Stamford Court, University of Leicester, Manor Road, Oadby,
Stamford Court is conveniently located within easy reach of the M1 and M69 motorways. For Sat Navs please use LE2 2LH. There are complimentary car parking spaces.
Our host this year is Jenny Hincks at the University of Leicester.
Thanks to our Sponsors
10:00-12:30 Session 1.
10:00-10:10 – Introduction by Andrew Fry, College of Life Sciences, University of Leicester
10:10-11:10 – The Many Faces of Flow: Diverse Applications of Flow Cytometry - Early Careers Presentations
10:10-10:30 – Reem Ali Targeting DNA polymerase β for platinum sensitization and synthetic lethality in epithelial ovarian cancers
10:30-10:50 – Nonantzin Beristain-Covarrubias Asynchronic development of Salmonella-induced thrombi in spleen and liver
10:50-11:10 – Paulina Tomaszewska Measuring and exploiting biodiversity in Brachiaria and Panicum tropical forage grasses
11:10-12:10 – Immune Focus: B-Cells in Flow
11:10-11:30 – Raul Maqueda Antibody Secreting Cells Dynamics Following Cutaneous Dengue Virus Infection in Immune-Competent Mice
11:30-11:50 – Inara Liepina EBF1 is an important component of pro-B cell structural matrix
11:50-12:10 – Juan Carlos Yam-Puc The enigmatic role of IgD still veiled: effects of chimeric IgD-IgG1 BCR on B cell development and B cell activation
12:10-12:30 – Flash Poster Presentations
13:30-15:00 Session 2. Commercial Workshops
1. Advanced Flow Cytometry and Data Analysis.
13:30-14:15 – John Lawry, BD Biosciences – Multi-colour Problem Solving
14:15-15:00 – Thomas Adejumo, Fluidigm – Simplifying High dimensional Flow cytometry
2. Basic Introduction to Flow Cytometry.
13:30-14:00 – Mike Blundell, Biorad – To Boldly Flow…
14:00-14:30 – Aimee Tyler, Miltenyi Biotech - The Importance of Sample Preparation for Flow
14:30-15:00 – Sun Yung, Nexcelom – Cellometer Spectrum
15:30-16:30 Session 3. Keynote Speaker
Derek Davies The Changing Landscape of Cytometry Training
16:15-16:30 - Presentation of Prizes
16:30-17:00 Drinks Reception
10:10-10:30 – Reem Ali
Targeting DNA polymerase β for platinum sensitization and synthetic lethality in epithelial ovarian cancers
Reem Ali, Adel Alblihy, Islam M Miligy, Muslim L Alabdullah, Mansour Alsaleem, Michael S Toss, Tarek Abdel-Fatah, Paul Moseley, Stephen Chan, Nigel Mongan, Satya Narayan, Emad A Rakha and Srinivasan Madhusudan
Ovarian cancer (OC) is the fifth most common cause of cancer death in women worldwide. Standard treatment available for ovarian cancer is surgery and platinum-based chemotherapy. However, Tumours mostly recur after 6 months prior to platinum therapy. Targeting PARP1 for synthetic lethality (SL) is an exciting strategy for BRCA germ-line mutated ovarian cancers. However, not all patients respond due to intrinsic or acquired resistance. Development of alternative synthetic lethality approaches is an area of unmet need. Polb, a critical player in BER, interacts with PARP1 during DNA repair.
Here we show that polb deficiency is a predictor of platinum sensitivity in human tumours. Polb deficiency not only promoted platinum sensitivity but also reduced invasion, migration and impaired EMT of ovarian cancer cells. We implied flow cytometry to characterize the DNA damage of Polb small molecular inhibitors (Pamoic acid & NSC666719) in ovarian cancer cell lines. Pamoic acid & NSC666719) were selectively toxic to BRCA2 deficient cells including in 3-D spheroid models. Increased toxicity was associated with double strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis. Interestingly, PARG inhibitor (PDD00017273) but not PARP1 inhibitor (Olaparib) was synthetically lethal in polb deficient cells including in 3-D spheroid models. Increased cytotoxicity to PDD00017273 treatment was associated with poly (ADP-ribose) (PAR) accumulation, reduced NAD+ level, DSB accumulation, cell cycle arrest and increased apoptosis. Our data provides evidence that polb targeting is attractive and warrants further pharmaceutical development.
