miR-23a contributes to T cellular redox metabolism in juvenile idiopathic oligoarthritis

Rheumatology ◽  
2021 ◽  
Author(s):  
Anandhi Rajendiran ◽  
Patricia Klemm ◽  
Anastasia Schippers ◽  
Anja Scheufen ◽  
Tobias Schwarz ◽  
...  
Keyword(s):  
T Cells ◽  
Expression Analysis ◽  
Mirna Expression ◽  
Peripheral Blood ◽  
Target Genes ◽  
Ex Vivo ◽  
Chronic Inflammatory Disease ◽  
Pcr Analysis ◽  
Redox Metabolism ◽  
Mitochondrial Ros

Abstract Objective JIA is a chronic inflammatory disease of unknown origin. The regulation of inflammatory processes involves multiple cellular steps including mRNA transcription and translation. Different miRNAs control these processes tightly. We aimed to determine the roles of specific miRNAs within JIA pathogenesis. Methods We performed a global miRNA expression analysis in parallel in cells from the arthritic joint and peripheral blood of oligoarticular JIA patients and healthy controls. Quantitative RT-PCR analysis was used to verify expression of miRNA in T cells. Ex vivo experiments and flow cytometric analyses were used to analyse proliferation and redox metabolism. Results Global miRNA expression analysis demonstrated a different composition of miRNA expression at the site of inflammation compared with peripheral blood. Bioinformatic analysis of predicted miRNA target genes suggest a huge overrepresentation of genes involved in metabolic and oxidative stress pathways in the inflamed joint. Despite enhanced reactive oxygen species (ROS) levels within the local inflammatory milieu, JIA T cells are hyperproliferative and reveal an overexpression of miR-23a, which is an inhibitor of Peptidyl-prolyl isomerase F (PPIF), the regulator of mitochondrial ROS escape. Mitochondrial ROS escape is diminished in JIA T cells, resulting in their prolonged survival. Conclusion Our data suggest that miRNA-dependent mitochondrial ROS shuttling might be a mechanism that contributes to T cell regulation in JIA at the site of inflammation.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 4953-4960 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Aloisio Felipe-Silva ◽  
Bianca Heemskerk ◽  
Daniel J. Powell ◽  
John R. Wunderlich ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Metastatic Melanoma ◽  
Treg Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Production Capacity ◽  
Biological Marker ◽  
Foxp3 Expression ◽  
Treg Cells

Abstract Regulatory T (Treg) cells are often found in human tumors; however, their functional characteristics have been difficult to evaluate due to low cell numbers and the inability to adequately distinguish between activated and Treg cell populations. Using a novel approach, we examined the intracellular cytokine production capacity of tumor-infiltrating T cells in the single-cell suspensions of enzymatically digested tumors to differentiate Treg cells from effector T cells. Similar to Treg cells in the peripheral blood of healthy individuals, tumor-infiltrating FOXP3+CD4 T cells, unlike FOXP3− T cells, were unable to produce IL-2 and IFN-γ upon ex vivo stimulation, indicating that FOXP3 expression is a valid biological marker for human Treg cells even in the tumor microenvironment. Accordingly, we enumerated FOXP3+CD4 Treg cells in intratumoral and peritumoral sections of metastatic melanoma tumors and found a significant increase in proportion of FOXP3+CD4 Treg cells in the intratumoral compared with peritumoral areas. Moreover, their frequencies were 3- to 5-fold higher in tumors than in peripheral blood from the same patients or healthy donors, respectively. These findings demonstrate that the tumor-infiltrating CD4 Treg cell population is accurately depicted by FOXP3 expression, they selectively accumulate in tumors, and their frequency in peripheral blood does not properly reflect tumor microenvironment.

scholarly journals Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

2021 ◽  
Vol 67 (2) ◽  
pp. 95-101
Author(s):  
Monica Vuță ◽  
Ionela-Maria Cotoi ◽  
Ion Bogdan Mănescu ◽  
Doina Ramona Manu ◽  
Minodora Dobreanu
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Gradient Centrifugation ◽  
Intracellular Cytokine Staining ◽  
Interleukin 2 ◽  
Middle Aged

