[CANCER RESEARCH 44, 1946-1953, May 1984] Lymphokine-activated Killer Cells: Lysis of Fresh Syngeneic Natural Killer-resistant Murine Tumor Cells by Lymphocytes Cultured in Interleukin 2 Maury Rosenstein, llana Yron, Yael Kaufmann, and Steven A. Rosenberg1 Surgery Branch, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20205 ABSTRACT optimal conditions for the production of IL-2 and long-term in vitro growth of both murine and human T-lymphoid cells (17, 27- Normal splenocytes that are cultured in the lymphokine, inter- 32, 37). In human studies, the LAK cells may be induced with leukin 2 (IL-2), for as short as 2 days develop lytic activity for only a 2-day incubation of autologous lymphoid cells in the fresh syngeneic natural killer-resistant tumor cells as well as presence of IL-2. These cells expressed the phenotypic charac natural killer-sensitive YAC cells in a 4-hr 51Cr release assay. teristics of allogeneic CTL based on their susceptibility to lysis Lymphokine-activated killer (LAK) cells do not lyse syngeneic by OKT-3 and OKT-8 monoclonal antibody and complement, fresh lymphocytes but do lyse syngeneic concanavalin A-induced and lysed a variety of fresh autologous human tumor cells as lymphocyte blasts. Lysis is not due to the presence of lectin or well as fresh allogeneic tumor cells, but not normal lymphocytes xenogeneic serum and appears to be an intrinsic property of (8-10). Little information exists, however, as to the presence of lymphocytes activated in IL-2. The activation appears universal LAK cells and their characterization in the mouse (37). In this in that lymphocytes from all strains of mice activated in this paper, we report on the presence of LAK cells in a variety of manner exhibited similar patterns of lysis for fresh tumor target mouse strains, the precursor and effector phenotypes of the cells. murine LAK cells, and the specificity of lysis of these cells. The To characterize the cells responsible for this lysis, we analyzed high levels of lysis by LAK cells of syngeneic NK-resistant tumor the phenotypic expression of surface markers on these cells cells may be of value in studying interactions between lymphoid with depletion techniques using monoclonal antibody and com cells and tumors as well as for adoptive immunotherapy. plement. These studies indicate that the precursor of the LAK cell is Thy-1+ and nonadherent to plastic or nylon wool. Lysis of syngeneic tumor was inhibited when LAK cells were treated with MATERIALS AND METHODS an anti-Thy-1.2, or anti-Lyt-2.2 monoclonal antibody and comple Animals. C57BL/6, BALB/c, DBA/2, AKR, C3H, and B10.D2 mice ment but not with anti-Lyt-1.2 monoclonal antibody and comple were obtained from the Animal Production Colonies of the NIH, Bethesda, ment, indicating that the observed lytic activity was due to a Thy-1+ Lyt-1~2+ cell. Furthermore, LAK cell-mediated lysis could MD. Tumors. Two sarcomas, MCA-102 and MCA-106, were induced by be inhibited by the addition of anti-Lyt-2 or LFA-1 monoclonal the injection of 0.1 ml of 1% 3-methylcholanthrene in sesame oil into the antibody to cytotoxicity assays. Cold target inhibition analysis right hind limb of female C57BL/6 mice as described previously (24). The revealed that the syngeneic tumor cells were lysed by recognition tumors were maintained by repeated passage in syngeneic hosts and of a determinant not present on normal lymphocytes or lympho used in these experiments in the fourth to seventh transplant generation. P-815 (H-2d) was passaged in ascites form in DBA/2 syngeneic mice, cyte blasts. This lysis of fresh solid tumor cells by lymphoid cells and EL-4 (H-26) was passaged in ascites form in C57BL/6 mice. grown in IL-2 may be of value in the study of tumor-host Preparation of Tumor Cell Suspensions. Tumor cell suspensions immunological interactions. The biological significance of tumor were prepared by short trypsinization as described previously (37). In lysis by IL-2-activated cells requires further study. brief, tumors were cut into 5-cu mm fragments in HBSS, washed once with 0.25% trypsin in Dulbeco's phosphate-buffered saline without Ca2+ and Mg2+ (NIH Media Unit), and then trypsinized at room temperature INTRODUCTION for 10 min. The supernatant containing released cells was removed into The ability to generate T-cells with lytic activity for fresh an equal volume of cold RPMI Medium 1640 (Grand Island Biological autologous tumor after specific in vitro sensitization has met Co., Grand Island, NY), penicillin (100 units/ml), and streptomycin (100 with limited success in both murine and human studies. In an ¿ig/ml).This medium is referred to in this paper as