10:30-10:50 – Nonantzin Beristain-Covarrubias
Asynchronic development of Salmonella-induced thrombi in spleen and liver
Nonantzin Beristain-Covarrubias*, Marisol Perez-Toledo**, Julie Rayes*, Steve P. Watson*, Adam F. Cunningham**.
*Institute of Cardiovascular Sciences and **Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, UK
Thrombosis is a life threatening consequence of infection. We have shown that 7 days after systemic infection with attenuated Salmonella Typhimurium (STm) there is a widespread inflammation-driven thrombosis in the liver. We demonstrated that in liver, thrombosis is mediated by a CLEC-2 (platelets)-podoplanin (PDPN) (monocytes/macrophages)-dependent mechanism. Being the spleen rich in platelets and monocyte-lineage cells, and a major site of bacterial colonisation; it was surprising not to observe extensive thrombosis concurrently with the liver. Here, we further assessed development of splenic thrombi in C57BL/6J mice infected with STm, by imaging thrombosis and bacteria in spleen sections at different time-points post-infection. Furthermore, PDPN expression on splenocytes and platelets activation was assessed by FACS. In contrast to the liver, within 24h after infection there were large and numerous thrombi in the spleen that largely resolved 48h post-infection, despite total bacterial numbers continuing to rise. Although, there was an associated upregulation of PDPN on splenic monocytes/macrophages, thrombi were maintained in the absence of either CLEC-2 on platelets or PDPN on hematopoietic cells. Thus, thrombosis in spleen and liver does not share similar mechanisms. Moreover, most of thrombi analysed contained low numbers or no bacteria not being associated with bacterial capture. Together, we show that Salmonella infection induces thrombosis in two different organs following completely different kinetics and presumably under distinct mechanisms. A better understanding of the mechanisms underlying infection-mediated thrombosis offer to improve the current therapeutic approaches for treating this life-threatening complication.
10:50-11:10 – Paulina Tomaszewska
Measuring and exploiting biodiversity in Brachiaria and Panicum tropical forage grasses.
Paulina Tomaszewska, University of Leicester
Flow cytometry is applied to plant research for the estimation of ploidy, cell cycle activity, endoreplication level and genome size. Ploidy screening is one of the most frequent applications of FCM in plants. Up to 70% of extant vascular plant species are believed to be recent polyploids. FCM allows for an accurate and facilitated detection of subpopulations of different ploidy levels. The method is rapid, convenient and suitable for many plant species, organs and tissues. Our project is a collaboration between International Center for Tropical Agriculture (CIAT, Columbia) and UK scientists to deliver state-of-the-art knowledge and tools to breed better forage grasses. We established flow cytometry protocol for dried, imported leaves of Brachiaria and Panicum forage grasses and identified ploidy levels of 342 accessions (60% of entire CIAT germplasm collection). The samples were analyzed using an Accuri C6 Flow Cytometer (Becton Dickinson). We found ploidy varied widely (2x, 4x, 5x, 6x). These data are valuable to support genomic selection and allow use of the accessions in crossing or breeding programmes. Newly identified tetraploid genotypes are candidates to enrich the diversity of breeding populations, while diploids with promising phenotypic characteristics could be subjected to ploidy duplications.
11:10-11:30 – Raul Maqueda
Antibody Secreting Cells Dynamics Following Cutaneous Dengue Virus Infection in Immune-Competent Mice
Maqueda-Alfaro RA. 1, Marcial-Juárez E. 1, Calderón-Amador J. 1, García-Cordero J.2, Cedillo-Barrón L. 2, Flores-Romo L. 1. Department of Cell Biology1 and Molecular Biomedicine 2, Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV-IPN), Zacatenco, Mexico City.
