Clin. cxp. Immunol. (1978) 34, 274-280.
Heterogeneity of human lymphocyte Fc receptors II. RELATIONSHIP TO ANTIBODY-DEPENDENT, CELL-MEDIATED CYTOTOXICITY B. J. GORMUS, MILDRED WOODSON & M. E. KAPLAN Hematology Section, Department of Medicine V.A. Hospital, Minneapolis, Minnesota, and the Departments of Microbiology and Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA
(Received 10 April 1978) SUMMARY
Human peripheral blood lymphocytes (PBL) were incubated (stripped) with pronase or papain and compared with unstripped lymphocytes for their ability to mediate antibody-dependent, cellmediated cytotoxicity (ADCC). Despite marked removal or inactivation of receptors for heataggregated IgG (aggG) by proteolytic digestion, and pronounced changes in the percentages of cells rosetting with IgG-sensitized erythrocytes (EA) (decreased by papain, increased by pronase), stripped PBL functioned normally in ADCC. Stripped and unstripped lymphocytes were pre-treated with aggG to determine the role ofaggG receptors in ADCC. AggG almost totally abolished ADCC by unstripped PBL, but inhibited ADCC by enzyme-stripped lymphocytes relatively poorly. Neither untreated nor stripped PBL were able to induce cytotoxicity of chicken erythrocyte (CRBC) target cells sensitized with the Fab'2 fragment of anti-CRBC IgG antibody (CRBC-A). Exposure of PBL to EA monolayers composed of CRBC-A or of sheep erythrocytes (SRBC) sensitized with rabbit anti-SRBC IgG antibody (SRBC-A) depleted PBL of cells that rosetted with CRBC-A and with human Rh-positive, type 0 erythrocytes sensitized with the human antiRh serum Ripley (HRBC-A Ripley). Non-adherent cells were incapable of binding aggG and had markedly diminished cytotoxicity in ADCC. Similarly, exposure of PBL to HRBC-A Ripley monolayers resulted in non-adherent cells that were incapable of rosette formation with HRBC-A or CRBC-A, failed to bind aggG, and exhibited significantly diminished ADCC activity. These studies indicated that: (1) cytotoxic effector PBL active in ADCC (K cells) have receptors for aggG and for EA; (2) PBL deficient in functional aggG receptors (enzymatically inactivated or removed) are capable of inducing normal levels of ADCC; (3) aggG and EA receptors appear to be closely associated on native K-cell membranes; (4) there is no clear-cut relationship in a given lymphocyte population between the presence of either aggG or EA receptors and ADCC activity; and (5) populations of PBL binding HRBC-A Ripley overlap with, and may be identical to, those binding aggG and other types of EA complexes.
INTRODUCTION The mechanisms involved in antibody-dependent, cell-mediated cytotoxicity (ADCC) are not completely understood. However, it is clear that membrane Fc receptors of effector killer (K) lymphocytes must interact with the Fc component of IgG antibody specifically bound to target cells (Larsson & Perlmann, 1972; Moller & Svehag, 1972; Hallberg, 1974; MacLennan et al., 1973; MacLennan, Connel & Gotch, 1973; Wisl0ff, Michaelsen & Fr0land, 1974; Dickler, 1974; Michaelsen, Wisl0ff & Natvig, 1974). After this interaction has occurred, K cells lyse the target cells. ADCC is easily demonstrable in vitro and may Correspondence: Dr B. J. Gormus, (151) Research, V.A. Hospital, Minneapolis, Minnesota 55417, USA. 0099-9104/78/0110-0274$02.00 ©1978 Blackwell Scientific Publications
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Heterogeneity of human lymphocyte Fc receptors. II.
