High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis

Tsai, Wanxia Li; Vian, Laura; Giudice, Valentina; Kieltyka, Jacqueline; Liu, Christine; Fonseca, Victoria; Gazaniga, Nathalia; Gao, Shaojian; Young, Neal S.; Biancotto, Angélique; Gadina, Massimo

doi:10.1016/j.jim.2019.112667

Cited by 8 publications

(12 citation statements)

References 41 publications

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“…Each subject had STAT4 risk allele genotyping prior to screening visit using commercially available TaqMan ® Single Nucleotide Polymorphism Genotyping. Detailed reagents and methods for PBMC isolation, stimulation, and the fluorescent barcoding method used for the staining and multiplexed phosphoflow analysis are described in Supplementary Methods and elsewhere 47 . Pathway analysis and gene annotation were completed using Toppgene (toppgene.cchmc.org).…”

Section: Methodsmentioning

confidence: 99%

“…The study protocol and statistical analysis plan are submitted as part of the Supplementary File. In addition, the source data for fluorescent cell barcoding is provided as a separate file and can be accessed as referenced 54 . The RNA-Seq and fluorescent cell barcoding data are available without any material transfer and sharing agreement.…”

Section: Statistical Analysis the Sample Size Chosen Was Based On What Is Commonlymentioning

confidence: 99%

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Phase 1 double-blind randomized safety trial of the Janus kinase inhibitor tofacitinib in systemic lupus erythematosus

et al. 2021

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Increased risk of premature cardiovascular disease (CVD) is well recognized in systemic lupus erythematosus (SLE). Aberrant type I-Interferon (IFN)-neutrophil interactions contribute to this enhanced CVD risk. In lupus animal models, the Janus kinase (JAK) inhibitor tofacitinib improves clinical features, immune dysregulation and vascular dysfunction. We conducted a randomized, double-blind, placebo-controlled clinical trial of tofacitinib in SLE subjects (ClinicalTrials.gov NCT02535689). In this study, 30 subjects are randomized to tofacitinib (5 mg twice daily) or placebo in 2:1 block. The primary outcome of this study is safety and tolerability of tofacitinib. The secondary outcomes include clinical response and mechanistic studies. The tofacitinib is found to be safe in SLE meeting study’s primary endpoint. We also show that tofacitinib improves cardiometabolic and immunologic parameters associated with the premature atherosclerosis in SLE. Tofacitinib improves high-density lipoprotein cholesterol levels (p = 0.0006, CI 95%: 4.12, 13.32) and particle number (p = 0.0008, CI 95%: 1.58, 5.33); lecithin: cholesterol acyltransferase concentration (p = 0.024, CI 95%: 1.1, −26.5), cholesterol efflux capacity (p = 0.08, CI 95%: −0.01, 0.24), improvements in arterial stiffness and endothelium-dependent vasorelaxation and decrease in type I IFN gene signature, low-density granulocytes and circulating NETs. Some of these improvements are more robust in subjects with STAT4 risk allele.

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Section: Methodsmentioning

confidence: 99%

Section: Statistical Analysis the Sample Size Chosen Was Based On What Is Commonlymentioning

confidence: 99%

Phase 1 double-blind randomized safety trial of the Janus kinase inhibitor tofacitinib in systemic lupus erythematosus

et al. 2021

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“…S5 , 20 out of 60 stimulation conditions). Most of such responses were induced by IFNα, which is indeed expected to induce not only phosphorylation of STAT1, but also STAT3 and pSTAT5 through the type I IFN receptor [ 17 , 28 , 29 ]. Other responses described in literature that we observed were IL2-STAT5 responses in T cells [ 12 ], IFNγ-STAT1 responses in monocytes and B cells [ 12 ], and IL6- and IL10- induced STAT3 responses in monocytes and CD4 + T cells.…”