Dengue virus (DENV) is an important viral pathogen affecting ~390 million people worldwide yearly. Recent studies describe a massive and rapid appearance of Antibody Secreting Cells (ASCs) in peripheral blood, by days 4 to 7, after Dengue fever onset in humans. Despite of antibodies produced by these cells might be involved in Dengue immunopathology, ASCs responses to acute DENV infection are poorly characterized. We have previously described the induction of large DENV-specific germinal centers in the draining lymph node (DLN) of immune-competent mice cutaneously infected with DENV-2. However, it remains so far unknown whether DENV promotes the generation of ASCs in this model. By FACS, we have evaluated the kinetics of ASCs generation; their transit through the different stages of the cell cycle, apoptosis rate and the proportion of class-switched cells. Mice inoculated with PBS or inactivated DENV-2 (iDENV) were used as controls. ASCs peaked at day 10 after DENV-infection, abruptly decreasing by day 14. Their characteristics indicate they are class-switched proliferating plasmablasts. Although we found ASCs in response to iDENV, the extent of this response was significantly lower compared to the one to active DENV. Further evaluation of the specificity of these ASCs to DENV will help us to clarify whether they are DENV-specific or the virus itself provokes a polyclonal antigen-independent response. Our results resemble the observations in human patients with Dengue. We consider this animal model can be importantly used to understand the in vivo biology of the humoral immune response to DENV.
11:30-11:50 – Inara Liepina
EBF1 is an important component of pro-B cell structural matrix
Inara Liepina, University of Leicester
EBF1 is a transcription factor which activates B-cell development at pre-pro-B cell phase and maintains B-cell development by suppressing cell differentiation into alternative lineages. Tamoxifen-induced EBF1 depletion in pro-B-cells, which expresses floxed Ebf1 and the Cre recombinase under the control of a tamoxifen-inducible promoter (EBF1fl/flRERTCre) results in nuclear matrix collapse, suggesting that EBF1 is an important component of pro-B cell structural matrix. Here we use flow cytometry as a tool to verify EBF1 depletion and EBF1fl/flRERTCre pro-B cell arrest at G1 phase upon EBF1 depletion. As well as a tool to test HDAC inhibitory effect on the EBF1 depleted pro-B cells in order to better understand the EBF1 role in the pro-B cell structural matrix.
11:50-12:10 – Juan Carlos Yam-Puc
The enigmatic role of IgD still veiled: effects of chimeric IgD-IgG1 BCR on B cell development and B cell activation
Juan Carlos Yam-Puc1, Lingling Zhang1, Yang Zhang1, Gillian Grafton2, See Heng Wong3, Mike Snaith3, and Kai-Michael Toellner1. 1Institute of Immunology and Immunotherapy, 2Institute of Clinical Sciences, College of Medical & Dental Sciences, University of Birmingham, Birmingham, UK
3Antibody Discovery and Protein Engineering, MedImmune, Granta Park, Cambridge, UK.
B-cell receptors (BCRs) have unique heavy chain structures and cytoplasmic domains. While membrane IgD (mIgD) and mIgM have short cytoplasmic regions and need Igα/Igβ for signalling, mIgG cytoplasmic tail is able to transmit signals. B-cells begin their development dependent on IgM expression and start to express mIgD when they finish this development in the spleen. To test whether modified mIgD signalling leads to changes in B-cell fate decisions during development and the immune response, we have generated IgDg1 mice by adding the IgG1 cytoplasmic tail to the C-terminus of mIgD. IgDg1 B-cells still contain the normal VDJ gene repertoire and are able to undergo class switch recombination. By FACS, we show that IgD-BCR changes leads to compensatory reduction of surface IgD in all B-cell subsets and a 50% larger splenic marginal zone B-cells. Numbers of other B-cell subsets are less affected. In vitro anti-IgD stimulation of mature IgDg1 B-cells leads to prolonged calcium influx and reduced phosphorylation of SYK and BLNK, while IgM signalling is unaffected. There are no major effects on antigen-dependent B cell differentiation. After antigen-induced activation, B-cells lose mIgDg1 expression presenting a conventional immune response and undergoing normal class-switch recombination and affinity maturation. Considered an enigmatic molecule and being present in all vertebrate taxa, most likely as old as IgM, IgD role is still veiled. Thus, further research is needed to understand the function of IgD.
