scholarly journals Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1)

2020 ◽  
Author(s):  
Patricia Letondal ◽  
Chrystel Marton ◽  
Yann Godet ◽  
Jeanne Galaine
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Internal Control ◽  
Jurkat Cell ◽  
Cellular Therapy ◽  
Adoptive Cell Therapy ◽  
Metabolic Potential ◽  
Experimental Conditions ◽  
Jurkat Cell Line

Abstract Background Metabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization. Results We aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and FCCP to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions. Conclusion The validation of the method consisting in a JURKAT cell line experimental incorporation as internal control contributes to improve the Seahorse technology’s robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.

scholarly journals Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1)

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Patricia Mercier-Letondal ◽  
Chrystel Marton ◽  
Yann Godet ◽  
Jeanne Galaine
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Jurkat Cell ◽  
Cellular Therapy ◽  
Adoptive Cell Therapy ◽  
Metabolic Potential ◽  
Experimental Conditions ◽  
Jurkat Cell Line ◽  
Set Up

Abstract Background Metabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization. Results We aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions. Conclusion The validation of the method consisting in a JURKAT cell line experimental incorporation as a control material contributes to improve the Seahorse technology’s robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.

NTAL Potentiates Glucocorticosteroid-Induced Apoptosis in T-ALL – in Vivo Observations and Experimental Model

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5043-5043
Author(s):  
Karel Svojgr ◽  
Tatiana Burjanivova ◽  
Martina Vaskova ◽  
Tomas Kalina ◽  
Tomas Brdicka ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Jurkat Cell ◽  
Annexin V ◽  
Signal Propagation ◽  
Living Cells ◽  
Negative Control ◽  
Jurkat Cells ◽  
Wild Type ◽  
Jurkat Cell Line

Abstract PAG, LAT, NTAL and LIME belong to category of transmembrane adaptor proteins (TRAPs). They do not possess an enzymatic or kinase function, but they are involved in mediation of signal transmission from surface receptors to cell nucleus. We propose that some of them are engaged in development or maintenance of leukaemia. We have previously demonstrated that expression status of TRAPs at mRNA level is specific in some subgroups of childhood ALL, particularly in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) with the TEL/AML1 fusion gene. Furthermore, we have described that variable expression of NTAL mRNA is related to the response to initial glucocorticosteroid pre-phase in the treatment of childhood T-ALL; patients with high NTAL levels show better treatment response compared to the low-NTAL cases. In the current study, we aimed to prove experimentally that different levels of NTAL protein influence response of leukaemic T cells to glucocorticosteroids. In the wild type Jurkat cells (human T-cell leukaemia cell line) the NTAL protein is undetectable. For the in-vitro experiments we created a derivative Jurkat cell line transfected with the NTAL construct. In the derivative cell line, the NTAL positivity at both mRNA and protein level was verified using RT-PCR and Western blotting. The derivative cell line in a cell culture behaves similar as wild type Jurkat cell line. Transfectants and wild type Jurkat cells were incubated with methylprednisolone, dexamethasone or with solvent alone (H2O) as a negative control. Using flow cytometry we determined a percentage of surviving cells after 24, 48 and 72 hours of treatment. Cells were stained with Annexin V and DAPI and living cells were defined as Annexin V, DAPI negative. At each time point, the number of living cells in the negative control was set to 100%. After 48 hours of dexamethasone treatment the number of surviving cells in the Jurkat wild-type was higher by 12% compared to the Jurkat cells expressing NTAL. After 72 hours this difference was even more prominent reaching 46%. The same effect of methylprednisolone treatment was less pronounced (non-significant difference at 48 hours and 5% difference at 72 hours). Based on our experimental data we propose that NTAL acts in T-cells as a putative tumour suppressor. A plausible mechanism of its function is that NTAL competitively inhibits another adaptor LAT, which is required for signal propagation to the nucleus in T-cells. NTAL and LAT can compete for its localisation in proper “lipid rafts” or for palmitoylation. Our data can be used for subsequent functional analysis of signaling pathways in leukaemic blasts as well as in physiological lymphoid cells.

scholarly journals Dose-Dependent Responses of I3C and DIM on T-Cell Activation in the Human T Lymphocyte Jurkat Cell Line

2017 ◽  
Vol 18 (7) ◽  
pp. 1409 ◽  
Author(s):  
Man Liu ◽  
Rumana Yasmeen ◽  
Naomi Fukagawa ◽  
Liangli Yu ◽  
Young Kim ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
T Cell Activation ◽  
T Lymphocyte ◽  
Jurkat Cell ◽  
Cell Activation ◽  
Jurkat Cell Line ◽  
Human T Lymphocyte ◽  
Dose Dependent

scholarly journals A Genome-wide Survey of Mutations in the Jurkat Cell Line

2017 ◽  
Author(s):  
Louis Gioia ◽  
Azeem Siddique ◽  
Steven R. Head ◽  
Daniel R. Salomon ◽  
Andrew I. Su
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Jurkat Cell ◽  
Past Research ◽  
Cell Receptor ◽  
Genomic Variation ◽  
T Cell Signaling ◽  
Jurkat Cell Line ◽  
Human T Cells ◽  
A Genome

