A Better Strategy To Extend the Proliferative Lifespan of Human T-Cells In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2398-2398
Author(s):  
Alexander Roeth ◽  
Lisa Schneider ◽  
Gabriela Baerlocher ◽  
Ulrich Duehrsen
Keyword(s):  
T Cells ◽  
Telomerase Activity ◽  
Culture Conditions ◽  
Autologous Serum ◽  
Feeder Cells ◽  
Human T Cells ◽  
Proliferative Lifespan ◽  
Pha Stimulation ◽  
Cells Cultured

Abstract Telomerase maintains telomere length by adding TTAGGG repeats to the 3′-ends of chromosomes. In human T-cells telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control their lifespan. To understand the effects of culture parameters on telomerase activity and cell lifespan is of critical importance to improve T-cell expansion. Here, we investigated the influence of culture conditions and of stimulation on lifespan, clonogenicity (number of positive wells), cell cycle and telomerase activity in T-cells in vitro. Isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured in RPMI 1640 medium containing IL-2, different sera (10%) (fetal calf serum (FCS), AB serum and autologous serum) and stimulated with phytohaemagglutinin (PHA) and irradiated allogeneic mononuclear feeder cells or anti-CD3/CD28 beads. T-cells were counted and passaged weekly until senescence. Expanded T-cells were analyzed for cell cycle by DNA staining with propidium iodide (PI) at various time points after stimulation and telomerase activity was measured using the telomerase PCR ELISA. Clonogenicity was determined by limiting dilution using different culture conditions. The proliferative lifespan of T-cells expanded with PHA/feeder cells and autologous serum from two different donors was significantly increased compared to T-cells stimulated with PHA/feeder cells and AB serum (44.7 vs. 14.2 PDs; 42.4 vs. 27.4 PDs). In contrast, no or only little difference was found for T-cells expanded with anti-CD3/CD28 beads and autologous or AB serum (8.7 vs. 8.1 PDs; 15.5 vs. 9.6 PDs). Most likely the latter findings reflect the loss of CD28 expression on T-cells. Furthermore, the use of autologous serum also increased the clonogenicity to about 4-fold compared to the use of AB serum or FCS without any signs for differences in the fractions of cycling cells. Interestingly, T-cells cultured with autologous serum exhibited a higher telomerase activity at day 3 after stimulation and a reduced decline of telomerase activity at day 6 and 9 compared to cultures with AB serum. Our results demonstrate that the use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity of human T-cells in vitro. In addition, T-cells cultured in medium containing autologous serum exhibit higher and prolonged telomerase activity compared to cultures with AB serum. Further studies are ongoing to elucidate the underlying factors involved in those observations. Nevertheless, we suggest that autologous serum together with PHA stimulation and feeder cells is superior to expand human T-cells especially for clinical applications where large numbers of specific T-cells are required for adoptive transfer strategies in cancer patients with for example severe viral infections.

Impact of culture conditions on the proliferative lifespan of human T cells in vitro

Cytotherapy ◽  
2007 ◽  
Vol 9 (1) ◽  
pp. 91-98 ◽  
Author(s):  
A. Röth ◽  
L. Schneider ◽  
H. Himmelreich ◽  
G.M. Baerlocher ◽  
U. Dührsen
Keyword(s):  
T Cells ◽  
Culture Conditions ◽  
Human T Cells ◽  
Proliferative Lifespan

scholarly journals A CRISPR-Cas9 Genome Engineering Platform in Primary CD4+ T Cells for the Interrogation of HIV Host Factors

2017 ◽  
Author(s):  
Judd F. Hultquist ◽  
Joseph Hiatt ◽  
Kathrin Schumann ◽  
Michael J. McGregor ◽  
Theodore L. Roth ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Genome Engineering ◽  
Culture Conditions ◽  
Host Factors ◽  
Biological Processes ◽  
Screen Design ◽  
Human T Cells

ABSTRACTCRISPR-Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human T cells to generate a high-throughput platform for analyzing the role of host factors in pathogen infection and lifecycle. Here, we describe applications of this system to investigate HIV pathogenesis in CD4+ T cells. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated primary human CD4+ T cells by nucleofection. These edited cells are then validated and expanded for use in downstream cellular, genetic, or protein-based assays. Our platform supports the arrayed generation of several gene manipulations in only a few hours’ time and is widely adaptable across culture conditions, infection protocols, and downstream applications. We present detailed protocols for crRNP synthesis, primary T cell culture, 96-well nucleofection, molecular validation, and HIV infection with additional considerations for guide and screen design as well as crRNP multiplexing.

