Approaching Mass Cytometry Translational Studies by Experimental and Data Curation Settings.

Clinical studies are conducted to better understand the pathological mechanism of diseases and to find biomarkers associated with disease activity, drug response, or outcome prediction. Mass cytometry (MC) is a high-throughput single-cell technology that measures hundreds of cells per second with more than 40 markers per cell. Thus, it is a suitable tool for immune monitoring and biomarker discovery studies. Working in translational and clinical settings requires a careful experimental design to minimize, monitor, and correct the variations introduced during sample collection, preparation, acquisition, and analysis. In this review, we will focus on these important aspects of MC-related experiments and data curation in the context of translational clinical research projects.

Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model.

With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.

Single-cell immune profiling of Meniere Disease patients.

BACKGROUND: Meniere Disease (MD) is an inner ear syndrome, characterized by episodes of vertigo, tinnitus and fluctuating sensorineural hearing loss. The pathological mechanism leading to sporadic MD is still poorly understood, however an allergic inflammatory response seems to be involved in some patients with MD. OBJECTIVE: Decipher an immune signature associated with the syndrome. METHODS: We performed mass cytometry immune profiling on peripheral blood from MD patients and controls. We analyzed differences in state and differences in abundance of the different cellular subsets. IgE levels were quantified through ELISA on supernatant of cultured whole blood. RESULTS: We have identified two clusters of individuals according to the single cell cytokine profile. These clusters presented differences in IgE levels, immune cell population abundance, including a reduction of CD56dim NK-cells, and changes in cytokine expression with a different response to bacterial and fungal antigens. CONCLUSION: Our results support a systemic inflammatory response in some MD patients that show a type 2 response with allergic phenotype, which could benefit from personalized IL-4 blockers.

Protocol for large scale whole blood immune monitoring by mass cytometry and Cyto Quality Pipeline.

Mass cytometry (MC) is a powerful large-scale immune monitoring technology. To maximize MC data quality, we present a protocol for whole blood analysis together with an R package, Cyto Quality Pipeline (CytoQP), which minimizes the experimental artifacts and batch effects to ensure data reproducibility. We describe the steps to stimulate, fix, and freeze blood samples before acquisition to make them suitable for retrospective studies. We then detail the use of barcoding and reference samples to facilitate multicenter and multi-batch experiments. For complete details on the use and execution of this protocol, please refer to Rybakowska et al. (2021a) and (2021b).

PeacoQC: Peak-based selection of high quality cytometry data.

In cytometry analysis, a large number of markers is measured for thousands or millions of cells, resulting in high-dimensional datasets. During the measurement of these samples, erroneous events can occur such as clogs, speed changes, slow uptake of the sample etc., which can influence the downstream analysis and can even lead to false discoveries. As these issues can be difficult to detect manually, an automated approach is recommended. In order to filter these erroneous events out, we created a novel quality control algorithm, Peak Extraction And Cleaning Oriented Quality Control (PeacoQC), that allows for automated cleaning of cytometry data. The algorithm will determine density peaks per channel on which it will remove low quality events based on their position in the isolation tree and on their mean absolute deviation distance to these density peaks. To evaluate PeacoQC's cleaning capability, it was compared to three other existing quality control algorithms (flowAI, flowClean and flowCut) on a wide variety of datasets. In comparison to the other algorithms, PeacoQC was able to filter out all different types of anomalies in flow, mass and spectral cytometry data, while the other methods struggled with at least one type. In the quantitative comparison, PeacoQC obtained the highest median balanced accuracy and a similar running time compared to the other algorithms while having a better scalability for large files. To ensure that the parameters chosen in the PeacoQC algorithm are robust, the cleaning tool was run on 16 public datasets. After inspection, only one sample was found where the parameters should be further optimized. The other 15 datasets were analyzed correctly indicating a robust parameter choice. Overall, we present a fast and accurate quality control algorithm that outperforms existing tools and ensures high-quality data that can be used for further downstream analysis. An R implementation is available.

Data processing workflow for large-scale immune monitoring studies by mass cytometry.

Mass cytometry is a powerful tool for deep immune monitoring studies. To ensure maximal data quality, a careful experimental and analytical design is required. However even in well-controlled experiments variability caused by either operator or instrument can introduce artifacts that need to be corrected or removed from the data. Here we present a data processing pipeline which ensures the minimization of experimental artifacts and batch effects, while improving data quality. Data preprocessing and quality controls are carried out using an R pipeline and packages like CATALYST for bead-normalization and debarcoding, flowAI and flowCut for signal anomaly cleaning, AOF for files quality control, flowClean and flowDensity for gating, CytoNorm for batch normalization and FlowSOM and UMAP for data exploration. As proper experimental design is key in obtaining good quality events, we also include the sample processing protocol used to generate the data. Both, analysis and experimental pipelines are easy to scale-up, thus the workflow presented here is particularly suitable for large-scale, multicenter, multibatch and retrospective studies.

Stabilization of Human Whole Blood Samples for Multicenter and Retrospective Immunophenotyping Studies.

