- Email: [email protected]
The nature of the effector cell in antibody-dependent, cell-mediated cytolysis (ADCC): The cytotoxic activity of murine tumor cells and peritoneal macrophages
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
5,
272-281 (1976)
The Nature of the Effector Cell in Antibody-Dependent, Cell-Mediated Cytolysis (ADCC): The Cytotoxic Activity of Murine Tumor Cells and Peritoneal Macrophages BARRY S. HANDWER~XR Departments
of Medicine and Microbiology. Medicine, Minneapolis,
HILLEL Immunology
Branch,
University Minrwsoia
of Minnesotu 55455
School
oj
AND S. KOREN
National Cancer Institute, National Bethesda. Maryland 20014
Institutes
of Health,
Received November 13. 1975 The nature of the effector cell in the antibody-dependent. cell-mediated cytolysis of anti-trinitrophenyl coated, TNP-modified chicken erythrocytes was evaluated. P388D, a macrophage-like murine tumor cell line, and adherent cells derived from normal peritoneal cells and from tumor-containing ascites cells were active in mediating cytolysis. In vitro cultured murine tumor cell lines with cell marker characteristics of T lymphocytes, B lymphocytes,“null” lymphoid cells, and mast cells lacked effector cell activity. Ascites cells obtained from tumor-bearing animals were active as cytotoxic cells, but that activity was due to “contaminating” adherent cells of host origin rather than to the activity of the tumor cells themselves.
INTRODUCTION We have recently described an in vitro cultured murine macrophage tumor (P388D,) and have demonstrated that this cell is active as an effector cell in the antibody-dependent, cell-mediated cytolysis of TNP-modified chicken erythrocytes in the presence of mouse anti-TNP antibodies (l).’ To investigate whether the cytotoxic potential of this cell is related to its tumor cell nature or to its macrophage-like characteristics, we have evaluated the ADCC effector cell activity of several in vitro cultured and in vivo ascites-passaged murine tumors. Tumor cells with characteristics of T lymphocytes, B lymphocytes, “null” lymphoid cells, mast cells and macrophages have been studied. In addition the cytotoxic activity of normal, noninduced peritoneal cells has been evaluated. The experiments suggest that macrophages are active effector cells in mediating ADCC lysis * Abbreviations used: ADCC, antibody-dependent, cell-mediated cytolysis; TNP, trinitrophenyl; T, thymus derived; B, bone marrow derived; CRBC, chicken red blood cell; E-IO% FCS, Eagle’s minimal essential medium supplemented with penicillin, streptomycin and 10% fetal calf serum; S-lg, surface immunoglobulin; BA6, brain-associated theta; Ig, immunoglobulin; 7S EA, sheep erythrocytes coated with 7s (IgG) anti-sheep erythrocyte antibodies; SRBC, sheep red blood cells; BSS. balanced salt solution; Fc, receptor for heat-aggregated human Ig or antibody-antigen complexes; SE, standard error. 272 Copyright All rights
@ 1976 by Academx Pre\\. Inc of reproduction in any form revervrd
NATURE
OF
EFFECTOR
CELL
IN
273
ADCC
of TNP-modified CRBC in the presence of anti-TNP serum and that the cytotoxic potential of P388D, is related to its macrophage origin rather than to its tumor cell nature. METHODS AND MATERIALS Sources and Preparation of Effector Cells I. Tumor cells. Tumor cells studied include: P388D,,
EL 4, LSTRA, P815Y, P388 (parent), L1210 and RDM 4. The histocompatibility (H-2) type, strain of origin, mode of induction and strain used for in vivo passage of each of these tumors is presented in Table 1. Tumors were maintained in vivo, in the ascites form, by serial intraperitoneal passage. Ascites cells were tested for ADCC activity 3-10 days after intraperitoneal transfer. In vivo passaged tumors were adapted to in vitro culture in Eagle’s minimal essential medium supplemented with penicillin (100 units/ml), streptomycin (100 pg/ml), glutamine (200 mM) and 10% FCS (E-10% FCS) (Grand Island Biological Co., Grand Island, N. Y.). Except in the case of P388D,, plastic-adherent cells were completely removed from tumor-containing ascites prior to the establishment of long-term culture. The cells were maintained at a cell concentration of 2 x 105- 1 x 106 cells/ml. All tumors were maintained in suspension culture in 75cm3 plastic tissue culture flasks (#3024, Falcon Plastics, Oxnard, Calif.). In addition, P388D, cells were grown in spinner culture. 2. Noninduced peritoneal cells. Normal noninduced peritoneal cells were obtained by washing the peritoneal cavity of normal DBA/2 mice with 5 ml of E-10% FCS. 3. Macrophage-depleted and enriched peritoneal cells. A modification of the adherent method of Mosier (2) was used to deplete macrophages from peritoneal cells obtained from normal and tumor-bearing mice. Peritoneal cells were adjusted to a concentration of lo6 cells/ml in E-10% FCS. Twenty milliliters of cells were added to a 75cm3 plastic tissue culture flask (Falcon). The cells were incubated at 37°C in a humidified 10% CO,-90% air atmosphere for l-3 hr. The nonadherent, macrophage-depleted population was then recovered by gentle decantation.
TABLE 1 MURINE TUMOR CELLS Tumor P338D, EL4 LSTRA P815Y P388 (parent) L1210 RDM 4
H-2 type
Strain of origin
Mode of induction
DBA/Z C57BLl6 BALBJc DBAl2 DBN2 DBA/2 AKR
Carcinogen Carcinogen Moloney virus Carcinogen Carcinogen Carcinogen Spontaneous
(2 P388D, was maintained only as an ascites form.
in vitro
Strain used for passage
in viva -a
C57BLJ6 BALBlc DBAl2 DBA/2 DBA/2 AKR
Reference 20 20 20 21 20 20 22
cultured tumor cell line. It was not studied in the
in viva
274
HANDWERGER
AND
KOREN
Following two washes in E-IO% FCS the adherent macrophage-enriched tion was removed with a rubber policeman.