275
have important in vivo functions (Hakala et al., 1974; O'Toole et al., 1974; Yu & Peter, 1974; d'Apice & Morris, 1974; Schneider et al., 1975; Shore etal., 1976; Pearson & Orr, 1976). Since ADCC may subserve an important role in immunological defence mechanisms (Fr0land & Natvig, 1973), it is important to define this phenomenon as precisely as possible. We have recently found that lymphocyte membrane receptors for heat-aggregated IgG (aggG) could be readily distinguished from the receptors that bind erythrocytes sensitized with IgG antibody (EA), since they differed markedly in their susceptibility to proteolysis (stripping) and in their re-expression in tissue culture (Gormus et al., 1978). These observations, and observations by others (Hallberg, 1974; Wisl0ff et al., 1974; Dickler, 1974) indicating that ADCC can be almost totally inhibited by pre-treatment of effector peripheral blood lymphocytes (PBL) with aggG, raisedquestions regarding the identity of Fc receptors required for ADCC. Thus, in the present report, studies were undertaken to compare the abilities of native (untreated) and of stripped PBL (having markedly altered abilities to bind aggG and EA) to mediate ADCC before and after aggG pre-treatment. Since our previous observations (Gormus et al., 1978) suggested that binding of human Rh-positive, type 0 erythrocytes (HRBC-A) sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley) was similar to binding of chicken erythrocytes (CRBC) sensitized with rabbit anti-CRBC IgG (CRBC-A), experiments were conducted to test the suggestion of others that HRBC-ARipley complexes have PBL-binding specificity different from other EA complexes (Fr0land, Natvig & Michaelson, 1974a, Fr0land, Wisloff & Michaelson, 1974b; Kurnick & Grey, 1975; Dickler, 1976).
MATERIALS AND METHODS Methods for isolation of human PBL free of monocytes, detection of membrane receptors, proteolytic stripping of PBL with pronase and papain, and preparation of Fab'2 fragments of rabbit anti-CRBC IgG have been described in an accompanying report (Gormus et al., 1978). Preparation of rabbit anti-CRBC serum. Rabbits were immunized by intravenous injection of 1 ml of a 10% washed CRBC suspension three times weekly for 3 weeks. Antisera were harvested from animals bled 1 week after the third injection. 5 1Cr-labelling ofCRBC. Blood was collected aseptically in modified Alsever's solution from female white Leghorn chickens, stored at 4°C, and used within 5 days. An aliquot of blood containing 1 x 108 CRBC was removed and washed three times in RPMI-1640. The washed cell pellet was resuspended in 100 pCi of Na51CrO4 (1 mCi/ml, New England Nuclear Corp., Boston, Massachusetts) and incubated for 60 min at 37°C. The labelled cells were washed three times in RPMI-1640 to remove unbound 51Cr and resuspended in 5 ml RPMI containing 6% heat-inactivated foetal calf serum (FCS) that had been absorbed previously with CRBC. After counting, 2 x 106 cells were removed and diluted to 12-5 ml with RPMI-6% FCS. ADCC. The assay was performed as described by Perlmann & Perlmann (1971). Briefly, 8x 105 lymphocytes in 0-2 ml RPMI-6% FCS were mixed with 0-2 ml 51Cr-labelled CRBC suspension (3-2 x 104 CRBC in RPMI-6% FCS) and 0-4 ml of a 1:50,000 dilution of rabbit anti-CRBC serum added. This dilution of anti-CRBC serum had been determined to give optimal cytotoxicity. Various controls were included: (1) a 1: 50,000 dilution of normal rabbit serum (NRS) instead