Plasma dynamics of neutrophil extracellular traps and cell-free DNA in septic and non-septic vasoplegic shock: a prospective comparative observational cohort study.

BACKGROUND: The association between neutrophil extracellular traps (NETs) and the requirement for vasopressor and inotropic support in vasoplegic shock is unclear. This study aimed to investigate the dynamics of plasma levels of NETs and cell-free DNA (cfDNA) up to 48 hours after the admission to the intensive care unit (ICU) for management of vasoplegic shock of infectious (SEPSIS) or non-infectious (following cardiac surgery, CARDIAC) origin. METHODS: Prospective, observational study of NETs and cfDNA plasma levels at 0H (admission) and then at 12H, 24H and 48H in SEPSIS and CARDIAC patients. The Vasopressor Inotropic Score (VIS), the Sequential Organ Failure Assessment (SOFA) score and time spent with invasive ventilation, in ICU and in hospital were recorded. Associations between NETs/cfDNA and VIS and SOFA were analysed by Spearman's correlation (rho), and between NETs/cfDNA and ventilation/ICU/hospitalisation times by generalised linear regression. RESULTS: Both NETs and cfDNA remained elevated over 48 hours in SEPSIS (n = 46) and CARDIAC (n = 30) patients, with time weighted average concentrations greatest in SEPSIS (NETs median difference 0.06 [0.02-0.11], p = 0.005; cfDNA median difference 0.48 [0.20-1.02], p < 0.001). The VIS correlated to NETs (rho = 0.3-0.60 in SEPSIS, p < 0.01, rho = 0.36-0.57 in CARDIAC, p ≤ 0.01) and cfDNA (rho = 0.40-0.56 in SEPSIS, p < 0.01, rho = 0.38-0.47 in CARDIAC, p < 0.05). NETs correlated with SOFA. Neither NETs nor cfDNA were independently associated with ventilator/ICU/hospitalisation times. CONCLUSION: Plasma levels of NETs and cfDNA correlated with the dose of vasopressors and inotropes administered over 48 hours in patients with vasoplegic shock from sepsis or following cardiac surgery. NETs levels also correlated with organ dysfunction. These findings suggest that similar mechanisms involving release of NETs are involved in the pathophysiology of vasoplegic shock irrespective of an infectious or non-infectious etiology.

Machine learning predicts cancer subtypes and progression from blood immune signatures.

Clinical adoption of immune checkpoint inhibitors in cancer management has highlighted the interconnection between carcinogenesis and the immune system. Immune cells are integral to the tumour microenvironment and can influence the outcome of therapies. Better understanding of an individual's immune landscape may play an important role in treatment personalisation. Peripheral blood is a readily accessible source of information to study an individual's immune landscape compared to more complex and invasive tumour bioipsies, and may hold immense diagnostic and prognostic potential. Identifying the critical components of these immune signatures in peripheral blood presents an attractive alternative to tumour biopsy-based immune phenotyping strategies. We used two syngeneic solid tumour models, a 4T1 breast cancer model and a CT26 colorectal cancer model, in a longitudinal study of the peripheral blood immune landscape. Our strategy combined two highly accessible approaches, blood leukocyte immune phenotyping and plasma soluble immune factor characterisation, to identify distinguishing immune signatures of the CT26 and 4T1 tumour models using machine learning. Myeloid cells, specifically neutrophils and PD-L1-expressing myeloid cells, were found to correlate with tumour size in both the models. Elevated levels of G-CSF, IL-6 and CXCL13, and B cell counts were associated with 4T1 growth, whereas CCL17, CXCL10, total myeloid cells, CCL2, IL-10, CXCL1, and Ly6Cintermediate monocytes were associated with CT26 tumour development. Peripheral blood appears to be an accessible means to interrogate tumour-dependent changes to the host immune landscape, and to identify blood immune phenotypes for future treatment stratification.

Neutrophil extracellular traps and their histones promote Th17 cell differentiation directly via TLR2.

Neutrophils perform critical functions in the innate response to infection, including through the production of neutrophil extracellular traps (NETs) - web-like DNA structures which are extruded from neutrophils upon activation. Elevated levels of NETs have been linked to autoimmunity but this association is poorly understood. By contrast, IL-17 producing Th17 cells are a key player in various autoimmune diseases but are also crucial for immunity against fungal and bacterial infections. Here we show that NETs, through their protein component histones, directly activate T cells and specifically enhance Th17 cell differentiation. This modulatory role of neutrophils, NETs and their histones is mediated downstream of TLR2 in T cells, resulting in phosphorylation of STAT3. The innate stimulation of a specific adaptive immune cell subset provides an additional mechanism demonstrating a direct link between neutrophils, NETs and T cell autoimmunity.

