scholarly journals Saturated Fatty Acid Activates T Cell Inflammation Through a Nicotinamide Nucleotide Transhydrogenase (NNT)-Dependent Mechanism

Biomolecules ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 79 ◽  
Author(s):  
Grace McCambridge ◽  
Madhur Agrawal ◽  
Alanna Keady ◽  
Philip Kern ◽  
Hatice Hasturk ◽  
...  
Keyword(s):  
Fatty Acids ◽  
T Cell ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Redox Homeostasis ◽  
Mitochondrial Protein ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Redox Balance ◽  
Specific Stimulus ◽  
Nicotinamide Nucleotide Transhydrogenase

Circulating fatty acids (FAs) increase with obesity and can drive mitochondrial damage and inflammation. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that positively regulates nicotinamide adenine dinucleotide phosphate (NADPH), a key mediator of energy transduction and redox homeostasis. The role that NNT-regulated bioenergetics play in the inflammatory response of immune cells in obesity is untested. Our objective was to determine how free fatty acids (FFAs) regulate inflammation through impacts on mitochondria and redox homeostasis of peripheral blood mononuclear cells (PBMCs). PBMCs from lean subjects were activated with a T cell-specific stimulus in the presence or absence of generally pro-inflammatory palmitate and/or non-inflammatory oleate. Palmitate decreased immune cell expression of NNT, NADPH, and anti-oxidant glutathione, but increased reactive oxygen and proinflammatory Th17 cytokines. Oleate had no effect on these outcomes. Genetic inhibition of NNT recapitulated the effects of palmitate. PBMCs from obese (BMI >30) compared to lean subjects had lower NNT and glutathione expression, and higher Th17 cytokine expression, none of which were changed by exogenous palmitate. Our data identify NNT as a palmitate-regulated rheostat of redox balance that regulates immune cell function in obesity and suggest that dietary or therapeutic strategies aimed at increasing NNT expression may restore redox balance to ameliorate obesity-associated inflammation.

IMPACT: Immunotherapy in patients with metastatic cancers and CDK12 mutations.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. TPS5091-TPS5091 ◽  
Author(s):  
Melissa Andrea Reimers ◽  
Wassim Abida ◽  
Jonathan Chou ◽  
Daniel J. George ◽  
Elisabeth I. Heath ◽  
...  
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Castration Resistant Prostate Cancer ◽  
Open Label ◽  
Histologic Diagnosis ◽  
Peripheral Blood Mononuclear ◽  
Diversity Assessment ◽  
Potential Biomarker

TPS5091 Background: Tumors with biallelic CDK12 loss have been identified as a distinct subtype in metastatic castration resistant prostate cancer (mCRPC) and other cancer types. The CDK12 biallelic loss mCRPC genomic signature, distinct from homologous recombination deficient (HRD) and ETS fusion signatures, is characterized by excessive tandem duplications, genomic instability, gene fusion-caused putative neoantigens, and increased tumor T cell infiltration. Early clinical experience with anti-PD-1 immunotherapy in CDK12 loss mCRPC patients (pts) is notable for deep and sustained PSA as well as radiographic responses. We hypothesize that CDK12 biallelic loss is a potential biomarker of immune checkpoint immunotherapy (ICI) efficacy in mCRPC and other cancers. Methods: IMPACT (NCT03570619) is a multi-center, open label, phase 2 study of pts with metastatic cancers that harbor CDK12 biallelic loss. mCRPC pts will be enrolled in cohort A (n = 25) in a Mini-Max Simon Two-Stage design, and all other pts in single-stage cohort B (n = 15). All pts will receive induction therapy with nivolumab 3 mg/kg IV and ipilimumab 1 mg/kg IV q3 weeks for up to 4 cycles, followed by maintenance nivolumab at 480 mg IV q4 weeks (up to 52 weeks in total). Eligible pts must have identified biallelic CDK12 loss on any CLIA/CAP approved next generation sequencing assay and a histologic diagnosis of metastatic prostate adenocarcinoma or other metastatic carcinoma. No prior ICI is allowed. The primary endpoint is the overall response rate (ORR) in cohort A per PCWG3 criteria. An ORR of 30% is targeted in cohort A. Secondary endpoints include safety, secondary efficacy measures, quality of life, and survival measures. Exploratory objectives include tumor whole exome analysis and changes in immune profiles with therapy. Comprehensive and serial monitoring of peripheral blood immune cell populations will be performed via T cell clonal diversity assessment and multi-parametric flow cytometry. Changes in myeloid and lymphoid populations will be assessed from whole blood. Polarization and effector function of T cells and activation of antigen presenting cells will be further characterized from isolated peripheral blood mononuclear cells. Study accrual is ongoing. Clinical trial information: NCT03570619.

