T cell receptor zeta reconstitution fails to restore responses of T cells rendered hyporesponsive by tumor necrosis factor alpha.

Expression and function of the antigen T cell receptor (TCR) play a central role in regulating immune responsiveness. Accordingly, targeting the expression of TCRalphabeta or its associated CD3 subunits profoundly influences T cell development and adaptive immunity. Down-regulation of the invariant TCRzeta chain has been documented in a wide variety of chronic inflammatory and infectious diseases, and is thought to contribute to the paradoxical immune suppression observed in these diseases. Previously, we reported that prolonged exposure of T cell hybridoma clones to tumor necrosis factor alpha (TNF) induces nondeletional and reversible hyporesponsiveness to TCR engagement, associated with down-regulation of TCRzeta chain expression, impaired TCR/CD3 complex assembly, and attenuation of TCR-induced membrane proximal tyrosine phosphorylation. Here, we have tested whether receptor specific T cell responses are rescued in TNF-treated T cell hybridomas by retroviral-mediated expression of zeta-chimeric (C2zeta) receptors or wild-type TCRzeta. Expression of C2zeta receptors at the cell surface is relatively refractory to chronic TNF stimulation. However, C2zeta receptor function depends on association with endogenous TCRzeta chains, whose expression is down-regulated by TNF, and so C2 receptor specific responses are attenuated in TNF-treated T cells. Unexpectedly, overexpression of wild-type TCRzeta maintains cell surface TCR/CD3 complex expression but fails to rescue receptor proximal signaling in TNF-treated T cells, suggesting the existence of hitherto unrecognized mechanisms through which TNF regulates T cell responsiveness. We provide additional evidence that TNF also uncouples distal TCR signaling pathways independently of its effects on TCRzeta expression.

Clustering of immunoreceptor tyrosine-based activation motif-containing signalling subunits in CD4(+) T cells is an optimal signal for IFN-gamma production, but not for the production of IL-4.

CD4(+) T cells with pre-defined MHC-unrestricted specificity to type II collagen (CII) were engineered for cell-based anti-inflammatory gene therapy of autoimmune arthritis. To this end, recombinant chimeric immunoreceptors, C2gamma or C2zeta, were expressed in primary mouse keyhole limpet hemocyanin (KLH)-specific T(h)1 and T(h)2 cells using retrovirus vector-based somatic cell gene transfer. The ectodomain of these tyrosine-based activation motif (ITAM)-containing immunoreceptors is a single-chain IgG variable domain of an anti-CII mAb. The engineered cells might arrest migration when they encounter CII in articular cartilage. Up to 19 and 55% of transduced CD4(+) T cells expressed respectively C2gamma and C2zeta. The expression of C2gamma or C2zeta on the surface of CD4(+) T cells was down-regulated upon binding CII, and cells activated in such a way proliferated, up-regulated CD25 expression and produced cytokines. Comparison of cytokine levels normalized by the number of producer cells revealed that C2gamma and C2zeta were as potent as TCR in the induction of IFN-gamma, but induced lower levels of IL-4. It appears that the reason why CD4(+) T cells stimulated through C2gamma and C2zeta produce low levels of IL-4 is a lack of integration between co-stimulatory signals required for the optimal production of this cytokine and the ITAM-dependent signals generated by the immunoreceptors. The significance of these data for the development of anti-inflammatory gene therapy based on CD4(+) T cells targeted to a tissue-specific protein is discussed.

Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein.

BACKGROUND: Introduction of recombinant genes in the genome of primary lymphocytes by virtue of a replication-deficient retrovirus can be used in immunological studies and for cell-based gene therapy. METHODS: Packaging cells GP+E86 producing replication-deficient retrovirus incorporating the genes of enhanced green fluorescent protein (eGFP), C2gamma or C2xi, were generated by calcium phosphate-mediated transfection. Clones with the highest titres of retrovirus vector were isolated from them and their supernatants were used for transduction of PT67 cells. Primary mouse lymphocytes and T-cell hybridoma MD.45 were transduced by centrifugation with retroviral stock. The retroviral content of packaging cell supernatants was determined by dot blotting and hybridization with a DNA probe. RESULTS: PT67 cells produced approximately 50 times more retrovirus vector than the original GP+E86 clones. When retroviral stocks of PT67 and GP+E86 cells were used at 1/50 dilution and undiluted, respectively (to normalize them for retroviral RNA content), the transduction efficiency of mouse T-cell hybridoma was 40% and 5%, respectively. Centrifugation of target cells with retroviral stock at 2000 g for 60 min increased the percentage of transduced cells two- to three-fold. Within a population of cells isolated from the draining lymph nodes of an immunized mouse and reactivated with an antigen, up to 60% of CD4(+) T cells and up to 80% of B cells could be transduced with a transgene in replication-deficient retrovirus packaged by PT67 cells using the optimized gene transfer protocol. CONCLUSIONS: This protocol allows for the generation of packaging cells producing high titres of retrovirus vector. The 10A1 envelope protein is superior to the ecotropic one for the transduction of mouse lymphocytes.