Telomerase levels control the lifespan of human T lymphocytes

Blood ◽  
2003 ◽  
Vol 102 (3) ◽  
pp. 849-857 ◽  
Author(s):  
Alexander Röth ◽  
Hans Yssel ◽  
Jérôme Pène ◽  
Elizabeth A. Chavez ◽  
Mike Schertzer ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Major Influence ◽  
Telomeric Dna ◽  
Human Telomerase Reverse Transcriptase ◽  
Human T Lymphocytes ◽  
Human T Cells

Abstract The loss of telomeric DNA with each cell division contributes to the limited replicative lifespan of human T lymphocytes. Although telomerase is transiently expressed in T lymphocytes upon activation, it is insufficient to confer immortality. We have previously shown that immortalization of human CD8+ T lymphocytes can be achieved by ectopic expression of the human telomerase reverse transcriptase (hTERT) gene, which encodes for the catalytic component of the telomerase complex. To study the role of endogenous hTERT in the lifespan of human T cells, we blocked endogenous hTERT expression by ectopic expression of dominant-negative (DN) hTERT. Cells expressing DN-hTERT had a decreased lifespan and showed cytogenetic abnormalities, including chromosome ends without detectable telomeric DNA as well as chromosome fusions. These results indicate that while endogenous hTERT cannot prevent overall telomere shortening, it has a major influence on the longevity of human T cells. Furthermore, we show that up-regulation of hTERT in T cells upon activation decreases over time in culture. Long-term–cultured T cells also show a decreased expression of c-myc upon activation, resulting in less c-myc–induced transcription of hTERT. Moreover, memory T cells, which have expanded in vivo upon antigen encounter, expressed a lower level of hTERT upon activation than naive cells from the same donor. The observed inverse correlation between telomerase levels and replicative history suggests that telomerase levels in T cells are limiting and increasingly insufficient to sustain their proliferation.

In Vitro analysis of Suicide Gene Expression and Function of 3 Suicide Gene-Prodrug Combinations In Human T Lymphocytes Further Modified to Express the CD19 Targeted 19–28z Chimeric Antigen Receptor.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3771-3771
Author(s):  
Jae H. Park ◽  
Raymond Yeh ◽  
Isabelle Rivière ◽  
Michel Sadelain ◽  
Renier J. Brentjens
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Suicide Gene ◽  
Comparable Efficacy ◽  
Suicide Genes ◽  
Human T Lymphocytes ◽  
Drug Concentrations

Abstract Abstract 3771 Adoptive infusion of T cells genetically modified to express chimeric antigen receptors (CARs) targeted to tumor associated antigens (TAAs) is a promising approach to cancer therapy. However, since TAAs are often expressed by normal tissues, safeguards are needed in the form of additional transduced suicide genes to allow for the efficient in vivo abrogation of infused T cells in case of unanticipated adverse events which may develop in the clinical setting. To this end, we have investigated the in vitro function of 3 different suicide genes each inserted distal to a CAR gene targeted against CD19 (19-28z) and a 2A linker peptide cloned into the SFG gammaretroviral vector. Specifically, we have tested the herpes simplex virus thymidine kinase (HSV-TK SR39) with the prodrug ganciclovir, inducible caspase 9 (iCasp9) with the chemical inducer of dimerization (CID), and the E.coli derived nitroreductase (NTR) with the prodrug metronidazole. Cell growth of PG13 murine fibroblasts transduced to express 19–28z CAR with NTR, HSV-TK, and iCasp9 was inhibited by 80% at 1mM of metronidazole, 85% at 1μM of ganciclovir, and 90% at 10nM of CID, respectively, when compared to control PG-13 fibroblasts. The drug concentrations tested in these assays were at physiologically achievable concentrations in humans, and did not affect the growth rate of control PG13 fibroblasts. Consistent with these findings in PG13 fibroblasts, we found that human T cells transduced with either 1928z.2A.NTR or 1928z.2A.HSV-TK demonstrated 90% and 88% inhibition, respectively, at similar substrate concentrations. Furthermore, we demonstrate that expression of these suicide genes does not affect the phenotype or function of the 19–28z CAR+ T cells, as assessed in vitro by T cell proliferation and cytotoxicity against CD19-expressing tumor cells. Our studies demonstrate highly effective suicide genes for human T lymphocytes transduced with a tumor targeted CAR, and a novel suicide gene/prodrug (NTR/metronidazole) combination with a comparable efficacy that can potentially serve as a reliable safety mechanism for adoptive T cell immunotherapy. While HSV-TK/ganciclovir has been utilized in various clinical settings, the NTR suicide gene has yet to be used in combination with gene modified tumor-targeted T cells. Furthermore, the NTR suicide gene holds several advantages over the HSV-TK and iCasp9 vectors. First, unlike HSV-TK, the NTR suicide gene is effective in both proliferating and non-proliferating cells. Second, unlike CID that is not commercially available, metronidazole is a widely available antibiotic that is relatively non-toxic. Lastly, metronidazole can be used in patients who may already be taking ganciclovir for cytomegalovirus (CMV) prophylaxis or treatment therefore limiting the application of T cells modified to express the HSV-TK suicide gene. Based on this in vitro data, we are currently testing the function of this suicide gene in vivo in two different animal models. Ultimately we anticipate that further studies with this novel suicide gene/prodrug combination will allow us to enhance safety in future clinical trials utilizing gene modified tumor targeted T cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells

2011 ◽  
Vol 9 (1) ◽  
pp. 4 ◽  
Author(s):  
Hendrik Schmidt ◽  
Christoph Gelhaus ◽  
Melanie Nebendahl ◽  
Marcus Lettau ◽  
Ralph Lucius ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Human T Lymphocytes ◽  
Human T Cells ◽  
Secretory Lysosomes

Functional Analyses of Human T Cell Extravasation in a Humanized NOD/SCID/IL2Rγcnull Transplantation Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2448-2448
Author(s):  
Ariane Brunk ◽  
Marion Nonn ◽  
Victoria Lang ◽  
Reinhard Henschler ◽  
Wolfgang Herr ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Skin Substitutes ◽  
Nsg Mice ◽  
Human T Cell ◽  
Human T Cells ◽  
Graft Engineering ◽  
Transplantation Model

Abstract Abstract 2448 Poster Board II-425 Donor lymphocyte graft engineering to avoid graft-versus-host (GVH) reactivity while improving graft-versus-leukemia (GVL) immunity remains of central interest in allogeneic hematopoietic stem cell transplantation (HSCT). However, appropriate models to evaluate experimental concepts of donor lymphocyte allograft engineering in vivo are missing. We, therefore, established a human-murine chimeric transplantation model using immunodeficient NOD/SCID/IL2Rγcnull (NSG) mice to evaluate GVH reactivity of human T cell grafts in vivo. Moreover, since mechanisms of immune functions resembling human GVH immunity have not yet been addressed in detail in these humanized mice we started to analyse T cell trafficking and homing to lymphoid tissues across species barrier to examine the clinical relevance of T cell activity observed in this model. To this end, skin substitutes composed of human primary allogeneic fibroblasts embedded in a collagen-based matrix were subcutaneously implanted into NSG mice to detect alloreactive specificities within the implant post adoptive transfer of MHC-mismatched or haploidentical donor T lymphocytes. The skin substitutes revealed murine vascularisation two weeks after implantation as demonstrated by immunohistological studies. Following transfer of human HLA-mismatched or haploidentical T lymphocytes, up to 23% of the T cells migrated into skin substitutes explanted 21 days post injection. As this T cell migration and homing involves both murine and human adhesion molecules we further analyzed the specific adhesion mechanisms underlying the egress of human T cells from the murine bloodstream. Using laminar flow chamber experiments and real time video recordings we could first demonstrate that human anti-CD3/anti-CD28 preactivated T lymphocytes but not naive T cells bound and firmly adhered to the murine endothelial cell line bEND.3 (BEND3.EC) at shear stresses of up to 3.5 dynes/cm2. As controls, human umbilical vein endothelial cells (HUVEC) were used. Adhesion and transmigration was significally enhanced when both human and murine endothelial cells (ECs) were prestimulated with low doses of TNF-α (5-20ng/ml) to resemble an activated phenotype. Firm adhesion of activated T lymphocytes was suppressed following pretreatment with function-blocking anti-integrin-alpha 4 (CD49d, subunit of VLA-4) or anti-integrin-alpha L (CD11a, subunit of LFA-1) antibody (Ab) or when ECs were preincubated with anti-VCAM-1 (CD106). No inhibitory effects were observed when anti-Endoglin (CD105) Ab was included as specificity control suggesting that the integrin dimer VLA-4 and its counter-receptor VCAM-1 as well as the integrin dimer LFA-1 are required for the transmigration of human T cells across murine ECs. Primary ECs derived from murine aorta are currently used to confirm our results obtained with bEND.3 cells. As the same ligand-receptor pairs are described for human T-EC interaction these findings indicate a closely related mechanism of T cell extravasation in human and murine endothelium at least in our transplantation model. In conclusion these results suggest that intravenously transferred activated human T cells migrate into allogeneic skin substitutes involving VCAM-1 and integrin-alpha-4 for firm adhesion followed by transmigration. In vivo studies investigating the effects of the function-blocking antibodies against VCAM-1 and integrin-alpha-4 in our model to confirm the in vitro results are in progress and will be reported. In addition, our human-murine chimeric NSG transplantation model may represent a promising tool to study human GVH biology and to evaluate T cell graft engineering in allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Genomic stability and functional activity may be lost in telomerase-transduced human CD8+ T lymphocytes