scholarly journals Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes 

2020 ◽  
Author(s):  
Han Chen ◽  
Christian M Schürch ◽  
Kevin Noble ◽  
Kenneth Kim ◽  
Peter Krutzik ◽  
...  
Keyword(s):  
T Cell ◽  
Density Gradient ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Human Peripheral Blood ◽  
Sample Type ◽  
Cell Preparation ◽  
Cell Recovery ◽  
Density Gradient Medium ◽  
Gradient Medium

Abstract Background: Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. To address potential differences in sample outcome, we isolated, cryopreserved, and compared PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation. Methods: Whole blood was processed in parallel using both Cell Preparation Tubesä (CPT, BD Biosciences) and Lymphoprepä Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of ten cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production. Results: No significant differences in cell recovery, viability, frequency of immune cell subsets, or T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were identified.Conclusion: CPT and Lymphoprep tubes are effective and comparable methods for PBMC isolation for immunological studies.

scholarly journals The differential immune responses to COVID-19 in peripheral and lung revealed by single-cell RNA sequencing

2020 ◽  
Author(s):  
Gang Xu ◽  
Furong Qi ◽  
Hanjie Li ◽  
Qianting Yang ◽  
Haiyan Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Close Association ◽  
Suppressor Cells ◽  
T Cell Subsets ◽  
Peripheral Blood Mononuclear ◽  
Peripheral T Cells

Understanding the mechanism that leads to immune dysfunction induced by SARS-CoV2 virus is crucial to develop treatment for severe COVID-19. Here, using single cell RNA-seq, we characterized the peripheral blood mononuclear cells (PBMC) from uninfected controls and COVID-19 patients, and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DC) and increased monocytes resembling myeloid-derived suppressor cells (MDSC) which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8+ T cell subsets, compared to health controls. In contrast, the proportions of various activated CD4+ T cell subsets, including Th1, Th2 and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients' peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG, TNF, CCL4 and CCL5 etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the patients' lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.

scholarly journals Using peripheral blood immune signatures to stratify patients with adult and juvenile inflammatory myopathies

Rheumatology ◽  
2019 ◽  
Author(s):  
Meredyth G Ll Wilkinson ◽  
Anna Radziszewska ◽  
Chris Wincup ◽  
Yiannis Ioannou ◽  
David A Isenberg ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Disease Activity ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Assessment Tool ◽  
Immune Cell ◽  
Memory B Cells ◽  
Healthy Controls ◽  
Immune Signatures

AbstractObjectiveThe inflammatory idiopathic myopathies (IIM) are a group of rare autoimmune diseases defined by muscle weakness and characterized by pro-inflammatory infiltrates in muscle. Little is known about the immunological profile in peripheral blood of these patients and how this relates to IIM subtypes. This study aimed to stratify adult and juvenile-onset IIM patients according to immune cell profile.MethodsPeripheral blood mononuclear cells from 44 patients with adult myositis (AM), 15 adolescent-onset juvenile dermatomyositis (a-JDM), and 40 age-matched healthy controls were analysed by flow cytometry to quantify 33 immune cell subsets. Adult myositis patients were grouped according to myositis subtype; DM and polymyositis; and also autoantibody specificity. Disease activity was determined by the myositis disease activity assessment tool and clinicians’ decision on treatment.ResultsUnique immune signatures were identified for DM, polymyositis and a-JDM compared with healthy controls. DM patients had a T-cell signature comprising increased CD4+ and TH17 cell frequencies and increased immune cell expression of IL-6. Polymyositis patients had a B-cell signature with reduced memory B cells. A-JDM had decreased naïve B cells and increased CD4+T cells. All patient groups had decreased CD8+central memory T-cell frequencies. The distinct immune signatures were also seen when adult myositis patients were stratified according to auto-antibody expression; patients with anti-synthetase-antibodies had reduced memory B cells and patients with autoimmune rheumatic disease overlap had an elevated Th17 profile.ConclusionUnique immune signatures were associated with adult vs juvenile disease. The Th17 signature in DM patients supports the potential use of IL-17 inhibitors in treatment of IIMs.