popula-
Cell Marker Studies 1. Surface immunoglobulin
(S-lg). S-Ig was identified by the use of fluoresceinconjugated polyvalent rabbit anti-mouse Ig (Cappel Laboratories, Downington, Pa., Lot 6377) as previously described (3). This reagent stained 47% of spleen cells, 30% of mesenteric lymph node cells and less than 0.5% of thymocytes from normal BALB/c mice. 2. Brain-associated theta (BAO). BAB was detected by both direct and indirect fluorescence using rabbit anti-mouse brain serum prepared by the method of Golub (4) and used as previously described (3). This reagent stained 34% of spleen cells, 57% of mesenteric lymph node cells and 98% of thymocytes from normal BALB/c mice. 3. Aggregated lg. Unlabeled and fluorescein-conjugated, heat-aggregated human Ig was prepared from human Cohn fraction II (HGII, Sigma Chemical Co., St. Louis, MO.) by the method of Dickler (5). Both direct and indirect fluorescence techniques were used to detect cells bearing a receptor for aggregated Ig. 4. Antibody-antigen complexes (7s EA). Sheep erythrocytes (SRBC) coated with 7s anti-SRBC antibodies (Cordis Laboratories, Miami, Fla.) were prepared and used according to the method of Shevach et al. (6). Controls consisted of tumor cells incubated with uncoated SRBC or SRBC coated with 19s Forssman antibody (Cordis). 5. Latex phagocytosis. Phagocytes were identified by their ability to ingest latex beads. One million cells were incubated in a shaking water-bath at 37°C for 45 min in E-20% FCS containing 6 x IO7 latex beads (Dow Chemical, Indianapolis, Ind.). The cells were then washed three times and wet-mount slides were prepared. A minimum of 200 cells was counted. Cells ingesting three or more beads were considered positive. Preparation
qf Anti-TNP
Serum
BDF, and C57BL/6 mice were injected in the foot pads with trinitrophenyl (TNP)-modified M.vcobacterium tuberculosis H 37 Ra incorporated in incomplete Freund’s adjuvant. After 2 weeks the mice were boosted with 100 pg of TNPmodified M. tuberculosis, intraperitoneally, and bled 2-3 weeks later. Sera were also obtained from mice immunized with TNP-modified keyhole limpet hemocyanin (TNP-KLH). All sera were heat inactivated at 56°C for 40 min. ADCC
Assa)
Chicken erythrocytes were labeled with 51Cr as described by Perlman and Perlman (7) washed and resuspended in phosphate-buffered saline (0.01 M P04, 0.14 M NaCl. pH 7.2) at a concentration of 4 x 10’ CRBC/ml. One milliliter of cells was added to 3 ml of 0.28 M cacodylate buffer containing 25 mg of trinitrobenzene sulfonate (TNP) and incubated at 4°C for 30 min. The cells were then washed three times in BSS-10% FCS. The 51Cr-labeled, TNP-modified CRBC were added in 50 ~1 to 1 ml of effector cells in 12 x 75-mm round-bottomed plastic tubes (Falcon #2052). Mouse anti-TNP serum at a final dilution of IO-3 was then
NATURE
OF
EFFECTOR
CELL
IN
275
ADCC
added in 50 ~1 and the cells incubated at 37°C for 3 hr. All samples were run in triplicate. Control tubes to determine spontaneous release contained effector cells and Wr-labeled, TNP-modified CRBC without added anti-TNP serum. At the end of the incubation, 1 ml of BSS-10% FCS at 4°C was added to each tube. After centrifuging at 700g for 10 min at 4”C, 1 ml of supernatant fluid was removed for counting in a well-type gamma scintillation counter (AmershamSearle, Chicago, Ill.). The percentage of specific lysis was calculated using the following formula: (E - S
1x
Specific lysis (%) =[ T - BG)
100
where E = mean counts of experimental group; S = mean counts from spontaneous release tubes; T = mean counts released following repetitive freeze-thawing; and BG = machine background counts. The effector cell dependency of the assay was assessed by measuring the percentage of 51Cr released in tubes containing antibody and target cells in the absence of effector cells. At no time did this value differ significantly from the mean counts of the spontaneous release tubes and never (even at 20 hr) did it exceed by greater than 5% the amount of 51Cr released by hapten-modified targets in the absence of both effecters and antibody. RESULTS
Cell Marker Characteristics of Tumor Cell Lines As illustrated in Table 2, two of the in vitro cultured tumor cell lines (EL 4 and RDM 4) are &bearing, Ig-negative, nonphagocytic lymphoid cells, and, therefore, are considered to be T lymphocytes; L1210 cells are Ig bearing, 0 negative and nonphagocytic and, therefore, presumably are B cells; P388 (parent) and LSTRA TABLE CELL MARKER
Tumor P388D, EL4 RDM 4 L1210 P388 (parent) LSTRA PSlSY
CHARACTERISTICS
Latex phagocytosisn fP -
2
OF CULTURED
TUMOR
CELL LINES
s-1g
BA@-
Fed
Ceil classification
-c + -
+ + -
+ + + + + +
Macrophage T lymphocyte T lymphocyte B lymphocyte “Null” lymphoid cell “Null” lymphoid cell Mast cell
a Latex phagocytosis, ability to phagocytize latex beads during 45-mitt incubation at 37°C. Cells ingesting more than three beads were considered positive. b S-Ig, surface Ig bearing; stains with fluorescein-conjugated polyvalent rabbit anti-mouse Ig. c BAB, Brain-associated theta bearing; reacts with rabbit anti-mouse brain serum. * Fc, Bears a receptor for heat-aggregated human Ig and/or antibody-antigen complexes (7s EA). c (+), Possess the cell marker characteristic under study; (-), does not possess the cell marker under study. For each cell marker a minimum of 200 cells was scored.
276
HANDWERGER
4NII
KOREN
are non-8 and non-Ig-bearing, non-phagocytic “null” lymphoid cells; P815Y is a mast cell; and as we have previously demonstrated (I), P388D, is a macrophagelike tumor. All of the in vitro cultured tumor cell lines except EL 4 bear a receptor for heat-aggregated human Ig and/or antibody-antigen complexes (7s EA). If cells of host origin (i.e.. nontumor cells) are excluded, the cell marker characteristics of the in vivo ascites tumors are identical to those of the corresponding in vitro cultured tumor cell lines. ADCC
Effector
Cell Ac,tivity
of Tumor-Containing
A.scite.v Cells
The ability of ascites cells obtained from tumor-bearing animals to act as effectors in the antibody-dependent cell-mediated cytolysis of anti-TNP coated, TNP-modified CRBC is demonstrated in Table 3. All of the tumor-containing ascites cell preparations were active as effector cells against antibody-coated target cells. No significant lysis of TNP-modified CRBC occurred in the absence of anti-TNP sera, in the absence of effector cells. or in the presence of normal C57BL/6 or BDF, serum. Although in Table 3 the cytotoxicity mediated by LSTRA-containing ascites cells was distinctly less than that of other tumorcontaining ascites cells, the percentage of specific lysis nevertheless was highly significant (P < 0.005 at an effector to target ratio of 10: 1). These data suggest that either most, if not all, tumor cell lines are capable of killing antibody-coated, hapten-modified CRBC or that the cytotoxic activity exhibited by the tumor-containing ascites cells is due not to the tumor cells themselves but rather to contaminating cells of host origin. These ascites tumor cell preparations, in fact, contained 4-18% macrophages by the criterion of latex ingestion. ADCC Effector Cell Activity of Plastic-Adherent und Nonadherent Peritoneal Cells Obtained from Tumor-Containing Ascites To explore whether the activity of the in \+vo passaged tumor cell preparations
was due to contaminating macrophages, macrophages were removed from P815Y-containing ascites by adherence methods. Under the conditions employed the tumor cells were nonadherent and macrophages were adherent to the plastic
Tumor
cell
P388 (parent) EL4 LSTRA P815Y RDM 4
13.2 11.4 2.5 12.6 14.8
a Three-hour assay, IO5 target b Effector to target cell ratio.
ir 0.3 IO.2 2 0.1 -t 0.0 -c 0.6 cells.
