Lymphokine-Activated Killer Activity in Long-Term Cultures with Anti-CD3 Plus Interleukin 2: Identification and Isolation of Effector Subsets1
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(CANCER RESEARCH 49. 963-968. February 15. 1989] Lymphokine-activated Killer Activity in Long-Term Cultures with Anti-CD3 plus Interleukin 2: Identification and Isolation of Effector Subsets1 Augusto C. Ochoa,2 Diane E. Hasz, Rebecca Rezonzew, Peter M. Anderson, and Fritz H. Bach Immunobiology Research Center, University of Minnesota Hospital and Clinic, Minneapolis, Minnesota 55455 ABSTRACT short-term LAK cultures, while CD3+ cells from such cultures have low lytic activity against NK-resistant targets (8-11). Peripheral blood lymphocytes cultured in recombinant interleukin 2 We have recently reported the generation of large numbers during 3 to 5 days (short-term cultures) develop the ability to lyse natural of cells with LAK activity using long-term (14 to 21 day) killer-resistant tumor lines and fresh tumor cells, i.e., express lympho- cultures of cells stimulated with anti-CD3 (OKT3) moAb and kine-activated killer (LAR) function. Phenotypic analysis has shown these cells to be natural killer cells, i.e., CD16+ and/or Leu 19+ cells. IL2 (6). Cells stimulated with OKT3 + IL2 undergo a high CD3*,CD16~ T-cells, instead, develop very low LAK function in these increase in cell number while maintaining specific LAK activity cultures. comparable to that of cells cultured for 3 to 5 days. LAK We recently reported the development of long-term (up to 21 days) activity of cells stimulated with OKT3 + IL2 for 14 days is cultured cells with LAK activity by stimulation with OKT3 + interleukin significantly increased when incubated for the last 48 h of 2 (11.2).These culture conditions repeatedly resulted in a several hundred culture in medium containing ILl-ß,IFN-7, or -ßinaddition fold expansion in cell number. Specific LAK activity on Day 14 of culture to IL2. was comparable to that of 3-day LAK cultures and could be further Results of isolation and identification of the effectors medi enhanced by the addition of ¡nterleukin1/3, ß-,or -y-interferon. Total ating LAK activity in long-term cultures with OKT3 + IL2 LAK activity was greatly increased in OKT3 + IL2 cultures over that found in short-term cultures. are presented in this report. In contrast to LAK effector cells Isolation of effectors mediating LAK function in long-term cultures in short-term cultures (3 to 5 days), which predominantly stimulated with OKT3 + IL2 showed that both CD3+,CD16" cells and are CD3~ cells, i.e., CD16+,Leul9+, effectors in long-term cul CD16*,CD3~ cells tested on Day 14 of culture expressed equivalent levels tures include at least two additional subpopulations, of LAK activity as shown by lysis of natural killer-resistant targets, CD3+,CD4-,CD8- cells and Leu 19+,CD3~,CD16- cells. The III 60 and Daudi. Further dissection of the subpopulations developing two major T-cell subpopulations, CD3*,CD4+ and CD3+,CD8+ LAK activity demonstrated that, in addition to CD16*,CD3~ cells, CD3+, lymphocytes, develop significantly lower LAK activity. CD4-,CD8- cells and Leu 19+,CD3-,CD16- cells also developed high LAK activity in long-term cultures with OKT3 + IL2. Further, long-term culture with OKT3 + IL2 induced increases in the numbers not only MATERIALS AND METHODS of CD3*,CD4-,CD8- cells but also of CD16%CD3" and Leu 19+,CD3~,CD16~ cells. Although there is a significant increase in the Isolation and Culture of LAK Cells. PBL were isolated from heparin- number of CD3*,CD8+ cells, neither these, nor the CD3+,CD4+ cells, ized venous blood by centrifugaron over Ficoll-Hypaque. Isolated mediate LAK activity to the same extent as the populations mentioned mononuclear cells were washed 3 times with PBS (GIBCO Laborato above. ries, Grand Island, NY) and counted. Cells (1 x IO6)were cultured in 16-mm wells (Costar No. 3424; Costar, Cambridge, MA) in 2 ml of TCM consisting of RPMI 1640 supplemented with 25 mM 4-(2-hy- INTRODUCTION droxyethyl)-l-piperazineethanesulfonic acid, 2 mM L-glutamine, 100 PBL3 stimulated by alloantigens (1, 2) or cultured in either units per ml of penicillin. 100 ng/ml of streptomycin (GIBCO, Grand Island. NY), and 6% pooled heat-inactivated human serum. Highly interleukin 2-containing supernatants or medium with IL2 for purified IL2 from Escherichia coli (12, 13) (Cetus Corporation, Em 3 to 5 days (3-5) develop lytic activity against fresh tumor cells eryville, CA) was used at 1000 units/ml. Cultures were incubated at and several NK-resistant targets, such as Daudi and HL60 (6). 