scholarly journals A252 FUNCTIONAL ANALYSIS OF GENETIC AETIOLOGIES IN A DOWNSTREAM OF TYROSINE KINASE (DOK) PROTEIN IN THE PATHOGENESIS OF PEDIATRIC INFLAMMATORY BOWEL DISEASE (IBD)

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 129-130
Author(s):  
V Batura ◽  
C Guo ◽  
N Warner ◽  
G Leung ◽  
A Ricciuto ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Immune Cell ◽  
Imaging Techniques ◽  
Inflammatory Disorder ◽  
Zebrafish Model ◽  
Peripheral Blood Mononuclear

Abstract Background IBD is a form of chronic inflammatory disorder of the gastrointestinal tract that arises due to genetic, environmental, immunological and microbial factors. The precise pathological mechanisms remain elusive. It is thought that the onset of pediatric IBD can largely be attributed to genetics. Muise lab, at SickKids, regularly screens children at the SickKids IBD clinic and through an international consortium to find possible genetic links to the disease. We report a patient at SickKids with biallelic mutations in DOK4 who has severe Crohn’s Disease along with other inflammatory conditions. Downstream of kinase (DOK) proteins are a family of adaptor molecules that serve as scaffolding proteins important in regulating cell signaling, especially in T cells. DOK4 has been shown to have negative regulatory effects on T cell activation but is also expressed across various other tissues where its function is yet to be determined. We predict that these mutations are causing immune cell dysregulation, which may be contributing to the patients IBD. Aims Through this study, we aim to enhance our understanding of the pathobiological mechanism of novel mutations in DOK4. Methods We have established T cell lines, expressing wild type and mutated DOK4, which will be used to perform functional tests, such as localization analysis through immunofluorescence and cytokine profiling, to check for T cell function. We have patient derived organoids, which will be used to assess changes in gut morphology using imaging techniques. We will also generate mutant zebrafish model that will be used to determine the susceptibility to colitis related to this mutation, disease progression and gut peristalsis using live imaging technology. Results Preliminary data shows variation in expression of the protein within patient derived peripheral blood mononuclear cells (PBMCs) compared to a healthy donor. Conclusions With this study, we hope to identify new therapeutic targets for patients with DOK4 mutations. Funding Agencies CIHRThe Leona M. and Harry B. Helmsley Charitable Trust

scholarly journals Lipofection with Synthetic mRNA as a Simple Method for T-Cell Immunomonitoring

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1232
Author(s):  
Natalia Teresa Jarzebska ◽  
Julia Frei ◽  
Severin Lauchli ◽  
Lars E. French ◽  
Emmanuella Guenova ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Simple Method ◽  
Murine Splenocytes ◽  
T Cell Immune Responses ◽  
Synthetic Mrna ◽  
The Impact

The quantification of T-cell immune responses is crucial for the monitoring of natural and treatment-induced immunity, as well as for the validation of new immunotherapeutic approaches. The present study presents a simple method based on lipofection of synthetic mRNA in mononuclear cells as a method to determine in vitro T-cell responses. We compared several commercially available transfection reagents for their potential to transfect mRNA into human peripheral blood mononuclear cells and murine splenocytes. We also investigated the impact of RNA modifications in improving this method. Our results demonstrate that antigen-specific T-cell immunomonitoring can be easily and quickly performed by simple lipofection of antigen-coding mRNA in complex immune cell populations. Thus, our work discloses a convenient solution for the in vitro monitoring of natural or therapy-induced T-cell immune responses.