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2663-2670 ◽  
Author(s):  
Marco W. J. Schreurs ◽  
Mario A. J. A. Hermsen ◽  
Ramon I. Klein Geltink ◽  
Kirsten B. J. Scholten ◽  
Antoinette A. T. P. Brink ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Clinical Application ◽  
Chromosomal Aberrations ◽  
Functional Activity ◽  
Constitutive Expression ◽  
Comparative Genomic ◽  
Human Telomerase Reverse Transcriptase

AbstractTo obtain the large amount of T cells required for adoptive immunotherapy in a clinical setting, T-cell lifespan extension by human telomerase reverse transcriptase (hTERT) transduction is of particular interest. However, constitutive expression of hTERT is associated with malignant transformation and thus warrants a detailed evaluation of the safety of hTERT-transduced T cells before clinical application. In view of this, we performed an extensive cytogenetic analysis of hTERT-transduced MART-1 (melanoma antigen recognized by T cell 1)–and human papillomavirus type 16 (HPV16) E7–specific human CD8+ cytotoxic T lymphocytes (CTLs), reactive against melanoma and cervical carcinoma, respectively. Our results, obtained by (spectral) karyotyping and array comparative genomic hybridization, showed the development of minor chromosomal aberrations in an hTERT-transduced MART-1–specific CTL clone, whereas severe clonal aberrations were detected in an hTERT-transduced HPV16 E7–specific CTL clone. Furthermore, hTERT transduction did not protect CTLs from immunosenescence, because the HPV16 E7–specific, hTERT-transduced CTL clone showed a decreased functional activity on prolonged culture. Although the general frequency of major chromosomal aberrations in hTERT-transduced CTLs and the in vivo significance of our observations remain still unclear at this point, the currently available data suggest that clinical application of hTERT-transduced CTLs should proceed with caution.

scholarly journals Coexpression of FOXP3 and a Helios isoform enhances the effectiveness of human engineered regulatory T cells

2020 ◽  
Vol 4 (7) ◽  
pp. 1325-1339 ◽  
Author(s):  
Amara Seng ◽  
Kelsey L. Krausz ◽  
Dong Pei ◽  
Devin C. Koestler ◽  
Ryan T. Fischer ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Inflammatory Diseases ◽  
Disease Model ◽  
Ectopic Expression ◽  
Full Length ◽  
Human T Cells ◽  
Cellular Pathways

