SEQUENTIAL TNF AND TGF-BETA REGULATION OF EXPANSION AND INDUCTION OF CYTOTOXICITY IN LONG-TERM CULTURES OF LYMPHOKINE-ACTIVATED KILLER-CELLS

Therapeutic use of lymphokine-activated killer (LAK) cells frequently requires higher numbers of effector cells than can be produced in shor t-term cultures. Extended stimulation of these cells by interleukin-2 (IL-2) past 2 weeks leads to a decrease in cytolytic activity and cell proliferation. To determine how known regulatory cytokines are involv ed in the mechanism responsible for loss of IL-2 responsiveness, adher ent LAK (A-LAK), nonadherent LAK (NA-LAK), monocyte-depleted (MD-LAK), and unmanipulated LAK (UN-LAK) cells derived from human peripheral bl ood were stimulated with high-dose IL-2 for 4 weeks, and cytokine prod uction was measured serially. Despite continued supplementation with I L-2, cell number plaeaued at 2 weeks with a 2.5-3.0 log increase in A- LAK cultures and a 1.0 log increase in NA-LAK, MD-LAK, and UN-LAK cult ures. Cytolytic activity had decreased significantly in all four cultu re systems after only 14 days of stimulation with IL-2 as assessed by the chromium release assay using K562, Daudi, and RP-mel tumor cell li nes as targets, and LAK activity was barely detectable after 28 days o f stimulation. Bioactive tumor necrosis factor (TNF) was present in co ncentrations of 15-55 U/ml during the first week of culture and at les s than 10 U/ml thereafter. Bioactive transforming growth factor-beta ( TGF-beta) was detected at 1-36 U/ml from 5 to 14 days of culture and d ecreased thereafter. Immunoreactive TGF-beta(2) was still present at c oncentrations of 20-90 pg/ml after 21 days of culture. Cytokine profil es were similar in all four culture systems, although TNF levels were higher and TGF levels lower in the A-LAK cultures. Supplementation of UN-LAK cultures with TNF failed to reverse the decrease in cell prolif eration and cytolytic activity in the long-term system, but IL-2 respo nsiveness was partially restored when cultures were supplemented with a neutralizing anti-TGF antibody whether or not TNF and lymphotoxin (L T) were also added. These results indicate that production of TGF, a n egative regulatory cytokine, is mainly responsible for the loss of IL- 2 responsiveness in the long-term A-LAK, NA-LAK, MD-LAK, and UN-LAK cu ltures, although the inability of TGF-neutralizing antibody to complet ely reverse the inhibition suggests that other negative regulatory cyt okines not investigated here may also have a role.