of antiCRBC; (2) graded concentrations of Fab'2 fragments of anti-CRBC IgG above, below, and including 1:50,000 instead of anti-CRBC serum; (3) RPMI-6% FCS instead of anti-CRBC serum; and (4) 8 x 105 unlabelled CRBC instead of lymphocytes. All cells and reagents were prepared in RPMI-6y/ FCS and each tube was set up in duplicate or triplicate. Tubes were incubated for 4 hours at 37°C in 5% C02-air. Thereafter, they were chilled in ice and centrifuged (150 x g) for 10 min at 4°C. Supernatant fluids were separated from pelleted cells and both were counted in a Packard gamma spectrometer. Cytotoxic indices (CI) were calculated as follows: CI= cpm (anti-CRBC)- cpm (NRS) X 100. cpm (Total)- cpm (NRS)
Total radioactivity/tube was determined by counting a diluted aliquot of the 5'Cr-labelled CRBC suspension. Pre-treatment of lymphocytes with aggG. Normal and pronase-, or papain-stripped lymphocytes (1 x 106/mi) from normal donors were incubated with 1 mg/ml of aggG in 0-05M PBS, pH 8-0 at room temperature for 30 min as described by Dickler (1974). One mg/ml of aggG was determined to be excess of the optimal concentration required to give maximal inhibition of ADCC and EA rosetting. Control cells (stripped and unstripped) were treated with buffer alone. PBL were then washed thrice in cold RPMI with centrifugation at 150g for 10 min at 4°C. Thereafter, lymphocytes were resuspended to the appropriate concentrations in RPMI-6% FCS, and studied for their ability to mediate ADCC. Depletion oflymphocytes adhering to EA monolayers. The method of Kedar, Ortiz de Landazuri & Bonavida (1974a) was used. Sheep erythrocytes (SRBC), CRBC, or HRBC were permitted to form monolayers by adherence to poly-L-lysine (PLL) coated, 60 x 15 mm polystyrene tissue-culture dishes (Falcon Plastics, Oxnard, California). After washing to remove excess
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B. J. Gormus, Mildred Woodson
M. E. Kaplan
RBC, the monolayers were sensitized with the IgG fraction of rabbit anti-SRBC antiserum (Cappel Laboratories, Downington, PA.), rabbit anti-CRBC, or Ripley anti-Rh serum, respectively. Control plates were treated with NRS that had been absorbed with appropriate RBC. All sera were heat-inactivated and centrifuged at 150,000g just prior to use, to remove aggG. Aliquots of 2-5 ml containing 107 PBL/ml were added to each dish and the lymphocytes allowed to adhere to the EA monolayers using the centrifugation procedure as described by Kedar et al. (1974a). After incubation, non-adherent lymphocytes were removed, washed twice and immediately studied for surface markers and assayed for cytolytic activity in ADCC.
RESULTS Stripping of lymphocytes with either pronase or papain failed to significantly alter their cytotoxic activities, despite significant removal or inactivation of aggG receptors and pronounced changes in the percentages of cells bearing EA receptors (Table 1). These results indicated that isolated PBL can, TABLE 1. Effects of pronase and papain treatment on PBL receptors and ADCC % Receptor-Positive cells
Lymphocytes
Control Pronase-stripped Papain-stripped
AggG
HRBC-A Ripley
CRBC-A
CI (%)*
13.8+0.9 5 5±0 9t 1 9+ 0-5t
1141+ F0 245+±2-5t 5 6+ 10t
11-4+1-1 17-1+ It 5-9+ 1-6t
41-6+2-0 43-8+4-9 36-2+ 2-7
* Each valve represents the mean+ 1 s.e. of at least twelve observations (see Gormus et al., 1978). t Experimental results that differ significantly (P< 0-005) from control values
A
(Student's t-test).