The Jurkat cell line has an extensive history as a model of T cell signaling. But at the turn of the 21st century, some irregularities were observed in Jurkat’s expression of central regulators of T cell receptor signaling, which raised doubts about how closely the cell line paralleled normal human T cells. While numerous expression deficiencies have been described in Jurkat, genetic explanations have only been provided for a handful of defects. Here, we report a comprehensive catolog of genomic variation in the Jurkat cell line based on whole-genome sequencing. With this list of all detectable, non-reference sequences, we prioritize potentially damaging mutations by mining public databases for functional effects. We confirm the majority of documented mutations in Jurkat and propose links from detrimental gene variants to observed expression abnormalities in the cell line. This work ties together decades of molecular experiments and serves as a resource that will streamline both the interpretation of past research and the design of future Jurkat studies.

scholarly journals Systemic and intravesical adoptive cell therapy of tumor-reactive T cells can decrease bladder tumor growth in vivo

2020 ◽  
Vol 8 (2) ◽  
pp. e001673
Author(s):  
Brittany L Bunch ◽  
Jennifer Morse ◽  
Sarah Asby ◽  
Jamie Blauvelt ◽  
Ahmet M Aydin ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cellular Therapy ◽  
Checkpoint Inhibitors ◽  
Adoptive Cell Therapy ◽  
Tumor Cell Line ◽  
Cell Infiltration ◽  
T Cell Infiltration

BackgroundThe therapeutic armamentarium of bladder cancer has been recently enriched with the introduction of new therapies including immune checkpoint inhibitors, receptor tyrosine kinase inhibitors and antibody drug conjugates, however treatment responses and duration of responses are still less than expected. Adoptive cellular therapy (ACT) using tumor-infiltrating lymphocytes (TILs) has potential to treat bladder cancer, as previously demonstrated by successful expansion of tumor reactive T cells from human bladder tumors.MethodsA model system using OT-I T cells and an ovalbumin expressing MB49 tumor cell line (MB49OVA) was developed to study ACT in bladder cancer. Systemic ACT-treated mice were given T cells intravenously after lymphodepleting chemotherapy and followed by interleukin (IL)-2 administration. Intravesical ACT treated mice were given T cells directly into the bladder, without chemotherapy or IL-2. TILs were isolated from MB49 orthotopic tumors and expanded ex vivo in IL-2. Immune cell infiltrates were analyzed by flow cytometry. T cell infiltration was studied using a CXCR3 blocking antibody.ResultsSystemic ACT-treated mice had a decrease in tumor growth, increase in T cell infiltration and long-term immune protection compared with control-treated mice. OT-I T cells delivered intravesically were able to control tumor growth without lymphodepleting chemotherapy or IL-2 in MB49OVA orthotopic tumors. Intravesical delivery of TIL expanded from MB49 tumors was also able to decrease tumor growth in mice with MB49 orthotopic tumors. Blocking CXCR3 on OT-I T cells prior to intravesical delivery decreased T cell infiltration into the tumor and prevented the control of tumor growth.ConclusionsThis study demonstrates how TIL therapy can be used in treating different stages of bladder cancer.

scholarly journals Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line

2020 ◽  
Vol 21 (23) ◽  
pp. 9163
Author(s):  
Chung Hyo Kang ◽  
Yeongrin Kim ◽  
Heung Kyoung Lee ◽  
So Myoung Lee ◽  
Hye Gwang Jeong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Jurkat Cell ◽  
Single Chain ◽  
Car T Cells ◽  
Car T ◽  
Nk T Cell

CD19 is the most promising target for developing chimeric-antigen receptor (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.

scholarly journals Comparative Analysis for Differential Drug Response between Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) and T-Cell Acute Lymphoblastic Leukemia (T-ALL) Patients Using the Cellworks Omics Biology Model (CBM): Mycare-021-03

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-19
Author(s):  
Scott Howard ◽  
Ansu Kumar ◽  
Anusha Pampana ◽  
Yashaswini S Ullal ◽  
Anuj Tyagi ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Jurkat Cell ◽  
Lymphoblastic Leukemia ◽  
Patient Specific ◽  
Therapy Options ◽  
Jurkat Cell Line