scholarly journals High-throughput investigation of molecular and cellular biomarkers in NMOSD

2020 ◽  
Vol 7 (5) ◽  
pp. e852
Author(s):  
Soumya S. Yandamuri ◽  
Ruoyi Jiang ◽  
Aditi Sharma ◽  
Elizabeth Cotzomi ◽  
Chrysoula Zografou ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Nk Cells ◽  
High Throughput ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Nk Cell ◽  
Peripheral Blood Mononuclear ◽  
Cytokines And Chemokines ◽  
Multivariable Regression Models

ObjectiveTo identify candidate biomarkers associated with neuromyelitis optica spectrum disorder (NMOSD) using high-throughput technologies that broadly assay the concentrations of serum analytes and frequencies of immune cell subsets.MethodsSera, peripheral blood mononuclear cells (PBMCs), and matched clinical data from participants with NMOSD and healthy controls (HCs) were obtained from the Collaborative International Research in Clinical and Longitudinal Experience Study NMOSD biorepository. Flow cytometry panels were used to measure the frequencies of 39 T-cell, B-cell, regulatory T-cell, monocyte, natural killer (NK) cell, and dendritic cell subsets in unstimulated PBMCs. In parallel, multiplex proteomics assays were used to measure 46 serum cytokines and chemokines in 2 independent NMOSD and HC cohorts. Multivariable regression models were used to assess molecular and cellular profiles in NMOSD compared with HC.ResultsNMOSD samples had a lower frequency of CD16+CD56+ NK cells. Both serum cohorts and multivariable logistic regression revealed increased levels of B-cell activating factor associated with NMOSD. Interleukin 6, CCL22, and CCL3 were also elevated in 1 NMOSD cohort of the 2 analyzed. Multivariable linear regression of serum analyte levels revealed a correlation between CX3CL1 (fractalkine) levels and the number of days since most recent disease relapse.ConclusionsIntegrative analyses of cytokines, chemokines, and immune cells in participants with NMOSD and HCs provide congruence with previously identified biomarkers of NMOSD and highlight CD16+CD56+ NK cells and CX3CL1 as potential novel biomarker candidates.

scholarly journals Elevated expression of miR-146a correlates with high levels of immune cell exhaustion markers and suppresses cellular immune function in chronic HIV-1-infected patients

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ting Yu ◽  
Zhao Ju ◽  
Mingqi Luo ◽  
Ronghua Hu ◽  
Yan Teng ◽  
...  
Keyword(s):  
T Cell ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Inhibitory Receptors ◽  
Chronic Infections ◽  
Cell Exhaustion ◽  
Cell Functions ◽  
Cellular Immune ◽  
Hiv 1

AbstractFunctional exhaustion of immune cells is a defining characteristic of HIV-1 chronic infections, exhibiting dysregulation of cellular immune responses and expression of co-inhibitory receptors. Although the molecular mechanisms controlling immune-cell exhaustion retains largely unknown, immune checkpoint blockade strategy has shown inspiring potential to reinvigorate T cell functions in chronic infections. In this study, we investigated peripheral blood mononuclear cells (PBMCs) exhaustion markers from 109 chronic HIV-1-infected patients and found they correlated positively with microRNA-146a, which was inversely correlated with CD4+ T cell count. Intriguingly, ex vivo neutralization of miR-146a in PBMCs from chronic HIV-1 infection exhibited an elevated antiviral cytokines production as well as the expression of GZMB and perforin, while simultaneously, decreased the inhibitory receptors expression such as PD-1, CTLA-4, TIM-3 and LAG-3. These results highlight the importance of miR-146a to HIV-1 induced immune cell exhaustion, and uncover a novel layer of HIV/AIDS pathogenesis and provide potential targets for improved immune intervention.

scholarly journals Identification of Human Immune Cell Subtypes Most Vulnerable to IL-1β-induced Inflammatory Signaling Using Mass Cytometry

