended application — here, flow cytometry — is the desired target, using one or more of the approaches covered in our workshop.
Purpose of workshop
This workshop will provide an overview of the problems associated with research antibodies and provide guidance on good practice for their choice and use. The focus of this workshop will be on flow cytometry, both when used to study mixed cell populations, such as leukocytes, using surface markers; but also approaches to validation of intracellular targets. The objective is to share with early career and training scientists:
The workshop will be informed by our analyses of more than 800 antibodies using a knockout characterisation pipeline including 3 or 4 staining protocols.
Further information
About our community: www.onlygoodantibodies.com
Our approaches to antibody validation for flow cytometry: https://blog.addgene.org/antibody-validation-for-flow-cytometry
Example of our open antibody characterisation data for SYT-1: https://doi.org/10.12688/f1000research.154034.1
An antibody characterisation infrastructure to accelerate flow cytometry applications
Michael Biddle, Carolyn Jones, Jemma Cooper, Panayiota Stylianou, Malgorzata Rekas, Barbara Ferreira, Riham Ayoubi, Aled Edwards, Peter McPherson, David Cousins, Carl Laflamme, Harvinder Virk
Antibody performance is critical for the reliability, reproducibility and robustness of research. Our platform (called “YCharOS”), evaluates the comparative performance of antibodies from 13 manufacturing partners, representing approximately 60% of the market. To characterise antibodies, we create and use knockout cell lines to assess their performance in different applications.
From a sample of 614 antibodies (used widely in neuroscience) more than 50% showed poor selectivity in western blot, immunoprecipitation, and immunocytochemistry (immunofluorescence). We estimate that approximately 12 publications per protein likely contains unreliable data. The first sample of antibodies were not tested in flow cytometry.
The immunology and flow cytometry community use antibodies extensively in multiplexed panels to define cell populations. The most commonly used antibodies are directed at leukocyte cell surface antigens, some of the clones used have been through characterisation by the Human Leukocyte Differentiation Antigen workshop process (CD markers). This workshop has characterised around 400 cell surface markers, so far. However, many cell surface molecules do not have HLDA approved clones, and the workshop does not characterise intracellular antigens.
For flow cytometry, we have evaluated antibodies (using three standardised staining protocols) against the traditional cell marker CD44 and six non-CD proteins (FUS, KCa3.1, SERPINA1, SOD1, SYT1, and TGFBR2). For CD44, 21 out of 26 antibodies demonstrated high selectivity for their target. For the six non-CD targets, selective antibodies were identified for all proteins with the exception of KCa3.1. Of the 68 antibodies raised against non-CD targets, only 10 exhibited selectivity.
Our platform enables the identification of antibodies suitable for flow cytometry for a variety of targets. These include intracellular targets where the majority of antibodies on the market perform poorly.
CAR-T cell adaptation to tumour microenvironment
Carmela de Santo – University of Birmingham
Cancer cells rely on extracellular amino acids to fuel proliferation and survival. Similarly, immune cells, including conventional T cells and chimeric antigen receptor (CAR) T cells, depend on these nutrients, leading to metabolic competition within the tumour microenvironment (TME). This nutrient scarcity presents a challenge to CAR-T cell therapy, potentially limiting its efficacy.
To address this, we engineered CAR-T cells to express the transmembrane amino acid transporters SLC7A5 and SLC7A11 alongside CAR constructs. These modifications enhanced CAR-T cell proliferation under conditions of low tryptophan or cystine availability without compromising cytotoxicity or increasing exhaustion. Transcriptomic and phenotypic analyses revealed that transporter-enhanced CAR-T cells upregulated intracellular arginase expression and activity, a key adaptation to amino acid scarcity. Building on this finding, we further engineered CAR-T cells to express functional arginase 1 and arginase 2 enzymes, enhancing both proliferation and antitumor activity.
