Efficient gene transfer of lymphocytes has been shown to be extremely difficult. The molecular background for this gene transfer resistance is not completely understood. We reasoned that apoptosis may play a role in this gene transfer resistance of lymphocytes. We show that transfection of lymphocytes via nonviral vectors leads to induction of apoptosis in a significant proportion of cells. Since apoptosis may be mediated via the TNF alpha and TNF alpha receptor pathway, we studied the amount of TNF secreted by transfected lymphocytes. The percentage of apoptotic lymphocytes correlated well with TNF alpha secretion. TNF secretion was dependent on the gene transfection method used. High amounts of TNF secretion were detected using receptor-mediated gene transfer and lipofection. In contrast, only low amounts of TNF were detected after electroporation and retroviral gene transfer. In receptor-mediated gene transfer, TNF secretion was due to the use of anti-CD3 antibody. Induction of apoptosis and increase in necrosis was blocked using an anti-TNF antibody. This blockage led to a significant increase in the proliferation rate of lymphocytes transfected with the interleukin-2 or interleukin-7 gene. In conclusion, gene transfer techniques led to TNF secretion, apoptosis and necrosis of lymphocytes. This could be blocked using an anti-TNF antibody. Blockage of apoptosis after gene transfer should have an impact on the use of lymphocytes transfected with cytokine genes as immunologic effector cells in cancer gene therapy protocols.