In-vitro assessment of Jurkat T-cells response to 1966 MHz electromagnetic fields in a GTEM cell

2015 ◽  
Author(s):  
Nektarios Moraitis ◽  
Maria Christopoulou ◽  
Konstantina S. Nikita ◽  
Georgia-Persephoni Voulgaridou ◽  
Ioannis Anestopoulos ◽  
...  
Keyword(s):  
T Cells ◽  
Electromagnetic Fields ◽  
Jurkat T Cells ◽  
In Vitro Assessment ◽  
Gtem Cell

scholarly journals Amorphous–Crystalline Calcium Phosphate Coating Promotes In Vitro Growth of Tumor-Derived Jurkat T Cells Activated by Anti-CD2/CD3/CD28 Antibodies

Materials ◽  
2021 ◽  
Vol 14 (13) ◽  
pp. 3693
Author(s):  
Yurii P. Sharkeev ◽  
Ekaterina G. Komarova ◽  
Valentina V. Chebodaeva ◽  
Mariya B. Sedelnikova ◽  
Aleksandr M. Zakharenko ◽  
...  
Keyword(s):  
T Cells ◽  
Calcium Phosphate ◽  
Biological Effects ◽  
Electrical Potential ◽  
Biological Properties ◽  
Modern Trend ◽  
Calcium Phosphate Coating ◽  
Jurkat T Cells ◽  
Micro Arc Oxidation

A modern trend in traumatology, orthopedics, and implantology is the development of materials and coatings with an amorphous–crystalline structure that exhibits excellent biocopatibility. The structure and physico–chemical and biological properties of calcium phosphate (CaP) coatings deposited on Ti plates using the micro-arc oxidation (MAO) method under different voltages (200, 250, and 300 V) were studied. Amorphous, nanocrystalline, and microcrystalline statesof CaHPO4 and β-Ca2P2O7were observed in the coatings using TEM and XRD. The increase in MAO voltage resulted in augmentation of the surface roughness Ra from 2.5 to 6.5 µm, mass from 10 to 25 mg, thickness from 50 to 105 µm, and Ca/P ratio from 0.3 to 0.6. The electrical potential (EP) of the CaP coatings changed from −456 to −535 mV, while the zeta potential (ZP) decreased from −53 to −40 mV following an increase in the values of the MAO voltage. Numerous correlations of physical and chemical indices of CaP coatings were estimated. A decrease in the ZP magnitudes of CaP coatings deposited at 200–250 V was strongly associated with elevated hTERT expression in tumor-derived Jurkat T cells preliminarily activated with anti-CD2/CD3/CD28 antibodies and then contacted in vitro with CaP-coated samples for 14 days. In turn, in vitro survival of CD4+ subsets was enhanced, with proinflammatory cytokine secretion of activated Jurkat T cells. Thus, the applied MAO voltage allowed the regulation of the physicochemical properties of amorphous–crystalline CaP-coatings on Ti substrates to a certain extent. This method may be used as a technological mechanism to trigger the behavior of cells through contact with micro-arc CaP coatings. The possible role of negative ZP and Ca2+ as effectors of the biological effects of amorphous–crystalline CaP coatings is discussed. Micro-arc CaP coatings should be carefully tested to determine their suitability for use in patients with chronic lymphoid malignancies.

scholarly journals Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Matthias Kästle ◽  
Camilla Merten ◽  
Roland Hartig ◽  
Thilo Kaehne ◽  
Ardiyanto Liaunardy-Jopeace ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Enzymatic Activity ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Sh2 Domain ◽  
Jurkat T Cells

Abstract Background Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.

scholarly journals PD-L1 chimeric costimulatory receptor improves the efficacy of CAR-T cells for PD-L1-positive solid tumors and reduces toxicity in vivo

