title: Construction of retroviral vectors carrying human CD3 gamma cDNA and reconstitution of CD3 gamma expression and T cell receptor surface expression and function in a CD3 gamma-deficient mutant T cell line.
abstract: CD3 gamma, a subunit of the T cell receptor-CD3 (TCR/CD3) complex, helps to support surface TCR/CD3 expression and participates in signal transduction for gene induction after antigen recognition by T lymphocytes, and in TCR/CD3 down-modulation. Humans with primary immunodeficiencies caused by inherited mutations in the CD3 gamma gene or in the gene encoding epsilon CD3é, another subunit of TCR/CD3 complex, have been previously reported. To develop a gene therapy protocol for CD3-deficient patients, CD3 gamma cDNA was orientationally inserted into two retroviral vectors (LNCX and LXSN), which resulted in recombinant vectors LNCG and LGSN, respectively. Two vector producer cell lines Am12/LNCG and Am12/LGSN were established from packaging cells GP+envAm12. Their mean viral titers were 6.5 x 10(6) and 2.0 x 10(7) cfu/ml, respectively, as shown by an improved retroviral vector production and transduction method that increases titers around five-fold over conventional methods. The presence of helper virus in vector stocks was tested by marker rescue assay and found to be < 1 cfu/ml. Southern blot analysis showed that multiple copies of the vectors were present in the genome of high-titer producers and that both vectors could transfer CD3 gamma cDNA into the genome of 3T3 cells. The vectors were used to correct in vitro a CD3 gamma-deficient Jurkat mutant cell line lacking TCR/CD3 expression and termed JGN (for Jurkat gamma negative). Both vectors increased TCR/CD3 expression in JGN (normally 2% using WT31 monoclonal antibody) to 34% and 37%, respectively, in G418-selected 3-week bulk cultures. Two clones from transduced JGN cells termed JGN/LNCG13 and JGN/LNCG15, with high TCR/CD3 expression (88% and 79%, respectively), were selected for further analyses. First, CD3 gamma protein reconstitution was demonstrated by immunoprecipitation. Second, interleukin-2 production after TCR/CD3 engagement and TCR/CD3 down-modulation in response to phorbol myristate acetate were shown to be comparable to wild-type Jurkat cells. We conclude that LNCG and LGSN may be useful for gene therapy purposes.
authors.affiliation: Immunología, Facultad de Medicina, Universidad Complutense, Madrid, Spain.
authors.forename: J Y
authors.initials: JY
authors.lastname: Sun
authors.forename: A
authors.initials: A
authors.lastname: Pacheco-Castro
authors.forename: A
authors.initials: A
authors.lastname: Borroto
authors.forename: B
authors.initials: B
authors.lastname: Alarcon
authors.forename: D
authors.initials: D
authors.lastname: Alvarez-Zapata
authors.forename: J R
authors.initials: JR
authors.lastname: Regueiro
mesh_terms: D018076:DNA, Complementary; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D019169:Jurkat Cells / Q000378:metabolism / Q000821:virology*; D009154:Mutation; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011994:Recombinant Proteins