Signaling through a CD3 gamma-deficient TCR/CD3 complex in immortalized mature CD4+ and CD8+ T lymphocytes.

The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD4+ and CD8+ T lymphocytes from a human selective CD3 gamma deficiency using Herpesvirus saimiri. The accessibility of the mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD8+ cells when compared with CD4+ cells, relative to their corresponding CD3 gamma-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-alpha) and late (proliferation and secretion of TNF-alpha), were normal in gamma-deficient cells, despite the fact that their TCR/CD3 complexes were significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 triggering was severely impaired in gamma-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in gamma-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when gamma is absent. They also provide evidence for the specific participation of the CD3 gamma chain in the induction of certain cytokine genes in both CD4+ and CD8+ human mature T cells. These immortalized mutant cells may prove to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex.

Phenotypical and functional characterization of Herpesvirus saimiri-immortalized human major histocompatibility complex class II-deficient T lymphocytes.

CD8+ T lymphocytes from two unrelated cases of MHC class II deficiency were immortalized in vitro using Herpesvirus saimiri. In both cases, a lack of expression of surface MHC class II molecules was ascertained, whereas variable defects were shown for MHC class I, CD74 (invariant chain) and LAG-3 (an MHC class II ligand). The functional analysis of both H. saimiri-immortalized T-cell lines revealed the existence of a proliferation impairment in response to anti-CD3 but not to other surface or transmembrane stimuli. Further characterization of this functional defect indicated that it was not associated with impaired early activation events (like calcium flux) but, rather, with certain late events, like the induction of IL-2. H. saimiri-immortalized T cells may be valuable in studying the biological role of MHC class II molecules in activated human T cells.

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.

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.