Capture of antigen-specific T lymphocytes from human blood by selective immortalization to establish long-term T-cell lines maintaining primary cell characteristics☆

Barsov, Eugene V.; Andersen, Hanné; Coalter, Vicky; Carrington, Mary; Lifson, Jeffrey D.; Ott, David E.

doi:10.1016/j.imlet.2005.11.028

Cited by 13 publications

(14 citation statements)

References 60 publications

(58 reference statements)

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“…Replication incompetent retroviral vectors have been used to deliver genes such as Bcl-2, Bcl-X L , IL-2, hTERT, and Tax that could prolong the survival of primary T cells. [49][50][51][52][53][54] However, none of these studies report prolific, clonal, cell expansion in the absence of repeated stimulation and exogenous cytokine as demonstrated by LC15. The LC15 cell line appears to be the result of an infrequent vector integration event and possibly synergy between constitutive IL-15 transgene expression and telomerase activity.…”

Section: Discussionmentioning

confidence: 99%

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

Hsu

Jones

Cohen

et al. 2007

Blood

100

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Malignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8 ؉ Tcell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus-based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28 ؊ , CD45RA ؊ , CD45RO ؉ , and CD62L ؊ , a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigenspecific T-cell receptors, the clone secreted IFN-␥ upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. IntroductionHuman gene therapy strategies using replication-incompetent retroviral vectors were implemented cautiously because of many concerns, including the potential for adverse events resulting from vector insertions in the human genome. 1 Theoretical concerns about insertional mutagenesis became reality with the report that 3 of 11 patients with severe combined immunodeficiency-X1 (SCID-X1) developed T-cell leukemia after treatment with hematopoietic stem cells (HSCs) engineered with the common ␥-chain cytokine receptor gene. These leukemias resulted from integration events which activated the LMO2 proto-oncogene. 2 Nevertheless, after more than 17 years of clinical gene therapy trials this remains the only report of overt cancer development resulting from genemodified cell therapy. Our understanding of the pathogenesis of human malignancy resulting from insertional mutagenesis remains limited to this specific clinical scenario.Several observations from the SCID-X1 gene therapy trial fall in line with general principles established through animal models and other human clinical trials investigating retroviral gene transfer. The well-characterized murine leukemia virus (MLV)-based gene transfer vectors integrate preferentially in the vicinity of cellular promoters, 3,4 potentially increasing the probability of activating or inactivating vector insertions which may dysregulate gene expression. 5,6 The target cells most at risk of insertional mutagenesis are believed to be primitive progenitor cells. 7 Potentially, therapeutic transgenes may have leukemogenic properties in the appropriate setting. 6 The interactions between retroviral gene transfer vectors and hematopoietic cells are being investigated intensely.In this report, we have characterized a primary human T-cell clone, which exhibited continuous growth after transduction with an MLV-based retroviral vector encoding the T-cell growth factor, IL-15. This was an unexpected and intriguing finding for several reasons. First, human cells are inherently difficult to immortalize, and the process of transformation in human cells is believed to be considerably more complex than that of experimental animals. Second, the cells that were transduced consisted of...

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Section: Discussionmentioning

confidence: 99%

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

Hsu

Jones

Cohen

et al. 2007

Blood

100

View full text Add to dashboard Cite

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“…When cells were actively dividing (day 5–8) they were immortalized using a murine leukemia virus based retroviral vector carrying the telomerase gene (hTERT) [59]. This vector preferentially transduces cells that are dividing, and the procedure results in the selective transduction of cells that are proliferating in response to antigen stimulation.…”

Section: Methodsmentioning

confidence: 99%

GM-CSF Production Allows the Identification of Immunoprevalent Antigens Recognized by Human CD4+ T Cells Following Smallpox Vaccination

Judkowski

Bunying

et al. 2011

PLoS ONE

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The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a “T cell–driven” methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens.

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“…Infection with Moloney murine leukemia virus-derived retroviral vectors, expressing TERT, allows selective capture and immortalizing of human antigen-specific T cells from a complex cell population such as blood [109,110]. Such vectors can only integrate in dividing cells [111,112], thus, being able to selectively transduce only those T cells in a population that are specifically activated and dividing.…”

Section: Tert-immortalized Human T Cells Maintain Antigen Specificitymentioning

confidence: 99%

“…T cells, constitutively expressing TERT, maintain characteristics of primary lymphocytes: they specifically recognize and respond to cognate antigens and maintain their natural effector functions, such as production of physiologically appropriate cytokines and antigen-specific cytolytic activity. We and others were able to maintain human and RM TERT-transduced antigen-specific T-cell lines and clones in culture for periods over 1 year without the loss of specific T-cell functions, which far exceeds the normal replicative lifespan of primary human and NHP T cells [107–109,114]. However, it remains to be established that TERT-transduced T-cell lines and clones are truly immortal, rather than protected from senescence for an extended, but still finite, period of time.…”

Section: Conclusion and Future Perspectivementioning

confidence: 99%

“…Human and RM T-cell lines overexpressing TERT display a behavior that is characteristic for normal primary T lymphocytes, for long periods of time [109]. Importantly, similar to their nontransduced counterparts, and in contrast to transformed T-cell lines, proliferation of TERT-transduced T cells can be induced by activation and their growth is strictly dependent on cytokines.…”

Section: Conclusion and Future Perspectivementioning

confidence: 99%

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Telomerase and Primary T Cells: Biology and Immortalization for Adoptive Immunotherapy

Barsov

2011

Immunotherapy

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Telomeres are specialized repeats, present at the end of chromosomes, whose loss during cell division is followed by growth arrest, a central mechanism of replicative senescence in human cells. Telomere length in stem cells is maintained by telomerase, a specialized reverse transcriptase, whose function is to restore shortening telomeres. Unlike most somatic cell types, human T lymphocytes are capable of briefly reactivating telomerase expression at the time of stimulation. Telomerase expression in T lymphocytes is modulated by a variety of external stimuli and by viral infections. However, telomerase reactivation in stimulated, proliferating human T lymphocytes is limited and cannot prevent the ultimate onset of senescence. Ectopic telomerase expression can rescue human and macaque antigen-specific T cells from senescence. Primary T cells have been engineered with telomerase to have substantially extended replicative lifespans without the loss of primary cell functions or malignant transformation. ‘Immortal’ antigen-specific T-cell lines and clones overexpressing telomerase are an invaluable source of well-characterized quasi-primary T cells for research of T-cell biology and are potentially useful for immunotherapy of cancer and AIDS.

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Capture of antigen-specific T lymphocytes from human blood by selective immortalization to establish long-term T-cell lines maintaining primary cell characteristics☆

Cited by 13 publications

References 60 publications

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

GM-CSF Production Allows the Identification of Immunoprevalent Antigens Recognized by Human CD4+ T Cells Following Smallpox Vaccination

Telomerase and Primary T Cells: Biology and Immortalization for Adoptive Immunotherapy

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