Background:Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), an orphan disease, is a sub-type of T-Cell Acute Lymphoblastic Leukemia (T-ALL) with very poor prognosis and limited therapy options. ETP-ALL is a heterogeneous disease with many distinct genomic profiles, often with more myeloid than lymphoid characteristics. However, standard of care (SOC) drugs for acute myeloid leukemia (AML) have shown limited efficacy for ETP-ALL (PMID: 32733662, 25435716). The genomic profiles of ETP-ALL patients have more complex cytogenetics and larger numbers of genomic aberrations when compared to non-ETP-ALL (T-ALL) profiles (PMID: 22237106, 30641417). We present an alternative multi-gene analysis approach using the Cellworks Omics Biology Model (CBM) workflow to identify unique, intersecting protein pathways in patient-specific disease profiles. The CBM predictive workflow was used to design novel personalized therapy options for an ETP-ALL representative PEER human lymphoid cell line in comparison to a T-ALL JURKAT cell line. The predicted combination therapies were then validated in a lab model. Methods:A PEER cell line was selected to represent ETP-ALL and a JURKAT cell line was selected as a representative for non-ETP T-ALL. Next Generation Sequencing (NGS) was performed for the PEER cell line. For the JURKAT cell line, publicly available NGS whole exome sequencing from cBioPortal and Sanger, along with array CGH from Agilent, were used. The genomic data for the PEER and JURKAT cell lines were used as inputs to the CBM to generate dynamic patient-specific disease protein network maps. Biomarkers and pathway characteristics unique to the PEER and JURKAT cell lines were identified. A digital drug library of targeted FDA-approved agents was simulated on the disease models using both single drug agents and drug combinations at varying doses. The treatment impact was assessed by quantitatively measuring drug effect on a cell growth score, which is a composite of the quantified values of cell proliferation, survival and apoptosis along with impact on the patient-specific disease biomarker score. Comparative dose response studies were run to assess IC50 differences for both cell lines. Cellworks VenturaTM predicted novel therapy combinations for the ETP-ALL representative PEER cell line, which were then prospectively validated by in vitro experiments. The same therapy options were predicted to be less effective in the T-ALL representative JURKAT cell line, which was also confirmed by in vitro studies. Results:The CBM predicted three novel combination therapies for the ETP-ALL representative PEER cell line: nilotinib + cytarabine, bortezomib + cytarabine and bortezomib + idarubicin. All three therapies were predicted to be less effective in JURKAT cells. In vitro, PEER cells were sensitive to all 3 combinations, as predicted by the CBM; whereas, JURKAT cell lines were not sensitive to the first 2 combinations (as predicted), but were sensitive to bortezomib + idarubicin. The CBM analysis is supported by scientific rationales for these combinations based on the genomics-driven disease characteristics of the cell-line. The reasons for drug sensitivity and resistance were determined. These combinations were then prospectively validated in vitro on both cell lines and the experimental responses matched the predicted outcomes. Conclusion:The Cellworks Omics Biology Model integrates the multiple genomic abnormalities in a patient to identify disease network characteristics unlike other NGS analytic tools that attempt to interpret the impact of each genomic alteration in isolation. CBM identified 3 novel therapy options for ETP-ALL that were validated in vitro, similar to anecdotal experience in vivo. This predictive technology can improve clinical decision-making and identify novel treatment options. Disclosures Howard: Cellworks:Consultancy;Servier:Consultancy, Other: Speaker;EUSA Pharma:Consultancy;Sanofi:Consultancy, Other: Speaker;Boston Scientific:Consultancy.Kumar:Cellworks Research India Private Limited:Current Employment.Pampana:Cellworks Research India Private Limited:Current Employment.Ullal:Cellworks Research India Private Limited:Current Employment.Tyagi:Cellworks Research India Private Limited:Current Employment.Lala:Cellworks Research India Private Limited:Current Employment.Kumari:Cellworks Research India Private Limited:Current Employment.Joseph:Cellworks Research India Private Limited:Current Employment.Raju:Cellworks Research India Private Limited:Current Employment.Balakrishnan:Cellworks Research India Private Limited:Current Employment.Mundkur:Cellworks Group Inc.:Current Employment.Macpherson:Cellworks Group Inc.:Current Employment.Nair:Cellworks Research India Private Limited:Current Employment.Kapoor:Cellworks Research India Private Limited:Current Employment.

scholarly journals Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

2021 ◽  
Vol 9 (3) ◽  
pp. e001877
Author(s):  
Irfan N Bandey ◽  
Jay R T Adolacion ◽  
Gabrielle Romain ◽  
Melisa Martinez Paniagua ◽  
Xingyue An ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Adoptive Cell Therapy ◽  
Population Heterogeneity ◽  
Serial Killer ◽  
Car T Cells ◽  
Car T ◽  
Single Cell Profiling ◽  
Killer T Cells

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

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