2020 ◽  
Author(s):  
Hema Kothari ◽  
Corey M. Williams ◽  
Chantel McSkimming ◽  
Mythili Vigneshwar ◽  
Eli R. Zunder ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
T Cell Subsets ◽  
Effector Memory ◽  
High Morbidity ◽  
Cytokine Storm ◽  
Mass Cytometry ◽  
Immune Cell Subsets

ABSTRACTIL-1β has emerged as a key mediator of the cytokine storm linked to high morbidity and mortality from COVID-19 and blockade of the IL-1 receptor (IL-1R) with Anakinra has entered clinical trials in COVID-19 subjects. Yet, knowledge of the specific immune cell subsets targeted by IL-1β and IL-1β-induced signaling pathways in humans is limited. Utilizing mass cytometry (CyTOF) of human peripheral blood mononuclear cells, we identified effector memory CD4 T cells and CD4−CD8low/-CD161+ T cells as the circulating immune subtypes with the greatest expression of p-NF-κB in response to IL-1β stimulation. Notably, CCR6 distinctly identified T cells most responsive to IL-1β. Other subsets including CD11c myeloid dendritic cells (mDCs), classical monocytes (CM), two subsets of natural killer cells (CD16−CD56brightCD161− and CD16−CD56dimCD161+) and a population of lineage−(Lin-) cells expressing CD161 and CD25 also showed IL-1β-induced expression of p-NF-kB. The IL-1R antagonist, Anakinra significantly inhibited IL-1β-induced p-NF-kB in the CCR6+ T cells and CD11c mDCs with a trending inhibition in CD14 monocytes and Lin−CD161+CD25+ cells. IL-1β also induced a rapid but much less robust increase in p-p38 expression as compared to p-NF-kB in the majority of these same immune cell subsets. Prolonged IL-1β stimulation greatly increased p-STAT3 and to a much lesser extent p-STAT1 and p-STAT5 in T cell subsets, monocytes, DCs and the Lin−CD161+CD25+ cells suggesting IL-1β-induced production of downstream STAT-activating cytokines, consistent with its role in cytokine storm. Interindividual heterogeneity and inhibition of this activation by Anakinra raises the intriguing possibility that assays to measure IL-1β-induced p-NF-kB in CCR6+ T cell subtypes could identify those at higher risk of cytokine storm and those most likely to benefit from Anakinra therapy.

scholarly journals Functional comaparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs Lymphoprep Tubes

2020 ◽  
Author(s):  
Han Chen ◽  
Christian M Schürch ◽  
Kevin Noble ◽  
Kenneth Kim ◽  
Peter Krutzik ◽  
...  
Keyword(s):  
T Cell ◽  
Density Gradient ◽  
T Cell Activation ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Human Peripheral Blood ◽  
Sample Type ◽  
Cell Preparation ◽  
Density Gradient Medium ◽  
Gradient Medium

Abstract Background : Cryopreserved human peripheral blood mononuclear cells (PBMCs) are a commonly used sample type for a variety of immunological assays. Many factors can affect the quality of PBMCs, and careful consideration and validation of an appropriate PBMC isolation and cryopreservation method is important for well-designed clinical studies. A major point of divergence in PBMC isolation protocols is the collection of blood, either directly into vacutainers pre-filled with density gradient medium or the use of conical tubes containing a porous barrier to separate the density gradient medium from blood. Results : To address potential differences in sample outcome, we isolated, cryopreserved, and compared frequency and functionality of PBMCs using parallel protocols differing only in the use of one of two common tube types for isolation. Whole blood was processed in parallel using both Cell Preparation Tubesä (CPT, BD Biosciences) and Lymphoprepä Tubes (Axis-Shield) and assessed for yield and viability prior to cryopreservation. After thawing, samples were further examined by flow cytometry for cell yield, cell viability, frequency of ten cell subsets, and capacity for stimulation-dependent CD4+ and CD8+ T cell intracellular cytokine production. Cell recovery, viability, frequency of immune cell subsets, and T cell functionality between PBMC samples isolated using CPT or Lymphoprep tubes were comparable. Conclusion : CPT and Lymphoprep tubes are both effective for PBMC isolation and may be used interchangeably for immunological studies involving T cell activation.

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