Gene transfer of human TCR in primary murine T cells is improved by pseudo-typing with amphotropic and ecotropic envelopes.
BACKGROUND
T cell receptor (TCR) gene therapy represents an attractive anti-cancer treatment but requires further optimization of its efficacy and safety in clinically relevant models, such as those using a tumor antigen and TCR of human origin. Currently, however, there is no consensus as to what protocol is most optimal for retroviral human TCR gene transfer into primary murine T cells, most notably with respect to virus pseudo-type.


METHODS
Primary murine T cells were transduced, expanded and subsequently tested for transgene expression, proliferation and antigen-specific function. To this end, murine leukemia virus (MLV) retroviruses were produced upon transfection of various packaging cells with genes encoding either green fluorescent protein (GFP) or TCRalphabeta specific for human melanoma antigen gp100(280-288) and the helper elements GAG/POL and ENV. Next to viral pseudotyping, the following parameters were studied: T cell densities; T cell activation; the amounts of IL-2 and the source of serum used to supplement medium.


RESULTS
The pseudo-type of virus produced by packaging cells critically determines T cell transduction efficiencies. In fact, MLV-A and MLV-E pseudo-typed viruses derived from a co-culture of Phoenix-A and 293T cells resulted in T cell transduction efficiencies that were two-fold higher than those based on retroviruses expressing either VSV-G, GALV, MLV-A or MLV-E envelopes. In addition, T cell densities during transduction were inversely related to transduction efficiencies. Further optimization resulted in transduction efficiencies of over 90% for GFP, and 68% for both a murine and a human (i.e. murinized) TCR. Importantly, TCR-transduced T cells proliferate (i.e. showing a log increase in cell number in a few days) and show antigen-specific function.


CONCLUSIONS
We set up a quick and versatile method to genetically modify primary murine T cells based on transient production of TCR-positive retroviruses, and show that retroviral gene transfer of a human TCR into primary murine T cells is critically improved by viral pseudo-typing with both MLV-A and MLV-E envelopes.
Pouw
Nadine M C
NM
Laboratory of Tumor Immunology, Department of Medical Oncology, Erasmus MC-Daniel den Hoed Oncology Center, Rotterdam, The Netherlands.
Westerlaken
Elike J
EJ
Willemsen
Ralph A
RA
Debets
Reno
R
D000818:Animals; D000911:Antibodies, Monoclonal; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D003208:Concanavalin A / Q000494:pharmacology; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000378:metabolism; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000821:virology*; D014161:Transduction, Genetic / Q000379:methods*; D019065:Virus Assembly / Q000187:drug effects
D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D017252:CD3 Complex; D008562:Membrane Glycoproteins; D011948:Receptors, Antigen, T-Cell; D011991:Receptors, Virus; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; C070506:ecotropic murine leukemia virus receptor; D003208:Concanavalin A