9.7 7.5 1.4 9.0 11.5
1 t 2 t 2
0.2 0.1 0.1 0.0 0.1
NATURE
OF
EFFECTOR
CELL
IN
277
ADCC
tissue culture flasks. As illustrated in Table 4, when adherent cells were removed from P815Y-containing, ADCC-active peritoneal cell preparations, effector activity was markedly diminished; moreover, that activity was recoverable in the adherent cell population. These data suggest that the cytotoxic effector activity of PSlSY-containing ascites is due to a contaminating adherent cell population and not the tumor cells themselves. ACDD Effector Cell Activity of In Vitro Cultured Tumor Cell Lines To explore further whether the activity of tumor-containing ascites populations was due to contaminating macrophages, long-term in vitro cultures of each of the in vivo passaged tumors were established. During the establishment of the cultures, macrophages were removed from the tumor cell populations by adherence methods. Table 5 illustrates the effector cell activity of the cultured tumor cell TABLE ADCC
EFFECTOR
CELL
4
ACTIVITY OF PLASTIC-NONADHERENT OBTAINED FROM P8lSY-CONTAINING
Duration of adherence Cell fraction
(hr)
Unseparated Nonadherent Nonadherent Nonadherent Adherent
None 1r T P 3r
AND -ADHERENT ASCITES
PERITONEAL
CELLS
Specific lysis (% 2 SE)a 5:1* 22.5 4.5 1.9 1.8 23.5
k + 2 r r
2.5: lb 1.3 0.2 0.1 0.2 1.2
12.8 k 1.5 5 0.8 2 0.6 2 13.1 -t
1.25:l”
0.7 0.1 0.1 0.1 0.8
5.9 0.7 0.3 0.0 6.1
2 + 2 2 2
0.1 0.1 0.1 0.9 0.1
a Three-hour assay, 5 x IO5 target cells. b Effector to target ratio. c Tumor-containing peritoneal cells were incubated in plastic tissue culture flasks at 37°C for 1, 2 or 3 hr. Aliquots of the nonadherent cells as well as the 3-hr adherent population were tested for ADCC effector activity. TABLE ADCC
EFFECTER
CELL
ACTIVITY
5 OF CULTURED
TUMOR
CELL
LINES
Specific lysis (% ? SE)” Tumor cell P388D, P388 (parent) EL4 LSTRA P815Y RDM 4 Ll210
10: lb 34.1 0.5 0.0 0.5 0.0 - I.0 0.8
-r- 2.8 + 0.7 2 0.3 2 0.3 f 0.2 2 0.1 k 0.2
a Three-hour assay, I x IO5 target cells. b Effector to target cell ratio.
5:1* 28.7 0.1 0.1 0.5 0.0 - 0.8 0.3
zk 1.3 r 0.2 + 0.2 2 0.3 f 0.2 2 0.1 2 0.1
178
HANDWERCER
AND
KOREN
lines in a 3-hr cytotoxicity assay. In contrast to the activity of the tumorcontaining ascites, none of the cultured tumors, other than P388D,, was active in mediating cytolysis. The lack of effector activity was not a consequence of a shift in the kinetics of killing, for cytotoxicity was not apparent even after 20 hr of incubation of effector cells with antibody-coated targets (Table 6). P388D, was active as an effector when cultured in either spinner or suspension culture. Water-lysis studies (9) and time-lapse microcinematography indicate that the interaction of P388D, cells with hapten-modified CRBC involves both phagocytosis and contactual lysis (data not shown). When ADCC-inactive, macrophage-depleted, in vitro cultured tumor cells were injected intraperitoneally into syngeneic hosts, the tumor-containing ascites collected 6 days later again possessed effector cell activity, while tumor cells maintained in cultures still lacked cytotoxicity capability (Table 7). This experiment demonstrated that the lack of ADCC activity of in \+tro cultured tumors was not simply due to an irreversible change in the tumor cells and suggested once again that the effector cell activity of the ascites populations was due to “contaminating” cells of host origin. ADCC Effector DBAIZ Mice
Cell Acti\!ity
of Noninduced
Peritoneal
Cells Ji-orn Normd
If macrophages are the cells in tumor-containing ascites that are active in the ADCC lysis of hapten-modified CRBC, then normal peritoneal macrophages should also possess cytotoxic activity in our system. As illustrated in Table 8. normal noninduced peritoneal cells (consisting of 45-60s latex-ingesting cells) were active as cytotoxic effecters. Removal of adherent cells resulted in a marked diminution of effector cell activity. The adherent, macrophage-enriched population retained cytotoxic capability. DISCUSSION
The nature of the effector cell in antibody-dependent, cell-mediated cytolysis remains controversial. The use of highly purified cell populations should aid in resolving this controversy. We have recently described a macrophage-like tumor cell line, P388D,, which is active as an effector in the ADCC lysis of anti-TNP TABLE
6
Specific Cells EL4 LSTRA P815Y RDM 4 Normal C57BW6 a Effector
3 Hours
spleen
to target
cells
cell ratio
- 0.6 0.2 d.2 0.3 12.4 was 20: 1; 1 x 105 target
+ + t t -+
cells.
0.4 0.1 0.5 0.2 0.8
lysis
(% -C SE)” 20 Hours 0.0 0.8 I.1 0.9 40.5
i -c _t it
0.4 0.3 0.4 0.8 0.9
NATURE
OF
EFFECTOR
CELL
TABLE ADCC EFFECXOR
CELL ACTIVITY
IN
7
OF ASCITES CELLS OBTAINED CULTURED TUMOR CELL LINES
Effector to target Tumor cell EL4 P815Y u Three-hour
assay, 1
x
FROM ANIMALS
INJKXD
WITH
Specific lysis (% + SE)”
cell ratio
Cultured
20: 1 10: 1 20: 1 10: I
-0.8 2 0.3 0.1 -t 0.2 -0. I k 0.0
Ascites 11.9 8.4 14.7 6.5
-t 0.5 2 0.6 -c 0.1 2 0.8
lo5 target cells.
TABLE ADCC EFFECTOR
279
ADCC
CELL ACTIVITY
OF NONINDUCED
8 PERITONEAL
Duration of adherence
CELLS FROM NORMAL
DBA/2 MICE
Specific lysis (% ? SE)”
Cell fraction
(hr)
10: I*
5:1*
2.5: I*
Unseparated Nonadherent Adherent
None T 3’
10.6 t 1.1 1.8 ” 0.5 8.7 t 0.5
3.6 k 0.4 -1.0 * 0.5 3.5 2 0.2
2.9 2 0.4 -4.2 5 0.5 1.8 + 0.3
a Three-hour assay, 1 x IO5 target cells. * Effector to target cell ratio. r Tumor-containing peritoneal cells were incubated in plastic tissue culture flasks at 37°C for 3 hr. Aliquots of the nonadherent cells as well as the 3-hr adherent population were tested for ADCC effector activity.
coated, TNP-modified CRBC (1). The present communication demonstrates that plastic-adherent cells derived from normal noninduced peritoneal cells and from tumor-containing ascites cells are also active as effector cells in the ADCC lysis of antibody-coated, hapten-modified CRBC. Tumor cells with cell marker characteristics of T lymphocytes, B lymphocytes, “null” lymphoid cells and mast cells do not possess cytotoxic capability. These data suggest that adherent cells, presumably macrophages, can act as effector cells in antibody-dependent, cellmediated cytolysis and that the activity of P388D, is probably due to its macrophage-like characteristics rather than to its tumor cell nature. This conclusion is further supported by the observation that a murine reticulum cell sarcoma with macrophage-like properties (8) and an in vitro established line of mouse peritoneal macrophages (9) also have the ability to mediate ADCC lysis. Scomik and Cosenza (10) have recently reported that proteose-peptone induced peritoneal macrophages and that splenic adherent cells possess effector cell activity in ADCC. Holm and Hammarstrom (11) and Holm (12), furthermore, have demonstrated that human peripheral blood monocytes and human peritoneal macrophages are cytotoxic to antibody-coated human erythrocytes.
