Human Natural Killer Cells, Activated Lymphocyte Killer Cells, And

Proc. Nati. Acad. Sci. USA Vol. 80, pp. 7606-7610, December 1983 Immunology

Human natural killer cells, activated lymphocyte killer cells, and monocytes possess similar cytotoxic mechanisms (monoclonal antibody/cytotoxic factor/inhibition of killing) GORDON F. BURNS, TONY TRIGLIA, PERRY F. BARTLETT, AND IAN R. MACKAY The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia Communicated by F. M. Burnet, August 19, 1983 ABSTRACT The relationship between the killing mechanisms killing and then tested for its effect on the cytolytic activity of of human natural killer (NK) cells, mitogen- and mixed-lympho- ALK cells and, for comparison, MHC-restricted cytotoxic T cells cyte-culture-induced activated lymphocyte killer (ALK) cells, and and cultured monocytes. The results suggest that killing by ALK monocytes was investigated with a monoclonal antibody. The IgG2 cells, NK cells, and monocytes depends on similar mecha- antibody 9.1C3 was prepared from mice immunized with purified nisms. human large granular lymphocytes and selected from clones that inhibited NK cell killing. The 9.1C3 antibody bound to all mono- MATERIALS AND METHODS nuclear cells but not to granulocytes or K562 cells, and it selec- tively blocked killing of K562 targets by both NK and ALK cells Preparation of Lymphocytes and Large Granular Lympho- without affecting the binding of effector to target cells. The an- cytes (LGL). Peripheral blood mononuclear cells (MNC) were tibody blocked killing when present from time zero and-it still in- obtained from the blood of normal subjects by Ficoll/Hypaque hibited partially even when added 1 hr after initiation of the lytic centrifugation. Monocytes were removed from the MNC by reaction. Killing of Epstein-Barr virus-transformed B lympho- plastic adherence, and enrichment for LGL was carried out by blasts by classical cytotoxic T lymphocytes was not inhibited. Of centrifugation over discontinuous Percoll gradients by a mod- interest, 9.1C3 did block the killing of K562 target cells by cul- ification of a standard method (12). tured peripheral blood monocytes. Other monoclonal antibodies Preparation of Monoclonal Antibodies. BALB/c mice (main- that bound to monocytes did not block killing, and a nonspecific tained at The Walter and Eliza Hall Institute) were immunized effect of the antibody on monocytes was excluded. These data sug- by three intraperitoneal injections of 107 LGL over a period of gest that NK cells, ALK cells, and monocytes can kill tumor cell 30 days and a further intravenous boost 1 week later. After 3 targets by using similar lytic mechanisms. days spleen cells were fused with NS-1 cells by using 50% (vol/ vol) polyethylene glycol 4000 and hybrids were selected with The concept of immune surveillance over the emergence of hypoxanthine, aminopterin, and thymidine (HAT). The hybri- cancer has stimulated much investigation into the possible un- doma supernatants were tested for blocking antibodies by re- derlying mechanisms (1, 2). Since the realization that natural duction in 51Cr release in cytotoxic assays (see below). The cells killer (NK) cells may play an important role in combating virus secreting supernatant showing significant blocking of K562 lysis infections and tumor cell growth there has been considerable by NK cells were cloned at 1 cell per well, then recloned twice interest in these cells and, as a result, several mechanisms of at 0.3 cell per well on a feeder layer of thymocytes. Antibody NK-mediated lysis of tumor cells have been proposed (3-6). 9. 