Illuminating T Cell Signalling: Optogenetics and FRET Sensors in Flow Cytometry
Emma Davey and John James
Warwick Medical School, University of Warwick
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.
These biosensors can be combined with optogenetic receptors, providing much greater control and higher temporal resolution of the cellular inputs, in comparison to chemical methods. This approach can be extended by multiplexing multiple biosensors, leveraging the power of spectral detection, to simultaneously measure key signalling pathways. We hope that this system will provide new insights into how signalling dynamics are decoded by the T cell signalling network.
Using Two High Parameter Spectral Flow Cytometry Panels to Understand the Role gd T Cells Play in Clinical Immunity to Malaria
Rosie Sanders1, Bridget Penman2, and Martin Davey1
1Warwick Medical School, University of Warwick, 2Department of Biology, University of Oxford.
Malaria is a life-threatening disease spread by mosquitos infected with a parasite from the Plasmodium species. The greatest burden of malaria is in sub-Saharan Africa, where it has profound social, health, and economic impacts, making it a critical focus for ongoing research. The immune response to malaria is multi-faceted, with recent research having identified γδ T cells as playing a pivotal role. This study aims to build on this knowledge, utilising two high-parameter spectral panels to characterise the γδ immune profile influenced by endemic malaria exposure.
γδ T cells are unique lymphocytes capable of bridging innate and adaptive immunity, with two primary subsets: innate-like Vδ2+ and adaptive-like Vδ2−. To better understand their role in malaria immunity, we designed and optimised two spectral panels: a 37-color surface panel and a 31-color intracellular panel run on the Sony ID7000. These panels allow detailed analysis of activation, memory function, effector function, cytotoxic potential and more in γδ T cells. Thus, helping us to uncover mechanisms through which γδ T cells may confer protection against malaria. Peripheral blood mononuclear cells (PBMCs) were collected from a Malian cohort in West Africa, including donors aged 7–16 sampled over four years, and Malian adults who were clinically free of malaria at the point of donation. These samples were compared with PBMCs from malaria-naïve donors in the UK, USA, and Australia. Using spectral cytometry, we observed a higher frequency of γδ T cells and an expansion of adaptive-like Vδ2− cells in malaria-endemic samples. These cells also displayed increased effector and cytotoxic characteristics in response to malaria, and potentially the development of clinical immunity. This study demonstrates the power of high-parameter spectral cytometry in profiling rare and unconventional immune cells, and sheds light on the critical role of γδ T cells in malaria immunity.
Novel 355nm (and Lower) Excitable and Tuneable Emission (Blue through Red) Fluorophores for Utility in Flow Cytometry
Sareena Sund1, Sunil Claire1, Michael Butlin1, Parvez Iqbal1, Jevgenijs Petrovs1,
Karolis Virzbickas1, Jon A Preece1
1 ChromaTwist Ltd, Science Centre, University of Wolverhampton Science Park.
The chemical modularity of ChromaTwist’s fluorescent dye technology has allowed the development of 70+ innovative patented UV molecular excitable dyes, which emit from blue to red, with potential extension into the near-IR, making them suitable for applications in flow cytometry.
ChromaTwist dyes can achieve these long Stokes shift emissions without the need for tandem polymer dye based approaches with their associated drawbacks in manufacture and for end-users.
ChromaTwist has already developed the dye chemistry to enable conjugation to antibodies and demonstrated their utility in flow cytometry with deep UV (320 nm) and UV (355 nm) excitation in multicolour and spectral flow cytometers.
Multicolour Flow Cytometry: The chemical tunability of ChromaTwist dyes allows us to tailor their fluorescent emission outputs to align with the six visible flow cytometry filters, and ultimately the two infrared filters. This tunability is enabling the development of a portfolio of (deep) UV-excitable, molecular-based dyes which will serve the multicolour flow cytometry market.