280
HANDM’ERGER
ASD
KOKtN
It should not be concluded, though, that the macrophage is the only cell capable of mediating ADCC lysis. In systems utilizing target cells other than haptenmodified CRBC. evidence has been presented which suggests that “null” cells or K cells (19,25-27), B lymphocytes (28-32). human lymphoblastoid cell lines (33), and human polymorphonuclear leukocytes (34) may be active as effecters in antibody-dependent, cell-mediated cytolysis. The present study demonstrates the need for highly purified cell populations in evaluating the nature of the effector cell in ADCC. When the cell population being studied is not totally pure. then the observed activity of that population may be due to a minor “contaminating” cell population and not to the major cell type. Thus, peritoneal cells obtained from the ascites of animals bearing nonmacrophage tumors were active as cytotoxic effecters. Though tumor cells comprised greater than 80% of the peritoneal cells. the effector cell activity was not due to the tumor cells themselves, but rather to contaminating adherent cells of host origin. In this regard the use of in t?tro cultured tumor cell lines with different cell marker characteristics as purified cell populations offers a unique model for the evaluation of antibody dependent, cell-mediated cytolysis. Finally, several investigators have documented that the cytotoxic effector cell in ADCC possesses a receptor for the Fc portion of IgG (2,13- 19). The studies reported here demonstrate that although an Fc receptor may be required, its mere presence is not sufficient to endow a cell with cytotoxic capability. Thus P388D,, L1210, P388 (parent), LSTRA, P815Y and RDM 4 cells all possess a receptor for heat-aggregated human Ig and/or antibody-antigen complexes, yet P388D, was the only tumor cell active as an effector in the ADCC lysis of antibody-coated. hapten-modified CRBC. ACKNOWLEDGMENTS The authors are grateful to Drs. J. R. Wunderlich, William Terry, and Steven D. Douglas for their help and advice. We thank Ms. Terry Peters for her excellent technical assistance and Mr. Walter Lyles for the supervision of animal facilities. This research was supported by the National Institutes of Health. USPHS grants, No. AI-12478-01 and HL-06314-15. and by grants from the National Leukemia Association, the Arthritis Foundation. Minnesota Chapter, and the Minnesota Medical Foundation. Addendum to tert. The work reported here was presented at the Annual Meeting of the American Association for Cancer Research in April 1975 (23). Subsequently, Tracey, et al. (24). using different murine tumors, have independently confirmed that the ADCC activity of ascites cells obtained from mice bearing nonmacrophage tumors is due to an adherent cell of host origin.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11.
Koren, H. S.. Handwerger, B. S.. and Wunderlich. J. R., .I. fmmunol. 114, 894. 1975. Mosier, D. E., Science 1.58, 1573, 1967. Handwerger. B. S.. and Schwartz, R. H.. Transplantation 18, 544. 1974. Golub, E., Cell. Immunol. 2, 353, 1971. Dickler. H. B., .I. hp. Med. 140, 508. 1974. Shevach, E. M., Ellman, L., Davie, Y. M., and Green, I., Blood 39. I. 1972. Perlmann, P.. and Perlmann. H., Cell. Immunol. 1, 300, 1970. Ralph, P., Prichard, J., and Cohn, M.. J. Immunol. 115, 898, 1975. Walker, W. S., and Demus. A.. J. lmmunot. 115, 765. 1975. Scomik, J. C., and Cosenza, H., J. Zmmunol. 113, 1527. 1974. Holm, G., and Hammarstrom, S.. Clin. Exp. Immunol. 13, 29. 1973.
NATURE
OF
EFFECTOR
CELL
IN
ADCC
281
Holm, G., Int. Arch. Allergy 43, 671, 1972. 13. Cerottini, J. C., and Brunner, K. T., Advan. fmmuno/. 18, 67. 1974. 14. Perlmann, P., Perlmann, H., and Wigzell, H.. Transplant. Rev. 13, 91, 1972. 1.5. Larrson, A., and Perlmann, P., Znt. Arch. Allergy 43, 80, 1972. 16. Moller, G., and Svehag, S. E., Cell. Immunol. 4, 1, 1972. 17. Hallberg, T., Stand. J. Immunol. 3, 117. 1974. 18. Wisloff, F., Michaelson, T. W., and Froland, S. S., Stand. J. Immunol. 3, 29, 1974. 19. Calder, E. A., Urbaniak, S. J., Penhole. W. J., and Irvine, W. J., C/in. Exp. Immunol. 18, 579. 1974. 20. Shevach, E. M., Stobo, J. D., and Green, I., J. Immunol. 108, 1146, 1972. 21. Dunn, T. B. and Potter, M., J. Nat. Cancer Inst. 18, 587, 1957. 22. Wunderlich, J. R., personal communication. 23. Koren, H. S. and Handwerger, B. S., Abstr. Amer. Ass. Cancer Res. 501, 126. 1975. 24. Tracey, D. E., Pross, H. F., Jondal, M., and Witz, I. P., submitted for publication. 25. Greenberg, A. H., Hudson, L., Shen, L., and Roitt, I. M., Nature New’ Viol. 242, 111, 1973. 26. Greenberg, A. H., Shen, L., Walker, L., Amaiz-Villena, A., and Roitt, I. M. Eur. J. Immunol. 5, 474, 1975. 27. Wisloff, F., and Froland, S. S., &and. J. Immunol. 2, 151, 1973. 28. Forman, J., and Moller, G., Transplant. Rev. 17, 108, 1973. 29. Yust, I., Wunderlich, J. R., Mann, D. L., and Buell, D. N., J. Zmmunol. 110, 1672, 1973. 30. Van Boxel. J. A., Stobo, J. D., Paul, W. E., and Green, I., Science 175, 194, 1972. 31. Zighelboim, J., Gale, R. P., Chiu, A., Bonavida. B., Ossorio, R. C., and Fahey, J. L., C/in. Immunol. Immunopathol. 3, 193, 1974. 32. F’I’OSS. H. F., Tracey, D. E.. Wigzell, H., and Schirrmacher, V., &and. J. Immune/. 3,769, 1974. 33. Lefebre, J-C., Petitprez, A., Juy, D., and Bona, C.. Eur. J. Zmmunol. 5, 266, 1975. 34. Gale, R. P.. Zighelboim. J., Ossorio, C., and Fahey, J. L., Clin. Res. 22, 180A. 1974. 12.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
5,
272-281 (1976)
The Nature of the Effector Cell in Antibody-Dependent, Cell-Mediated Cytolysis (ADCC): The Cytotoxic Activity of Murine Tumor Cells and Peritoneal Macrophages BARRY S. HANDWER~XR Departments
of Medicine and Microbiology. Medicine, Minneapolis,
HILLEL Immunology
Branch,
University Minrwsoia
of Minnesotu 55455
School
oj
AND S. KOREN
National Cancer Institute, National Bethesda. Maryland 20014
Institutes
of Health,
Received November 13. 1975 The nature of the effector cell in the antibody-dependent. cell-mediated cytolysis of anti-trinitrophenyl coated, TNP-modified chicken erythrocytes was evaluated. P388D, a macrophage-like murine tumor cell line, and adherent cells derived from normal peritoneal cells and from tumor-containing ascites cells were active in mediating cytolysis. In vitro cultured murine tumor cell lines with cell marker characteristics of T lymphocytes, B lymphocytes,“null” lymphoid cells, and mast cells lacked effector cell activity. Ascites cells obtained from tumor-bearing animals were active as cytotoxic cells, but that activity was due to “contaminating” adherent cells of host origin rather than to the activity of the tumor cells themselves.