627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A663-A663
Author(s):  
Keegan Cooke ◽  
Juan Estrada ◽  
Jinghui Zhan ◽  
Jonathan Werner ◽  
Fei Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Mouse Models ◽  
T Cell Activation ◽  
Phase 1 ◽  
Phase 1 Study ◽  
Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

scholarly journals In vitro response of human T cells to Pseudomonas aeruginosa.

1983 ◽  
Vol 40 (2) ◽  
pp. 670-674 ◽  
Author(s):  
J M Porwoll ◽  
H M Gebel ◽  
G E Rodey ◽  
R B Markham
Keyword(s):  
Pseudomonas Aeruginosa ◽  
T Cells ◽  
Human T Cells ◽  
In Vitro Response

scholarly journals Chimeric Antigen Receptor T Cells Specific for CLL-1 for Treatment of Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2205-2205 ◽  
Author(s):  
Elisa De Togni ◽  
Miriam Y Kim ◽  
Matt L Cooper ◽  
Julie Ritchey ◽  
Julie O'Neal ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Cytotoxic Effects ◽  
Car T Cells ◽  
Human T Cells ◽  
Car T ◽  
Acute Myeloid

Abstract Chimeric antigen receptor (CAR) T cells are a novel therapeutic approach which have shown good clinical outcomes in patients receiving CD19 CAR T cells for B cell acute lymphoblastic leukemia. CAR T cells are made to express a CAR that recognizes a specific surface antigen on a cell upon which they can then exert cytotoxic effects. We aim to extend the success of this therapy to acute myeloid leukemia (AML), a disease with generally poor clinical outcomes. However, due to the genetic heterogeneity characteristic of AML and the limited number of distinctive tumor markers, it has been difficult to find effective targets for CAR T cells on AML. C-type lectin like molecule-1 (CLL-1), also known as CD371, is a transmembrane glycoprotein that is expressed on about 90% of AML patient samples. CLL-1 may function as an inhibitory signaling receptor, as it contains an intracellular immunoreceptor tyrosine based inhibitory motif (ITIM). CLL-1 is primarily expressed on myeloid lineage cells in the bone marrow and in peripheral blood. While CLL-1 has been shown to be expressed on some granulocytes in the spleen, it is not reported to be expressed in non-hematopoietic tissues or on hematopoietic stem cells, which make CLL-1 a potential therapeutic target for AML. We generated two types of CLL-1 CARs, termed A and B, by using two different single chain variable fragments (scFvs) recognizing CLL-1. We used second generation CARs containing the scFvs, CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3 zeta signaling domains. Using a lentiviral vector, we transferred the CAR gene into healthy donor human T cells and detected CAR expression by flow cytometry. We then tested the specific cytotoxic effects of CLL-1 CART-A and B on a CLL-1-expressing AML cell line, U937, by conducting a 4-hour chromium release assay. We found that both CAR T cells exhibited a dose-dependent killing of U937 (CLL-1 positive), while the untransduced (UTD) T cells had no cytotoxic effect (Figure 1A). We also found that U937 induces degranulation of CLL-1 CAR T cells as measured by CD107a expression by flow cytometry, while Ramos, a CLL-1 negative cell line, does not (Figure 1B). We then proceeded to investigate the in vivo efficacy of the CAR T cells. We injected NOD/SCID/IL2RG-null (NSG) mice with 1 x 106 THP-1 cells, a CLL-1 positive cell line. We confirmed engraftment by bioluminescent imaging (BLI) after 7 days and then injected 4 x 106 UTD, CLL-1 CART-A or CLL-1 CART-B. Surprisingly, only one of the CAR constructs, CLL-1 CART-A, showed significant activity in vivo, although both CARs had shown comparable activity in vitro. CLL-1 CART-A treated mice had delayed tumor progression and significantly increased length of survival (85 days vs. 63 days, p = 0.0021) compared to mice injected with UTD (Figure 1C and D). While CLL-1 CART-B treated mice also exhibited slower tumor growth and a trend towards better survival (72 days vs. 63 days, p=0.0547) this was not statistically significant. Post-mortem analysis showed that human T cells that continued to express CAR were present in the tumor, bone marrow and spleen of mice treated with CLL-1 CART-A only, while the UTD and CLL-1 CART-B treated mice showed tumor in all organs and no T cells. In summary, we show that CLL-1 CAR T cells can selectively eliminate CLL-1 positive target cells in vitro and in vivo, albeit with different degrees of efficacy modulated by the scFv. Studies are ongoing to investigate the mechanism behind the differential activity of these CAR constructs and to increase the long-term antitumor efficacy. Our results demonstrate that targeting CLL-1 using CAR T cell therapy holds promise for the treatment of AML. Disclosures Cooper: WUGEN: Consultancy, Equity Ownership.

