scholarly journals Single-cell characterization of a model of poly I:C-stimulated peripheral blood mononuclear cells in severe asthma

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Ailu Chen ◽  
Maria P. Diaz-Soto ◽  
Miguel F. Sanmamed ◽  
Taylor Adams ◽  
Jonas C. Schupp ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Severe Asthma ◽  
Mononuclear Cells ◽  
Effector T Cells ◽  
Poly I:C ◽  
Peripheral Blood Mononuclear ◽  
Main Cell ◽  
Blood Mononuclear Cells ◽  
Quantitative Changes

Abstract Background Asthma has been associated with impaired interferon response. Multiple cell types have been implicated in such response impairment and may be responsible for asthma immunopathology. However, existing models to study the immune response in asthma are limited by bulk profiling of cells. Our objective was to Characterize a model of peripheral blood mononuclear cells (PBMCs) of patients with severe asthma (SA) and its response to the TLR3 agonist Poly I:C using two single-cell methods. Methods Two complementary single-cell methods, DropSeq for single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF), were used to profile PBMCs of SA patients and healthy controls (HC). Poly I:C-stimulated and unstimulated cells were analyzed in this study. Results PBMCs (n = 9414) from five SA (n = 6099) and three HC (n = 3315) were profiled using scRNA-Seq. Six main cell subsets, namely CD4 + T cells, CD8 + T cells, natural killer (NK) cells, B cells, dendritic cells (DCs), and monocytes, were identified. CD4 + T cells were the main cell type in SA and demonstrated a pro-inflammatory profile characterized by increased JAK1 expression. Following Poly I:C stimulation, PBMCs from SA had a robust induction of interferon pathways compared with HC. CyTOF profiling of Poly I:C stimulated and unstimulated PBMCs (n = 160,000) from the same individuals (SA = 5; HC = 3) demonstrated higher CD8 + and CD8 + effector T cells in SA at baseline, followed by a decrease of CD8 + effector T cells after poly I:C stimulation. Conclusions Single-cell profiling of an in vitro model using PBMCs in patients with SA identified activation of pro-inflammatory pathways at baseline and strong response to Poly I:C, as well as quantitative changes in CD8 + effector cells. Thus, transcriptomic and cell quantitative changes are associated with immune cell heterogeneity in this model to evaluate interferon responses in severe asthma.

scholarly journals Single-cell Profiling of the Response to Poly I:C in Peripheral Blood Mononuclear Cells in Severe Asthma

2020 ◽  
Author(s):  
Ailu Chen ◽  
Maria P Diaz-Soto ◽  
Miguel F Sanmamed ◽  
Taylor Adams ◽  
Jonas Christian Schupp ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Severe Asthma ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Poly I:C ◽  
Peripheral Blood Mononuclear ◽  
Effector Cells ◽  
Single Cell Profiling ◽  
Quantitative Changes

Background: Asthma has been associated with impaired interferon responses. Multiple cell types have been implicated in these impaired responses and may be responsible for increased exacerbations and immunopathology of asthma. Objective: Characterize the single-cell response to Poly I:C of peripheral blood mononuclear cells (PBMCs) of patients with severe asthma (SA). Methods: Two complementary single-cell methods, DropSeq for single-cell RNA sequencing (scRNA-Seq) and mass cytometry (CyTOF), were used to profile PBMCs of SA and healthy controls (HC). Poly I:C and unstimulated cells were analyzed in this study. Results: PBMCs (n=9,414) from five SA (n=6,099) and three HC (n=3,315) were profiled using scRNA-Seq. Six main cell subsets, including CD4+ T cells, CD8+ T cells, natural killer (NK) cells, B cells, dendritic cells (DCs), and monocytes, were identified. CD4+ T cells were the main cell type and demonstrated a pro-inflammatory profile characterized by increased JAK1 expression in unstimulated cells. Following Poly I:C stimulation, PBMCs from SA had a robust induction of interferon pathways compared with HC. Additional analyses to identify core regulators of the enhanced interferon response in SA identified IRF1, STAT1, IRF7, STAT2, and IRF9. CyTOF profiling of Poly I:C and unstimulated PBMCs (n=120,000) from the same individuals (SA=4; HC=2) demonstrated higher numbers of CD8+ effector cells and Th1 CD4+ T cells in unstimulated conditions, followed by a decrease of these two cell subsets after poly I:C stimulation. Conclusion: Single-cell profiling of PBMCs with scRNA-seq and CyTOF in patients with SA identified activation of pro-inflammatory pathways at baseline and strong response to Poly I:C, as well as quantitative changes in CD8+ effector cells and Th1 cells. Thus, transcriptomic and cell quantitative changes are associated with immune cell heterogeneity in severe asthma.

scholarly journals Single-cell RNA sequencing of peripheral blood mononuclear cells from acute Kawasaki disease patients

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhen Wang ◽  
Lijian Xie ◽  
Guohui Ding ◽  
Sirui Song ◽  
Liqin Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Kawasaki Disease ◽  
Immune Responses ◽  
Single Cell ◽  
Rna Sequencing ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Single Cell Rna Sequencing ◽  
Blood Mononuclear Cells