2020 ◽  
Author(s):  
Qibin Liao ◽  
Yunyu Mao ◽  
Huan He ◽  
Xiangqing Ding ◽  
Xiaoyan Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Solid Tumors ◽  
Jurkat T Cells ◽  
Costimulatory Receptor ◽  
Car T Cells ◽  
Car T ◽  
Engineered T Cells ◽  
Therapeutic Safety

Abstract Background: On-target off-tumor toxicity impedes the clinical application of chimeric antigen receptor-modified T cells (CAR-T cells) in the treatment of solid tumors. The combinatorial antigen recognition strategy can improve the therapeutic safety of CAR-T cells by targeting two different tumor-associated antigens (TAAs) using a CAR and a chimeric costimulatory receptor (CCR). Although programmed death-ligand 1 (PD-L1, also known as B7-H1) is expressed on multiple tumors, the potential of PD-L1 as a universal target for designing CCR remains unknown.Methods: A first-generation CD19 or HER2 CAR and a PD-L1 CCR containing the CD28 signaling domain were constructed and delivered into Jurkat T cells or primary T cells by a pseudotyped lentivirus. The release of cytokines, including IL-2, IFN-γ and TNF-α, was quantified using enzyme-linked immunosorbent assay (ELISA) kits or a cytometric bead array (CBA). The in vitro cytotoxicity of CAR-T cells was detected with a luciferase-based killing assay. The in vitro proliferation of CAR-T cells was assessed by flow cytometry. The therapeutic safety and efficacy of CAR-T cells was evaluated using a subcutaneous dual-tumor model in vivo.Results: Jurkat T cells or primary T cells expressing both the CD19/HER2 CAR and PD-L1 CCR produced higher levels of cytokines in the presence of CD19/HER2 and PD-L1 than in the presence of HER2/CD19. Compared to HER2-z-engineered T cells, HER2-z-PD-L1-28-engineered T cells had higher in vitro cytotoxicity potential against PD-L1-positive tumor cells. CD19/HER2-z-PD-L1-28-engineered T cells showed higher proliferation potential in the presence of CD19/HER2 and PD-L1 than in the absence of PD-L1. CD19/HER2-z-PD-L1-28-engineered T cells preferably destroyed xenograft tumors expressing CD19/HER2 and PD-L1 in vivo and did not significantly affect CD19/HER2-expressing tumors. The PD-L1 CCR improved the antitumor efficacy of low-affinity HER2 CAR-T cells against PD-L1-positive tumors expressing high levels of HER2.Conclusion: Our findings confirmed that PD-L1 can be used as a universal target antigen for designing CCR, improving the efficacy of CAR-T cells in the treatment of PD-L1-positive solid tumors but reducing toxicity within PD-L1-negative normal tissues expressing low levels of TAA in vivo.

scholarly journals Cytokine Levels in the In Vitro Response of T Cells to Planktonic and Biofilm Corynebacterium amycolatum

2019 ◽  
Vol 68 (4) ◽  
pp. 457-464
Author(s):  
ALINA OLENDER ◽  
AGNIESZKA BOGUT ◽  
AGNIESZKA MAGRYŚ ◽  
JACEK TABARKIEWICZ
Keyword(s):  
T Cells ◽  
Conditioned Medium ◽  
Statistical Significance ◽  
Jurkat T Cells ◽  
Cytokine Levels ◽  
Planktonic Form ◽  
In Vitro Response ◽  
Anti Inflammatory ◽  
Culture Supernatants