Synthesis, Characterization and anti-HIV and Antitumor Activities of New Coumarin Derivatives

2008 ◽  
Vol 63 (1) ◽  
pp. 83-89 ◽  
Author(s):  
Yaseen A. Al-Soud ◽  
Haitham H. Al-Sa’doni ◽  
Houssain A. S. Amajaour ◽  
Kifah S. M. Salih ◽  
Mohammad S. Mubarakb ◽  
...  
Keyword(s):  
T Cells ◽  
In Vitro Cytotoxicity ◽  
Tumor Cell Lines ◽  
Coumarin Derivatives ◽  
Antitumor Activities ◽  
Reverse Transcriptase Inhibitors ◽  
Human T Cells ◽  
Anti Hiv ◽  
Hiv 1

A new series of coumarin and benzofuran derivatives were synthesized as potential non-nucleoside reverse transcriptase inhibitors (NNRTIs) by reacting, separately, 4-bromomethylcoumarins, their sulphonyl chlorides, and ethyl 3-(bromomethyl)-6-methoxy-1-benzofuran-2-carboxylate with different imidazoles and their benzo analogs. The antiviral (HIV-1, HIV-2) properties of the newly synthesized compounds were investigated in vitro and all compounds were found to be inactive, except 10 which showed inhibition of HIV-2 with EC50 > 0.51 μgmL−1. The in vitro cytotoxicity of 17 and 19 was assayed against a panel of tumor cell lines consisting of CD4 human T-cells.

CD4+ Foxp3+ regulatory T cell-mediated immunomodulation by anti-depressants inhibiting acid sphingomyelinase

2018 ◽  
Vol 399 (10) ◽  
pp. 1175-1182 ◽  
Author(s):  
Jürgen Schneider-Schaulies ◽  
Niklas Beyersdorf
Keyword(s):  
T Cells ◽  
T Cell ◽  
Treg Cells ◽  
Tricyclic Antidepressant ◽  
Acid Sphingomyelinase ◽  
Human T Cells ◽  
And Function ◽  
Rate Limiting ◽  
Deficient Mice

AbstractAcid sphingomyelinase (ASM) is the rate-limiting enzyme cleaving sphingomyelin into ceramide and phosphorylcholin. CD4+Foxp3+regulatory T (Treg) cells depend on CD28 signaling for their survival and function, a receptor that activates the ASM. Both, basal and CD28-induced ASM activities are higher in Treg cells than in conventional CD4+T (Tconv) cells. In ASM-deficient (Smpd1−/−) as compared to wt mice, membranes of T cells contain 7–10-fold more sphingomyelin and two- to three-fold more ceramide, and are in a state of higher order than membranes of T cells from wt mice, which may facilitate their activation. Indeed, the frequency of Treg cells among CD4+T cells in ASM-deficient mice and their suppressive activityin vitroare increased. Moreover,in vitrostimulation of ASM-deficient T cells in the presence of TGF-β and IL-2 leads to higher numbers of induced Treg cells. Pharmacological inhibition of the ASM with a clinically used tricyclic antidepressant such as amitriptyline in mice or in tissue culture of murine or human T cells induces higher frequencies of Treg cells among CD4+T cells within a few days. This fast alteration of the balance between T cell populationsin vitrois due to the elevated cell death of Tconv cells and protection of the CD25highTreg cells by IL-2. Together, these findings suggest that ASM-inhibiting antidepressants, including a fraction of the serotonin re-uptake inhibitors (SSRIs), are moderately immunosuppressive and should be considered for the therapy of inflammatory and autoimmune disorders.

Sign in / Sign up

Export Citation Format

Share Document