Spectral Flow Cytometry: The chemical tunability of ChromaTwist dyes positions us to develop a portfolio of 70+ UV-excitable dyes (320 nm and/or 355 nm), each featuring a distinct spectral fingerprint. This advancement paves the way for potentially 70+ ChromaTwist-dye antibody conjugates, all excited by 320 nm and/or 355 nm UV lasers, transforming possibilities in UV-based spectral flow cytometry applications.
We present data showcasing the use of ChromaTwist dyes on multiple spectral instruments (SONY ID7000, Cytek Aurora and BD Biosciences S8), successfully obtaining distinct positively stained populations. Moreover, similarity indices indicate the potential for multiplexing with the current commercial tandem polymer UV-excitable dyes, demonstrating the utility of the ChromaTwist dye conjugates for spectral panels.
ChromaTwist Dye Technology: A Modular and Scalable Chemical Approach to Produce a Large Bio-conjugatable Portfolio of (Deep) UV Excitable Dyes With Emission Across the Visible Spectrum and Potentially into the Near-IR
Sunil Claire, Sareena Sund, Michael Butlin, Parvez Iqbal, Jevgenijs Petrovs, Karolis Virzbickas, Jon A Preece
ChromaTwist Ltd, Science Centre, University of Wolverhampton Science Park.
To date, ChromaTwist has synthesised 70+ UV excitable fluorescent dyes, via a modular and scalable chemical methodology which enables various chemical functionalities to be introduced for bioconjugation.
This chemical versatility has not only led to a comprehensive understanding of the molecular factors that influence the photophysics, but also a technology that can be scaled for imaging and sensing technologies, such as flow cytometry.
Thus, the ChromaTwist dyes are characterised by:
We will discuss the molecular design concepts and chemistry which led to such a versatile portfolio of dyes for biomedical applications, in particular flow cytometry.
T cell dysfunction as a biomarker of chemo-immunotherapy response in early Triple Negative Breast Cancer
Bhagani S1, Allsopp R1, Page K1, Achempong E1, Ayodele O2, Ahmed S2, Varadhan B2, Reza S2, Shaw J1
Background: Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer diagnosed; that lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) protein. It is the most aggressive subtype, with higher propensity to progress to metastatic disease and with a rapid disease trajectory. Moreover, it predominately affects women under 40, and those of ethnic minorities, consequently is currently an area of clinical unmet need.
Combination chemo-immunotherapy (pembrolizumab) in the neoadjuvant (NAT) setting is now the standard of care due to the favourable outcome of the Keynote 522 trial. This trial demonstrated improvements in pathological complete response (hazard ratio, 0.63, 95% CI, 0.43-0.93), as well as prolonged event free survival (EFS) (hazard ratio, 0.63, 95% CI, 0.48-0.82) and overall survival (OS) (hazard ratio, 0.66, 95% CI, 0.50-0.87). Despite, the overwhelmingly supportive survival advantage, toxicity of the regime is high. In the subgroup analysis, PD-L1 positivity was not able to distinguish EFS advantage; as such, no biomarkers are currently in use in the early/NAT TNBC setting to discriminate those with a survival advantage, as well as those likely to discontinue treatment due to toxicity.
Due to their effector mechanisms, T cells have been the focus of many of developments in the field. T cell exhaustion and senescence are two dominant dysfunctional T cell states, both of which share the progressive repression of T cell effector function. The aim of this study was to assess whether the accumulation of exhausted and senescent T cells may be responsible for cancer progression, and disparities in response to immunotherapy in TNBC.
Methods: Thirty early TNBC patients undergoing the Keynote 522 regime were recruited through the UHL11274-UoL Cancer research biobank, and followed up with serial blood sampling throughout their treatment. Whole blood samples were collected in K2 EDTA tubes, kept on ice prior to processing, and processed within 2 hours of collection, as previously described. The uninterrupted whole blood tubes with remaining buffy coat and red blood cell layer were used for peripheral blood mononuclear cells (PBMC) isolation using density gradient centrifugation. Panels for T cell exhaustion and senescence were developed, and used for flow cytometry analysis using the Attune CytPix Flow cytometer. The panel was further validated using CD3/CD28 stimulation protocol in healthy volunteer PBMC.