37°Cin a humidified atmosphere of 5% CÛ2.Cell density was deter These cells have been named LAK cells. Initial reports sug mined by counting cells every 48 h. Cells were subcultured in fresh gested that LAK precursor cells did not express the T-cell TCM plus IL2 at 0.5 x IO6cells/ml. Cultures which were treated with receptor as determined by anti-CD3 binding, but that after anti-CD3 moAb (OKT3; Ortho, Raritan, NJ) were initiated as above, i.e., 0.5 x IO6 PBL/ml plus 1000 units/ml of IL2; however, 10 ng/ml incubation in IL2, the effector population expressed CD3 (7). of anti-CD3 were present in culture during the first 48 h. At this time More recent reports have clearly demonstrated that cells ex the cells were counted and subcultured at 0.1 to 0.2 x IO6cells/ml in pressing NK markers, such as CD 16 (Leu 11) and/or Leu 19, TCM containing IL2; i.e., the anti-CD3 moAb was added only during are responsible for the great majority of the LAK activity of the initial 48 h of culture and not thereafter. Tumor Lines. Tumor lines K562, HL60, and Daudi were maintained Received 4/1/88; revised 8/1/88. 11/7/88; accepted 11/14/88. The costs of publication of this article were defrayed in part by the payment in culture in RPMI 1640 with 10% fetal bovine serum (GIBCO, Grand of page charges. This article must therefore be hereby marked advertisement in Island, NY). Cells were subcultured at 0.5 x 106/ml in fresh medium accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This is Paper 478 from the Jordan Bazelon Laboratories of the Immuno- twice a week. Cells of line HL60 are not lysed by unstimulated PBL and are therefore considered NK resistant. Although cells of some biology Research Center, Box 724, University of Minnesota, Hospital and Clinic, Harvard Street at East River Road. Minneapolis. MN 55455. This work was donors show the ability to use the Daudi cells to a low extent (generally supported in part by NIH Grants AI 17687. AI 18326. AI 19007. AI 22682. AI <10% lysis), it is usually also considered as NK resistant. 72626. and CA 47097. Cell-mediated Lympholysis. This was done as described elsewhere 2To whom requests for reprints should be addressed at: Immunobiology (14). In summary, tumor cell line targets were labeled with 250 to 750 Research Center. Box 724 UMHC, 420 Delaware St. S.E., Minneapolis. MN tiC'i of Na5'CrO4 (5000 /jCi/ml; New England Nuclear, Boston, MA) 55455. 3The abbreviations used are: PBL, peripheral blood lymphocytes; IL2, inter for 1 to 1'/2 h at 37°C.Cells were washed once in TCM, resuspended leukin 2; rIL2. recombinant IL2; moAb. monoclonal antibody: LAK, lymphokine- in culture medium and held at room temperature for 60 min. Cells were activated killer; NK. natural killer; PBS, phosphate-buffered saline: TCM, tissue then washed twice, resuspended in fresh medium, counted, and ali- culture medium: FITC. fluorescein isothiocyanate: PE. phycoerythrin; ILI. inter leukin 1; 1FN. interferon; MHC, major histocompatibility complex; CTL, cyto- quoted at 500 targets/well in a 96-well V-bottomed plates (Costar) into toxic T-lymphocytes. which the effectors had been previously aliquoted at set concentrations. 963 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. LAK ACTIVITY IN T-CELLS AND NK CELLS The effectortarget cell ratios ranged from 30:1 to 0.1:1. Plates were 70 Daudi centrifuged at 65 x g for 5 min and incubated in 5% CO: at 37°Cfor 60 4 h, after which 150 p\ of medium were harvested from each well into a scintillation vial with 3 ml of scintillation fluid (Biofluor; New 50 England Nuclear, Boston, MA); radioactivity was counted in a liquid 40 scintillation counter (LKB 1216). Cytotoxicity was determined by 30 % of cytotoxicity 20 _ experimental mean cpm-spontaneous release mean cpm 10 x 100 maximal mean cpm-spontaneous mean cpm 0 10:1 3:1 0.3:1 Cell Sorting by Immunofluorescence. PBL were cultured in OKT3 + 70 HL60 IL2 as described elsewhere (6). After 12 days in culture, cells were counted and adjusted to 1 x 107/ml. As many as 60 x IO6cells were 60 incubated with FITC-conjugated or PE-conjugated moAbs [OKT3, 50 OKT8, OKT4 (Ortho Diagnostics, Raritan, NJ) or Leu 2 (CDS), Leu 40 3 (CD4), Leu 4 (CD3), Leu 5b (CD2), Leu 11 (CD 16), Leu 19 (NKH1) (Becton Dickinson, Mt. View, CA)] for 30 min at 4'C. Cells were then o 30 washed twice with PBS containing 2% fetal calf serum. Cells were * 20 sorted on a FACS IV (Becton Dickinson, Mt. View, CA). Sorted cells were centrifuged, and an aliquot was restained to test the purity of the 10 populations. All of the sorted subpopulations used for determining 0 LAK activity were more than 97% positive for the desired cluster 10:1 3:1 1:1 0.3:1 determinant (see "Results"). Some experiments included three-color analysis using biotin-conjugated Leu 4 (CD3).