With Cytotoxicity and Lymphocyte Phenotype (Natural Killer Cell/Cytotoxic T Lymphocyte/Lymphokine-Activated Killer Cell/Mechanism of Cellular Cytotoxicity) DALE E
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Proc. Nati. Acad. Sci. USA Vol. 84, pp. 2946-2950, May 1987 Immunology Induction of synthesis of the cytolytic C9 (ninth component of complement)-related protein in human peripheral mononuclear cells by monoclonal antibody OKT3 or interleukin 2: Correlation with cytotoxicity and lymphocyte phenotype (natural killer cell/cytotoxic T lymphocyte/lymphokine-activated killer cell/mechanism of cellular cytotoxicity) DALE E. MARTIN, LEORA S. ZALMAN, GUNDRAM JUNG, AND HANS J. MULLER-EBERHARD Division of Molecular Immunology, Research Institute of Scripps Clinic, La Jolla, CA 92037 Contributed by Hans J. MuIller-Eberhard, January 15, 1987 ABSTRACT Synthesis of the cytolytic C9-related protein major histocompatibility complex (MHC)-unrestricted cyto- (C9RP) was induced by activation of resting human peripheral toxicity may be demonstrated by use of OKT3-anti-target T lymphocytes with the anti-CD3 antibody OKT3 or interleu- cell mAb conjugates. When PBMCs were depleted of CD16+ kin 2. Comparison ofcellular cytotoxicity and C9RP content at cells, representing the majority of the LGLs and NK cells, various times during activation yielded a coefficient of corre- and subsequently activated with OKT3, the resulting popu- lation r = 0.92. During OKT3 stimulation of peripheral lation consisted primarily of CD4+ and CD8+ large lympho- mononuclear cells, maximal C9RP content and cytotoxicity blasts. The cytolytic, pore-forming protein of these cells was were observed by day 2 or 3, with subsequent decline to identical with C9RP of LGLs with respect to molecular baseline values by day 5, whereas during interleukin 2 stimu- weight, reactivity with anti-C9RP, and specific cytolytic lation, both parameters reached the maximal level at days 3-5. activity toward human melanoma cells and K562 cells (8). It After fluorescence-activated cell sorting, C9RP and cytotoxic- was concluded, therefore, that both populations, the OKT3- ity were quantitated in CD41, CD81, and Leu-19' subsets. In activated CD3+ CTLs and the IL-2-stimulated LGLs, em- OKT3-activated CD81 cells, C9RP increased to =3 x 106 ployed C9RP as their pore-forming, cytolytic protein. molecules per cell, with a corresponding increase in lysis of Having established that C9RP is the cytolytic protein of human melanoma cells mediated by anti-CD3-anti-melanoma both types of human killer lymphocytes, the opportunity monoclonal antibody conjugates. Interleukin 2-stimulated arose to study the induction of C9RP synthesis upon lym- CD8+ cells showed similar increases, but cytotoxicity was phocyte activation and to correlate C9RP content with conjugate-independent. Activated CD4+ cells showed minimal expression of cellular cytotoxicity both in total PBMCs and increase in C9RP content. Leu-19+ cells, which exhibit natural in subpopulations. killer cell activity, had a high C9RP content (=2.5 X 106 molecules per cell) before stimulation. MATERIALS AND METHODS Human large granular lymphocytes (LGLs), which include Human Peripheral Blood Mononuclear Cells. PBMCs were the natural killer (NK) cell population, have been shown in isolated from heparin-treated normal blood by Ficoll-Hy- this laboratory to contain a Mr 70,000 protein that is im- paque (Pharmacia) density gradient centrifugation and were munochemically related to C9, the ninth component of cultured in RPMI 1640 medium (M. A. Bioproducts, Walk- complement (1-5). The C9-related protein (C9RP) was iso- ersville, MD) supplemented with 2 mM L-glutamine, 50 ,g of lated from the cytoplasmic granules of these cells by anti-C9 gentamicin per ml, and 10% heat-inactivated fetal bovine affinity chromatography and shown to form, in the presence serum. Certain cultures were stimulated with human recom- of Ca2 , transmembrane channels in liposomes and to lyse binant IL-2 (Amgen Biologicals, Thousand Oaks, CA) at 100 K562 erythroleukemia cells. Certain monoclonal antibodies units/ml and others with OKT3 at 100 ng/ml. The antibody (mAbs) capable of reacting with C9RP inhibited the killing of was purified from hybridomas (American Type Culture K562 cells by LGLs, indicating that the protein was directly Collection). operative in the cytotoxic mechanism of LGLs. These cells Phenotypic Analysis and Cell Sorting. PBMCs were phe- were largely Leu-7- and Leu-11 (CD16)-positive and prolif- notyped by using phycoerythrin- or fluorescein isothiocya- erated in the presence of interleukin 2 (IL-2). nate (FITC)-conjugated mAbs: OKT4 (anti-CD4) and OKT8 Identification of C9RP as the cytolytic effector protein of (anti-CD8) (Ortho Diagnostics); anti-Leu-11a (anti-CD16) human LGLs raised the question as to whether human and anti-Leu-19 (NKH-1) (Becton Dickinson); and IL-2R1 cytotoxic T lymphocytes (CTLs) also utilize this protein for (anti-IL-2 receptor) (Coulter). OKT3 (anti-CD3) was used as the execution of target membrane attack. Human peripheral a primary