RESUMEN
When peripheral blood lymphocytes (PBL) are incubated with interleukin 2 (IL 2), a novel cytotoxic lymphocyte subpopulation, termed lymphokine activated killers (LAK), arises. LAK are functionally defined as IL 2 responsive cells demonstrating major histocompatibility antigen-unrestricted cell-mediated cytotoxicity against fresh solid tumors and other natural killer cell-resistant and -sensitive tumor targets in the absence of prior antigen priming. Flow cytometric analysis of IL 2 activated PBL using forward and right angle light scatter and fluorescence intensity identified the emergence of a large, optically dense, autofluorescent cell population which paralleled the generation of LAK activity. These unique IL 2 induced lymphocytes have been named giant autofluorescent lymphocytes (GAL). These cells are readily distinguished from the small nonfluorescent lymphocytes (SNL) observed in fresh PBL, unstimulated cultured PBL, and those cells remaining after incubation with IL 2 which have not acquired GAL characteristics. In this investigation, LAK cultures were sorted on days 4, 5, and 6 into GAL and SNL populations and were tested for oncolytic activity against the natural killer-resistant Daudi and RC-1 tumor targets. Against these targets, lymphocytes from non-IL 2 activated PBL or the sorted SNL population expressed less than 2% of the oncolytic activity (measured in lytic units) exhibited by GAL effectors. The SNL and GAL populations were cultured in IL 2 for up to 48 h following the sorting procedure and then reassayed for tumor cytolytic activity. During this culture period, GAL but not SNL continued to express LAK killing against natural killer-resistant tumor targets. Using gamma-irradiation to prevent further cell cycling, it was shown that the functional half-life of the LAK effector was approximately 8.5 h. Therefore, the cytotoxicity expressed by the sorted GAL population after 48 h in culture (equivalent to five functional half-lives) must be expressed by progeny of the originally plated lymphocytes. These results indicate that in addition to the LAK effector, the GAL population contains a self-sustaining, recycling intermediate responsible for generating new LAK. Our data indicate that analysis of IL 2 activated PBL using GAL light-scattering properties has application in phenotyping LAK, monitoring of cellular kinetics, cell sorting, and enrichment of the LAK effector population, and in the clinical monitoring of IL 2 therapy.
Asunto(s)
Interleucina-2/farmacología , Células Asesinas Naturales/inmunología , Ciclo Celular , Citotoxicidad Inmunológica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Células Asesinas Naturales/efectos de los fármacos , Luz , Linfocitos/clasificación , Linfocitos/inmunología , Linfocinas/farmacología , Fenotipo , Dispersión de RadiaciónRESUMEN
The proliferation kinetics and population renewal of recombinant interleukin-2 (rIL-2)-induced murine lymphokine activated killers (LAK) arising from splenic precursors was studied. Extensive proliferation has been shown to accompany the de novo generation of LAK cytotoxicity. In this report, a thymidine 'hot pulse' suicide technique was employed to examine the sensitivity of LAK progenitors during various time periods following culture initiation. Hot pulse during the first 24 hr of culture resulted in a 30-35% reduction in lytic activity when assayed on day 5. Pulse periods between days 1 and 4 resulted in almost complete inhibition (90-95%) of lytic function when assayed on day 5. Proliferation of LAK progenitors was documented by limiting dilution analysis comparison of splenic precursors and functionally mature LAK cultures. These studies showed a 75- to 80-fold enrichment of LAK progenitors after 3 days culture in rIL-2. By flow cytometric cell cycle analysis, we demonstrated that the number of cells in the S/G2/M phase increased with the length of rIL-2 culture and represented approximately 40% of the cells by day 4. Finally, we used the rate of decay of lytic activity following irradiation as a factor to define the mean life span of a cytotoxic effector in the absence of cellular input. An exponential decrease to approximately 50% of controls was observed within 8-9 hr after irradiation. Taken together, these results suggest that the LAK system is highly dynamic and requires continuous cellular proliferation for its maintenance.