Costimulation of transduced T lymphocytes via T cell receptor-CD3 complex and CD28 leads to increased transcription of integrated retrovirus

Primary human T lymphocytes were transduced at high efficiency with the Mol oney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an interna l cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with solu ble antibodies to CD3 or CD28, transgene expression was significantly incre ased after activation on immobilized anti-CD3 antibodies (CD3i) or by simul taneous activation on immobilized anti-CDS and anti-CD28 antibodies (CD3i/C D28i). A similar pattern of transgene expression was observed in T cells tr ansduced with Mo-MuLV LNC-EGFP. Proviral copy number was maintained in LNC- mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Sub stantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocy tes, compared with those maintained in IL-2, correlated with increased tran scription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced wi th the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady s tate ZIPPGK-mADA RNA on reactivation. High levels of transgene expression w ere evident irrespective of cell cycle position in both CD4(+) and CD8(+) l ymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed i n the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i sho wed a dose-dependent decrease in transgene expression when incubated with s elective protein kinase inhibitors. These data provide new insights into th e mechanisms governing transgene expression driven by Mo-MuLV constructs co ntaining internal promoters in transduced primary T lymphocytes.