Neutralizing the pathological effects of extracellular histones with small polyanions.

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9-1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.

Induction of Genotype Cross-Reactive, Hepatitis C Virus-Specific, Cell-Mediated Immunity in DNA-Vaccinated Mice.

A universal hepatitis C virus (HCV) vaccine should elicit multiantigenic, multigenotypic responses, which are more likely to protect against challenge with the range of genotypes and subtypes circulating in the community. A vaccine cocktail and vaccines encoding consensus HCV sequences are attractive approaches to achieve this goal. Consequently, in a series of mouse vaccination studies, we compared the immunogenicity of a DNA vaccine encoding a consensus HCV nonstructural 5B (NS5B) protein to that of a cocktail of DNA plasmids encoding the genotype 1b (Gt1b) and Gt3a NS5B proteins. To complement this study, we assessed responses to a multiantigenic cocktail regimen by comparing a DNA vaccine cocktail encoding Gt1b and Gt3a NS3, NS4, and NS5B proteins to a single-genotype NS3/4/5B DNA vaccine. To thoroughly evaluate in vivo cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses against Gt1b and Gt3a HCV peptide-pulsed target cells, we exploited a novel fluorescent-target array (FTA). FTA and enzyme-linked immunosorbent spot (ELISpot) analyses collectively indicated that the cocktail regimens elicited higher responses to Gt1b and Gt3a NS5B proteins than those with the consensus vaccine, while the multiantigenic DNA cocktail significantly increased the responses to NS3 and NS5B compared to those elicited by the single-genotype vaccines. Thus, a DNA cocktail vaccination regimen is more effective than a consensus vaccine or a monovalent vaccine at increasing the breadth of multigenotypic T cell responses, which has implications for the development of vaccines for communities where multiple HCV genotypes circulate.IMPORTANCE Despite the development of highly effective direct-acting antivirals (DAA), infections with hepatitis C virus (HCV) continue, particularly in countries where the supply of DAA is limited. Furthermore, patients who eliminate the virus as a result of DAA therapy can still be reinfected. Thus, a vaccine for HCV is urgently required, but the heterogeneity of HCV strains makes the development of a universal vaccine difficult. To address this, we developed a novel cytolytic DNA vaccine which elicits robust cell-mediated immunity (CMI) to the nonstructural (NS) proteins in vaccinated animals. We compared the immune responses against genotypes 1 and 3 that were elicited by a consensus DNA vaccine or a DNA vaccine cocktail and showed that the cocktail induced higher levels of CMI to the NS proteins of both genotypes. This study suggests that a universal HCV vaccine can most readily be achieved by use of a DNA vaccine cocktail.

Antigen presenting capacity of murine splenic myeloid cells.

BACKGROUND: The spleen is an important site for hematopoiesis. It supports development of myeloid cells from bone marrow-derived precursors entering from blood. Myeloid subsets in spleen are not well characterised although dendritic cell (DC) subsets are clearly defined in terms of phenotype, development and functional role. Recently a novel dendritic-like cell type in spleen named 'L-DC' was distinguished from other known dendritic and myeloid cells by its distinct phenotype and developmental origin. That study also redefined splenic eosinophils as well as resident and inflammatory monocytes in spleen. RESULTS: L-DC are shown to be distinct from known splenic macrophages and monocyte subsets. Using a new flow cytometric procedure, it has been possible to identify and isolate L-DC in order to assess their functional competence and ability to activate T cells both in vivo and in vitro. L-DC are readily accessible to antigen given intravenously through receptor-mediated endocytosis. They are also capable of CD8+ T cell activation through antigen cross presentation, with subsequent induction of cytotoxic effector T cells. L-DC are MHCII- cells and unable to activate CD4+ T cells, a property which clearly distinguishes them from conventional DC. The myeloid subsets of resident monocytes, inflammatory monocytes, neutrophils and eosinophils, were found to have varying capacities to take up antigen, but were uniformly unable to activate either CD4+ T cells or CD8+ T cells. CONCLUSION: The results presented here demonstrate that L-DC in spleen are distinct from other myeloid cells in that they can process antigen for CD8+ T cell activation and induction of cytotoxic effector function, while both L-DC and myeloid subsets remain unable to activate CD4+ T cells. The L-DC subset in spleen is therefore distinct as an antigen presenting cell.

