Midlands Innovation Flow Cytometry Meeting
Birmingham 2018
Poster Abstracts
A research poster session will take place during the lunch break, posters will however be up all day. There will be a first and second poster prize!
1. Limb-specific training adaptations and a reduced active muscle mass can attenuate the acute inflammatory response to interval exercise
Hoekstra SP.1, Westerman M.1, Beke F.1, Bishop NC.1, Leicht CA.1
1. The Peter Harrison Centre for Disability Sport, School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, United Kingdom
This study compares the inflammatory response to upper- and lower-body interval exercise in individuals chronically trained in these respective modalities to investigate the influence of limb-specific training status and the amount of active muscle mass on the inflammatory response to exercise. 90 min of work-matched interval exercise on an arm-crank (ARM) as well as a cycle ergometer (LEG) was performed by 8 trained paddlers and 8 trained cyclists. Blood samples were taken pre-, post- and 2h post-exercise. The expression of intracellular Hsp72 in monocytes (iHsp72) and the relative distribution of CD14++CD16- classical, CD14++CD16+ intermediate and CD14+CD16++ non-classical monocytes were assessed using flow cytometry. ARM induced a significantly smaller iHsp72 response compared to LEG (ARM p=0.10; LEG p<0.001; Time x Modality p=0.05). Following ARM, only the cyclists increased iHsp72 expression (cyclists p=0.02; paddlers p=0.64), while iHsp72 expression following LEG increased in both groups (cyclists p=0.027; paddlers p<0.001). ARM and LEG induced a similar, significant increase in the percentage classical monocytes at 2h post-exercise (p<0.001), accompanied by a decrease in percentage intermediate (p<0.01) and non-classical monocytes (p<0.001) (Time x Modality p>0.13). This pattern was present in both groups. Adaptations to upper-body exercise training dampened the iHsp72 response to this modality, while upper-and lower-body exercise were equally effective in altering the monocyte subset distribution, regardless of training status. Together, this implies that regular exercise training can attenuate the stress response in an exercise modality specific manner, making exercise possibly less effective in reducing chronic low-grade inflammation in individuals relying on their upper body.
2. The effect of pulmonary rehabilitation on markers of neutrophil activation in COPD
Alex R. Jenkins, Neil S. Holden, Arwel W. Jones (University of Lincoln)
Rationale – Neutrophils play a key role in the inflammatory processes that underpin the progression of Chronic Obstructive Pulmonary Disease (COPD). Exercise, an integral component of pulmonary rehabilitation, has been shown to confer anti-inflammatory properties and modulate neutrophil activation in healthy individuals. However, the effects of exercise on neutrophil activation in COPD are unclear.
Methods – 22 mild to severe COPD patients (FEV1pred 49±15%) provided blood samples at the beginning and end of pulmonary rehabilitation. Neutrophil counts were obtained using an automated haematology analyser (Horiba ABX Pentra 60, Horiba Medical). Established markers of neutrophil activation (CD11b, CD62L & CD66b) were assessed in blood neutrophils (CD45+/CD16b+) using flow cytometry (BD FACS Verse, BD Biosciences). Plasma C-reactive protein (CRP) concentrations were measured in a subgroup of patients (n=15) a further biomarker of sub-clinical inflammation (Human CRP ELISA kit, AssayPro).
Results – Small increases in the expression of CD11b and CD62L were seen at the end of pulmonary rehabilitation compared to the beginning (CD11b, 397±1670, d=0.2; CD62L, 2181±5432, d=0.4) whereas CD66b expression remained relatively unchanged (87±1706; d<0.1). A small decrease was observed in plasma CRP (-5.4±23.5; d=0.2) and absolute blood neutrophil count (-0.26±1.44; d=0.2) at the end of pulmonary rehabilitation.
Conclusions – Preliminary findings suggest that exercise training, delivered as part of pulmonary rehabilitation, may modulate the expression of blood neutrophil activation markers (CD62L and CD11b) and concentration of CRP in COPD. CD66b expression on blood neutrophils does not appear to be altered by pulmonary rehabilitation.
3. The effect of NOX regulatory compounds on CD4+ T cell activation
Ali Remtulla1, Irundika Dias1, Kiran Shabir1, Malin Hultqvist2, Peter Olofson2 and Helen Griffiths1, 3.