INTRODUCTION We have recently described an in vitro cultured murine macrophage tumor (P388D,) and have demonstrated that this cell is active as an effector cell in the antibody-dependent, cell-mediated cytolysis of TNP-modified chicken erythrocytes in the presence of mouse anti-TNP antibodies (l).’ To investigate whether the cytotoxic potential of this cell is related to its tumor cell nature or to its macrophage-like characteristics, we have evaluated the ADCC effector cell activity of several in vitro cultured and in vivo ascites-passaged murine tumors. Tumor cells with characteristics of T lymphocytes, B lymphocytes, “null” lymphoid cells, mast cells and macrophages have been studied. In addition the cytotoxic activity of normal, noninduced peritoneal cells has been evaluated. The experiments suggest that macrophages are active effector cells in mediating ADCC lysis * Abbreviations used: ADCC, antibody-dependent, cell-mediated cytolysis; TNP, trinitrophenyl; T, thymus derived; B, bone marrow derived; CRBC, chicken red blood cell; E-IO% FCS, Eagle’s minimal essential medium supplemented with penicillin, streptomycin and 10% fetal calf serum; S-lg, surface immunoglobulin; BA6, brain-associated theta; Ig, immunoglobulin; 7S EA, sheep erythrocytes coated with 7s (IgG) anti-sheep erythrocyte antibodies; SRBC, sheep red blood cells; BSS. balanced salt solution; Fc, receptor for heat-aggregated human Ig or antibody-antigen complexes; SE, standard error. 272 Copyright All rights
@ 1976 by Academx Pre\\. Inc of reproduction in any form revervrd
NATURE
OF
EFFECTOR
CELL
IN
273
ADCC
of TNP-modified CRBC in the presence of anti-TNP serum and that the cytotoxic potential of P388D, is related to its macrophage origin rather than to its tumor cell nature. METHODS AND MATERIALS Sources and Preparation of Effector Cells I. Tumor cells. Tumor cells studied include: P388D,,
EL 4, LSTRA, P815Y, P388 (parent), L1210 and RDM 4. The histocompatibility (H-2) type, strain of origin, mode of induction and strain used for in vivo passage of each of these tumors is presented in Table 1. Tumors were maintained in vivo, in the ascites form, by serial intraperitoneal passage. Ascites cells were tested for ADCC activity 3-10 days after intraperitoneal transfer. In vivo passaged tumors were adapted to in vitro culture in Eagle’s minimal essential medium supplemented with penicillin (100 units/ml), streptomycin (100 pg/ml), glutamine (200 mM) and 10% FCS (E-10% FCS) (Grand Island Biological Co., Grand Island, N. Y.). Except in the case of P388D,, plastic-adherent cells were completely removed from tumor-containing ascites prior to the establishment of long-term culture. The cells were maintained at a cell concentration of 2 x 105- 1 x 106 cells/ml. All tumors were maintained in suspension culture in 75cm3 plastic tissue culture flasks (#3024, Falcon Plastics, Oxnard, Calif.). In addition, P388D, cells were grown in spinner culture. 2. Noninduced peritoneal cells. Normal noninduced peritoneal cells were obtained by washing the peritoneal cavity of normal DBA/2 mice with 5 ml of E-10% FCS. 3. Macrophage-depleted and enriched peritoneal cells. A modification of the adherent method of Mosier (2) was used to deplete macrophages from peritoneal cells obtained from normal and tumor-bearing mice. Peritoneal cells were adjusted to a concentration of lo6 cells/ml in E-10% FCS. Twenty milliliters of cells were added to a 75cm3 plastic tissue culture flask (Falcon). The cells were incubated at 37°C in a humidified 10% CO,-90% air atmosphere for l-3 hr. The nonadherent, macrophage-depleted population was then recovered by gentle decantation.
TABLE 1 MURINE TUMOR CELLS Tumor P338D, EL4 LSTRA P815Y P388 (parent) L1210 RDM 4
H-2 type
Strain of origin
Mode of induction
DBA/Z C57BLl6 BALBJc DBAl2 DBN2 DBA/2 AKR
Carcinogen Carcinogen Moloney virus Carcinogen Carcinogen Carcinogen Spontaneous
(2 P388D, was maintained only as an ascites form.
in vitro
Strain used for passage
in viva -a
C57BLJ6 BALBlc DBAl2 DBA/2 DBA/2 AKR
Reference 20 20 20 21 20 20 22
cultured tumor cell line. It was not studied in the
in viva
274
HANDWERGER
AND
KOREN
Following two washes in E-IO% FCS the adherent macrophage-enriched tion was removed with a rubber policeman.
