Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules

The adoptive transfer of tumor-draining lymph node (LN)T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56(lck) and p59(fyn) and proteins of the NF-kappa B family were analyzed in tumor-draining LN T cells. The levels of P56(lck) and p59(fyn) were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56(lck) and p59(fyn) and protein tyrosine kinase activity increased. Interestingly, the levels of p56(lck), p59(fyn), and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear kappa B DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-kappa B. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function.