Whole blood is often collected for large-scale immune monitoring studies to track changes in cell frequencies and responses using flow (FC) or mass cytometry (MC). In order to preserve sample composition and phenotype, blood samples should be analyzed within 24 h after bleeding, restricting the recruitment, analysis protocols, as well as biobanking. Herein, we have evaluated two whole blood preservation protocols that allow rapid sample processing and long-term stability. Two fixation buffers were used, Phosphoflow Fix and Lyse (BD) and Proteomic Stabilizer (PROT) to fix and freeze whole blood samples for up to 6 months. After analysis by an 8-plex panel by FC and a 26-plex panel by MC, manual gating of circulating leukocyte populations and cytokines was performed. Additionally, we tested the stability of a single sample over a 13-months period using 45 consecutive aliquots and a 34-plex panel by MC. We observed high correlation and low bias toward any cell population when comparing fresh and 6 months frozen blood with FC and MC. This correlation was confirmed by hierarchical clustering. Low coefficients of variation (CV) across studied time points indicate good sample preservation for up to 6 months. Cytokine detection stability was confirmed by low CVs, with some differences between fresh and fixed conditions. Thirteen months regular follow-up of PROT samples showed remarkable sample stability. Whole blood can be preserved for phenotyping and cytokine-response studies provided the careful selection of a compatible antibody panel. However, possible changes in cell morphology, differences in antibody affinity, and changes in cytokine-positive cell frequencies when compared to fresh blood should be considered. Our setting constitutes a valuable tool for multicentric and retrospective studies. © 2020 International Society for Advancement of Cytometry.

Key steps and methods in the experimental design and data analysis of highly multi-parametric flow and mass cytometry.

High-dimensional, single-cell cell technologies revolutionized the way to study biological systems, and polychromatic flow cytometry (FC) and mass cytometry (MC) are two of the drivers of this revolution. As up to 30-50 dimensions respectively can be measured per single-cell, they allow deep phenotyping combined with cellular functions studies, like cytokine production or protein phosphorylation. In parallel, the bioinformatics field develops algorithms that are able to process incoming data and extract the most useful and meaningful biological information. However, the success of automated analysis tools depends on the generation of high-quality data. In this review we present the most recent FC and MC computational approaches that are used to prepare, process and interpret high-content cytometry data. We also underscore proper experimental design as a key step for obtaining good quality data.

Brief Report: Patients With Primary Sjögren's Syndrome Who Are Positive for Autoantibodies to Tripartite Motif-Containing Protein 38 Show Greater Disease Severity.

OBJECTIVE: Autoantibodies reactive with Ro52 (tripartite motif-containing protein 21 [TRIM21]) are detected in 70% of patients with primary Sjögren's syndrome (SS). TRIM21 belongs to a 34-member C-IV family of TRIM proteins. Although autoantibodies against other TRIM proteins within the C-IV family have been detected in the sera of patients with primary SS, their clinical relevance remains unclear. This study was undertaken to investigate the frequency of anti-TRIM38 in patients with primary SS and evaluate its association with various clinical measures of the disease. METHODS: Serum samples from patients with primary SS (n = 235) and controls (n = 50) were analyzed for reactivity with in vitro-transcribed and -translated (35) S-methionine-labeled TRIM38 protein. The associations of anti-TRIM38 with various laboratory and clinical measures of primary SS were evaluated. Reactivity of anti-TRIM38 with different structural domains of TRIM38 was analyzed. Affinity-purified anti-TRIM38 antibodies were used to immunoprecipitate TRIM21. RESULTS: TRIM38-reactive autoantibodies were detected in the sera of 24 of the 235 patients with primary SS and 2 of the 50 controls. Anti-TRIM38 positivity was significantly associated with the presence of anti-Ro60, anti-Ro52, anti-La, rheumatoid factor, and hypergammaglobulinemia. Clinically, anti-TRIM38 was associated with significantly higher ocular surface staining scores, lower Schirmer's test scores, and minor labial salivary gland biopsy focus scores of ≥3.0. Anti-TRIM38 antibodies mainly recognized the cortactin-binding protein 2 (CortBP-2; amino acids 128-238) and the B30.2/SPRY (amino acids 268-465) domains on TRIM38. Affinity-purified antibodies to TRIM38-CortBP-2 and TRIM38-B30.2/SPRY domains reacted with TRIM21. CONCLUSION: Our data demonstrate that anti-TRIM38 specificity arising in a subset of patients with primary SS is associated with increased severity of the disease.

Interaction between innate immunity and Ro52-induced antibody causes Sjögren's syndrome-like disorder in mice.

OBJECTIVES: Autoantibodies reactive with Ro52 are often found in sera of patients with Sjögren's syndrome (SS). This study was undertaken to investigate the role of Ro52-induced immune responses in pathogenesis of SS. METHODS: New Zealand Mixed (NZM) 2758 mice were immunised with Ro52 in alum adjuvant. Control mice were immunised either with maltose-binding protein or injected with alum alone. Mice were monitored for anti-Ro52 antibody, sialoadenitis and pilocarpine-induced salivation. Antibody binding to salivary gland (SG) cells was analysed in vivo and in vitro by immunofluorescence. Sera from immunised mice were passively transferred into untreated or alum injected NZM2758 mice. RESULTS: By day 30 post-immunisation, Ro52 immunised mice generated immunoprecipitating anti-Ro52 antibodies and they had the maximum drop in saliva production. Both Ro52 immunised and control mice showed evidence of mild sialoadenitis. However, only Ro52 immunised mice had antibody deposition in their SG. Passive transfer of Ro52-immune sera induced SG dysfunction in recipient mice, only if the recipients were primed with alum. In vitro, antibodies from Ro52-immune sera were internalised by a SG cell line and this uptake was inhibited by cytochalasin D treatment. CONCLUSIONS: Our data show for the first time that antibodies induced by Ro52 are capable of inducing SG dysfunction, and that this phenomenon is dependent on the activation of innate immunity. The mouse model described in this study implies that autoantibody deposition in the SG might be an important step in the induction of xerostomia and pathogenesis of SS.