Section: Resultsmentioning

confidence: 99%

Impaired JAK-STAT pathway signaling in leukocytes of the frail elderly

et al. 2022

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Background Elderly often show reduced immune functioning and can develop chronic low-grade inflammation. Why some elderly are more prone to become frail is unknown. We investigated whether frailty is associated with altered cytokine signaling through the JAK-STAT pathway in leukocytes of 34 individuals aged 65–74 years. In addition, we investigated how this relation is affected by chronic low-grade inflammation during the previous 20 years. Cytokine signaling was quantified by measuring intracellular STAT1, STAT3, and STAT5 phosphorylation in monocytes, B cells, CD4+ T cells and CD8+ T cells upon stimulation with IL-2, IL-6, IL-10, IFNα and IFNγ, using phospho-flow cytometry. Presence of chronic low-grade inflammation was investigated by evaluating 18 different plasma inflammatory markers that had been measured repeatedly in the same individuals over the previous 20 years. Frailty was assessed as a score on a frailty index. Results We found that lower cytokine-induced pSTAT responsiveness in the various cell subsets was seen with higher frailty scores in both men and women, indicative of dysfunctional pSTAT responses in frailer individuals. Associations differed between men and women, with frailer women showing lower pSTAT1 responses in monocytes and frailer men showing lower pSTAT5 responses in CD4+ and CD8+ T cells. Notably, lower IL-10-induced pSTAT3 responses in men were related to both higher frailty scores and higher CRP levels over the past 20 years. This might indicate poor resolution of low-grade inflammation due to defective regulatory pSTAT signaling in older men. Conclusions Our results emphasize the importance of preserved JAK-STAT pathway signaling in healthy aging and reveal cellular pSTAT levels as a candidate biomarker of frailty.

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“…Previous approaches to computational deconvolution aimed to automate biaxial gating [5]. Though this approach decreases analysis time and improves reproducibility, the sample multiplexing capacity remains limited to $24 samples using two barcoding dyes, a scheme designed for manual gating by a human expert.…”

mentioning

confidence: 99%

“…One approach for overcoming this is to use additional cell type markers to gate populations of interest that might variably uptake dye prior to deconvolution [5][6][7]. A limitation of this approach is that it requires prior knowledge and thus can introduce bias, especially in high dimensional schemes.…”

mentioning

confidence: 99%

DebarcodeR increases fluorescent cell barcoding capacity and accuracy

et al. 2021

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Fluorescent cell barcoding (FCB) enables efficient collection of tens to hundreds of flow cytometry samples by covalently marking cells with varying concentration of spectrally distinct dyes. A key consideration in FCB is to balance the density of dye barcodes, the complexity of cells in the sample, and the desired accuracy of the debarcoding. Unfortunately, barcoding bench and computational methods have not benefited from the high dimensional revolution in cytometry due to a lack of automated computational tools that effectively balance these common cytometry needs.DebarcodeR addresses these unmet needs by providing a framework for computational debarcoding augmented by improvements to experimental methods. Adaptive regression modeling accounted for differential dye uptake between different cell types and Gaussian mixture modeling provided a robust method to probabilistically assign cells to samples. Assignment tolerance parameters are available to allow users to balance high cell recovery with accurate assignments. Improvements to experimental methods include: (1) inclusion of an "external standard" control where a pool of all cells was stained a single level of each barcoding dyes and (2) an "internal standard" where each cell is stained with a single level of a separate dye. DebarcodeR significantly improved speed, accuracy, and reproducibility of FCB while avoiding selective loss of unusual cell subsets when debarcoding microtiter plates of cell lines and heterogenous mixtures of primary cells. DebarcodeR is available on Github as an R package that works with flowCore and Cytoverse packages at github.com/cytolab/ DebarcodeR.

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High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis

Cited by 8 publications

References 41 publications

Phase 1 double-blind randomized safety trial of the Janus kinase inhibitor tofacitinib in systemic lupus erythematosus

Phase 1 double-blind randomized safety trial of the Janus kinase inhibitor tofacitinib in systemic lupus erythematosus

Impaired JAK-STAT pathway signaling in leukocytes of the frail elderly

DebarcodeR increases fluorescent cell barcoding capacity and accuracy

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