1C3 was purified from ascites fluid by affinity chromatog- Also of possible relevance in the prevention of tumor cell raphy with Sepharose-coupled staphylococcal protein A, and a growth in vivo is the activated (lymphocyte) killer (ALK) cell radioimmunometric assay employing "2I-labeled class-specific (7). Like NK cells, ALK cells can recognize and kill certain tu- sheep antibodies and control mouse monoclonal antibodies of mor cell targets without prior sensitization and without major known isotype (supplied by G. Morahan) was used to type 9.1C3 histocompatibility complex (MHC) restriction; however, at a as an IgG2 antibody. given effector-to-target ratio ALK cells are much more effective Induction of Cytotoxic T Cells (CTC) and ALK. CTC were killers and in addition they can rapidly lyse certain tumor cell established from MNC of normal subjects by stimulating 2 X targets, including autologous tumor cells, that are resistant to 106 MNC in 2 ml of RPMI 1640 medium containing 10% fetal freshly isolated NK cells (8). The nature and origin of ALK cells calf serum and 50 ,uM 2-mercaptoethanol with irradiated al- is disputed. They can be generated from blood mononuclear logeneic or autologous B lymphoblastoid cells at a MNC-to- cells in vitro by mixed cell culture, or with mitogens, and can stimulator-cell ratio of 50:1 for 10 days (13). Previous studies be maintained in culture with lymphokines, probably T-cell have demonstrated that this latter protocol results in the recall growth factor (7, 8). ALK cells express the T-cell membrane of memory CTC specific for Epstein-Barr virus (EBV)-trans- antigens T3 and T8 and can be generated in vitro from NK-de- formed lymphoblasts (13, 14). The definition of ALK cells is pleted mononuclear cells (7-9). Because of this they have been essentially operational, and the cultures that were used to in- described as activated T lymphocytes (10). However, studies on duce CTC also generated high levels of ALK cells; CTC were precursor cell depletion suggest that ALK cells are derived from identified by their killing of the stimulating B lymphoblasts and NK cells (11) or from a cell lineage distinct from both T lym- ALK cells were measured by the killing of K562 target cells. phocytes and NK cells (8). Mitogen-activated MNC were also used to generate ALK cells To investigate the mechanism of tumor cell killing by ALK in the absence of specific CTC by stimulating MNC with con- cells we prepared a monoclonal antibody that blocked NK cell Abbreviations: ALK, activated lymphocyte killer; CTC, cytotoxic T cell(s); The publication costs of this article were defrayed in part by page charge EBV, Epstein-Barr virus; LGL, large granular lymphocytes; MHC, ma- payment. This article must therefore be hereby marked "advertise- jor histocompatibility complex; MNC, mononuclear cells; NK, natural ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. killer. 7606 Downloaded by guest on September 28, 2021 Immunology: Bums et al. Proc. Nati. Acad. Sci. USA 80 (1983) 7607

ditioned medium containing trace amounts of phytohemagglutinin and maintaining the cells for 14 days with phytohemagglutinin-free T-cell growth factor; such ALK cells have a T-cell phenotype and there are no identifiable cells with the markers of NK cells (15). 70 ------Cytotoxicity Assay. A 51Cr release assay was employed (14). Target cells were labeled with 51Cr, 100 j1l at 105 cells per ml 601- was placed into the wells of V-bottomed microtiter plates, and different concentrations of effector cells were added in 100 ul. 50 F Plates were centrifuged (400 X g) for 1 min and incubated at 370C for 3-4 hr before recentrifugation. One hundred microli- ters of supernatant was removed from each well and the re- 30 leased radioactivity was measured in a gamma counter, and the 0O results of triplicate tests were calculated as mean percent spe- CL 0 30 cific lysis (14). In antibody blocking tests the effector cells were (n added to the wells in 50 S4 together with 50 .l of test or control 20V antibody before the addition of labeled target cells. Cell Lines. The cell lines used as target cells were K562, an erythroleukemic cell line, the melanoma cell