Unravelling of the interplay between the immune system and non-diphtheria corynebacteria would contribute to understanding their increasing role as medically important microorganisms. We aimed at the analysis of pro- (TNF, IL-1β, IL-6, IL-8, and IL-12p70) and anti-inflammatory (IL-10) cytokines produced by Jurkat T cells in response to planktonic and biofilm Corynebacterium amycolatum. Two reference strains: C. amycolatum ATCC 700207 (R-CA), Staphylococcus aureus ATCC 25923 (R-SA), and ten clinical strains of C. amycolatum (C-CA) were used in the study. Jurkat T cells were stimulated in vitro by the planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM) derived from the relevant cultures of the strains tested. The cytokine concentrations were determined in the cell culture supernatants using the flow cytometry. The levels of the cytokines analyzed were lower after stimulation with the BCM when compared to the PCM derived from the cultures of C-CA; statistical significance (p < 0.05) was observed for IL-1β, IL-12 p70, and IL-10. Similarly, planktonic R-CA and R-SA stimulated a higher cytokine production than their biofilm counterparts. The highest levels of pro-inflammatory IL-8, IL-1β, and IL-12p70 were observed after stimulation with planktonic R-SA whereas the strongest stimulation of anti-inflammatory IL-10 was noted for the BCM derived from the mixed culture of both reference species. Our results are indicative of weaker immunostimulatory properties of the biofilm C. amycolatum compared to its planktonic form. It may play a role in the persistence of biofilm-related infections. The extent of the cytokine response can be dependent on the inherent virulence of the infecting microorganism.

Novel Mechansims for Pk (Gb3) Inhibition of HIV Infection.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1430-1430
Author(s):  
Nicole Lund ◽  
Clifford A. Lingwood ◽  
Jeffrey A. Medin ◽  
Martin L. Olsson ◽  
Cyril Levene ◽  
...  
Keyword(s):  
T Cells ◽  
Hiv Infection ◽  
Cell Viability ◽  
Membrane Fusion ◽  
Blood Group ◽  
Jurkat T Cells ◽  
Hiv 1 ◽  
Receptor Ccr5 ◽  
P Blood Group

Abstract Many blood group antigens belong to a class of lipid-sugar conjugate molecules known as glycosphingolipids (GSLs), including ABH, Lewis and P blood group systems. GSLs are implicated in HIV-host cell fusion, and several bind to HIV-gp120 in vitro. In addition, inhibiting glycolipid biosynthesis has been shown to prevent HIV membrane fusion. The Pk blood group antigen, also known as globotriaosylceramide (Gb3), has been shown to augment HIV fusion in in vitro models; however its exact role in HIV infection is not known. We have previously developed a soluble analogue of Pk, a highly effective gp120 ligand, which inhibits HIV-1 and HIV-2 membrane fusion. We hypothesize that Pk influences susceptibility to HIV infection, inhibiting at the level of membrane fusion and entry. We have presently investigated the effects of soluble and expressed Pk on HIV-1 infection in vitro. Soluble Pk effects were considered by pre-treating monocyte (M)-tropic HIV-1Ba-L or T-cell (T)-tropic HIV-1IIIB with soluble Pk prior to infection of PHA or PHA/IL2 activated peripheral blood-derived mononuclear cells (PBMCs) or Jurkat T-cells. Productive M-tropic and/or T-tropic HIV-1 infection of PBMCs and Jurkat cells was inhibited by more than 90%. Inhibition of membrane fusion and entry was visualised by transmission electron microscopy. Soluble Pk had minimal effects on cell viability or HIV receptor (CD4) and co-receptor (CCR5 or CXCR4) expression as measured by flow cytometry. Exogenously introduced Pk in Jurkat T-cells was accomplished by liposome fusion, and susceptibility to T-tropic HIV-1IIIB infection was determined. Pk transfer in Jurkat T-cells also reduced productive HIV-1 infection by 50%, without affecting cell viability or HIV receptor and co-receptor expression. Effects of differential Pk expression on HIV susceptibility was investigated using PBMCs with Pk accumulation, from patients with Fabry disease and P1k blood group individuals, or which lack Pk, from p blood group individuals. PBMCs representative of these two groups were infected with M-tropic HIV-1Ba-L or T-tropic HIV-1IIIB. PBMCs with Pk accumulation from patients with Fabry disease were resistant to productive M-tropic, but not T-tropic HIV-1 infection. These PBMCs had significantly decreased expression levels of the M-tropic HIV-1 co-receptor CCR5. Pk accumulation in P1k blood group PBMCs shows resistance to T-tropic and M-tropic HIV-1 infection, unrelated to levels of chemokine receptors. PBMCs lacking Pk from p blood group individuals were hypersensitive to M-tropic HIV-1 infection, while T-tropic HIV-1 infection levels were variable. Interestingly, p blood group PBMCs showed significantly higher levels of the CCR5 co-receptor. Our findings suggest Pk is inhibitory to HIV infection, which may translate into natural resistance in individuals that express high levels of Pk. This may be linked to interactions with CCR5, through receptor trafficking to the cell surface, or associations within lipid rafts. Furthermore, soluble Pk or potential mechanisms to increase cellular Pk expression, provide novel strategies for prevention of HIV infection.

