A retroviral vector that directs simultaneous expression of alpha and beta T cell receptor genes.
The transfer of alpha/beta T cell receptor (TCR) genes into T lymphocytes or their precursors could provide a means to increase frequency of tumor- or pathogen-specific cytotoxic T lymphocytes. To begin to address this possibility, we have used class I MHC-restricted alpha/beta TCR cDNAs to develop a retroviral TCR expression vector. Alpha- and beta-chain cDNAs were inserted into a derivative of the LN series of retroviral vectors, with the retroviral LTR directing expression of TCR-beta and an internal cytomegalovirus promoter/enhancer driving TCR-alpha expression. The variable region fragments can be replaced using unique restriction sites that have been introduced into the proximal constant regions. We have used this vector system to transfer two different pairs of alpha/beta TCR genes into an alpha- and beta-chain-deficient T cell hybridoma. TCR- hybridoma cells were transduced by coculture with pools of virus-producing cells, and fluorescence-activated cell sorting was used to enrich for cells expressing the transduced TCR. Transduction with either alpha/beta TCR restores stable, long-lived expression of the alpha/beta TCR complex. TCR-mediated signal transduction is also reconstituted, as demonstrated by the ability of transduced cells to secrete IL-2 following stimulation with Vbeta-specific antibodies. Our results suggest that alpha/beta T cell receptor gene transfer could provide a basis for new approaches to immunotherapy, and that further studies examining the in vivo fate of transduced TCR are possible.
Pogulis
R J
RJ
Department of Immunology, Mayo Foundation for Education and Research, Rochester, MN 55905, USA.
Pease
L R
LR
D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D019673:Genes, T-Cell Receptor alpha; D019674:Genes, T-Cell Receptor beta; D005822:Genetic Vectors; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007376:Interleukin-2 / Q000378:metabolism; D008213:Lymphocyte Activation; D011401:Promoter Regions, Genetic; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis* / Q000276:immunology; D012190:Retroviridae / Q000235:genetics*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology*; D020079:Terminal Repeat Sequences / Q000235:genetics
D018076:DNA, Complementary; D007376:Interleukin-2; D016693:Receptors, Antigen, T-Cell, alpha-beta