line LiBr, and B 10 lymphoblastoid cells grown from the peripheral blood of normal subjects by infection with EBV. All of the cell lines were 5x105 5x104 5x103 500 50 5 0.5 demonstrated to be free of mycoplasma (16). Antibody Concentration (ng/ml) Antibodies. The broad specificities of the monoclonal antibodies used are given later. The OK reagents were purchased FIG. 1. Inhibition of NK cell-mediated killing of K562 targt cells from Ortho Pharmaceutical (Raritan, NJ) and the Leu reagents by antibody 9.1C3. Shown is the mean specific lysis (±SD) of 'Cr-la- from Becton Dickinson (Sunnyvale, CA). The NIMP-R10 and beled target cells by 5 x 10' effector MNC at an effector-to-target ratio of 50:1 in the presence of dilutions of purified 9.1C3 antibody to the MAC-1 antibodies were a gift from A. L6pez (Walter and Eliza final concentration given. The broken line is the control in the absence Hall Institute), 3A1 from B. F. Haynes (Duke University, Dur- of antibody. The dose-response experiment was repeated with tissue ham, NC) and A. S. Fauci (National Institutes of Health, Be- culture supernatant and with ascites fluid containing 9.1C3, and the thesda, MD), FMC 17 from H. Zola (Flinders Medical Centre, results were similar. Adelaide, Australia), A2 and T200 from I. F. C. McKenzie (University of Melbourne, Melbourne, Australia), and UCHT1 treated with 9.1C3 and washed before assaying in the standard and UCHT3 from P. C. L. Beverley (University College Hos- cytotoxicity test, but it did block when the effector cells were pital, London). allowed to react with antibody and washed prior to testing. When purified LGL that contained most of the NK cell activity of RESULTS MNC were tested for conjugate formation, visual examination Blocking of NK Cell Function. Purified 9. 1C3 antibody in showed that the presence of 9.1C3 did not inhibit binding to the absence of complement was tested for its ability to block the targets; indeed, in one experiment, enhanced effector-tar- killing of K562 cells by freshly isolated MNC. A representative get cell conjugates were observed (Table 1 and Fig. 2). By con- experiment is shown in Fig. 1, where it can be seen that contrast, the control antibody A2 that blocked NK cell killing did centrations as low as 50 ng/ml of antibody resulted in a 50% cause some inhibition of NK cell conjugate formation. inhibition of NK cell killing by 5 x 105 MNC. A range of mono- Antibody 9.1C3 Blocks the Killing of K562 by ALK Cells. clonal antibodies with broad specificity for LGL (NIMP-R1O, ALK cells were generated by in vitro coculture or with mitogen LUu 7), LGL and T cells (A2, OKT10, 3A1), T cells (UCHT1, and growth in T-cell growth factor and tested for cytolytic ac- UCHT3), monocytes (MAC-1, FMC17), f-microglobulin, and tivity in the presence of antibody. It was found that killing of the common leukocyte antigen (T200) was tested for blocking K562 cells by ALK cells was blocked by the presence of 9. 1C3 activity on NK cells. Each of the antibodies was tested at a con- antibody (Fig. 3 and Table 2). Of the other monoclonal anti- centration giving maximal staining by fluorescence analysis and, bodies tested, antibody A2 (range 5-10% inhibition) and an- apart from 9.1C3, only antibody A2 with specificity for the 50- kilodalton sheep erythrocyte receptor showed significant in- Table 1. Failure of antibody 9.1C3 to block conjugate formation hibition (18% ± 2%, SEM). Because fluorescence analysis between LGL cells and K562 target cells showed that most of the antibodies bound to potential NK cells % conjugate formation* with a greater intensity than 9. 1C3, it was clear that binding or cell agglutination alone was insufficient to cause blocking of Exp. No antibody 9.1C3 A2 killing. Others have reported that a monoclonal antibody with 1 45 82 26 the same specificity as A2 blocked NK cell killing and also kill- 2 26 22 14 ing by MHC-restricted T cells (17). Purified LGL were held for 30 mm at room temperature with an equal Distribution of the 9.1C3 Antigen and Failure of the An- volume of culture supernatant containing antibody. The treated effec- tibody to Block NK Cell-Target Cell Conjugate Formation. tor cells were then mixed directly with fluorescein diacetate-labeled When studied by indirect immunofluorescence and flow cy- K562 target cells at an effector-to-target cell ratio of100:1, centrifuged tometry, 9. 1C3 was found to stain all MNC with low intensity at 400 x g for 1 min, and held on ice until counted by using combined but did not bind to granulocytes or to the K562 cells used as phase-contrast and epifluorescence microscopy. The results are from targets in cytotoxicity assays (data not shown). Thus the dis- experiments performed with LGL from two different donors and the percentages were obtained by counting >100 target cells. tribution of 9.1C3 appeared to be different from that of NK- *Aconjugatewasscoredas + if1 ormore (nonfluorescent) effector cells specific or HLA-related antigens. The antibody did not block were attached to a (fluorescent) target cell after gentle resuspension NK killing when the 51Cr-labeled K562 target cells were pre- of the pellet formed by centriftigation. Downloaded by guest on September 28, 2021 7608 Immunology: Burns et al. Proc. Natl. Acad. Sci. USA 80 (1983)

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I I I I I v- 4 4x10-1 4x10-24xo3 4x10-,44x10-54X1O-6 Reciprocal Dilution of 9.1C3 Ascites FIG. 3. Blocking ofALK cell killing of K562 cells by antibody 9.103. Cytotoxicity assay as in Fig. 1. In this representative experiment the ALK cells were generated by 10-day coculture of MNC with irradiated autologous B lymphoblasts; similar results were obtained with ALK FIG. 2. Enhanced binding of purified LGL to K562 target cells in cells generated with mitogen. Each point represents the mean specific the presence of 9.103 (Exp. 1, Table 1). Numerous LGL can be seen at- lysis ± 1 SD of triplicate wells at an effector-to-target ratio of 10: 1. It tached to the two central, fluorescently labeled, K562 cells. Immuno- shouldbe noted that ALK cells are much more effective killers than NK fluorescence studies showed the 9.103 antibody did not bind to K562 cells (Fig. 1) andthe effector-to-target ratios selected forboth cell types, cells. (x500.) which were on the linear region of the cytotoxicity curve, reflect this point. tibody T200 (10-20% inhibition) showed slight inhibition, but none of the others, including UCHT1, Leu 7, OKM1, OKT4, Antibody 9.1C3 Inhibits the Killing of K562 by Monocytes and OKT8, gave any inhibition of killing (data for UCHT1 in but Has No Effect on the Phagocytic Function of These Cells. Table 2). Monocytes were obtained from MNC by adherence to plastic Antibody 9. 1C3 Does Not Block the Killing of EBV-Infected dishes for 2 days and the strongly adherent cells were harvested B Lymphoblasts by Induced T Cells. Specific CTC were tested with a rubber policeman and tested for their cytolytic activity for their cytolytic activity against the same clone of B lympho- against K562. Evidence that we were observing killing by blasts as was used for induction. By clone splitting experiments monocytes rather than by residual NK cells or ALK cells (both we showed previously that different effector cells kill B lym- of which are nonadherent) was as follows. All of the adherent phoblasts and ALK cell targets, and it is of interest that anti- cells were, morphologically, activated monocytes/macrophag- body 9. 1C3 did not block the killing of B lymphoblasts by al- es after cytocentrifugation and staining with May-Griinwald/ loinduced or virus-induced CTC, whereas the control antibody, Giemsa stain, and most expressed strong nonspecific esterase UCHT1, markedly did so (Table 2). This is in direct contrast to staining; over 80% expressed the FMC 17 antigen, which is the killing of K562 by ALK cells, which was blocked by 9. 1C3 specific for monocytes, whereas none expressed the NK cell but not by UCHT1 (Table 2). marker Leu 7; and, perhaps most significant, relatively high