scholarly journals Activation of the STAT6 transcription factor in Jurkat T-cells by the herpesvirus saimiri Tip protein

2012 ◽  
Vol 93 (2) ◽  
pp. 330-340 ◽  
Author(s):  
Yuri Kim ◽  
Eun-Kyung Kwon ◽  
Ju-Hong Jeon ◽  
Insuk So ◽  
In-Gyu Kim ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Cell Transformation ◽  
Herpesvirus Saimiri ◽  
Viral Gene ◽  
Jurkat T Cells

Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr127 residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS.

scholarly journals In vitro assessment of an engineered tBID-based safety switch system in human T lymphocytes

2021 ◽  
Author(s):  
Jiamiao Lu ◽  
Patrick Collins ◽  
Ki Jeong Lee ◽  
Chi-Ming Li ◽  
Songli Wang
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Therapeutic Modality ◽  
Caspase 9 ◽  
T Cell Therapy ◽  
Genetic Modifications ◽  
Switch System ◽  
In Vitro Assessment ◽  
Simplex Virus

ABSTRACTCell therapy as a promising therapeutic modality to treat cancer has been intensively studied for decades. However, the clinical trials have indicated that patients under T cell therapy may develop severe cytokine release syndrome resulting in hospitalization or even death. Furthermore, genetic modifications to promote proliferation and persistence of T cells could result in high numbers of long-lived engineered cells in patients after treatment. In this study we developed a novel tBID-based safety switch regulated through a small molecule inducible on switch to control undesired toxicity or ablate engineered cells as needed. We compared several tBID-based safety switch constructs with the clinically validated safety switches, HSV-TK (human herpes simplex virus thymidine kinase) and iCasp9 (inducible Caspase 9), few tBID-based safety switch constructs were systematically tested and investigated in vitro in Jurkat or human primary T cells. We demonstrated that our novel tBID based safety switch, with optimization, was able to eliminate up to ~90% of transduced human primary T cells within 72hr after activation, providing an alternative switch system to manage safety concerns for cell therapy.

scholarly journals Significance of nutrient media choice for the long-term cultures of leukemic T-lymphoblasts

2021 ◽  
Vol 23 (3) ◽  
pp. 593-604
Author(s):  
L. S. Litvinova ◽  
K. A. Yurova ◽  
V. V. Shchupletsova ◽  
N. D. Gazatova ◽  
O. G. Khaziakhmatova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nutrient Medium ◽  
The Body ◽  
In Vitro Cultivation ◽  
Jurkat T Cells ◽  
Nutrient Media ◽  
Toxicological Studies

