TNF-alpha secretion and apoptosis of lymphocytes mediated by gene transfer

Efficient gene transfer of lymphocytes is extremely difficult. Apoptosis ma y play a role in this gene transfer resistance of lymphocytes. Here we show that transfection of lymphocytes via non-viral vectors leads to induction of apoptosis in a significant proportion of cells. Since apoptosis may be m ediated via tumor necrosis factor d (TNF-alpha) and the TNF-alpha receptor pathway, we studied the amount of TNF-alpha secreted by lymphocytes transfe cted without gene insert. TNF-alpha secretion was dependent on the gene tra nsfer method used. High amounts were detected using receptor-mediated gene transfer and lipofection. In contrast, only low amounts of TNF-alpha were d etected after electroporation and retroviral gene transfer. In receptor-med iated gene transfer, TNF-alpha secretion was due to the use of anti-CD3 ant ibody. Transfection of lymphocytes led to selective decrease in CD120b/TNF alpha receptor II (TNFR-2)-positive cells. Induction of apoptosis and necro sis mediated by TNF-alpha via TNFR-beta (p80) was partially blocked using a neutralizing anti-TNF-alpha antibody. Blockage of apoptosis and necrosis c ould be further increased by adding anti-Fas-ligand (FasL) antibody, sugges ting that induction of apoptosis via Fast and Fas receptor (Apo-1/CD95) may also play a role. This blockage led to a significant increase in the proli feration rate of lymphocytes transfected with cytokine genes. In conclusion , various gene transfer techniques led to TNF-alpha secretion, apoptosis an d necrosis of lymphocytes. Apoptosis and necrosis could be partially blocke d using a neutralizing anti-TNF-alpha antibody.