979. Clonal Expansion of a CD8+ T Cell Clone after Retroviral Transduction of Mature T Lymphocytes with the IL-15 Gene

Abstract

Top of pageAbstract We have characterized a primary human T cell line, designated LC15, which has apparently been immortalized after transduction with an MSCV-based retroviral vector encoding the T cell growth factor, IL-15. This was an unexpected finding because, in previously reported animal and human studies, the target cells most at risk for insertional mutagenesis events were primitive progenitor cells. LC15 was generated by activating peripheral blood lymphocytes with OKT3 and IL-2. Subsequently, the cells were transduced with a retroviral vector encoding an IL-15 gene. Long-term culture of these cells demonstrated logarithmic growth for more than 365 days and constitutive expression of IL-15. In order to evaluate the LC15 cells for clonality, we utilized a 5'RACE technique to determine T cell receptor |[beta]|-chain usage; LC15 cells express only V|[beta]|13s.3 and all ofthe sequences contained identical D- and J-regions. Spectral karyotyping revealed a clonal abnormality with 4 translocations: 44, X, der(X)t(X;16)(p22.3;p10), der(13;16)(q10;p10), der(14;21)(q10;q10), |[minus]|16,+22,i(22)(q10). This result confirmed that LC15 is a clone and rules out chromosomal rearrangement as a mechanism for dysregulated growth of the cell line because all of the chromosomal breaks occurred at centromeres. We performed Southern blot analysis and found that the vector was intact and that the cells contained four vector integration sites. Phenotypically, the cells evolved into a pure population of CD8 lymphocytes with an effector memory phenotype (CD45 RA-, CD45RO+, and CD62L-). LC15 cells exhibited heterogeneous staining for CD5, CD25, and CD38, suggesting that multiple levels of activation existed within the population. Presumably, constitutive IL-15 expression contributes to the growth and maintenance of the cell line, however proliferation of the LC15 cell line could not be blocked by an anti-IL-15 monoclonal antibody. The cell line could not be grown by limiting dilution cloning and could not be established in immunodeficient mice, thus its malignant potential remains uncertain. Telomere length and telomerase activity were also examined. After 2 months in culture, the telomeres were approximately 4 kb in length. After 12 months in culture, the telomeres were approximately 2.3 kb. Telomerase was constitutively active in these cells in contrast to normal lymphocytes in which telomerase is upregulated by stimulation. We believe that this is the first report describing retroviral vector-induced transformation of a mature T lymphocyte. We have transduced a total of 20 different human T cell cultures with the IL-15 retroviral vector and LC15 represents the only incidence of clonal expansion; we hypothesize that this cell line is the result of an unusual and infrequent retroviral vector integration event. Current efforts are directed towards identifying the genomic integration sites in LC15 and evaluating the expression of genes in proximity of these sites. Potentially, this may reveal new insights into the genes that regulate T lymphocyte growth and survival. Furthermore, investigation of this cell line may identify genetic alterations involved in the transformation of mature T lymphocytes.