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

scholarly journals Persistent High Frequencies of Varicella-Zoster Virus ORF4 Protein-Specific CD4+ T Cells after Primary Infection

2006 ◽  
Vol 80 (19) ◽  
pp. 9772-9778 ◽  
Author(s):  
Louise Jones ◽  
Antony P. Black ◽  
Gathsaurie N. Malavige ◽  
Graham S. Ogg
Keyword(s):  
T Cells ◽  
Varicella Zoster Virus ◽  
Peripheral Blood ◽  
Primary Infection ◽  
Ex Vivo ◽  
High Frequencies ◽  
Varicella Zoster ◽  
Early Protein ◽  
Orf4 Protein ◽  
Ifn Γ

ABSTRACT Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+ T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+ T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-γ) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+ T cells. Indeed, CD4+ T cells were found to dominate the overall virus-specific IFN-γ cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+ T cells, with implications for disease pathogenesis and future vaccine development.

Expression of CD10 by Human T Cells That Undergo Apoptosis Both In Vitro and In Vivo

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3067-3076 ◽  
Author(s):  
Giovanna Cutrona ◽  
Nicolò Leanza ◽  
Massimo Ulivi ◽  
Giovanni Melioli ◽  
Vito L. Burgio ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Lymphoid Cells ◽  
Normal Peripheral Blood ◽  
Apoptotic Process ◽  
Cd10 Expression ◽  
T Cell Lines

Abstract This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10+ when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4+ and CD8+ T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV+ subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10+ as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

scholarly journals Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis

2020 ◽  
Author(s):  
Christina Gerstner ◽  
Sara Turcinov ◽  
Aase H Hensvold ◽  
Karine Chemin ◽  
Hannes Uchtenhagen ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
At Risk ◽  
T Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Class Ii ◽  
Hla Class Ii ◽  
Phenotypic Characterization ◽  
Early Ra

Abstract Background: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterization of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. Results: Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-b, vimentin as well as cartilage intermediate layer protein (CILP).First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n=7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n=14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n=5) and early RA patients (n=5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n=10) and found that the group of patients achieving minimum disease activity (DAS28 <2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. Conclusions: Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterization of antigen-specific T cells in ex vivo patient samples including RA ‘at risk’ subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.

scholarly journals Targeted Gene Candidates for Treatment and Early Diagnosis of Age-Related Macular Degeneration

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Emine Cinici ◽  
Ozge Caglar ◽  
Mehmet Enes Arslan ◽  
Nilay Dilekmen ◽  
Bahadır Utlu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Early Diagnosis ◽  
Macular Degeneration ◽  
Expression Analysis ◽  
Peripheral Blood ◽  
Gene Expression Analysis ◽  
Mononuclear Cells ◽  
Age Related Macular Degeneration ◽  
Pcr Analysis ◽  
Age Related

Age-related macular degeneration (AMD) is an eye disease that impairs the sharp and central vision need for daily activities. Recent advances in molecular biology research not only lead to a better understanding of the genetics and pathophysiology of AMD but also to the development of applications based on targeted gene expressions to treat the disease. Clarification of molecular pathways that causing to development and progression in dry and wet types of AMD needs comprehensive and comparative investigations in particular precious biopsies involving peripheral blood samples from the patients. Therefore, in this investigation, dry and wet types of AMD patients and healthy individuals were aimed at investigating in regard to targeted gene candidates by using gene expression analysis for the first time. 13 most potent candidate genes involved in neurodegeneration were selected via in silico approach and investigated through gene expression analysis to suggest new targets for disease therapy. For the analyses, 30 individuals (10 dry and 10 wet types AMD patients and 10 healthy people) were involved in the study. SYBR-Green based Real-Time PCR analysis was performed on isolated peripheral blood mononuclear cells (PBMCs) to analyze differentially expressed genes related to these cases. According to the investigations, only the CRP gene was found to be upregulated for both dry and wet disease types. When the downregulated genes were analyzed, it was found that 11 genes were commonly decreased for both dry and wet types in the aspect of expression pattern. From these genes, CFH, CX3CR1, FLT1, and TIMP3 were found to have the most downregulated gene expression properties for both diseases. From these results, it might be concluded that these common upregulated and downregulated genes could be used as targets for early diagnosis and treatment for AMD.