Table 2. Blocking of ALK-mediated lysis of K562 by antibody 9.103 but failure to inhibit the killing of LiBr targets or CTC killing of B lymphoblasts % specific lysis in presence of antibody* Effector- Allogeneic culture Autologous culture Mitogen culture to-target No No No Target cell ratio antibody 9.1C3 UCHT1 antibody 9.1C3 UCHT1 antibody 9.103 UCHT1 B lymphoblasts 10:1 20 20 14t 41 42 lot - 5:1 14 17 11 32 33 8t K562 10:1 63 40t 63 66 39t 66 69 42t 64 5:1 45 32t ND 46 25t 47 57 31t 55 LiBr 10:1 44 39 43 39 37 ND 46 43 ND 5:1 30 30 37 26 28 ND 32 34 ND ALK cells and specific CTC generatedby coculturing MNC with irradiated allogeneic or autologous EBV-transformed B lymphoblasts or by stim- ulation with phytohemagglutinin followed by culture in T-cell growth factor were tested for cytolytic activity against 61Cr-labeled target cells in the presence or absence of antibody in a standard 3-hr assay. The assay was repeated on two other occasions with similar results. * Antibody 9.1C3 was added to the effector cells as 50 ,ul of undiluted tissue culture supernatant and UCHT1 as 50 il of ascites fluid diluted 1:50 prior to the addition of target cells. Variances are not shown but SDs were always <2%, and individual values marked t hadP < 0.001 compared with control (no antibody) by Student's t test. ND, not determined. *Mitogen-activated ALK cells from most individuals usually do not kill B lymphoblasts, this apparently being a function ofinduced MHC-restricted CTC. Downloaded by guest on September 28, 2021 Immunology: Bums et al. Proc. Natl. Acad. Sci. USA 80 (1983) 7609 effector-to-target cell ratios and long (18-hr) incubation times 70 IF a - were required before any target cell killing was observed. Un- J6 der these conditions (Table 3) monocytes from different donors varied in their ability to kill K562, and this activity was greatly 60 t inhibited by 9. 1C3 but not by the anti-monocyte antibody FMC 17 or by A2, which blocked NK cell killing. 501 When monocytes prepared in the same way were incubated a with 9. 1C3 or control antibodies (FMC17 and NIMP-R10) for *i 40 I. 60 min at room temperature, washed, and then tested for their .2a ability to phagocytose opsonized ox erythrocytes, it was found I, 30 that none of the test antibodies inhibited this function and that Lo) some 70% of the cells contained phagocytosed erythrocytes after 0 20 1 hr at 37c. Kinetics of the Inhibitory Effect of Antibody 9.1C3 on the 10 Killing of K562 by NK Cells. NK cell killing was carried out in the presence of 9. 1C3 antibody when the antibody was added .I I I I I I to the effector cells at various periods prior to the addition of -2 -1 0 +1 +2 +3 K562 target cells, at the time of addition of the target cells, or Tim. of Antibody Addition (hours) at various periods after target cell addition and incubation at FIG. 4. Kinetics of 9.103 antibody blocking the K562 target cell 37TC. It can be seen from Fig. 4 that incubation of killers with killing by NK cells. Purified 9.1C3 antibody (0.2 mg/ml) was added to antibody prior to addition to target cells was no more effective each well containing effector cells at the time indicated. At time 0, tar- than addition at time zero, and that inhibition of killing was get cells were added (E:T, 50:1), then the plates were centrifuged to observed even when the antibody was added as long as 1 hr pellet the cells and incubated at 3700. The antibody additions after time 0 were carried out without disturbing the pellet of effector and target after the start of culture. By 2 hr addition of 9.1C3 had no effect cells and the +3-hr antibody was added at the termination ofthe