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 Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced 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. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in 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 showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.
Pollok
K E
KE
Section of Pediatric Hematology/Oncology, Herman B Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis 46202, USA.
van der Loo
J C
JC
Cooper
R J
RJ
Kennedy
L
L
Williams
D A
DA
D000906:Antibodies / Q000276:immunology; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D008979:Moloney murine leukemia virus / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D018106:CD28 Antigens; D007376:Interleukin-2; D012333:RNA, Messenger; D017260:Receptor-CD3 Complex, Antigen, T-Cell