1 Aston University 2 Redoxis 3 University of Surrey
A reduced cell surface protein redox state is required for receptor mediated T cell stimulation. Increases in reactive oxygen species (ROS) are predicted to inhibit T cell receptor signalling and activation. The main objective of this study was to investigate the effects of specific NOX regulatory compounds (from Redoxis) on T cell activation. Naïve CD4+ T cells were isolated from peripheral whole blood of healthy consenting volunteers using negative isolation. CD4+ T cells were activated using CD3/CD28 antibodies in the presence or absence of NOX compounds at non-cytotoxic concentrations for up to 24hours. CD3/CD28 activated CD4+ T cells in the presence of NOX compounds show no decrease in the viability and cell numbers were increased by ~20% over 24hours. Proliferation was associated with an increase in IL-2 secretion (ELISA). Surface Trx1 expression was increased in activated (Mdx=52.75±4.1) compared to un-activated (Mdx= 8.5±5) T cells after 24h. IL-2 secretion was higher in the absence of two different NOX-regulatory compounds; activated (1.76±0.02ng/ml) compared to activated in the presence of compound 1 (0.79±0.19) and activated in the presence of compound 2 (0.59±0.11ng/ml). The kinetics and nature of T cell surface redox change during activation is under investigation. In conclusion, the NOX compounds have a profound effect on T cell activation. Further identification of the proteins that change redox state during activation may provide novel targets for modulating T cell proliferation and IL-2 secretion.
4. Co-operative targeting of Bcl-2 and Mcl-1 in AML cells
Sahana Balakrishnan. University of Nottingham
Acute myeloid leukaemia (AML) cells often rely on up-regulating pro-survival members of the Bcl-2 protein family, such as Bcl-2 and Mcl-1, to avoid apoptosis. Venetoclax targets Bcl-2 and has shown promising efficacy in AML but resistance can be encountered by over-expression of Mcl-1. A co-operative approach, targeting both Bcl-2 and Mcl-1 may therefore be required. This study investigated potential co-operative relationships between Venetoclax and the novel Mcl-1 inhibitor, S63845, in MV4-11 cells and primary AML samples.
MV4-11 cells and primary AML samples were treated for 4 hours with Venetoclax, S63845 or a combination of both. A flow cytometric technique called co-operative profiling was used to assess synergy using cytochrome c release as a read out of response. This technique enabled us to identify response in the CD34+/CD38- leukaemic stem cell (LSC) fraction of primary samples.
Our data demonstrates that Venetoclax and S63845 act synergistically in an AML cell line and in primary samples (95% of bulk cells and 92% of LSCs). Primary samples with an IDH mutation were more sensitive to Venetoclax as a single agent (p=0.011) whereas samples with a FLT3-ITD mutation were more resistant (p=0.015). Conversely FLT3-ITD samples were more sensitive to S63485 as a single agent (p=0.029). All FLT3-ITD and NPM1 mutated samples were sensitive to cytochrome c release when treated with the combination of Venetoclax and S63485.
Our data demonstrates that co-operatively targeting Bcl-2 and Mcl-1 may be beneficial in AML and molecular genetics can identify patients who might best respond to this targeted treatment.
5. Measurement of pre-platelets using ImageStream flow cytometry
Sam Kemble. University of Birmingham.
Measuring platelet turnover is an essential tool for diagnosing and treating thrombocytopenic disorders. However, the current method for quantifying newly produced platelets by flow cytometry (i.e. immature platelet fraction, IPF) is based upon arbitrary gating. Immature platelets are generally larger, with a greater mRNA content than mature platelets. In response to enhanced peripheral destruction and feedback to the bone marrow, the number of large, RNA-rich platelets increases. It is now recognised young circulating platelets can undergo division via pre-platelet barbell shaped intermediates that are visible in citrated but not EDTA anticoagulated blood (Thon et al., 2010) due to platelet swelling. As EDTA is used routinely to anticoagulate blood samples for IPF determination, we hypothesize that this artefact has a significant impact upon the measurement. Image-based flow cytometry utilises high acquisition rates of conventional flow cytometry combined with microscopy to image all acquired events. With a robust image analysis platform (IDEAS) it is possible to discriminate between different shapes of cells that appear to be immunologically identical based on a comparison of complex morphological parameters. Using the ImageStream MKII (Merck Ltd) we have now designed a new method to quantify pre-platelets in whole blood. Normal blood samples were labelled with anti-CD61, P-selectin and a nucleic acid dye and analysed using the ImageStream. Preliminary data confirms that higher numbers of pre-platelets are present in citrate whole blood compared to EDTA under steady state production. Quantitative analysis of pre-platelets potentially offers a new parameter for measuring platelet turnover.
