title: High-efficiency gene transfer into rhesus macaque primary T lymphocytes by combining 32 degrees C centrifugation and CH-296-coated plates: effect of gene transfer protocol on T cell homing receptor expression.
abstract: Although steady progress has been made in transducing human T lymphocytes by Moloney murine leukemia virus (Mo-MuLV)-based vectors, few studies have been done to define ex vivo gene transfer protocols to transduce rhesus macaque primary T lymphocytes. Given the fact that simian immunodeficiency virus (SIV) infection in rhesus macaque is a well-characterized model for human immunodeficiency virus (HIV), it is of great interest to develop an efficient protocol to transduce rhesus macaque primary T cells. In this study, we have used MuLV-10A1-pseudotyped retrovirus expressing enhanced green fluorescent protein (EGFP) to evaluate a number of ex vivo gene transfer protocols in rhesus macaque primary T lymphocytes. Our objectives in designing these protocols were (1) to test whether higher efficiency gene transfer could be obtained by combining two previously defined protocols, centrifugation at 32 degrees C and the CH-296-coated plate; and (2) to study the effect of an ex vivo gene transfer protocol on the expression of lymphocyte homing receptors L-selectin and alpha 4 beta 7 and alpha 4 beta 1 integrins. From seven independent experiments we demonstrate by flow cytometry analyses of EGFP expression that whereas centrifugation at 32 degrees C or the fibronectin fragment CH-296-coated plate protocol alone yielded 10-14% transduction efficiency, combining these two protocols resulted in 28.1-51.2% transduction efficiency. EGFP in transduced cells was expressed highly throughout the 14 days of posttransduction expansion. Our results also demonstrate that whereas the transduction procedure per se did not significantly alter the expression of lymphocyte homing receptors, anti-CD3 and anti-CD28 antibody stimulation profoundly reduced the expression of L-selectin. The selective reduction of L-selectin may result in significant in vivo consequences if transduced cells are infused.
authors.affiliation: Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, TX 78227-5301, USA. pzhou@icarus.sfbr.org
authors.forename: P
authors.initials: P
authors.lastname: Zhou
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: P
authors.initials: P
authors.lastname: Moore
authors.forename: K M
authors.initials: KM
authors.lastname: Brasky
mesh_terms: D000818:Animals; D018106:CD28 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism; D016827:CD8 Antigens / Q000378:metabolism; D002460:Cell Line; D002469:Cell Separation; D002478:Cells, Cultured; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000378:metabolism; D019169:Jurkat Cells; D019041:L-Selectin / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D008253:Macaca mulatta; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D013601:T-Lymphocytes / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014161:Transduction, Genetic
chemical_list: D018106:CD28 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D003470:Culture Media; D005353:Fibronectins; D016023:Integrins; D008164:Luminescent Proteins; D011948:Receptors, Antigen, T-Cell; D019041:L-Selectin; D049452:Green Fluorescent Proteins