Suspension packaging cell lines for the simplified generation of T-cell receptor encoding retrovirus vector particles.
The transfer of T-cell receptor (TCR) genes into primary human T-cells to endow their specificity toward virus-infected and tumor cells is becoming an interesting tool for immunotherapy. TCR-modified T cells are mainly generated by retrovirus-mediated gene transfer. To produce TCR-retrovirus particles, fibroblast packaging cell lines are the most common tool. We constructed two packaging cell lines based on the human suspension T-cell lymphoma line Deltabeta-Jurkat, which lacks endogenous TCRbeta-chains and is therefore unable to express CD3 complexes on the cell surface. After supply of gag-pol (murine leukemia virus (Mo-MLV)) and env (GALV or MLV-10A1) genes, a green fluorescent protein (GFP)-encoding retrovirus vector was transduced into both packaging cell clones, which then stably produced GFP-retroviruses with titers of up to 4 x 10(5) infectious particles (IP)/ml. After transfer of a TCRalpha/beta-encoding retrovirus vector, Deltabeta-Jurkat/GALV and Deltabeta-Jurkat/10A1 cells expressed CD3 molecules on the cell surface. CD3-high expressing packaging cells were enriched by fluorescence-activated cell sorter sorting. In these cells, the CD3 expression level directly correlated with the titer of vector particles. TCR-retroviruses efficiently transduced human T-cell lines and primary T cells. In conclusion, the method allowed the fast and easy generation of high virus titer supernatants for TCR gene transfer.
Reuss
S
S
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
Biese
P
P
Cosset
F-L
FL
Takeuchi
Y
Y
Uckert
W
W
D019264:Adoptive Transfer / Q000379:methods*; D015153:Blotting, Western / Q000379:methods; D017252:CD3 Complex; D002999:Clone Cells; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000378:metabolism / Q000821:virology; D014161:Transduction, Genetic / Q000379:methods
D017252:CD3 Complex; D011948:Receptors, Antigen, T-Cell; D049452:Green Fluorescent Proteins