popula-
Cell Marker Studies 1. Surface immunoglobulin
(S-lg). S-Ig was identified by the use of fluoresceinconjugated polyvalent rabbit anti-mouse Ig (Cappel Laboratories, Downington, Pa., Lot 6377) as previously described (3). This reagent stained 47% of spleen cells, 30% of mesenteric lymph node cells and less than 0.5% of thymocytes from normal BALB/c mice. 2. Brain-associated theta (BAO). BAB was detected by both direct and indirect fluorescence using rabbit anti-mouse brain serum prepared by the method of Golub (4) and used as previously described (3). This reagent stained 34% of spleen cells, 57% of mesenteric lymph node cells and 98% of thymocytes from normal BALB/c mice. 3. Aggregated lg. Unlabeled and fluorescein-conjugated, heat-aggregated human Ig was prepared from human Cohn fraction II (HGII, Sigma Chemical Co., St. Louis, MO.) by the method of Dickler (5). Both direct and indirect fluorescence techniques were used to detect cells bearing a receptor for aggregated Ig. 4. Antibody-antigen complexes (7s EA). Sheep erythrocytes (SRBC) coated with 7s anti-SRBC antibodies (Cordis Laboratories, Miami, Fla.) were prepared and used according to the method of Shevach et al. (6). Controls consisted of tumor cells incubated with uncoated SRBC or SRBC coated with 19s Forssman antibody (Cordis). 5. Latex phagocytosis. Phagocytes were identified by their ability to ingest latex beads. One million cells were incubated in a shaking water-bath at 37°C for 45 min in E-20% FCS containing 6 x IO7 latex beads (Dow Chemical, Indianapolis, Ind.). The cells were then washed three times and wet-mount slides were prepared. A minimum of 200 cells was counted. Cells ingesting three or more beads were considered positive. Preparation
qf Anti-TNP
Serum
BDF, and C57BL/6 mice were injected in the foot pads with trinitrophenyl (TNP)-modified M.vcobacterium tuberculosis H 37 Ra incorporated in incomplete Freund’s adjuvant. After 2 weeks the mice were boosted with 100 pg of TNPmodified M. tuberculosis, intraperitoneally, and bled 2-3 weeks later. Sera were also obtained from mice immunized with TNP-modified keyhole limpet hemocyanin (TNP-KLH). All sera were heat inactivated at 56°C for 40 min. ADCC
Assa)
Chicken erythrocytes were labeled with 51Cr as described by Perlman and Perlman (7) washed and resuspended in phosphate-buffered saline (0.01 M P04, 0.14 M NaCl. pH 7.2) at a concentration of 4 x 10’ CRBC/ml. One milliliter of cells was added to 3 ml of 0.28 M cacodylate buffer containing 25 mg of trinitrobenzene sulfonate (TNP) and incubated at 4°C for 30 min. The cells were then washed three times in BSS-10% FCS. The 51Cr-labeled, TNP-modified CRBC were added in 50 ~1 to 1 ml of effector cells in 12 x 75-mm round-bottomed plastic tubes (Falcon #2052). Mouse anti-TNP serum at a final dilution of IO-3 was then
NATURE
OF
EFFECTOR
CELL
IN
275
ADCC
added in 50 ~1 and the cells incubated at 37°C for 3 hr. All samples were run in triplicate. Control tubes to determine spontaneous release contained effector cells and Wr-labeled, TNP-modified CRBC without added anti-TNP serum. At the end of the incubation, 1 ml of BSS-10% FCS at 4°C was added to each tube. After centrifuging at 700g for 10 min at 4”C, 1 ml of supernatant fluid was removed for counting in a well-type gamma scintillation counter (AmershamSearle, Chicago, Ill.). The percentage of specific lysis was calculated using the following formula: (E - S
1x
Specific lysis (%) =[ T - BG)
100
where E = mean counts of experimental group; S = mean counts from spontaneous release tubes; T = mean counts released following repetitive freeze-thawing; and BG = machine background counts. The effector cell dependency of the assay was assessed by measuring the percentage of 51Cr released in tubes containing antibody and target cells in the absence of effector cells. At no time did this value differ significantly from the mean counts of the spontaneous release tubes and never (even at 20 hr) did it exceed by greater than 5% the amount of 51Cr released by hapten-modified targets in the absence of both effecters and antibody. RESULTS
Cell Marker Characteristics of Tumor Cell Lines As illustrated in Table 2, two of the in vitro cultured tumor cell lines (EL 4 and RDM 4) are &bearing, Ig-negative, nonphagocytic lymphoid cells, and, therefore, are considered to be T lymphocytes; L1210 cells are Ig bearing, 0 negative and nonphagocytic and, therefore, presumably are B cells; P388 (parent) and LSTRA TABLE CELL MARKER
Tumor P388D, EL4 RDM 4 L1210 P388 (parent) LSTRA PSlSY
CHARACTERISTICS
Latex phagocytosisn fP -
2
OF CULTURED
TUMOR
CELL LINES
s-1g
BA@-
Fed
Ceil classification
-c + -
+ + -
+ + + + + +
Macrophage T lymphocyte T lymphocyte B lymphocyte “Null” lymphoid cell “Null” lymphoid cell Mast cell
a Latex phagocytosis, ability to phagocytize latex beads during 45-mitt incubation at 37°C. Cells ingesting more than three beads were considered positive. b S-Ig, surface Ig bearing; stains with fluorescein-conjugated polyvalent rabbit anti-mouse Ig. c BAB, Brain-associated theta bearing; reacts with rabbit anti-mouse brain serum. * Fc, Bears a receptor for heat-aggregated human Ig and/or antibody-antigen complexes (7s EA). c (+), Possess the cell marker characteristic under study; (-), does not possess the cell marker under study. For each cell marker a minimum of 200 cells was scored.
276
HANDWERGER
4NII
KOREN
are non-8 and non-Ig-bearing, non-phagocytic “null” lymphoid cells; P815Y is a mast cell; and as we have previously demonstrated (I), P388D, is a macrophagelike tumor. All of the in vitro cultured tumor cell lines except EL 4 bear a receptor for heat-aggregated human Ig and/or antibody-antigen complexes (7s EA). If cells of host origin (i.e.. nontumor cells) are excluded, the cell marker characteristics of the in vivo ascites tumors are identical to those of the corresponding in vitro cultured tumor cell lines. ADCC
Effector
Cell Ac,tivity
of Tumor-Containing
A.scite.v Cells
The ability of ascites cells obtained from tumor-bearing animals to act as effectors in the antibody-dependent cell-mediated cytolysis of anti-TNP coated, TNP-modified CRBC is demonstrated in Table 3. All of the tumor-containing ascites cell preparations were active as effector cells against antibody-coated target cells. No significant lysis of TNP-modified CRBC occurred in the absence of anti-TNP sera, in the absence of effector cells. or in the presence of normal C57BL/6 or BDF, serum. Although in Table 3 the cytotoxicity mediated by LSTRA-containing ascites cells was distinctly less than that of other tumorcontaining ascites cells, the percentage of specific lysis nevertheless was highly significant (P < 0.005 at an effector to target ratio of 10: 1). These data suggest that either most, if not all, tumor cell lines are capable of killing antibody-coated, hapten-modified CRBC or that the cytotoxic activity exhibited by the tumor-containing ascites cells is due not to the tumor cells themselves but rather to contaminating cells of host origin. These ascites tumor cell preparations, in fact, contained 4-18% macrophages by the criterion of latex ingestion. ADCC Effector Cell Activity of Plastic-Adherent und Nonadherent Peritoneal Cells Obtained from Tumor-Containing Ascites To explore whether the activity of the in \+vo passaged tumor cell preparations
was due to contaminating macrophages, macrophages were removed from P815Y-containing ascites by adherence methods. Under the conditions employed the tumor cells were nonadherent and macrophages were adherent to the plastic
Tumor
cell
P388 (parent) EL4 LSTRA P815Y RDM 4
13.2 11.4 2.5 12.6 14.8
a Three-hour assay, IO5 target b Effector to target cell ratio.
ir 0.3 IO.2 2 0.1 -t 0.0 -c 0.6 cells.