Results: Two flow cytometry panels were developed for T cell exhaustion and senescence using a healthy volunteer sample. Fluorescence minus one (FMO) control were used to ensure accuracy of the gating strategy.
Conclusion: A panel for T cell exhaustion and senescence has been developed and validated in healthy volunteers, with further analysis planned in stored PBMC from TNBC patients receiving NAT under the Keynote 522 regime for potential biomarker identification.
Using Fluorescence-Activated Cell Sorting to evolve therapeutics for Nipah Virus
Tanya S Masood1, Rachel Ireland2, Nicholas P J Brindle1
Departments of Cardiovascular Sciences and Biochemistry, University of Leicester1
Defence Science and Technology Laboratory2
Nipah virus, a paramyxovirus related to Hendra virus, was first identified in Malaysia in 1998. Two strains have been reported of Nipah virus, the Bangladesh strain (NiV B) and the Malaysian strain (NiV M). There are currently no licensed therapies or vaccines available to treat or prevent disease in either people or animals. Nipah Virus glycoprotein (NiVG) recognises and binds the host cell receptors EphrinB2 / EphrinB3. Soluble EphrinB2 ectodomain can therefore be used as a decoy receptor to bind NiVG on the viral surface preventing the virus from interacting and infecting host cells. However, physiologically EphrinB2 normally acts as a ligand for six members of the Eph receptor family, which constitutes the largest known subfamily of receptor tyrosine kinases (RTKs). This prevents use of EphrinB2 as a viral decoy, as it would interfere with the Ephrin-Eph signalling pathways that play important roles in regulating cell behaviour and tissue morphology. The aim of this project, therefore, is to use a Fluorescence-Activated Cell Sorting-based approach for directed protein evolution, to develop variants of EphrinB2 that selectively bind NiVG and that could be used to block Nipah virus infection, without binding and interfering with normal Eph:Ephrin signalling.
Optimising CAR T-Cell Therapy: Pulsatile Optogenetic Activation to Enhance Durability and Efficacy Against Tumours
Emma Jennings, Geroge Smith, Muna Fuyal, John James
University of Warwick
Chimeric Antigen Receptor (CAR) T-cell therapy, involving the extraction of patient T-cells and arming them with receptors designed to target tumour specific antigens, has revolutionised the treatment of haematological malignancies in recent years. Despite the notable successes, CAR T-cell therapy only confers a durable response in 30-40 % of patients and exhibits a limited efficacy against solid tumours. Rapid exhaustion of CAR T-cells in response to persistent antigen stimulation is a known contributor to this limitation.
Our work aims to explore the use optically controllable pulsatile activation, thereby reducing the total CAR T-cell stimulation time, as a method of reducing cell exhaustion. Primary CD4+ T-cells are transduced with a photoactivatable anti-CD19 CAR construct. These OptoCAR T-cells are then co-cultured with CD19+ Raji B-cells under varying pulsatile sequences of light before conventional flow cytometry and/or spectral cytometry was employed to explore their phenotype. Varied pulsatile signalling regimens led to altered expression of both co-inhibitory (e.g. PD-1) and co-stimulatory (e.g. ICOS) receptors on the surface of OptoCAR T-cells.
Recently, Th9 cells have been observed to have a more robust effectiveness against tumours, possessing an enhanced longevity. We observed that pulsatile activation of OptoCAR T-cells was able to induce a Th9 phenotype (IL-9+), in the absence of TGF-β and IL-4 cytokine culture. Our data suggests that pulsatile activation warrants further investigation as a novel method for enhanced polarization of conventional CAR T-cells to a potentially more durable and effective therapeutic modality in the form of Th9 CAR T-cells.
