Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette.
The transfer of T cell receptor (TCR) genes allows to endow T cells with a new antigen specificity. For clinical applications of TCR-redirected T cells, efficient functional expression of the transgenic TCR is a key prerequisite. Here, we compared the influence of the transgene cassette on the expression and function of the murine TCR P14 (recognizing a LCMV gp33 epitope) and the human TCR WT-1 (recognizing an epitope of the tumor-associated antigen WT-1). We constructed different vectors, in which TCRalpha- and beta-chain genes were either (a) linked by an internal ribosomal entry site (IRES), (b) combined by a 2A peptide, or (c) introduced into two individual retroviral constructs. While in a TCR-deficient T cell line TCR P14 was expressed equally well by all constructs, we found that IRES- but not 2A-employing TCR expression is hampered in a TCR-bearing cell line and in primary murine T cells where the transgenic TCR has to compete with endogenous TCR chains. Similarly, 2A-linked TCR WT-1 genes yielded highest expression and function as measured by tetramer binding and peptide-specific IFN-gamma secretion. Differences in expression were independent of copy number integration as shown by real-time PCR. Thus, linking TCRalpha- and beta-chain genes by a 2A peptide is superior to an IRES for TCR expression and T cell function.
Leisegang
Matthias
M
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092, Berlin, Germany.
Engels
Boris
B
Meyerhuber
Peter
P
Kieback
Elisa
E
Sommermeyer
Daniel
D
Xue
Shao-An
SA
Reuss
Simone
S
Stauss
Hans
H
Uckert
Wolfgang
W
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018628:Gene Dosage; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D012190:Retroviridae; D013601:T-Lymphocytes / Q000276:immunology*; D019076:Transgenes / Q000235:genetics*
D010455:Peptides; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell