Investigation of alpha nascent polypeptide-associated complex functions in a human CD8(+) T cell ex vivo expansion model using antisense oligonucleotides

In order to determine molecules involved in the differentiation and proliferation of human CD8(+) cells, two ex vivo expansion models were established: coculture of freshly purified human CD8(+) cells with irradiated autologous feeders (AF) or stimulation with anti-CD3. Two different proliferation kinetics of CD8(+) cells and expression patterns of CD57 were observed between these conditions. Differential display reverse transcriptase–polymerase chain reaction was applied to investigate the differential expression of mRNA species between CD8(+) CD57(+) and CD8(+) CD57(–) populations. A differentially expressed RNA species called alpha nascent polypeptide associated complex (α NAC) was found at a higher level in CD8(+) CD57(–) cells than in CD8(+) CD57(+) cells. In the presence of AF, the expression of α NAC was reduced on culturing whilst proliferation increased. Similarly, in cultures stimulated with anti-CD3, α NAC reverted to its inactive form and differentiation and proliferation increased. Using a phosphorothioate-modified oligodeoxynucleotide antisense directed specifically against α NAC mRNA, protein expression was inhibited and increased CD8(+) cell proliferation and CD25 expression were observed irrespective of the culture conditions. This suggests that α NAC protein is antiproliferative molecule. This is the first description of the function of the α NAC protein in human CD8(+) T cells

Cytomegalovirus (CMV) infection in AIDS patients is associated with a CD3 receptor-mediated T cell hyporesponsiveness

HIV+ individuals with human CMV (HCMV) reactivation have a CD3 receptor-mediated T cell hyporesponsiveness when compared with CD4-matched HIV+ and HCMV−control groups. The impairment of proliferation was not reversed by exogenous IL-2. A typical increase in NFκB expression was observed following cross-linking of the CD3 receptor, but did not lead to increased CD25 cell surface expression or cell proliferation. The HCMV-induced non-responsiveness was not observed when cells were stimulated with phorbol esters. Lymphocytes cultured with media collected from cell cultures infected with HCMV showed a dose-dependent inhibition in the total T cell population even though cells staining dually for CD8/57 increased in number. The altered growth factor requirements of CD8/57+ cells may therefore account for their presence in AIDS and patients following bone marrow transplantation

Optimization of functional efficacy of phosphorothioate-modified oligonucleotides in a human CD8+ T-Cell Ex Vivo expansion model

Antisense oligodeoxyribonucleotides (ODNs) can specifically inhibit gene expression, but their application to fresh human CD8+ T cells is limited by poor spontaneous uptake (<2%). We have examined and optimized the uptake of phosphorothioate-modified oligodeoxyribonucleotides (PS-ODNs) into these cells in an ex vivo expansion model. Optimal antisense treatments were found to be, for fresh CD8+ T cells, 1 µm PS-ODNs complexed with lipofectin (LF), which resulted in 35% uptake and 10 µm PS-ODNs in the absence of LF, for cultured cells, which resulted in 95% uptake. The delivered antisenses were functional, as determined by the inhibition of protein expression. In this respect, partially phosphorothioate-modified ODNs (PS-ODNs-P) were twice as effective as completely modified (PS-ODNs-C), and the antisense specific for the cap site showed the highest protein suppression of those tested (68%). Uptake mechanisms were also investigated. To our knowledge, this is the first optimization of the delivery of antisense oligonucleotides into human CD8+ T cells. This protocol could be used to study the function of a particular gene in cytotoxic T lymphocytes and also by those looking for a method to deliver short interfering RNA into cell lines to specifically suppress a gene of interest