Introducing the Northampton Advanced Imaging Facility (NAIF): A New Hub for Flow Cytometry and Advanced Imaging
Lewis Collins, Lee Machado, Karen Anthony
Centre for Physical Activity and Life Sciences, University of Northampton.
The Northampton Advanced Imaging Facility (NAIF) is the University of Northampton’s new core facility for cutting-edge biomedical imaging and flow cytometry, offering a wide range of equipment, expert training and analytical support for both internal and external researchers.
Our flow cytometry service is designed to support diverse applications in biomedical and life science research. The facility houses a Beckman Coulter CytoFLEX platform, equipped with three active lasers (violet [405 nm], blue [488 nm], and red [638 nm]) and nine fluorescence detection channels with 13 repositionable bandpass filters, enabling high precision multiparametric cell analysis.
Researchers can benefit from comprehensive training packages, access to dedicated cell culture facilities and advanced data analysis using FlowJo software. Our case studies include cell cycle and apoptosis assays for cancer research and evaluating the efficacy of novel RNA therapeutics for rare neurological diseases.
In addition to flow cytometry, NAIF provides access to a suite of imaging technologies, including scanning electron microscopy, fluorescence microscopy, infrared microscopy, digital and optical microscopy, and slide scanning.
NAIF addresses a geographic gap in access to advanced imaging and flow cytometry, bringing world-class research capabilities to underserved regions of Northamptonshire. This facility represents a significant step in levelling up scientific infrastructure across the entirety of the Midlands fostering new collaborations and driving innovation in life science research.
Low oxygen level changes macrophage phenotype and function.
Meryem Gultekin1,4, Cathy Merry2,4, Alan McIntyre3,4, Ian Spendlove4, Hester Franks4,
Andrew M Jackson1,4
1 Host-Tumour Interactions Group, 2 Stem Cell Glycobiology Group, 3 Hypoxia and Acidosis Group, Nottingham Breast Cancer Research Centre 4 Cancer Biology. Biodiscovery Institute, School of Medicine, University of Nottingham.
Background: Macrophages represent the most abundant leukocyte population infiltrating the tumour microenvironment (TME). Given their heterogeneous and highly plastic nature, understanding the macrophage response to hypoxia—a hallmark condition of solid tumours—is essential for identifying novel therapeutic targets to combat cancer effectively.
Methods: CD14+ monocytes were isolated from whole blood and differentiated into M1, M2, and M-NECA macrophages using GM-CSF, M-CSF, and M-CSF + the adenosine analogue NECA, respectively, under normoxia (21% O₂) and hypoxia (1% O₂). The expression of HIF-1α in macrophages was assessed via Western blot. Flow cytometry was employed to evaluate changes in macrophage surface markers, while soluble analytes were quantified using the LUMINEX platform. To investigate the impact of hypoxia on macrophage functionality, a mixed lymphocyte reaction assay was conducted to determine their role in T cell activation or suppression.
Results: Hypoxic macrophage showed elevated HIF-1α expression. While hypoxia did not affect IL-12p70 secretion, it significantly reduced the secretion of IL-23 and IL-10 by 50% and 75%, respectively. Hypoxia also attenuated the expression of HLA-DR, CD86, CD206, and CD163, whereas TREM-1 expression was upregulated, with no observed changes in TREM-2 levels. Moreover, hypoxic macrophages suppressed allogeneic T lymphocyte proliferation.
Conclusion: These findings demonstrate that the adaptation of macrophages to hypoxia leads to significant alterations in their phenotype and function.
Investigating Physiological Changes in Diatoms Under Phosphorus Limitation
Rushil Patel, Steven Servin-Gonzalez & Michaela A Mausz.