15:30-16:30 Session 3. Keynote Speaker - Derek Davies
The Changing Landscape of Cytometry Training
Flow cytometry is a well-established technique in clinical, research and industrial settings. Although the basic principles have remained almost the same over the past 50 years there have been alterations in the ways we approach flow experiments notably in the areas of signal detection and processing, and data analysis. Teaching cytometry has also changed in recent years. It is important that all end users understand the basics of the cytometer, how to plan and execute an optimal experiment and how to analyse that experiment effectively so that data can be displayed correctly and appropriate metrics derived. There is a clear distinction though between creating a cytometrist and someone who can run a cytometry experiment. In recent years, we have seen the emergence of specific centres that curate a National capability in a particular technology; this will allow access to state of the art equipment and be staffed by those not only able to operate and understand the technology but also be able to train others to use this - the concept of ‘training the trainer’ allows this knowledge to be disseminated. Currently this does not exist for flow cytometry. Here I will discuss some of the factors that might allow this to happen and how this would affect the way we consider training in cytometry.
Worms for immune regulation of multiple sclerosis (WIRMS): a randomised double-blinded placebo controlled trial
Radu Tanasescu, Nanci Frakich, Sonika Singh, David Onion, Cris S Constantinescu
Background. Epidemiological studies supporting the hygiene hypothesis show inverse relationship between Multiple Sclerosis (MS) and infections with gut helminths. Experimental and observational studies suggest gut worms can induce an immune response that protects against MS increasing regulatory T cells (Treg).
Methods. A phase II, randomized, double-blind, placebo controlled trial of Necator americanus was done in relapsing MS. Cumulative number of new and enlarging T2 lesions and gadolinium enhancing lesions (Gd+) at month 9 was assessed. In addition, the percentage of Treg as CD4+CD25++CD127- T cells and CD4+CD25++Foxp3+ -T cells at screening and 9 month visits were determined.
Results. Patients were randomly assigned to group 1 (hookworm; n=35) or group 2 (placebo; n=36).
Median cumulative number of new, enlarging and Gd+ lesions at month 9 were not significantly different by pre-planned Mann-Whitney test. However, a significance proportion of patients in the group 1 had no MRI activity.
An increase in CD25++CD127-T regulatory cells at month 9 was observed in the hookworm group (compared to placebo, p=0.013).
Conclusion. This is the first randomised controlled trial of hookworm infection in MS. Hookworm was safe and well-tolerated.
Based on the pre-planned Mann-Whitney test, the differences in the primary outcomes were not statistically significant. However, the zero detectable MRI activity, particularly in the treated group, suggests a therapeutic effect of hookworm.
Controlled hookworm infection also increased the proportion of regulatory T cells in people with MS. The potential of hookworm as immunomodulator in MS warrants further investigation.
Funding. Supported by the UK MS Society, an unrestricted grant from Bayer, and the Forman Hardy Charitable Trust via the University of Nottingham.
The development of multi-parameter flow cytometry panels to identify immune cells in renal transplant recipients and healthy controls
Ganisha J Fatania1, Alice C Smith1, John E Pearl2, Nicolette C Bishop2,3, Andrea M Cooper2
1Department of Health Sciences, University of Leicester, UK
2Department of Respiratory Sciences, University of Leicester, UK
3School of Sport, Exercise and Health Sciences, Loughborough University, UK
Introduction: Cardiovascular disease is a major cause of morbidity and mortality for renal transplant recipients (Collins, Foley, Gilbertson, & Chen, 2015), with infection and malignancy also limiting graft and patient survival (Bamoulid et al., 2016). Immunosuppressive medications alter innate and adaptive immunity and can result in immune dysfunction. Over-suppression of the immune system can result in open windows of opportunity for patients to contract an infection (Walsh et al., 2011), and under-suppression can result in graft rejection, limiting survival rate. Therefore, it is vital to monitor immune status in this population in clinical settings and in research. Our aim was to design multi-colour flow cytometry panels to assess lymphoid and myeloid cell populations from human peripheral blood mononuclear cells from renal transplant recipients. These panels cover cell types such as T cells, B cells, natural killer (NK cells), dendritic cells (DCs) and monocytes as well as phenotypic and chemokine migratory receptors of these populations.