assay. on the lytic reaction, but it is possible that lysis was complete Each point represents the mean ± 1 SD for three replicate wells and after this time. the broken line is for control wells not receiving antibody. The experiment was repeated, using 1:1,000 ascites fluid, with similar results. DISCUSSION Little is known of the method by which NK cells recognize or of ALK cells. Thus it has been shown that both UCHTI and kill their tumor cell targets, although many mechanisms have OKT3 antibodies, which recognize the same 19- to 20-kilodal- been proposed. The present study describes the blocking of ton glycoprotein complex on human T cells (13), are able to block NK cell function by a murine monoclonal antibody, 9.1C3, which CTC-mediated killing when present at the effector stage (13, bound to NK effector cells but did not inhibit attachment of 18), and this has led to suggestions that the UCHT1/T3 mo- these cells to the K562 target cells. Of particular interest, 9.1C3 lecular complex is intimately linked to an antigen recognition also blocked killing of K562 cells by ALK cells and monocytes structure or to an associated activation structure on human T without affecting other functional activities of these cells. cells (18, 19). The present studies confirmed that UCHTI was The antibody 9. 1C3 did not block the killing of autologous able to block specific CTC killing and further showed that this or allogeneic B lymphoblasts by sensitized CTC; this may be antibody did not block killing by ALK cells even though these related to the target cell specificity, but the method of target cells bear the UCHT1/T3 complex on their surface (8, 9). cell recognition by CTC does appear to be different from that Our antibody blocking studies point to a closer relationship between ALK cells and NK cells than between ALK cells and Table 3. Inhibition of monocyte-mediated killing of K562 cells by CTC. Also, antibody 9.1C3 blocked the killing of K562 by antibody 9.103 monocytes, a finding that might support claims (20) of a my- Antibody present % specific % eloid origin for NK cells. Arguments for cell lineage based on Exp. (dilution) lysis of K562 inhibition the cross specificity of monoclonal antibodies have been weak- ened in the case of NK cells because of their reactivity with 1 No antibody 10 ± 1.1 0 antibodies to T cells, monocytes, and granulocytes, but the 9.103 2 ± 0.1 80 demonstration of an antibody that blocks a common function A2 11±0.9 0 perhaps provides stronger evidence than simply the expression 2 No antibody 49 ± 6.9 0 of surface markers. 9.103 10 ± 0.5 80 Another possibility is that NK cells, ALK cells, and mono- FMC17 53 ± 0.9 0 cytes can utilize the same cytolytic mechanism against tumor 3 No antibody 71 ± 1.1 0 cells, regardless of their lineage and method of target cell rec- 9.1C3 (1:10) 28 ± 0.2 61 ognition. In favor of this is the similarity in some of the mech- 9.1C3 (1:100) 20 ± 2.1 72 anisms of tumor cell lysis reported for toxic products synthe- 9.1C3 (1:1,000) 20 ± 0.8 72 sized by monocytes and macrophages (21) and by NK cells (2- FMC17 (1:10) 62 ± 0.0 13 6). Thus cytolytic factor, serum proteases, and C3a complement Monocytes purified by plastic adherence were tested in an 18-hr 51Cr components have all been implicated in killing by both cell types, release assay with K562 target cells. Each experiment was carried out and our antibody blocking studies could suggest that these cells in triplicate with monocytes from different individual donors. In Exp. produce an antigenically related toxic factor. Since target cell 1, 10 ul of antibody (undiluted culture supernatant of 9.1C3 and 0.1 binding by NK cells was unaffected, and there was significant mg/ml of A2) was added to 200 ,ul of cells at a final effector-to-target inhibition of killing even when the antibody was added 1 hr (E:T) ratio of 20:1. In Exp. 2 the antibodies were