Abstract Regulatory T cells (Tregs) are a subset of immune cells that suppress the immune response. Treg therapy for inflammatory diseases is being tested in the clinic, with moderate success. However, it is difficult to isolate and expand Tregs to sufficient numbers. Engineered Tregs (eTregs) can be generated in larger quantities by genetically manipulating conventional T cells to express FOXP3. These eTregs can suppress in vitro and in vivo but not as effectively as endogenous Tregs. We hypothesized that ectopic expression of the transcription factor Helios along with FOXP3 is required for optimal eTreg immunosuppression. To test this theory, we generated eTregs by retrovirally transducing total human T cells (CD4+ and CD8+) with FOXP3 alone or with each of the 2 predominant isoforms of Helios. Expression of both FOXP3 and the full-length isoform of Helios was required for eTreg-mediated disease delay in a xenogeneic graft-versus-host disease model. In vitro, this corresponded with superior suppressive function of FOXP3 and full-length Helios-expressing CD4+ and CD8+ eTregs. RNA sequencing showed that the addition of full-length Helios changed gene expression in cellular pathways and the Treg signature compared with FOXP3 alone or the other major Helios isoform. Together, these results show that functional human CD4+ and CD8+ eTregs can be generated from total human T cells by coexpressing FOXP3 and full-length Helios.

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 Metabolic radiolabeling and in vivo PET imaging of cytotoxic T lymphocytes to guide combination adoptive cell transfer cancer therapy

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Dehua Lu ◽  
Yanpu Wang ◽  
Ting Zhang ◽  
Feng Wang ◽  
Kui Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Solid Tumors ◽  
Cytotoxic T Lymphocytes ◽  
Pet Imaging ◽  
Stage Migration ◽  
Cell Transfer ◽  
Tumor Infiltration ◽  
Antitumor Effects

Abstract Background Adoptive T cell transfer-based immunotherapy yields unsatisfactory results in the treatment of solid tumors, partially owing to limited tumor infiltration and the immunosuppressive microenvironment in solid tumors. Therefore, strategies for the noninvasive tracking of adoptive T cells are critical for monitoring tumor infiltration and for guiding the development of novel combination therapies. Methods We developed a radiolabeling method for cytotoxic T lymphocytes (CTLs) that comprises metabolically labeling the cell surface glycans with azidosugars and then covalently conjugating them with 64Cu-1,4,7-triazacyclononanetriacetic acid-dibenzo-cyclooctyne (64Cu-NOTA-DBCO) using bioorthogonal chemistry. 64Cu-labeled control-CTLs and ovalbumin-specific CTLs (OVA-CTLs) were tracked using positron emission tomography (PET) in B16-OVA tumor-bearing mice. We also investigated the effects of focal adhesion kinase (FAK) inhibition on the antitumor efficacy of OVA-CTLs using a poly(lactic-co-glycolic) acid (PLGA)-encapsulated nanodrug (PLGA-FAKi). Results CTLs can be stably radiolabeled with 64Cu with a minimal effect on cell viability. PET imaging of 64Cu-OVA-CTLs enables noninvasive mapping of their in vivo behavior. Moreover, 64Cu-OVA-CTLs PET imaging revealed that PLGA-FAKi induced a significant increase in OVA-CTL infiltration into tumors, suggesting the potential for a combined therapy comprising OVA-CTLs and PLGA-FAKi. Further combination therapy studies confirmed that the PLGA-FAKi nanodrug markedly improved the antitumor effects of adoptive OVA-CTLs transfer by multiple mechanisms. Conclusion These findings demonstrated that metabolic radiolabeling followed by PET imaging can be used to sensitively profile the early-stage migration and tumor-targeting efficiency of adoptive T cells in vivo. This strategy presents opportunities for predicting the efficacy of cell-based adoptive therapies and for guiding combination regimens. Graphic Abstract

Determination of in vivo protein synthesis in human T lymphocytes

2001 ◽  
Vol 20 (2) ◽  
pp. 181-182 ◽  
Author(s):  
A. JANUSZKIEWICZ ◽  
P. ESSÉN ◽  
M.A. McNURLAN ◽  
O. RINGDÉN ◽  
P.J. GARLICK ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
T Lymphocytes ◽  
Human T Lymphocytes ◽  
In Vivo Protein Synthesis
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