antibody at 10 ,g/ml for 30 min on ice, followed by CTLs are largely resting cells lacking cytotoxic activity. They FITC-conjugated goat anti-mouse F(ab')2 (Tago, Burlin- require activation, which is physiologically provided by the game, CA) at a 1:30 final dilution. One- or two-color fluo- binding of antigen to the T-cell antigen receptor. Antigen- rescence measurements were performed on a FACS IV independent activation of all CD3+ peripheral CTLs may be (Becton Dickinson) flow cytometer. Fluorochrome-conjugat- achieved with mAb to the CD3 structure ofthe receptor, such ed, isotype-matched controls were used to determine the as OKT3 (6). As shown in this laboratory, OKT3 stimulation percentage of positive cells. Cell sorting of PBMCs was ofperipheral blood mononuclear cells (PBMCs) results in the performed on a FACSTAR (Becton Dickinson) cell sorter. generation of potent killer cells within 2-3 days (7) and their Abbreviations: C9, ninth component of complement; C9RP, C9- related protein; CTL, cytotoxic T lymphocyte; FITC, fluorescein The publication costs of this article were defrayed in part by page charge isothiocyanate; IL-2, interleukin 2; LGL, large granular lymphocyte; payment. This article must therefore be hereby marked "advertisement" mAb, monoclonal antibody; NK cell, natural killer cell; PBMC, in accordance with 18 U.S.C. §1734 solely to indicate this fact. peripheral blood mononuclear cell. Downloaded by guest on September 28, 2021 2946 IImmunology: Martin et al. Proc. Natl. Acad. Sci. USA 84 (1987) 2947 Measurement of RNA and DNA Synthesis. RNA staining 1.0 r was performed as described (9). Cells were incubated with 4 AM pyronin Y (Sigma) for 45 min at 370C prior to analysis on the FACS IV. DNA synthesis was measured by 5-bromo-2'- 0.8 deoxyuridine incorporation (10). Cells were incubated with 10 ,M bromodeoxyuridine (Sigma) for 30 min at 37TC. After three washes in phosphate-buffered saline (PBS), cells were 0.6 k fixed in 70%6 ethanol for 30 min on ice, followed by DNA denaturation with 4 M HCl for 30 min. After neutralization 0 with 0.1 M Na2B407, cells were incubated with FITC- 0.4 F conjugated anti-bromodeoxyuridine mAb (Becton Dickin- son) for 30 min on ice, washed, and resuspended in PBS containing propidium iodide (5 ,g/ml; Sigma) for analysis on 0.2 F the FACS. Conjugation of Monoclonal Antibodies. mAb 9.2.27, which 5 1==5--20====21 is directed against a human melanoma-associated chondroitin 5 10 15 20 25 sulfate proteoglycan (11), was conjugated with OKT3 by use Protein, ng of N-succinimidyl 3-(2-pyridyldithio)propionate (Pierce) as described (12). FIG. 2. Standard curves, obtained with C9RP from CTLs (o) and Target Cells. M21 cells, a human melanoma cell line from LGLs (e) by ELISA, used for quantitation of C9RP in cell lysates. Specificity is shown by inhibition resulting from preincuba- originally provided by D. L. Morton (University ofCalifornia tion of anti-C9RP with an excess of isolated C9RP (broken lines). at Los Angeles), were maintained in RPMI 1640 medium supplemented as described above. Cytotoxicity Assays. M21 cells (1.5 x 106 per ml) were ELISA. Lymphocytes were washed in RPMI 1640, sus- labeled with 51Cr (200 ACi/ml; 1 Ci = 37 GBq) for 90 min at pended in 0.1 M-carbonate buffer (pH 9) containing 2 mM 370C and washed twice. Aliquots of 1.5 x 104 cells were EGTA, and disintegrated by freezing and thawing six times, added to 96-well microtiter plates and allowed to adhere and the cell lysates were obtained after centrifugation. Total overnight at 37°C. OKT3-9.2.27 mAb conjugate was added to protein was quantitated by the Bio-Rad assay (Bio-Rad), some wells at 3 ,g/ml for 30 min at 37°C. After washing, using bovine gamma-globulin as standard. Microtiter plates were were incubated at were coated with cell lysates at 4°C for 16 hr and blocked with effector cells added and the mixtures 0.2% (wt/vol) nonfat milk (Carnation) in PBS. Protein A- 37°C for 4 hr. After centrifugation, supernatants were re- purified F(ab')2 fragment of rabbit anti-human C9RP was moved for radioactivity measurement, and cytotoxicity was used at various dilutions, followed by peroxidase-conjugated calculated using a standard formula (7). Each experimental F(ab')2 fragment of goat anti-rabbit IgG (Tago) at 1:5000 point in the killing assays represents the average of duplicate dilution. 2,2'-Azinobis(3-ethylbenzthiazolinesulfonic acid) determinations. The effector/target cell ratio in all assays (Boehringer Mannheim) was used as substrate. Standard was 10:1. curves were derived from C9RP purified from human LGLs Purification of C9RP. C9RP was isolated from IL-2- or activated CTLs (5, 8). stimulated human LGLs (1, 5) and from CD16-depleted, OKT3-activated human PBMCs (8) by immunoaffinity chro- matography on a Sepharose-anti-human C9RP column. RESULTS Quantitation of C9RP in Cell Lysates. Samples of 5-15 x 106 cells were disrupted by freezing and thawing, and the C9RP content in the cell lysates was quantitated by ELISA using as standards C9RP isolated from IL-2-stimulated LGLs and from OKT3-activated CD3+ CTLs (Fig.