Construction of Retroviral Vectors Carrying Human CD3 γ cDNA and Reconstitution of CD3 γ Expression and T Cell Receptor Surface Expression and Function in a CD3 γ -Deficient Mutant T Cell Line

Abstract

CD3γ, 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 CD3γ gene or in the gene encoding CD3ϵ, another subunit of TCR/CD3 complex, have been previously reported. To develop a gene therapy protocol for CD3-deficient patients, CD3γ 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 × 10⁶ and 2.0 × 10⁷ 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 CD3γ cDNA into the genome of 3T3 cells. The vectors were used to correct in vitro a CD3γ-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, CD3γ 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.

Overview summary

Humans lacking the CD3γ or CD3ϵ chains of the T lymphocyte antigen receptor (TCR/CD3 complex) show impaired TCR/CD3 expression and defective immune responses that may be lethal. Because T lymphocytes are produced throughout life, it is possible that transducing the missing chains to mature T cells or to their progenitors reconstitutes TCR expression and restores immune function in vivo. Thus, reconstituted T cells should be easy to detect by flow cytometry. We have developed retroviral vectors containing CD3γ cDNA and shown that they can indeed reconstitute CD3γ and correct TCR/CD3 expression and function (interleukin-2 induction, phorbol ester-induced modulation) in a Jurkat mutant T lymphoblastoid cell lacking CD3γ. This is a first step toward gene therapy for CD3γ deficiency.