RPMI medium. An attempt to reproducibly generate T-cells with lytic activity to equal volume of fresh trypsin solution was added to the trypsinization flask, and the procedure was repeated once. The pooled cell suspension fresh autologous tumor, we have been studying the nonspecific supernatants were washed 3 times with cold RPMI medium by centrifu- activation of T-lymphoid cells caused by short-term culture in IL- 2.2 gation for 7 min at 100 x g. In order to lyse erythrocytes, cell suspensions were treated with In previous reports from our laboratory, we have described buffered ammonium chloride lysing solution. Preparation of Splenocyte Suspensions. Spleens were removed 1To whom requests for reprints should be addressed, at Surgery Branch, aseptically into HBSS and were passed gently through a 40 mesh screen National Cancer Institute, Building 10, Room 2B42, Bethesda, MD 20205. and through a single layer of 100 mesh nylon. The cells were centrifuged 2 The abbreviations used are: IL-2, interteukin 2; LAK, lymphokine-activated at 100 x g for 7 min, and the pellet was resuspended in a buffered killer; CTL, cytotoxic T-lymphocytes; Con A, concanavalin A; CM, complete me dium; HBSS, Hank's balanced salt solution; NK, natural killer; MCA, methylchol- ammonium chloride solution for 1 min at room temperature to lyse the anthrene-induced sarcoma. RBC. After 1 min, the suspension was diluted in RPMI medium and Received September 22, 1983; accepted December 21,1983. washed 3 times. For the growth of T-cells, the final cell pellet was 1946 CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. Tumor Lysis by Cells in IL-2 den, CT) for 30 min at 37°; 100 n\ of the 51Cr-labeled MCA-102 tumor suspended in complete RPMI Medium 1640, (Grand Island Biological Co.) supplemented with 0.03% fresh glutamine (NIH Media Unit), 1 UM target cells were then added to the wells. The plates were incubated for an additional 4 hr prior to harvest as described for the 51Cr release assay. sodium pyruvate (Microbiological Associates, Walkersville, MD), 0.1 ITIM nonessential amino acids (Microbiological Associates), 5 x 105 M 2- Role of Lyt-2 and LFA-1 in LAK Effector Cell Lysis. The effect of mercaptoethanol, penicillin (100 units/ml), streptomycin (100 /¿g/ml),and anti-Lyt-2 and anti-LFA-1 monoclonal antibodies on cytotoxicity of CTL 10% heat-inactivated fetal calf serum (Grand Island Biological Co.). and LAK was studied by incubating 5x10* effector cells in microtiter Preparation of Con A-stimulated Blast Cells (Con A Blasts). Spie- plates with various dilutions of the following monoclonal antibodies: (a) nocytes (4 x 106) were incubated for 48 hr in 2 ml of CM containing 10 anti-Lyt-2 53.6.7 (15), concentrated 10 times; and (b) anti-LFA-1 H35- ^g of Con A in the wells of Costar 24-well plates (Catalogue No. 3524; 89.9 (25) ammonium sulfate precipitate from ascites, kindly supplied by Dr. P. Golstein and Dr. M. Pierres, Centre d'Immunologie, France. After (Costar, Cambridge, MA). 30-min incubation at 37°in 150 n\ of medium, 5 x 103 51Cr-labeled target In Vitro Sensitization. Allogeneic in vitro sensitizations were per formed using conventional techniques (27, 29, 30). In brief, spleen cell cells were added in 50 p\. Plates were spun for 5 min at 500 rpm and suspensions were prepared as described above. Stimulator cells re incubated for 5 hr at 37°.Assays were harvested as described above. ceived 2000 rads in a 7-irradiator. Responder cells (4 x 106) and 106 Adherent Cell Depletion. Splenocytes were incubated at 2 x 10'' irradiated stimulator cells were mixed and added to each well of a Costar cells/ml in 45 ml of CM in supine 650-ml tissue culture flask (Costar) at 24-well tissue culture plate in 2 ml of CM. The cells were then incubated 37°for 1 hr. Nonadherent cells were removed and incubated for 1 hr at for 4 days at 37°, 5% CO2. In experiments where large numbers of 37° in a 50-ml centrifuge tube containing 0.8 g nylon wool, and the alloreactive cells were required for antibody and complement depletion, nonadherent cells were harvested and resuspended in CM. 75 x 106 responder cells were admixed with 15 x 106 irradiated stimu lator cells (2000 R) in 90 ml of CM in 250-ml upright culture flasks (Falcon RESULTS Plastics, Oxnard, CA). The cells were incubated at the culture conditions described previously. Cytotoxic Activity and Specificity of LAK. Normal fresh Preparation of IL-2. IL-2, free of lectin, was prepared as described previously (31). In brief, 2 x 109 BALB/c splenocytes were incubated in C57BL/6 lymphocytes or lymphocytes cultured for 5 days in CM 50 ml of CM containing Con A (10 ^g/ml) at 37°,5% CO2 for 2 hr.