CytoPipeline and CytoPipelineGUI: a Bioconductor R package suite for building and visualizing automated pre-processing pipelines for flow cytometry data.

BACKGROUND: With the increase of the dimensionality in flow cytometry data over the past years, there is a growing need to replace or complement traditional manual analysis (i.e. iterative 2D gating) with automated data analysis pipelines. A crucial part of these pipelines consists of pre-processing and applying quality control filtering to the raw data, in order to use high quality events in the downstream analyses. This part can in turn be split into a number of elementary steps: signal compensation or unmixing, scale transformation, debris, doublets and dead cells removal, batch effect correction, etc. However, assembling and assessing the pre-processing part can be challenging for a number of reasons. First, each of the involved elementary steps can be implemented using various methods and R packages. Second, the order of the steps can have an impact on the downstream analysis results. Finally, each method typically comes with its specific, non standardized diagnostic and visualizations, making objective comparison difficult for the end user. RESULTS: Here, we present CytoPipeline and CytoPipelineGUI, two R packages to build, compare and assess pre-processing pipelines for flow cytometry data. To exemplify these new tools, we present the steps involved in designing a pre-processing pipeline on a real life dataset and demonstrate different visual assessment use cases. We also set up a benchmarking comparing two pre-processing pipelines differing by their quality control methods, and show how the package visualization utilities can provide crucial user insight into the obtained benchmark metrics. CONCLUSION: CytoPipeline and CytoPipelineGUI are two Bioconductor R packages that help building, visualizing and assessing pre-processing pipelines for flow cytometry data. They increase productivity during pipeline development and testing, and complement benchmarking tools, by providing user intuitive insight into benchmarking results.

In depth comparative phenotyping of blood innate myeloid leukocytes from healthy humans and macaques using mass cytometry.

Comparative immune-profiling of innate responses in humans and non-human primates is important to understand the pathogenesis of infectious and chronic inflammatory diseases as well as for the preclinical development of vaccines and immune therapies. However, direct comparisons of the two species are rare and were never performed using mass cytometry. Here, whole-blood-derived leukocytes from healthy humans and cynomolgus macaques were analyzed with mass cytometry. Two similar panels of around 30 monoclonal antibodies targeting human markers associated with innate myeloid cells to stain fixed human and macaque leukocytes were constructed. To compare the circulating innate cells from the two primate species, an analysis pipeline combining a clustering analysis by the Spanning-tree Progression Analysis of Density-normalized Events (SPADE) algorithm with a two-step hierarchical clustering of cells nodes and markers was used. Identical SPADE settings were applied to both datasets, except for the 20 clustering markers which slightly differed. A correlation analysis designed to compare the phenotypes of human and macaque cell nodes and based on 16 markers, including 15 shared clustering markers and CD19 for humans or CD20 for macaques, revealed similarities and differences between staining patterns. This study unique by the number of individuals (26 humans and 5 macaques) and the use of mass cytometry certainly contributes to better assess the advantages and limits of the use of non-human primates in preclinical research. © 2017 International Society for Advancement of Cytometry.

Lipid-Polymer Hybrid Nanoparticles for mRNA Delivery to Dendritic Cells: Impact of Lipid Composition on Performance in Different Media.

To combine the excellent transfection properties of lipids with the high stability of polymeric nanoparticles, we designed a hybrid system with a polymeric core surrounded by a shell of different lipids. The aim is to use this technology for skin vaccination purposes where the transfection of dendritic cells is crucial. Based on a carrier made of PLGA and the positively charged lipid DOTMA, we prepared a panel of nanocarriers with increasing amounts of the zwitterionic phospholipid DOPE in the lipid layer to improve their cell tolerability. We selected a nomenclature accordingly with numbers in brackets to represent the used mol% of DOPE and DOTMA in the lipid layer, respectively. We loaded mRNA onto the surface and assessed the mRNA binding efficacy and the degree of protection against RNases. We investigated the influence of the lipid composition on the toxicity, uptake and transfection in the dendritic cell line DC 2.4 challenging the formulations with different medium supplements like fetal calf serum (FCS) and salts. After selecting the most promising candidate, we performed an immune stimulation assay with primary mouse derived dendritic cells. The experiments showed that all tested lipid-polymer nanoparticles (LPNs) have comparable hydrodynamic parameters with sizes between 200 and 250 nm and are able to bind mRNA electrostatically due to their positive zetapotential (20-40 mV for most formulations). The more of DOPE we add, the more free mRNA we find and the better the cellular uptake reaching approx. 100% for LPN(60/40)-LPN(90/10). This applies for all tested formulations leading to LPN(70/30) with the best performance, in terms of 67% of live cells with protein expression. In that case, the supplements of the medium did not influence the transfection efficacy (56% vs. 67% (suppl. medium) for live cells and 63% vs. 71% in total population). We finally confirmed this finding using mouse derived primary immune cells. We can conclude that a certain amount of DOTMA in the lipid coating of the polymer core is essential for complexation of the mRNA, but the zwitterionic phospholipid DOPE is also important for the particles' performance in supplemented media.

The Combination Vaccine Adjuvant System Alum/c-di-AMP Results in Quantitative and Qualitative Enhanced Immune Responses Post Immunization.

The development of new effective vaccines strongly depends on adjuvants and formulations able to stimulate not only strong humoral responses against a certain pathogen but also effector as well as memory CD4+ and CD8+ T cells (Dubensky et al., 2013). However, the majority of vaccines licensed for human use or currently under clinical investigation fail to stimulate efficient cellular responses. For example, vaccines against hepatitis B virus (HBV), human papillomavirus (HPV), diphtheria, tetanus and influenza are usually administered by intramuscular (i.m.) injection and contain aluminum salts (alum) as adjuvant. Alum has been shown to stimulate Th2 immune cells resulting in increased production of antigen-specific antibodies but to be incapable of stimulating robust Th1 or cytotoxic responses. To overcome such limitations recent research has focused on the development of adjuvant combinations (e.g., MF59, AS03 or AS04) to not only further strengthen antigen-specific immune responses but to also allow their modulation. We have shown previously that bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) constitutes a promising adjuvant candidate stimulating both effective Th1/Th2 and cytotoxic immune responses when included in mucosal or parenteral vaccine formulations. In the present work we demonstrate that c-di-AMP can be also combined with other adjuvants like alum resulting in increases in not only humoral responses but more striking also in cellular immune responses. This leads to improved vaccine efficacy against intracellular pathogens.

Prime and Boost Vaccination Elicit a Distinct Innate Myeloid Cell Immune Response.

Understanding the innate immune response to vaccination is critical in vaccine design. Here, we studied blood innate myeloid cells after first and second immunization of cynomolgus macaques with the modified vaccinia virus Ankara. The inflammation at the injection site was moderate and resolved faster after the boost. The blood concentration of inflammation markers increased after both injections but was lower after the boost. The numbers of neutrophils, monocytes, and dendritic cells were transiently affected by vaccination, but without any major difference between prime and boost. However, phenotyping deeper those cells with mass cytometry unveiled their high phenotypic diversity with subsets responding differently after each injection, some enriched only after the primary injection and others only after the boost. Actually, the composition in subphenotype already differed just before the boost as compared to just before the prime. Multivariate analysis identified the key features that contributed to these differences. Cell subpopulations best characterizing the post-boost response were more activated, with a stronger expression of markers involved in phagocytosis, antigen presentation, costimulation, chemotaxis, and inflammation. This study revisits innate immunity by demonstrating that, like adaptive immunity, innate myeloid responses differ after one or two immunizations.