9.7 7.5 1.4 9.0 11.5
1 t 2 t 2
0.2 0.1 0.1 0.0 0.1
NATURE
OF
EFFECTOR
CELL
IN
277
ADCC
tissue culture flasks. As illustrated in Table 4, when adherent cells were removed from P815Y-containing, ADCC-active peritoneal cell preparations, effector activity was markedly diminished; moreover, that activity was recoverable in the adherent cell population. These data suggest that the cytotoxic effector activity of PSlSY-containing ascites is due to a contaminating adherent cell population and not the tumor cells themselves. ACDD Effector Cell Activity of In Vitro Cultured Tumor Cell Lines To explore further whether the activity of tumor-containing ascites populations was due to contaminating macrophages, long-term in vitro cultures of each of the in vivo passaged tumors were established. During the establishment of the cultures, macrophages were removed from the tumor cell populations by adherence methods. Table 5 illustrates the effector cell activity of the cultured tumor cell TABLE ADCC
EFFECTOR
CELL
4
ACTIVITY OF PLASTIC-NONADHERENT OBTAINED FROM P8lSY-CONTAINING
Duration of adherence Cell fraction
(hr)
Unseparated Nonadherent Nonadherent Nonadherent Adherent
None 1r T P 3r
AND -ADHERENT ASCITES
PERITONEAL
CELLS
Specific lysis (% 2 SE)a 5:1* 22.5 4.5 1.9 1.8 23.5
k + 2 r r
2.5: lb 1.3 0.2 0.1 0.2 1.2
12.8 k 1.5 5 0.8 2 0.6 2 13.1 -t
1.25:l”
0.7 0.1 0.1 0.1 0.8
5.9 0.7 0.3 0.0 6.1
2 + 2 2 2
0.1 0.1 0.1 0.9 0.1
a Three-hour assay, 5 x IO5 target cells. b Effector to target ratio. c Tumor-containing peritoneal cells were incubated in plastic tissue culture flasks at 37°C for 1, 2 or 3 hr. Aliquots of the nonadherent cells as well as the 3-hr adherent population were tested for ADCC effector activity. TABLE ADCC
EFFECTER
CELL
ACTIVITY
5 OF CULTURED
TUMOR
CELL
LINES
Specific lysis (% ? SE)” Tumor cell P388D, P388 (parent) EL4 LSTRA P815Y RDM 4 Ll210
10: lb 34.1 0.5 0.0 0.5 0.0 - I.0 0.8
-r- 2.8 + 0.7 2 0.3 2 0.3 f 0.2 2 0.1 k 0.2
a Three-hour assay, I x IO5 target cells. b Effector to target cell ratio.
5:1* 28.7 0.1 0.1 0.5 0.0 - 0.8 0.3
zk 1.3 r 0.2 + 0.2 2 0.3 f 0.2 2 0.1 2 0.1
178
HANDWERCER
AND
KOREN
lines in a 3-hr cytotoxicity assay. In contrast to the activity of the tumorcontaining ascites, none of the cultured tumors, other than P388D,, was active in mediating cytolysis. The lack of effector activity was not a consequence of a shift in the kinetics of killing, for cytotoxicity was not apparent even after 20 hr of incubation of effector cells with antibody-coated targets (Table 6). P388D, was active as an effector when cultured in either spinner or suspension culture. Water-lysis studies (9) and time-lapse microcinematography indicate that the interaction of P388D, cells with hapten-modified CRBC involves both phagocytosis and contactual lysis (data not shown). When ADCC-inactive, macrophage-depleted, in vitro cultured tumor cells were injected intraperitoneally into syngeneic hosts, the tumor-containing ascites collected 6 days later again possessed effector cell activity, while tumor cells maintained in cultures still lacked cytotoxicity capability (Table 7). This experiment demonstrated that the lack of ADCC activity of in \+tro cultured tumors was not simply due to an irreversible change in the tumor cells and suggested once again that the effector cell activity of the ascites populations was due to “contaminating” cells of host origin. ADCC Effector DBAIZ Mice
Cell Acti\!ity
of Noninduced
Peritoneal
Cells Ji-orn Normd
If macrophages are the cells in tumor-containing ascites that are active in the ADCC lysis of hapten-modified CRBC, then normal peritoneal macrophages should also possess cytotoxic activity in our system. As illustrated in Table 8. normal noninduced peritoneal cells (consisting of 45-60s latex-ingesting cells) were active as cytotoxic effecters. Removal of adherent cells resulted in a marked diminution of effector cell activity. The adherent, macrophage-enriched population retained cytotoxic capability. DISCUSSION
The nature of the effector cell in antibody-dependent, cell-mediated cytolysis remains controversial. The use of highly purified cell populations should aid in resolving this controversy. We have recently described a macrophage-like tumor cell line, P388D,, which is active as an effector in the ADCC lysis of anti-TNP TABLE
6
Specific Cells EL4 LSTRA P815Y RDM 4 Normal C57BW6 a Effector
3 Hours
spleen
to target
cells
cell ratio
- 0.6 0.2 d.2 0.3 12.4 was 20: 1; 1 x 105 target
+ + t t -+
cells.
0.4 0.1 0.5 0.2 0.8
lysis
(% -C SE)” 20 Hours 0.0 0.8 I.1 0.9 40.5
i -c _t it
0.4 0.3 0.4 0.8 0.9
NATURE
OF
EFFECTOR
CELL
TABLE ADCC EFFECXOR
CELL ACTIVITY
IN
7
OF ASCITES CELLS OBTAINED CULTURED TUMOR CELL LINES
Effector to target Tumor cell EL4 P815Y u Three-hour
assay, 1
x
FROM ANIMALS
INJKXD
WITH
Specific lysis (% + SE)”
cell ratio
Cultured
20: 1 10: 1 20: 1 10: I
-0.8 2 0.3 0.1 -t 0.2 -0. I k 0.0
Ascites 11.9 8.4 14.7 6.5
-t 0.5 2 0.6 -c 0.1 2 0.8
lo5 target cells.
TABLE ADCC EFFECTOR
279
ADCC
CELL ACTIVITY
OF NONINDUCED
8 PERITONEAL
Duration of adherence
CELLS FROM NORMAL
DBA/2 MICE
Specific lysis (% ? SE)”
Cell fraction
(hr)
10: I*
5:1*
2.5: I*
Unseparated Nonadherent Adherent
None T 3’
10.6 t 1.1 1.8 ” 0.5 8.7 t 0.5
3.6 k 0.4 -1.0 * 0.5 3.5 2 0.2
2.9 2 0.4 -4.2 5 0.5 1.8 + 0.3
a Three-hour assay, 1 x IO5 target cells. * Effector to target cell ratio. r Tumor-containing peritoneal cells were incubated in plastic tissue culture flasks at 37°C for 3 hr. Aliquots of the nonadherent cells as well as the 3-hr adherent population were tested for ADCC effector activity.
coated, TNP-modified CRBC (1). The present communication demonstrates that plastic-adherent cells derived from normal noninduced peritoneal cells and from tumor-containing ascites cells are also active as effector cells in the ADCC lysis of antibody-coated, hapten-modified CRBC. Tumor cells with cell marker characteristics of T lymphocytes, B lymphocytes, “null” lymphoid cells and mast cells do not possess cytotoxic capability. These data suggest that adherent cells, presumably macrophages, can act as effector cells in antibody-dependent, cellmediated cytolysis and that the activity of P388D, is probably due to its macrophage-like characteristics rather than to its tumor cell nature. This conclusion is further supported by the observation that a murine reticulum cell sarcoma with macrophage-like properties (8) and an in vitro established line of mouse peritoneal macrophages (9) also have the ability to mediate ADCC lysis. Scomik and Cosenza (10) have recently reported that proteose-peptone induced peritoneal macrophages and that splenic adherent cells possess effector cell activity in ADCC. Holm and Hammarstrom (11) and Holm (12), furthermore, have demonstrated that human peripheral blood monocytes and human peritoneal macrophages are cytotoxic to antibody-coated human erythrocytes.