AbstractKawasaki disease (KD) is the most common cause of acquired heart disease in children in developed countries. Although functional and phenotypic changes of immune cells have been reported, a global understanding of immune responses underlying acute KD is unclear. Here, using single-cell RNA sequencing, we profile peripheral blood mononuclear cells from seven patients with acute KD before and after intravenous immunoglobulin therapy and from three age-matched healthy controls. The most differentially expressed genes are identified in monocytes, with high expression of pro-inflammatory mediators, immunoglobulin receptors and low expression of MHC class II genes in acute KD. Single-cell RNA sequencing and flow cytometry analyses, of cells from an additional 16 KD patients, show that although the percentage of total B cells is substantially decreased after therapy, the percentage of plasma cells among the B cells is significantly increased. The percentage of CD8+ T cells is decreased in acute KD, notably effector memory CD8+ T cells compared with healthy controls. Oligoclonal expansions of both B cell receptors and T cell receptors are observed after therapy. We identify biological processes potentially underlying the changes of each cell type. The single-cell landscape of both innate and adaptive immune responses provides insights into pathogenesis and therapy of KD.

scholarly journals OP0033 REGULATORY T CELL CD39 EXPRESSION AS A PREDICTOR OF EARLY REMISSION-INDUCTION WITH METHOTREXATE IN NEW-ONSET RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 18.2-18
Author(s):  
P. Brown ◽  
A. Anderson ◽  
B. Hargreaves ◽  
A. Morgan ◽  
J. D. Isaacs ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Disease Modifying Therapy ◽  
Blood Mononuclear Cells ◽  
Treatment Naïve ◽  
Disease Modifying ◽  
Pre Treatment

Background:The long term outcomes for patients with rheumatoid arthritis (RA) depend on early and effective disease control. Methotrexate remains the key first line disease modifying therapy for the majority of patients, with 40% achieving an ACR50 on monotherapy(1). There are at present no effective biomarkers to predict treatment response, preventing effective personalisation of therapy. A putative mechanism of action of methotrexate, the potentiation of anti-inflammatory adenosine signalling, may inform biomarker discovery. By antagonism of the ATIC enzyme in the purine synthesis pathway, methotrexate has been proposed to increase the release of adenosine moieties from cells, which exert an anti-inflammatory effect through interaction with ADORA2 receptors(2). Lower expression of CD39 (a cell surface 5-’ectonucleotidase required for the first step in the conversion of ATP to adenosine) on circulating regulatory T-Lymphocytes (Tregs) was previously identified in patients already established on methotrexate who were not responding (DAS28 >4.0 vs <3.0)(3). We therefore hypothesised that pre-treatment CD39 expression on these cells may have clinical utility as a predictor of early methotrexate efficacy.Objectives:To characterise CD39 expression in peripheral blood mononuclear cells in RA patients naïve to disease modifying therapy commencing methotrexate, and relate this expression to 4 variable DAS28CRP remission (<2.6) at 6 months.Methods:68 treatment naïve early RA patients starting methotrexate were recruited from the Newcastle Early Arthritis Clinic and followed up for 6 months. Serial blood samples were taken before and during methotrexate therapy with peripheral blood mononuclear cells isolated by density centrifugation. Expression of CD39 by major immune subsets (CD4+ and CD8+ T-cells, B-lymphocytes, natural killer cells and monocytes) was determined by flow cytometry. The statistical analysis used was binomial logistic regression with baseline DAS28CRP used as a covariate due to the significant association of baseline disease activity with treatment response.Results:Higher pre-treatment CD39 expression was observed in circulating CD4+ T-cells of patients who subsequently achieved clinical remission at 6 months versus those who did not (median fluorescence 4854.0 vs 3324.2; p = 0.0108; Figure 1-A). This CD39 expression pattern was primarily accounted for by the CD4+CD25 high sub-population (median fluorescence 9804.7 vs 6455.5; p = 0.0065; Figure 1-B). These CD25 high cells were observed to have higher FoxP3 and lower CD127 expression than their CD39 negative counterparts, indicating a Treg phenotype. No significant associations were observed with any other circulating subset. A ROC curve demonstrates the discriminative utility of differential CD39 expression in the CD4+CD25 high population for the prediction of DAS28CRP remission in this cohort, showing greater specificity than sensitivity for remission prediction(AUC: 0.725; 95% CI: 0.53 - 0.92; Figure 1-C). Longitudinally, no significant induction or suppression of the CD39 marker was observed amongst patients who did or did not achieve remission over the 6 months follow-up period.Figure 1.Six month DAS28CRP remission versus pre-treatment median fluorescence of CD39 expression on CD4+ T-cells (A); CD25 High expressing CD4+ T-cells (B); and ROC curve of predictive utility of pre-treatment CD39 expression on CD25 High CD4+ T-cells (C).Conclusion:These findings support the potential role of CD39 in the mechanism of methotrexate response. Expression of CD39 on circulating Tregs in treatment-naïve RA patients may have particular value in identifying early RA patients likely to respond to methotrexate, and hence add value to evolving multi-parameter discriminatory algorithms.References:[1]Hazlewood GS, et al. BMJ. 2016 21;353:i1777[2]Brown PM, et al. Nat Rev Rheumatol. 2016;12(12):731-742[3]Peres RS, et al. Proc Natl Acad Sci U S A. 2015;112(8):2509-2514Disclosure of Interests:None declared

scholarly journals Single-cell multi-omics analysis of the immune response in COVID-19

2021 ◽  
Author(s):  
Emily Stephenson ◽  
◽  
Gary Reynolds ◽  
Rachel A. Botting ◽  
Fernando J. Calero-Nieto ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Single Cell ◽  
Mononuclear Cells ◽  
Severe Disease ◽  
Effector Memory ◽  
Peripheral Blood Mononuclear ◽  
Cross Sectional ◽  
Hematopoietic Stem ◽  
Symptomatic Disease

AbstractAnalysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.

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