function normally in ADCC after essentially total removal, or inactivation of, their aggG receptors and after significant increases (pronase-induced) or decreases (papain-induced) in the percentages of cells bearing EA receptors. In studies utilizing Fab'2 fragments of rabbit anti-CRBC IgG for sensitization, no rosetting and little ADCC activity was observed before and after proteolytic treatment of PBL. Normal and stripped PBL failed to bind unsensitized RBC. To further test the efficiency of aggG-receptor removal and to study the relationship between aggG receptors and ADCC, ADCC activities were measured after normal and stripped lymphocytes were exposed to aggG. Pre-treatment of control lymphocytes with aggG resulted in 90%0 inhibition of ADCC, whereas, stripped lymphocytes were significantly more refractory to aggG inhibition (57%° and 28% inhibition of pronase- and papain-stripped lymphocytes, respectively) (Table 2). After aggG pre-treatment of pronase-stripped cells residual cytotoxicity (19.4%) was significantly less than that of pronase-stripped cells not treated with aggG (43-80%) (Table 3). However, residual cytoTABLE 2. Reduction in ADCC by aggG treatment of control and stripped lymphocytes
Effector cells
Control Pronase-stripped Papain-stripped
CI (% change)*
Pt
-903+ 2-6
-
- 57 0+ 5-7
< 0 0005 < 0 0005
-27-8+ 6-1
* Each value represents the mean+ 1 s.e. of at least twelve observations. 1 Significance of difference between stripped and control lymphocytes (Student's t-test).
277 Heterogeneity of human lymphocyte Fc receptors. II. toxicity remaining after aggG pre-treatment of papain-stripped cells (26 6%) differed only slightly from papain-stripped cells not treated with aggG (36.2%), but differed markedly from unstripped, aggGtreated control cells (4 5%4). Thus, papain treatment of PBL almost totally abolished their ability to bind aggG (Table 1) and almost completely prevented aggG inhibition of ADCC induced by these cells. Pronase treatment reduced the number of aggG-binding cells by only 60% (Table 1), and ADCC mediated by these cells remained partially inhibitable by aggG (Tables 2 and 3). However, this degree of inhibition was significantly less than that observed with unstripped, aggG-treated cells. TABLE 3. ADCC with and without aggG treatment of control and stripped lymphocytes
(CI, %)* - aggG
Lymphocytes
41V6+2-0 Control Pronase-stripped 438+ 4.9 Papain-stripped 36-2+ 2-7
+aggG
Pt
4-5+±06
< 00005 < 0-0005 < 0 025
19-4+ 3-3 26-6+ 3.4
* Each value represents the mean+ 1 s.e. of at least twelve observations. t Significance of differences between aggG-treated and the corresponding aggG-nontreated samples (Student's ttest).
TABL 4. Effects of depletion of PBL by adherence to CRBC-A monolayers
%Y of Cells binding* Lymphocytes
CRBC-A
HRBC-A Ripley
Unfractionated Controlt Non-adherent to CRBC-A
10-2+ 2-3 10 0+ 4 0 18+ 1-I
14-0+ 2-2 9.8+ 15 1 0+ 1 0T
aggG
CI (%O)*
11-1+0-9 67-5+ 3-1 10-7+ 24 59-6+ 6-0 1-9+ 0-7t 7 4± 19t
* Each value represents the mean+ 1 s.e. of three observations. t PBL non-adherent to E-NRS. I Experimental values differing significantly (P< 0.025) from corresponding controls (Student's t-test).
TABLE 5. Effects of depletion of PBL by adherence to HRBC-A Ripley monolayers
% of Cells binding* Lymphocytes
Unfractionated Controlt Non-adherent to HRBC-A Ripley
aggG
CI (%)*
9-7+ 2-7 9-7+ 3-8
11-3+0-7 8-0+ 2-6
49 7+ 5 2 36-7+ 6-4
1-5±12t
2-3+0-9t 14-3+4-41:
HRBC-A Ripley CRBC-A 9-0+ 3-1 10-3+ 5-5
1-3+0-3t
* Each value represents the mean+ 1 s.e. of three observations. t PBL non-adherent to E-NRS. I Experimental values differing significantly (P< 0 025) from corresponding controls (Student's t-test).