School of Life Sciences, The University of Warwick
Diatoms are unicellular photosynthetic microalgae responsible for ~20% of global primary production, which makes them integral to the global carbon cycle. Key to growth and survival is the macronutrient phosphorus, which is part of major cellular components, such as nucleic acids, adenosine triphosphate, a cellular energy resource, and phospholipids, but its bioavailability can be significantly limited in the marine environment. To evade phosphorus stress, diatoms can substitute phospholipids (a large reservoir of phosphorus) for non-phosphorus-containing lipids. This important strategy, which strongly alters the membrane composition, can spare diatoms up to 30% of their total cellular phosphorus demand. Nevertheless, it is currently unclear whether lipid substitution within membranes elicits further cellular changes impacting physiology.
To better understand the consequences of lipid substitution following phosphorus limitation on growth, cell physiology and morphology, we studied the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Diatoms were axenified via antibiotic treatment or cell sorting and consequently grown under phosphorus replete and deplete conditions. Growth was monitored via flow cytometry, showing a reduction in phosphorus deplete cells over the course of the experiment. In parallel, the ratio of non-phosphorus lipids to phospholipids increased, as confirmed by liquid chromatography-mass spectrometry. This coincided with changes in the photophysiological parameter Fv/Fm, a proxy for photosynthetic efficiency, and pigment content. Further, we aimed to determine changes in cell morphology such as surface area or in the case of P. tricornutum the abundance of the two observed morphotypes by imaging flow cytometry. Hence, this indicates that phosphorus-induced lipid substitution, a strategy to evade phosphorus stress, affects diatom physiology negatively.
In conclusion, with climate models predicting both an increase in atmospheric carbon and the spread of phosphorus limitation within the marine environment, lipid substitution in these integral phytoplankton could affect future carbon fixation and, consequently, global climate.
Duchenne muscular dystrophy gene product expression is associated with survival in Sarcoma
Sonika Divakar1, Lewis Collins1, Louis El-Khoury3; Karen Anthony1*, Lee Machado1 *
1Centre for Physical Activity and Life Sciences, University of Northampton, 2Faculty of Medical Sciences, Newcastle University, 3St. Jude’s Children’s Research Hospital, Memphis, Tennessee, USA. *Senior co-authors
Mutations in the Duchenne Muscular Dystrophy (DMD) gene are commonly associated with Duchenne and Becker muscular dystrophies. Alterations in DMD expression, and the expression of the 71 kDa dystrophin protein, Dp71, may also play a role in the tumourigenesis of myogenic and non-myogenic cancers. Here, we aimed to explore this association in soft tissue (SARC) and osteosarcoma (OS). Kaplan-Meier survival and pathway analysis was conducted on publicly available soft tissue (TCGA) and osteosarcoma (TARGET) RNAseq datasets using R. Patient data was dichotomised into low DMD and high DMD expressing groups. Functionally, we characterised Dp71 expression across a panel of sarcoma cell lines as well as the effect of over-expressing specific Dp71 isoforms on various cellular functions including proliferation, cell cycle and apoptosis.
For both SARC (P = 0.031) and OS (P = 0.0015) there was improved overall survival in patients expressing high DMD. Transcript level data was available for SARC and revealed Dp40, Dp71a, Dp71ab and Dp427m were highly expressed. Significant survival differences were found for Dp40 (P = 0.023) and Dp71a (P=0.034). In functional experiments, U2OS cells were transfected with plasmids containing either Dp71a or Dp71ab which differ by the presence of DMD exon 78. Dp71ab overexpression resulted in reduced cellular proliferation in WST-1 assays (P=0.0057). Cell cycle analysis with propidium iodide staining and apoptosis analysis using Annexin V and Sytox was performed using a CytoFlex 3-laser flow cytometer. Transfection revealed no significant differences in cell cycle distribution and apoptotic cell death between Dp71a and Dp71ab transfected cells.
Our findings suggest that high DMD expression is associated with improved survival in sarcoma, supporting a potential tumour-suppressive role. Notably, the differential expression and functional effects of Dp71 isoforms, particularly Dp71ab, indicate that these variants may contribute to DMD’s role in tumour biology. These insights may provide a foundation for identifying novel therapeutic targets.