6. Extracellular vesicles released by CD40/IL-4-stimulated CLL cells confer altered functional properties to CD4+ T cells.
Smallwood DT, Apollonio B, Willimott S, Lezina L, Alharthi A, Ambrose AR, De Rossi G, Ramsay AG, Wagner SD. De Montfort University
Extracellular vesicle (EV) release by stimulated chronic lymphocytic leukaemia B-cells alters the functional properties of CD4+ T-cells. The microRNA miR-363 was identified by microarray analysis and found to be enriched in EVs compared to parental cell microRNA expression profiles. Potential binding sites for miR-363 were identified using TargetScan software. Reporter assays were undertaken to confirm its target as T-cell immunoregulatory receptor CD69.
Flow cytometry showed repression of CD69 expression in human Jurkat T cells following transfection with a miR-363 mimic.
Incubation of primary CD4+ T cells with stimulated autologous CLL-EVs increased intracellular levels of T-cell miR-363. Flow cytometry analysis revealed that incubation of primary CD4+ T cells with stimulated autologous CLL-EVs led to significant suppression of CD69 expression compared with the treatment of T cells with control CLL-EVs from unstimulated CLL cells.
Further studies, confirmed by miR-363 knockdown, revealed increased CD4+ T-cell motility rate, increased immunological synapse signalling and interaction with tumour cells. These studies suggest EVs play an important role in crosstalk between tumour cells and the tumour micro environment.
7. Flow cytometry-based evaluation of the bacterial removal efficiency of a blackwater reuse treatment plant and the microbiological changes in the associated distribution network
Rachel Whitton, Sarah Fane, Peter Jarvis, Martyn Tupper, Marie Raffin, Frédéric Coulon and Andreas Nockera.
a Cranfield Water Sciences Institute, Cranfield University, United Kingdom
b Thames Water Utilities Ltd, United Kingdom
c IWW Water Centre, Germany
Flow cytometry (FCM) was applied as a rapid method for monitoring bacterial numbers across a full-scale membrane bioreactor (MBR) plant for blackwater reuse and the associated distribution network. The Old Ford Water Recycling Plant, operated by Thames Water, is the UK’s largest blackwater reuse plant. The plant abstracts and treats raw municipal and light commercial waste from the sewer. The treated water is then chlorinated and distribution through a dedicated network to venues and facilities within the Queen Elizabeth Park (which hosted the London Olympic Games in 2012) for the purpose of direct non-potable use (irrigation and toilet flushing). Total and intact bacterial concentrations across the treatment train and throughout the distribution network were analysed by FCM.
Treatment by the MBR resulted in a 5-log reduction in bacterial numbers resulting in coliform-free treated water. In the distribution network, the proportion of intact cells greatly depended on the free chlorine residual, with a decreasing residual enabling regrowth. Such loss of residual chlorine mainly applied to distant points in the distribution network from the MBR plant. Flushing these network points for 5 mins did not substantially reduce cell numbers. However, at points closer to the MBR plant, cell numbers fell by up to 1.5-log units. The outcomes of this study assisted Thames Water to adjust their network flushing events, particular in periods of low water demand, and define a threshold chlorine residual which should be maintained within the network to avoid the risk of deteriorating microbiological water quality.
8. Reduced Pro-coagulant Phenotype of Microparticles in Pancreatic Cancer Patients on Omega-3-Supplemented Gemcitabine Treatment
Martin N 1,2, Hackney AB 1, Isherwood J 3, Runau F 3, Arshad A 3, Chung WY 2,3, Dennison AR 3
1Health and Life Sciences, De Montfort University, Leicester, UK; 2Infection, Immunity and Inflammation, University of Leicester, Leicester, UK; 3Hepato-Pancreato-Biliary Unit, University Hospitals of Leicester, Leicester, UK
Pancreatic cancer (PC) is an aggressive cancer associated with advanced disease at diagnosis and lack of effective therapy. Microparticles (MP), biologically active nanoparticles shed from cells, are novel markers of inflammation and might contribute to the growth and spread of PC. The aim of this study is to determine the effects of omega-3 supplementation in PC patients.