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B. J. Gormus, Mildred Woodson & M. E. Kaplan
Depletion of EA-binding lymphocytes on PLL-EA (CRBC-anti-CRBC) monolayers essentially abolished ADCC and aggG binding by the resultant non-adherent fraction, and totally removed cells that rosetted with CRBC-A or with HRBC-A Ripley (Table 4). Essentially identical results were obtained by depletion on PLL-SRBC-A monolayers (data not shown). In similar experiments, when PBL were depleted of cells binding to HRBC-A Ripley-PLL monolayers, non-adherent cells showed significantly diminished aggG binding and ADCC activity, and failed to rosette with CRBC-A or HRBC-A Ripley (Table 5). In control experiments, lymphocytes exposed to CRBC, SRBC or HRBC monolayers treated with normal sera failed to show significantly diminished ADCC activity, rosetted normally with CRBC-A and HRBC-A Ripley, and bound aggG normally (Tables 4 and 5).
DISCUSSION ADCC is an extremely sensitive index of K-cell Fc-receptor activity (Moller & Svehag, 1972). The blocking of K-cell Fc receptors by aggG would be expected to result in significant inhibition of ADCC if the receptors for aggG and EA (CRBC-A) are identical. However, as we have demonstrated in an accompanying paper (Gormus et al., 1978), these receptors differ in their susceptibility to proteolytic digestion by pronase and papain. Stripping of PBL with either of these two enzymes resulted in marked diminution in aggG-receptor sites; concomitantly, papain induced a significant decrease in EA-receptor activity, whereas pronase digestion markedly enhanced EA-rosette formation. If there are different receptors for aggG and EA on individual peripheral blood K cells, it would be of interest to determine which, if either, receptor is involved in ADCC. The data indicate that (1) proteolysis leading to significant diminution in aggG receptors on PBL does not alter the ability of these cells to mediate ADCC (Table 1); (2) pre-treatment of PBL with aggG almost totally abrogates ADCC by normal lymphocytes, but inhibits ADCC by enzyme-stripped lymphocytes to a much lesser degree (Table 2). From these data we conclude that aggG receptors are not required for ADCC. However, removal of aggG receptors resulted in significant decreases in the susceptibility to ADCC inhibition by aggG. This observation suggests that aggG receptors almost certainly are present on normal K cells in close association with the Fc receptors involved in ADCC. These findings imply steric inhibition or modulation of Fc receptors that are involved in ADCC by membrane-bound aggG. If a reasonable assumption is made that Fc receptors of the EA type are required for ADCC (since the target cell itself is an EA, i.e. CRBC-A), then it is necessary to explain the poor correlation between the percentages of cells binding EA and the degree of ADCC induced by a given lymphocyte population. Thus, papain-stripping results in significant diminution in the percentages of PBL rosetting with EA but does not induce a parallel decrease in ADCC activity. Conversely, pronase induces marked increases in the percentages of PBL rosetting without a concomitant increase in ADCC activity. There are several possible explanations for these apparently conflicting findings: (1) EA receptors detected by the rosetting methodologies employed may be different from those that induce ADCC. However, the studies demonstrating depletion of K cells by EA monolayers provide strong evidence against this hypothesis; (2) it is possible that receptors that induce ADCC by stripped PBL are different (e.g., not Fc receptors) from those that mediate ADCC by untreated lymphocytes. However, the studies demonstrating lack of cytotoxic activity against unsensitized CRBC or CRBC sensitized with Fab'2 antibody by untreated, as well as by proteolytically stripped, lymphocytes strongly suggest that Fc receptors are involved; and (3) heterogeneity may exist among EA receptors, papain removing these receptors from non-K cells and pronase exposing EA receptors on a yet unidentified subpopulation of lymphocytes that are incapable of supporting ADCC. At present, the last hypothesis (number 3) offers the most reasonable explanation for the results obtained. Indeed, other investigators, in particular Cordier, Samarut & Revillard (1976), and Revillard et al. (1975) have published data strongly supporting the concept of EA-receptor heterogeneity. Thus, Cordier et al. (1976) have shown that PBL depleted of lymphoid cells having very high avidity EA receptors (1-2X% of total PBL) byrosetting, totally inhibited ADCC activity; the residual fraction of PBL having less avid Fc receptors were incapable of mediating ADCC. Revillard etal. (1975),
Heterogeneity ofhuman lymphocyte Fc receptors. II.