Methods: A 10-colour lymphocyte panel and two 6-colour myeloid panels (monocytes and dendritic cells) have been designed to assess changes in the frequency and phenotype of cells within the peripheral blood of renal transplant recipients undergoing defined exercise programmes. Peripheral blood mononuclear cells were isolated from whole blood of renal transplant recipients (n=5, age=50±6 years, eGFR=46±18 mL·min·1.73m2, BMI=27±1) and healthy controls (n=3, age=38±17, eGFR=>90 mL·min·1.73m2, BMI=29±1). These cells were stained with lymphoid lineage markers: CD3, CD4, CD8a, CD19, CD56, and function associated markers: CD45RA, CD45RO, CD127, CD25, and CD197. Monocyte subsets were identified using HLA-DR, CD14, CD16, and their migratory potential determined using CCR2, CCR5 and CX3CR1. Dendritic cell populations were identified using HLA-DR, CD1c, CD11c, CD141, CD123 and CD14.
Results/ Discussion: The panels allow detection of cell populations in peripheral blood cells from renal transplant recipients and healthy volunteers. These panels are now being used to assess immune cell populations in renal transplant recipients undergoing three distinct exercise protocols. Determining how cell populations change in response to exercise will prove information regarding the impact of exercise on immune status in this population.
Preliminary data observing dendritic cell populations in renal transplant recipients and healthy controls
Ganisha J Fatania1, Andrea M Cooper2 , Nicolette C Bishop2, 3, Alice C Smith1
1Department of Health Sciences, University of Leicester, UK
2Department of Respiratory Sciences, University of Leicester, UK
3School of Sport, Exercise and Health Sciences, Loughborough University, UK
Introduction: Infection and malignancy are the major causes of morbidity and mortality in renal transplant recipients, and can also limit graft survival (Bamoulid et al., 2016). Immunosuppressive medications alter innate and adaptive immunity and may lead to immune dysfunction. Over-suppression of the immune system can result in open windows of opportunity for infection (Walsh et al., 2011), and under-suppression can result in graft rejection. Dendritic cells (DCs) are recognised as important inducers and regulators of innate and adaptive immunity (Steinman & Cohn, 1973). Low frequency of circulating dendritic cells can result in a lack of activated DCs migrating to the lymph nodes (Alvarez, Vollmann, & von Andrian, 2008), limiting patient and graft survival. Therefore, our aim was to assess differences in dendritic cell populations in renal transplant recipients and healthy controls.
Methods: Venous blood samples were obtained from renal transplant patients (n=7, mean±SD: age=45±10, eGFR=46±18 mL·min·1.73m2, BMI=28±6) and healthy controls (n=3, age=38±17, eGFR=>90 mL·min·1.73m2, BMI=29±1). Peripheral blood mononuclear cells (PBMCs) were isolated using density-gradient centrifugation and frozen for analysis. A six-colour flow cytometry panel was designed to assess DC lineage and phenotypic markers: HLA-DR, CD14, CD11c, CD123, CD1c and CD141. Cells were also assessed for viability and doublet discrimination. Cells highly expressed for HLA-DR but not for CD14 were used to exclude monocytes. High expression of CD123 and low expression of CD11c were characterised as plasmacytoid dendritic cells. Cells with low expression of CD123 and high expression of CD11c were characterised as myeloid dendritic cells. Differential subsets of CD141 and CD1c were identified from myeloid dendritic cells. A multivariate analysis of variance was conducted (P<0.05, figure 1).
Results/ discussion: Preliminary results showed no apparent differences in myeloid dendritic and plasmacytoid dendritic cell populations or the differential CD141 and CD1c subsets between renal transplant recipients and healthy (P>0.05). Confirmation of these observations will require further analysis using a larger sample size.