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 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 An Imbalance between Frequency of CD4+CD25+FOXP3+ Regulatory T Cells and CCR4+ and CCR9+ Circulating Helper T Cells Is Associated with Active Perennial Allergic Conjunctivitis

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
J. Galicia-Carreón ◽  
C. Santacruz ◽  
J. Ayala-Balboa ◽  
A. Robles-Contreras ◽  
S. M. Perez-Tapia ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Allergic Conjunctivitis ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Effector Cells ◽  
Inflammatory Microenvironment ◽  
Surface Molecules ◽  
Intracellular Cytokines ◽  
Eye Disorders

Allergic conjunctivitis (AC) is one of the most common eye disorders in ophthalmology. In mice models, it has been suggested that control of allergic conjunctivitis is a delicate balance between Tregs and inflammatory migrating effector cells. Our aim was to evaluate the frequency of Tregs and the frequency of homing receptors expressing cells in peripheral blood mononuclear cells (PBMC) from patients with perennial allergic conjunctivitis (PAC). The analyses of phenotypic markers on CD4+ T cells and both soluble or intracellular cytokines were performed by flow cytometry. CD4+CD25+ cells were 15 times more frequent in PBMC from patients than HC; the vast majority of these CD4+CD25+ cells were FOXP3−, and most of CD4+ T cells were CCR4+ and CCR9+ cells. Upon allergen-stimulation, no significant changes were observed in frequency of Treg; however, an increased frequency of CD4+CCR4+CCR9+ cells, CD4+CD103+ cells and CD4+CD108+ cells with increased IL-5, IL-6, and IL-8 production was observed. These findings suggest an immune dysregulation in PAC, characterized by diminished frequency of Tregs and increased frequency of circulating activated CD4+ T cells; upon allergen-stimulation, these cells were expressing cell-surface molecules related to mucosa homing and were able to trigger an inflammatory microenvironment.

scholarly journals The influence of CD40 ligation and interferon-γ on functional properties of human monocyte-derived dendritic cells activated with polyinosinic-polycytidylic acid

2011 ◽  
Vol 68 (4) ◽  
pp. 301-308 ◽  
Author(s):  
Ana Dragicevic ◽  
Tanja Dzopalic ◽  
Sasa Vasilijic ◽  
Dragana Vucevic ◽  
Biljana Bozic ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Poly I:C ◽  
Peripheral Blood Mononuclear ◽  
Th1 Response ◽  
Polycytidylic Acid ◽  
Polyinosinic Polycytidylic Acid ◽  
Stimulation Of

Background/Aim. Ligation of a Toll-like receptor (TLR) by specific TLR agonists is a powerful tool for maturation induction of monocyte-derived dendritic cells (MoDCs). Studies so far have shown that the treatment of dendritic cells (DCs) with a TLR3 ligand, polyinosinic-polycytidylic acid [Poly(I:C)], may be an appropriate activation agent for obtaining mature MoDCs, competent to prime effective immune responses. However, little is known about how subsequent interaction of MoDCs with T cell-derived stimuli, such as CD40 or interferon-? (IFN-?), modulates MoDC functions. Therefore, this problem was the main objective of this study. Methods. Immature MoDCs were prepared by cultivation of monocytes from peripheral blood mononuclear cells with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 5 days. After that, maturation was induced by the treatment of these cells with Poly(I:C) for 2 days. At day 6, immature MoDCs and Poly(I:C)-activated MoDCs were incubated either with CD40 ligand (L)-transfected J558 cells or IFN-? for additional 24 hours. Cytokine production was measured by ELISA and FlowCytomix Human T helper Th1/Th2 11plex. Allostimulatory capability of MoDCs was tested using an allogeneic mixed leukocyte reaction (MLR) assay. Results. Immature MoDCs showed a moderate potential for stimulation of proliferation of CD4+ T cells, which was enhanced by the treatment with Poly(I:C). Ligation of CD40 or treatment with IFN-? of immature or Poly(I:C)-treated MoDCs significantly up-regulated their allostimulatory activity. MoDCs matured in the presence of Poly(I:C) up-regulated the production of IL- 12 and IL-10, which was followed by increased levels of IFN- ? and decreased levels of IL-5 in co-cultures with allogeneic CD4+ T cells. Ligation of CD40 on immature MoDCs upregulated the production of IL-12 and IL-23 which was accompanied by increased secretion of IFN-? in co-culture. Stimulation of CD40 on Poly(I:C)-treated MoDCs significantly enhanced the production of IL-12, IL-23 and IL-10. However, such treated MoDCs decreased the production of IFN-? and IL-10 and up-regulated the secretion of IL-17. Immature MoDCs treated with IFN-? up-regulated IL-12, but lowered the production of IL-5 and IL-17 by CD4+ T cells. Treatment of Poly(I:C)-activated MoDCs with IFN-? down-regulated the production of IL-12 and up-regulated IL- 10 by these cells and increased/decreased the levels of IL-10/ IFN-?, respectively, in co-culture with CD4+ T cells. Conclusion. Treatment with Poly(I:C) or ligation of CD40 on immature MoDCs induces maturation of these cells into a phenotype that supports Th1 response. Activation of CD40 on Poly(I:C)-treated MoDCs shifts the immune response towards Th17. Treatment of immature MoDCs with IFN-? down-regulated Th2 and Th17 responses. However, addition of IFN-? to Poly(I:C)-activated MoDCs down-regulated Th1 response and promote T regulatory mechanisms. Each of these results may have functional and therapeutic implications.