279
observed decreases in EA rosetting PBL with no change in ADCC activity after treatment with papain (1975). Perlmann et al. (1975), utilizing an ADCC plaque assay technique, found that approximately 1% of PBL are capable of mediating ADCC, whereas much larger percentages of PBL have demonstrable EA receptors by conventional rosetting techniques (Fr0land & Natvig, 1973; Revillard et al., 1975; Cordier et al., 1976). Thus, it appears from these and our data (1) that not all cells that have EA receptors are capable of inducing ADCC; (2) that EA receptors that are inactive in ADCC are more readily removed by papain digestion; and (3) the cryptic EA receptors exposed by pronase digestion are inactive in ADCC. Kedar, Ortiz de Landazuri & Fahey (1974b) utilizing rat-spleen lymphocytes, reported that EAreceptor modulation, induced by proteolysis with various concentrations of pronase and papain, resulted in generally poor correlations between ADCC and the percentages of EA-rosette-forming cells. Our data confirm these observations (Table 1). However, in contrast to the results herein reported, Kedar observed (1) moderate decreases in percentages of EA-rosetting cells and modest diminution in ADCC after lymphocytes were treated with pronase (or trypsin); (2) increased percentages of EA-rosetting cells and somewhat enhanced ADCC activities after treatment with papain (Kedar et al., 1974b). These results, which are totally different from ours, may be due to differences in experimental design and methods (e.g. use of rat-spleen lymphocytes rather than human PBL, differences in EA-rosetting methods, differences in enzyme concentrations or length ofproteolytic digestion, etc.). Indeed, Fr0land et al. (1974b) observed increases in EA rosetting by human PBL after trypsin treatment and Cordier et al. (1976) reported decreases in EA-rosette formation by human PBL after papain treatment. It has been suggested that HRBC-A Ripley rosette with a small subset of human PBL that do not rosette with other EA used to detect Fc receptors (Dickler, 1976). In studies designed to explore this possibility, we have shown that PBL incubated with SRBC-A or CRBC-A monolayers resulted in depletion of (1) cells rosetting with CRBC-A and HRBC-A Ripley; (2) cells binding aggG; and (3) K cells (Table 4). Conversely, exposure of PBL to HRBC-A Ripley monolayers depleted cells that (1) rosetted with HRBC-A Ripley and CRBC-A; (2) bound aggG; and (3) killed target cells in ADCC (Table 5). These data strongly suggest that the population of PBL binding HRBC-A Ripley is essentially identical to the population that binds other types of EA. Moreover, the same PBL appear to bind aggG (Tables 4 and 5). In summary, the data herein presented strongly suggest that: (1) PBL K cells have receptors for aggG and for EA; (2) PBL deficient in functional aggG receptors (enzymatically removed or inactivated) are capable of inducing normal levels of ADCC; (3) aggG and EA receptors appear to be closely associated topographically on K-cell membranes; (4) there is no clear-cut relationship in a given lymphocyte population between the presence ofeither aggG or EA receptors and ADCC activity; and (5) populations of PBL binding HRBC-A Ripley overlap with, and may be identical to, those binding aggG and other types of EA complexes, viz., CRBC-A and SRBC-A. This work was supported by a Research Grant from the Veterans Administration. We wish to express our appreciation Ms Linda B. Good and Juanita Olson for the typing of this manuscript.