added as 50 1l (su- after initiation of the this is unrelated pernatant 9.1C3 and 1:10 FMC17 ascites) in 200 ,u1 at a final E:T of assay, blocking probably 20:1. Exp. 3 was with 50 j4l ofthe dilution of ascites given for each an- to effector cell recognition. Moreover antibody 9.1C3 also blocks tibody in 200 ul at a final E:T of 50:1. Percent specific lysis is given the killing of K562 cells by antibody-dependent K cells (J. as mean ± SD. Werkmeister, personal communication), which clearly bind to Downloaded by guest on September 28, 2021 7610 Immunology: Bums et al. Proc. Natl. Acad. Sci. USA 80 (1983)

target cells by an independent mechanism. 5. Baum, L. L., James, K. K., Glaviano, R. R. & Gerwurz, H. (1983) Although there are reports of other monoclonal antibodies J. Exp. Med. 157, 301-311. blocking NK cell killing, these do not show whether this block- 6. Wright, S. C. & Bonavida, B. (1983) Proc. Nati. Acad. Sci. USA 80, 1688-1692. ing activity is specific for NK cells. One of these antibodies (22) 7. Seeley, J. K., Masucci, G., Poros, A., Klein, E. & Golub, S. H. appears to be similar to 9. 1C3 in its binding specificity, and it (1979) J. Immunol. 123, 1303-1311. is interesting that this antibody blocked the killing of only 5 of 8. Grimm, E. A., Ramsay, K. M., Mazumder, A., Wilson, D. J., 13 susceptible targets, including K562. It is clear from the pres- Djeu, J. Y. & Rosenberg, S. A. (1983)1. Exp. Med. 157, 884-897. ent report that our antibody also blocks the killing of only some 9. Pawelec, G. P., Hadam, M. R., Ziegler, A., Lohmeyer, J., Reh- target cells, since killing of a melanoma target cell, LiBr, was bein, A., Kumbier, I. & Wernet, P. (1982)J. Immunol. 128, 1892- 1896. not inhibited (Table 2). It is possible that some effector cells can 10. Masucci, M. G., Klein, E. & Argov, S. (1980) J. Immunol. 124, employ different cytotoxic mechanisms against diverse targets 2458-2463. that vary in their relative susceptibility or that there is a het- 11. Strassmann, G., Bach, F. H. & Zarling, J. M. (1983) J. Immunol. erogeneity of effector cell types with select target specificities, 130, 1556-1560. only some of which are blocked by 9. 1C3 antibody. Alterna- 12. Timonen, T. & Saksela, E. (1980) J. Immunol. Methods 36, 285- tively, if a cytotoxin is involved (6), the same toxin may be cou- 291. 13. Bums, G. F., Boyd, A. W. & Beverley, P. C. L. (1982)J. Immu- pled to a series of ligands, each with specificity for particular nol. 129, 1451-1457. target cell types; in this latter case there would be competition 14. Wallace, L. E., Rickinson, A. B., Rowe, M., Moss, D. J., Allen, for the ligand of the cytotoxic factor by 9. 1C3 and the receptor D. J. & Epstein, M. A. (1982) Cell. Immunol. 67, 129-140. present on the target cell membrane. 15. Bums, G. F., Battye, F. L. & Goldstein, G. (1982) Cell. Immunol. 71, 12-26. We acknowledge the expert technical assistance of Filomena Lau- 16. Triglia, T. & Bums, G. F. (1983)J. Immunol. Methods, in press. riola. This work was supported by the Lions-Sponsored Cancer Re- 17. Fast, L. D., Hansen, J. A. & Newman, W. (1981)J. Immunol. 127, search Fund. 448-452. 18. Chang, T. W, Kung, P. C. & Goldstein, G. (1981) Proc Natl. Acad. 1. Burnet, F. M. (1971) Transplant. Rev. 7, 3-25. Sci. USA 78, 1805-1808. 2. Bloom, B. R. (1982) Nature (London) 300, 214-215. 19. Reinherz, E. L., Meuer, S., Fitzgerald, K. A., Hussey, R. E., 3. Hudig, D., Haverty, T., Fulcher, C., Redelman, D. & Mendel- Levine, H. & Schlossman, S. F. (1982) Cell 30, 735-743. sohn, J. (1981)J. Immunol. 126, 1569-1574. 20. Kay, H. D. & Horwitz, D. A. (1980) J. Clin. Invest. 66, 847-851. 4. Charriaut, C., Senik, A., Kolb, J.-P., Barel, M. & Frade, R. (1982) 21. Mathews, N. (1983) Immunology 48, 321-327. Proc. Nati. Acad. Sci. USA 79, 6003-6007. 22. Newman, W. (1982) Proc. Natl. Acad. Sci. USA 79, 3858-3862. Downloaded by guest on September 28, 2021