Use of an in vivo FTA assay to assess the magnitude, functional avidity and epitope variant cross-reactivity of T cell responses following HIV-1 recombinant poxvirus vaccination.

Qualitative characteristics of cytotoxic CD8+ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.

The use of fluorescent target arrays for assessment of T cell responses in vivo.

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into > 250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8(+) and CD4(+) T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8(+) T cell-mediated killing of FTA target cells and CD4(+) T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since > 250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g. the cumulative magnitude of the response) and a qualitative (e.g. functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

Fluorescent target array T helper assay: a multiplex flow cytometry assay to measure antigen-specific CD4+ T cell-mediated B cell help in vivo.

CD4(+) T cells play a central role in regulating the immune response. Their effector function is commonly assessed by their capacity to secrete cytokines detected by ELISPOT and intracellular cytokine staining. However, one aspect of their effector function that is often overlooked is their ability to help activation of cognate B cells directly, a process that is initiated through the engagement of their T cell-receptor (TCR) with cognate peptide presented on major histocompatibility complex class II (MHC-II) molecules by B cells. Here we report a method to monitor CD4(+) T cell-mediated B cell help in vivo using a multiplex high throughput assay. This assay utilizes a fluorescent target array (FTA), which is composed of lymphocytes labeled with numerous (>200) unique fluorescence signatures that can be delineated in a single recipient animal based on combination labeling with the three vital dyes carboxyfluorescein diacetate succinimidyl ester (CFSE), CellTrace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD). By pulsing different B cell populations in a FTA with titrated amounts of cognate MHC-II binding peptides, CD4(+) T cell help could be assessed by measuring induction of the B cell activation markers CD69 and CD44 by antibody labeling and flow cytometry. We call this the "FTA T helper assay", and have found it to be a robust and sensitive assay to measure CD4(+) T cell helper activity across a multitude of peptide-pulsed B "target" cells in real time in vivo. Furthermore, the technique can be used simultaneously with the FTA killing assay that measures cytotoxic T cell function, to provide a comprehensive tool for measuring both CD4(+) and CD8(+) T cell activity during an immune response in vivo.

Fluorescent target array killing assay: a multiplex cytotoxic T-cell assay to measure detailed T-cell antigen specificity and avidity in vivo.

Here we describe a multiplex, fluorescence-based, in vivo cytotoxic T-cell assay using the three vital dyes carboxyfluorescein diacetate succinimidyl ester, cell trace violet, and cell proliferation dye efluor 670. When used to label cells in combination, these dyes can discriminate >200 different target cell populations in the one animal due to each target population having a unique fluorescence signature based on fluorescence intensity and the different emission wavelengths of the dyes. This allows the simultaneous measurement of the in vivo killing of target cells pulsed with numerous peptides at different concentrations and the inclusion of many replicates. This fluorescent target array killing assay can be used to measure the fine antigen specificity and avidity of polyclonal cytotoxic T-cell responses in vivo, immunological parameters that were previously impossible to monitor.

New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes.

The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure lymphocyte proliferation by flow cytometry has become one of the most widely utilised assays for assessing lymphocyte responses. The properties of CFSE make it ideal for such a task, covalently labelling cells with a long-lived fluorescence of high intensity and low variance with minimal cell toxicity. No dye in the last 20 years has been capable of replicating CFSE in these respects. However, currently CFSE is limited to following a maximum of 7 cell divisions and is not compatible for use with ubiquitously available fluorescein conjugates or other fluorescent molecules with spectral properties similar to fluorescein, such as EGFP. Here we characterise two new fluorescent dyes for measuring lymphocyte proliferation, Cell Trace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD), which have different excitation and emission spectra to CFSE and, consequently, are compatible with fluorescein conjugates. We found that while both CTV and CPD can label cells to a high fluorescence intensity, which is long-lived and has low variability and low toxicity and makes them ideal for long-term tracking of non-dividing lymphocytes in vivo, CTV offers possibly the best available alternative to CFSE in the analysis of cell divisions. We also describe how intercellular dye transfer and cell autofluorescence can affect division resolution with the three different dyes and describe labelling conditions for the three dyes that produce ultra-bright lymphocytes for in vivo tracking studies and allow up to 11 cell divisions to be detected when using CFSE and CTV as the fluorescent dyes.

The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor lymphocyte proliferation.

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.