MP were isolated from blood taken from PC patients undergoing Gemcitabine treatment (n=9, CON), patients undergoing treatment supplemented with omega-3 fatty acids (n=18, Ω-3) and from healthy controls (n=6, HC). MP were stained with AnV (PS-MP) and anti-CD142 to identify those expressing tissue factor (TF), acquired and analysed using flow cytometry.
Circulating MP in both groups of PC patients was significantly greater than HC (CON 84±17X105/ml (P<0.03), Ω-3 105±22x105/ml (P<0.05) vs HC 31±10x105/ml), these MP were also significantly more pro-coagulant than HC MP (CON 13795RFU and Ω-3 11232RFU vs HC 7587RFU, P<0.05). MP were significantly greater in number and size at later disease stage in CON (63±19x105/ml stage 3 vs 128±20x105/ml stage 4, P<0.03; HiFSC 156±15 stage 3 vs 533±213 stage 4, P<0.03), and significantly fewer in Ω-3 patients at early disease stage (136±36x105/ml, P<0.03). This effect on size of MP might be explained by in vitro experiments where incubation with rHuTF increased the size of MP released by a PANC1 cells (P<0.05).
The percentage of PS-MP was significantly decreased in Ω-3 patients (6.48% vs HC 13.48%, P<0.03). Significant increases in CD142-MP (P<0.05) and the increases in smaller CD142-MP at later disease stage (41±6 stage 3 vs 107±34 stage 4, P<0.03) seen in CON were not seen in Ω-3 patients, but overall Ω-3 patients had significantly greater numbers of smaller CD142-MP (117±24, P<0.03) at earlier disease stage.
Our results show that omega-3 supplementation affects the pro-coagulant nature and size of MP in PC patients. Further work is required to assess the effects of omega-3 and the role of MP in PC.
9. Differential effects of ethnic microparticles on endothelial cell death and apoptosis
Pritchard C 1, Henshaw M 1, Hussain A 1, Yousaf N 1, Akubueze A 1, Martin N 1,2
1Health and Life Sciences, De Montfort University, Leicester, UK; 2Infection, Immunity and Inflammation, University of Leicester, Leicester, UK
Individuals of South Asian and Black African origin are at a greater risk of diabetes, cancer and cardiovascular disease (CVD). Microparticles (MP), biologically active nanoparticles shed from cells, are novel markers of inflammation and might contribute to the pathogenicity of disease. The aim of this study is to assess the phenotypic differences of ethnic MP and their effects on endothelial cells in vitro.
In this pilot study, MP were isolated from blood taken from healthy volunteers of different ethnic groups (White n=6, Black n=7, Asian n=4; there were no differences in age or BMI of ethnic groups), and stained with AnV for PS-MP, CD142 (Tissue Factor) or CD295 (LeptinR); MP were also stimulated with 100ng/ml PMA and stained with DCFH-DA to assay ROS activity. Endothelial cell line EA.hy926 were seeded to confluence in 48-well plates, MP in media were added for 48 hours at 37⁰C 5% CO2. Supernatant MP were removed, and EA.hy926 were stained with AnV-PI for apoptosis. All MP and EA.hy926 were analysed using flow cytometry.
Circulating Asian MP stained significantly greater for pro-coagulant PS-MP (5.09±0.38% vs White 2.98±0.79%, Black 3.53±0.58%, P<0.03), pro-coagulant CD142-MP (11.57±1.10±0.38% vs White 8.24±1.23%, Black 7.56±0.40%, P<0.01) and tumourigenic CD295-MP (52.41±2.73±0.38% vs White 46.42±2.82%, Black 45.15±2.76%, P<0.01) than both White and Black MP. Black MP had a reduced, but not significant, change in ROS DCFH-DA-signal upon stimulation with PMA.
Interestingly, Asian MP induced a significantly greater proportion of smaller MP released from EA.hy926 cells (56.70±5.02% vs Black 39.70±3.10%, P<0.05), and greater apoptosis of EA.hy926 (19.80±1.97% vs Black 15.09±1.31%, P<0.05). Black MP induced significantly greater EA.hy926 death (1.07±0.15% vs White 0.92±0.08, P<0.005).