scholarly journals Single-cell transcriptomics reveals expansion of cytotoxic CD4 T cells in supercentenarians

2019 ◽  
Vol 116 (48) ◽  
pp. 24242-24251 ◽  
Author(s):  
Kosuke Hashimoto ◽  
Tsukasa Kouno ◽  
Tomokatsu Ikawa ◽  
Norihito Hayatsu ◽  
Yurina Miyajima ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd4 T Cells ◽  
Healthy Aging ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Peripheral Blood Mononuclear ◽  
Age Related

Supercentenarians, people who have reached 110 y of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from 7 supercentenarians and 5 younger controls. We identified a marked increase of cytotoxic CD4 T cells (CD4 cytotoxic T lymphocytes [CTLs]) as a signature of supercentenarians. Furthermore, single-cell T cell receptor sequencing of 2 supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15 to 35% of the entire CD4 T cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Indeed, CD4 CTLs extracted from supercentenarians produced IFN-γ and TNF-α upon ex vivo stimulation. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.

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.

scholarly journals Expression Cloning of an Immunodominant Family of Mycobacterium tuberculosis Antigens Using Human Cd4+ T Cells

2000 ◽  
Vol 191 (3) ◽  
pp. 551-560 ◽  
Author(s):  
Mark R. Alderson ◽  
Teresa Bement ◽  
Craig H. Day ◽  
Liqing Zhu ◽  
David Molesh ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Genomic Library ◽  
Cell Epitope ◽  
Single Amino Acid ◽  
Expression Cloning ◽  
Peripheral Blood Mononuclear

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon γ production from healthy purified protein derivative (PPD)+ donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4+ T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-γ production by peripheral blood mononuclear cells from PPD+ but not PPD− individuals. Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes. A T cell line from a PPD+ donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

scholarly journals Donor-Derived CD4+ T Cells and Human Herpesvirus 6B Detection After Allogeneic Hematopoietic Cell Transplantation

2020 ◽  
Author(s):  
Derek J Hanson ◽  
Hu Xie ◽  
Danielle M Zerr ◽  
Wendy M Leisenring ◽  
Keith R Jerome ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Transplantation ◽  
Cd4 T Cells ◽  
Hematopoietic Cell Transplantation ◽  
Mononuclear Cells ◽  
Human Herpesvirus ◽  
Hematopoietic Cell ◽  
Allogeneic Hematopoietic Cell Transplantation ◽  
Peripheral Blood Mononuclear

Abstract We sought to determine whether donor-derived human herpesvirus (HHV) 6B–specific CD4+ T-cell abundance is correlated with HHV-6B detection after allogeneic hematopoietic cell transplantation. We identified 33 patients who received HLA-matched, non–T-cell–depleted, myeloablative allogeneic hematopoietic cell transplantation and underwent weekly plasma polymerase chain reaction testing for HHV-6B for 100 days thereafter. We tested donor peripheral blood mononuclear cells for HHV-6B–specific CD4+ T cells. Patients with HHV-6B detection above the median peak viral load (200 copies/mL) received approximately 10-fold fewer donor-derived total or HHV-6B–specific CD4+ T cells than those with peak HHV-6B detection at ≤200 copies/mL or with no HHV-6B detection. These data suggest the importance of donor-derived immunity for controlling HHV-6B reactivation.

IRF5 Acts as a Potential Therapeutic Marker in Inflammatory Bowel Diseases

2020 ◽  
Author(s):  
Yonghong Yang ◽  
Cui Zhang ◽  
Dehuai Jing ◽  
Heng He ◽  
Xiaoyu Li ◽  
...  
Keyword(s):  
T Cells ◽  
Disease Activity ◽  
Inflammatory Bowel Diseases ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Orphan Receptor ◽  
Peripheral Blood Mononuclear ◽  
Bowel Diseases ◽  
Inflammatory Bowel

Abstract Background Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn’s disease (CD), are chronic inflammatory disorders. As is well known, interferon regulatory factor (IRF) 5 is closely associated with the pathogenesis of various inflammatory diseases. But the exact role of IRF5 in IBD remains unclear. Methods In this study, we detected IRF5 expression in peripheral blood mononuclear cells (PBMCs) and inflamed mucosa from IBD patients by immunohistochemistry, western blot, and quantitative real-time polymerase chain reaction. Peripheral blood CD4+ T cells were stimulated with inflammatory cytokines and transfected by lentivirus. Results In active IBD patients, the expression of IRF5 in PBMCs and inflamed colonic tissues was obviously increased and significantly associated with disease activity. Ectopic overexpression of IRF5 could promote the differentiation of IBD CD4+ T cells into Th1 and Th17 cells by regulating T-bet and RAR related orphan receptor C, whereas knockdown of IRF5 had the opposite effects. Tumor necrosis factor (TNF)-α upregulated expression of IRF5 in CD4+ T cells, but anti-TNF treatment with infliximab could markedly reduce IRF5 expression in CD4+ T cells and intestinal mucosa of CD patients. Conclusion Our study reveals a novel mechanism that IRF5 levels are correlated with disease activity in IBD and might function as a possible marker for the management of IBD via regulating Th1 and Th17 immune responses and cytokine production.

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