280
HANDM’ERGER
ASD
KOKtN
It should not be concluded, though, that the macrophage is the only cell capable of mediating ADCC lysis. In systems utilizing target cells other than haptenmodified CRBC. evidence has been presented which suggests that “null” cells or K cells (19,25-27), B lymphocytes (28-32). human lymphoblastoid cell lines (33), and human polymorphonuclear leukocytes (34) may be active as effecters in antibody-dependent, cell-mediated cytolysis. The present study demonstrates the need for highly purified cell populations in evaluating the nature of the effector cell in ADCC. When the cell population being studied is not totally pure. then the observed activity of that population may be due to a minor “contaminating” cell population and not to the major cell type. Thus, peritoneal cells obtained from the ascites of animals bearing nonmacrophage tumors were active as cytotoxic effecters. Though tumor cells comprised greater than 80% of the peritoneal cells. the effector cell activity was not due to the tumor cells themselves, but rather to contaminating adherent cells of host origin. In this regard the use of in t?tro cultured tumor cell lines with different cell marker characteristics as purified cell populations offers a unique model for the evaluation of antibody dependent, cell-mediated cytolysis. Finally, several investigators have documented that the cytotoxic effector cell in ADCC possesses a receptor for the Fc portion of IgG (2,13- 19). The studies reported here demonstrate that although an Fc receptor may be required, its mere presence is not sufficient to endow a cell with cytotoxic capability. Thus P388D,, L1210, P388 (parent), LSTRA, P815Y and RDM 4 cells all possess a receptor for heat-aggregated human Ig and/or antibody-antigen complexes, yet P388D, was the only tumor cell active as an effector in the ADCC lysis of antibody-coated. hapten-modified CRBC. ACKNOWLEDGMENTS The authors are grateful to Drs. J. R. Wunderlich, William Terry, and Steven D. Douglas for their help and advice. We thank Ms. Terry Peters for her excellent technical assistance and Mr. Walter Lyles for the supervision of animal facilities. This research was supported by the National Institutes of Health. USPHS grants, No. AI-12478-01 and HL-06314-15. and by grants from the National Leukemia Association, the Arthritis Foundation. Minnesota Chapter, and the Minnesota Medical Foundation. Addendum to tert. The work reported here was presented at the Annual Meeting of the American Association for Cancer Research in April 1975 (23). Subsequently, Tracey, et al. (24). using different murine tumors, have independently confirmed that the ADCC activity of ascites cells obtained from mice bearing nonmacrophage tumors is due to an adherent cell of host origin.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11.
Koren, H. S.. Handwerger, B. S.. and Wunderlich. J. R., .I. fmmunol. 114, 894. 1975. Mosier, D. E., Science 1.58, 1573, 1967. Handwerger. B. S.. and Schwartz, R. H.. Transplantation 18, 544. 1974. Golub, E., Cell. Immunol. 2, 353, 1971. Dickler. H. B., .I. hp. Med. 140, 508. 1974. Shevach, E. M., Ellman, L., Davie, Y. M., and Green, I., Blood 39. I. 1972. Perlmann, P.. and Perlmann. H., Cell. Immunol. 1, 300, 1970. Ralph, P., Prichard, J., and Cohn, M.. J. Immunol. 115, 898, 1975. Walker, W. S., and Demus. A.. J. lmmunot. 115, 765. 1975. Scomik, J. C., and Cosenza, H., J. Zmmunol. 113, 1527. 1974. Holm, G., and Hammarstrom, S.. Clin. Exp. Immunol. 13, 29. 1973.
NATURE
OF
EFFECTOR
CELL
IN
ADCC
281
Holm, G., Int. Arch. Allergy 43, 671, 1972. 13. Cerottini, J. C., and Brunner, K. T., Advan. fmmuno/. 18, 67. 1974. 14. Perlmann, P., Perlmann, H., and Wigzell, H.. Transplant. Rev. 13, 91, 1972. 1.5. Larrson, A., and Perlmann, P., Znt. Arch. Allergy 43, 80, 1972. 16. Moller, G., and Svehag, S. E., Cell. Immunol. 4, 1, 1972. 17. Hallberg, T., Stand. J. Immunol. 3, 117. 1974. 18. Wisloff, F., Michaelson, T. W., and Froland, S. S., Stand. J. Immunol. 3, 29, 1974. 19. Calder, E. A., Urbaniak, S. J., Penhole. W. J., and Irvine, W. J., C/in. Exp. Immunol. 18, 579. 1974. 20. Shevach, E. M., Stobo, J. D., and Green, I., J. Immunol. 108, 1146, 1972. 21. Dunn, T. B. and Potter, M., J. Nat. Cancer Inst. 18, 587, 1957. 22. Wunderlich, J. R., personal communication. 23. Koren, H. S. and Handwerger, B. S., Abstr. Amer. Ass. Cancer Res. 501, 126. 1975. 24. Tracey, D. E., Pross, H. F., Jondal, M., and Witz, I. P., submitted for publication. 25. Greenberg, A. H., Hudson, L., Shen, L., and Roitt, I. M., Nature New’ Viol. 242, 111, 1973. 26. Greenberg, A. H., Shen, L., Walker, L., Amaiz-Villena, A., and Roitt, I. M. Eur. J. Immunol. 5, 474, 1975. 27. Wisloff, F., and Froland, S. S., &and. J. Immunol. 2, 151, 1973. 28. Forman, J., and Moller, G., Transplant. Rev. 17, 108, 1973. 29. Yust, I., Wunderlich, J. R., Mann, D. L., and Buell, D. N., J. Zmmunol. 110, 1672, 1973. 30. Van Boxel. J. A., Stobo, J. D., Paul, W. E., and Green, I., Science 175, 194, 1972. 31. Zighelboim, J., Gale, R. P., Chiu, A., Bonavida. B., Ossorio, R. C., and Fahey, J. L., C/in. Immunol. Immunopathol. 3, 193, 1974. 32. F’I’OSS. H. F., Tracey, D. E.. Wigzell, H., and Schirrmacher, V., &and. J. Immune/. 3,769, 1974. 33. Lefebre, J-C., Petitprez, A., Juy, D., and Bona, C.. Eur. J. Zmmunol. 5, 266, 1975. 34. Gale, R. P.. Zighelboim. J., Ossorio, C., and Fahey, J. L., Clin. Res. 22, 180A. 1974. 12.