Our results suggest that the increased pro-coagulant and tumourigenic nature of Asian MP might affect disease pathology. Some of these effects include apoptosis and release of small MP from EA.hy926 in vitro, but the clinical significance of these findings is unclear. Black MP have reduced ROS upon stimulation, possibly due to higher baseline ROS levels and oxidative anergy; these MP also induce greater death in EA.hy926. These results might indicate a role of MP in the increased incidence of CVD in Black individuals. Further work on the pathophysiological role of ethnic MP is required to assess these initial findings.
10. Assessment of operator variation in flow cytometry measurements using Gauge R&R techniques
Rebecca Grant1, Nick Medcalf1, Karen Coopman1, Sebastian Mayer2, Bo Kara2, Sandro
Gomes2, Jonathan Campbell3, Julian Braybrook3, Tamara Lekishvili3, Jon Petzing1
1Centre for Biological Engineering, Loughborough University
Loughborough, Leicestershire, UK
2GlaxoSmithKline, Stevenage, Hertfordshire, UK
3LGC, Teddington, Middlesex, UK
An analysis of variation of HSCT starting materials has highlighted the potential for large deviations of practice in operator use of flow cytometers at clinical facilities. This has led to a variation of QC data within cell therapy bioprocessing, and at distinct stages various efforts are being pursued to minimise this variation and the impact on reported data. External Quality Assessment Schemes focus on inter-laboratory variation of results when a
standard sample and operator training are provided, whilst EuroFlow protocols provide standardised analysis to use with local samples. Furthermore, automated software is abundant for flow cytometry, but is often met with scepticism. Little has been published on intra-laboratory variation for flow cytometry, which is the basis of this developing research,
providing a bottom-up approach to quantification. Gauge R&R studies have been conducted with research and process based participants from Loughborough University and GlaxoSmithKline, analysing pre-defined Flow Cytometry data, from simple histogram gating to full derivation of certain cellular subsets. Analysis of the results has shown large variation of output data between operators and contributory variation. However, application of simple diagrammatical reference aids significantly reduced operator variation, providing supporting evidence for introducing more streamlined
protocols to improve flow cytometer results.
11. Pathogenesis of Epstein-Barr Virus Infections of T and NK cells
Paul Collins1, Gordon Ryan1, Christopher Fox2, Hayden Pearce1, Carmela Di Santo1, Heather Long1, Claire Shannon-Lowe1
1Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
2Nottingham University Hospitals NHS Trust, Nottingham City Hospital, Hucknall Road, Nottingham, NG5 1PB, UK
Background
Epstein-Barr virus (EBV) infects most people and is usually maintained as an asymptomatic infection of memory B cells for life. On rare occasions, however, EBV infects T or NK cells; resulting in a range of lymphoproliferations and malignancies. Chronic active EBV infection (CAEBV) is one such disease, characterized by persistent infectious mononucleosis-like symptoms and an inability to control the virus; CAEBV has a 50% mortality rate at 10 years.
Methods
Peripheral blood mononuclear cells (PBMC) were isolated from 3 patients with CAEBV. A novel flow cytometry-based fluorescence-in situ hybridization technique was used to detect EBV-encoded small RNAs (EBERs), a hallmark of EBV infection, in lymphocytes; 2 had EBV in their CD4+ T cells, while the third had EBV in their NK cells. EBV-infected and –uninfected counterparts were analysed for phenotype and T cell receptor usage.
Results
T and NK cells infected with EBV were a clonal population, indicating that they have originated from a single infected precursor. EBV-infected T cells displayed an altered immunophenotype, are highly activated and polarize toward distinct functional subsets. T helper 17 cell cytokines were elevated in the plasma of CAEBV patients while myeloid-derived suppressor cells (MDSC), which co-purified with PBMC, were expanded in these patients. MDSC were shown to potently suppress T cell responses, potentially revealing the mechanism by which EBV-infected cells escape immune control.
Conclusion
Use of a novel flow cytometry-based technique, which allows for the simultaneous detection of viral RNA species and host proteins, is revolutionizing our understanding of chronic active EBV infection.