Correct choice of nutrient media for culturing different types of cells in various applications is one of the most important aspects of modern biotechnology, since chemical composition of the culture media largely contains the necessary metabolites to support certain cells’ growth lines outside the body. Jurkat line of human leukemic T-lymphoblast-like cells (hereinafter Jurkat T-cells) is actively used for in vitro modeling of intracellular signaling and activation of normal blood T-lymphocytes mediated by the T-cell receptor/CD3/ CD4 complex in toxicological studies of immune and secretory responses, to test medicinal substances and ions. Also, Jurkat T-cells are widely used for ex vivo testing in immunology, oncology, toxicology, orthopedics, and traumatology. The existing standards and numerous studies are mainly based on short-term in vitro cultivation of Jurkat T-cells in RPMI 1640 nutrient medium. Meanwhile, the issues of long-term maintenance of the growth of Jurkat T-cells culture are poorly presented in the research literature. This study aimed for studying the activity of Jurkat T-cells over 7 to 14 days of in vitro culture and comparing the relative value of RPMI 1640 and αMEM media for the behavior of immunocompetent tumor cells. Using flow cytometry, multiplex analysis, and phase contrast Cell-IQ microscopy, the proportions of living cells and those dying by apoptosis and necrosis, secretion of cytokines and chemokines, and the dynamics of cell biomass propagation were studied. It was found that the αMEM medium in the complete nutrient medium, as compared with RPMI 1640, is more appropriate to in vitro promotion of cell viability (increased proportion of viable cells by 13.5% at the day 14), their secretory ability for 23 из 27 tested biomolecules, shortened adaptation time (на 32%) in culture before growth initiation, 5-fold increase of the Jurkat Т-cell cellularity by the day 7. Potential significance of the chemical components of nutrient media and secreted biomolecules for these results is discussed. As based on the results obtained, we concluded on superior properties of αMEM medium for long-term in vitro cultures of Jurkat T-cells. Consequently, the in vitro testing of medical devices intended for long-term contact with the body, including those for cancer patients, using Jurkat T-cell leukemia line in RPMI 1640 medium, may lead to wrong predictions on their biocompatibility and potential antitumor activity.

scholarly journals Bioactivities by a crude extract from the GreenlandicPseudomonassp. In5 involves the nonribosomal peptides, nunamycin and nunapeptin

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1476 ◽  
Author(s):  
Charlotte F. Michelsen ◽  
Helle Jensen ◽  
Vincent J. Venditto ◽  
Rosanna C. Hennessy ◽  
Peter Stougaard
Keyword(s):  
Antimicrobial Activity ◽  
T Cells ◽  
Crude Extract ◽  
Annexin V ◽  
Antimicrobial Activities ◽  
Nonribosomal Peptides ◽  
Microbial Metabolites ◽  
Suppressive Activity ◽  
Jurkat T Cells

Background.Bioactive microbial metabolites provide a successful source of novel compounds with pharmaceutical potentials. The bacteriumPseudomonassp. In5 is a biocontrol strain isolated from a plant disease suppressive soil in Greenland, which produces two antimicrobial nonribosomal peptides (NRPs), nunapeptin and nunamycin.Methods.In this study, we usedin vitroantimicrobial and anticancer bioassays to evaluate the potential bioactivities of both a crude extract derived fromPseudomonassp. In5 and NRPs purified from the crude extract.Results.We verified that the crude extract derived fromPseudomonassp. In5 showed suppressive activity against the basidiomyceteRhizoctonia solaniby inducing a mitochondrial stress-response. Furthermore, we confirmed suppressive activity against the oomycetePythium aphanidermatumby thePseudomonassp. In5 crude extract, and that the purified nunamycin and nunapeptin displayed distinct antimicrobial activities. In addition to the antimicrobial activity, we found that treatment of the cancer cell lines, Jurkat T-cells, Granta cells, and melanoma cells, with thePseudomonassp. In5 crude extract increased staining with the apoptotic marker Annexin V while no staining of healthy normal cells, i.e., naïve or activated CD4 T-cells, was observed. Treatment with either of the NRPs alone did not increase Annexin V staining of the Jurkat T-cells, despite individually showing robust antimicrobial activity, whereas an anticancer activity was detected when nunamycin and nunapeptin were used in combination.Discussion.Our results suggest that the bioactivity of a crude extract derived fromPseudomonassp. In5 involves the presence of both nunamycin and nunapeptin and highlight the possibility of synergy between multiple microbial metabolites.

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