scholarly journals Expansion of a unique subpopulation of cytotoxic T cells that express a C alpha V delta 1 T-cell receptor gene in a patient with severe persistent neutropenia

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3157-3163
Author(s):  
I Bank ◽  
M Book ◽  
L Cohen ◽  
A Kneller ◽  
E Rosental ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Receptor ◽  
T Lymphocyte ◽  
Peripheral Blood ◽  
Gene Segment ◽  
Cell Receptor ◽  
Severe Neutropenia ◽  
Pcr Analysis

CD8+ T-lymphocyte populations may be expanded in the peripheral blood of patients with chronic idiopathic neutropenia and may be involved in suppression of granulopoiesis. In this report, we have analyzed the T-cell receptor (TCR) used by the T lymphocytes of a patient with chronic severe neutropenia. Using specific oligonucleotides in the polymerase chain reaction (PCR) to amplify cDNA specific for the different families of the V alpha, V beta, and V delta TCR genes, and monoclonal antibodies (MoAbs) to examine T-lymphocyte subsets and their TCR, a persistent expansion of CD3+CD8+ T lymphocytes and a reduced repertoire of TCR V alpha and V beta genes were found in the patient's peripheral blood mononuclear cell (PBMC) preparations. A predominant portion of the T lymphocytes expressed a unique TCR structure. Thus, we found that, despite the fact that 98% of the T cells expressed alpha beta TCR on the surface membrane and less than 2% expressed tau delta TCR, nonetheless, 40% to 60% of the T cells stained positively with anti V delta 1 MoAb. Using the PCR analysis, the V delta 1 gene segment was found to be rearranged to C alpha, rather than to C delta genes. The expanded C alpha V delta 1+ cells, which are found only rarely in normal PB, expressed CD8 and were cytotoxic, and the C alpha V delta 1 receptor was functional in cytotoxicity. This constitutes the first description of an expansion of cytotoxic CD8+ lymphocytes expressing a functional “hybrid” C alpha V delta 1 gene in vivo, and suggests a pathogenic role for CD8+ C alpha V delta 1+ cells in some patients with idiopathic neutropenia.

scholarly journals Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis

2020 ◽  
Author(s):  
Amédée Renand ◽  
Iñaki Cervera-Marzal ◽  
Laurine Gil ◽  
Chuang Dong ◽  
Erwan Kervagoret ◽  
...  
Keyword(s):  
T Cells ◽  
Disease Activity ◽  
Autoimmune Hepatitis ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
B Cell Differentiation ◽  
Tcr Repertoire ◽  
First Time

AbstractBackground & AimsIn most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-Soluble Liver Antigen (SLA or SepSecs) autoantibodies is associated with significantly reduced overall survival, but the associated autoreactive CD4 T cells have not been characterized yet. Here we isolated and deeply characterized SLA-specific CD4 T cells in AIH patients.MethodsWe used brief ex vivo restimulation with overlapping SLA-derived peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterize their transcriptome and TCR repertoire in n=5 AIH patients. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing, to identify their phenotypic niche. We further characterized disease-associated peripheral blood T cells by high content flow cytometry in an additional cohort of n=46 AIH patients and n=18 non-alcoholic steatohepatitis (NASH) controls.ResultsAutoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1+CXCR5−CCR6−CD27+ phenotype. ScRNA-seq revealed their pro-inflammatory/B-Helper profile (IL21, IFNG, TIGIT, CTLA4, NR3C1, CD109, KLRB1 and CLEC2D). Autoreactive TCR clonotypes were restricted to the memory PD-1+CXCR5− CD4 T cells. This subset was significantly increased in the blood of AIH patients and supported B cell differentiation through IL-21. Finally, we identified a specific phenotype (PD-1+CD38+CD27+CD127−CXCR5−) of CD4 T cells linked to disease activity and IgG response during AIH.ConclusionsThis work provides for the first time a deep characterization of rare circulating autoreactive CD4 T cells and the identification of their peripheral reservoir in AIH. We also propose a generic phenotype of pathogenic CD4 T cells related to AIH disease activity.

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