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Heterogeneity of human lymphocyte Fc receptors II. RELATIONSHIP TO ANTIBODY-DEPENDENT, CELL-MEDIATED CYTOTOXICITY B. J. GORMUS, MILDRED WOODSON & M. E. KAPLAN Hematology Section, Department of Medicine V.A. Hospital, Minneapolis, Minnesota, and the Departments of Microbiology and Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA
(Received 10 April 1978) SUMMARY
Human peripheral blood lymphocytes (PBL) were incubated (stripped) with pronase or papain and compared with unstripped lymphocytes for their ability to mediate antibody-dependent, cellmediated cytotoxicity (ADCC). Despite marked removal or inactivation of receptors for heataggregated IgG (aggG) by proteolytic digestion, and pronounced changes in the percentages of cells rosetting with IgG-sensitized erythrocytes (EA) (decreased by papain, increased by pronase), stripped PBL functioned normally in ADCC. Stripped and unstripped lymphocytes were pre-treated with aggG to determine the role ofaggG receptors in ADCC. AggG almost totally abolished ADCC by unstripped PBL, but inhibited ADCC by enzyme-stripped lymphocytes relatively poorly. Neither untreated nor stripped PBL were able to induce cytotoxicity of chicken erythrocyte (CRBC) target cells sensitized with the Fab'2 fragment of anti-CRBC IgG antibody (CRBC-A). Exposure of PBL to EA monolayers composed of CRBC-A or of sheep erythrocytes (SRBC) sensitized with rabbit anti-SRBC IgG antibody (SRBC-A) depleted PBL of cells that rosetted with CRBC-A and with human Rh-positive, type 0 erythrocytes sensitized with the human antiRh serum Ripley (HRBC-A Ripley). Non-adherent cells were incapable of binding aggG and had markedly diminished cytotoxicity in ADCC. Similarly, exposure of PBL to HRBC-A Ripley monolayers resulted in non-adherent cells that were incapable of rosette formation with HRBC-A or CRBC-A, failed to bind aggG, and exhibited significantly diminished ADCC activity. These studies indicated that: (1) cytotoxic effector PBL active in ADCC (K cells) have receptors for aggG and for EA; (2) PBL deficient in functional aggG receptors (enzymatically inactivated or removed) are capable of inducing normal levels of ADCC; (3) aggG and EA receptors appear to be closely associated on native K-cell membranes; (4) there is no clear-cut relationship in a given lymphocyte population between the presence of either aggG or EA receptors and ADCC activity; and (5) populations of PBL binding HRBC-A Ripley overlap with, and may be identical to, those binding aggG and other types of EA complexes.
INTRODUCTION The mechanisms involved in antibody-dependent, cell-mediated cytotoxicity (ADCC) are not completely understood. However, it is clear that membrane Fc receptors of effector killer (K) lymphocytes must interact with the Fc component of IgG antibody specifically bound to target cells (Larsson & Perlmann, 1972; Moller & Svehag, 1972; Hallberg, 1974; MacLennan et al., 1973; MacLennan, Connel & Gotch, 1973; Wisl0ff, Michaelsen & Fr0land, 1974; Dickler, 1974; Michaelsen, Wisl0ff & Natvig, 1974). After this interaction has occurred, K cells lyse the target cells. ADCC is easily demonstrable in vitro and may Correspondence: Dr B. J. Gormus, (151) Research, V.A. Hospital, Minneapolis, Minnesota 55417, USA. 0099-9104/78/0110-0274$02.00 ©1978 Blackwell Scientific Publications
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Heterogeneity of human lymphocyte Fc receptors. II.
275
have important in vivo functions (Hakala et al., 1974; O'Toole et al., 1974; Yu & Peter, 1974; d'Apice & Morris, 1974; Schneider et al., 1975; Shore etal., 1976; Pearson & Orr, 1976). Since ADCC may subserve an important role in immunological defence mechanisms (Fr0land & Natvig, 1973), it is important to define this phenomenon as precisely as possible. We have recently found that lymphocyte membrane receptors for heat-aggregated IgG (aggG) could be readily distinguished from the receptors that bind erythrocytes sensitized with IgG antibody (EA), since they differed markedly in their susceptibility to proteolysis (stripping) and in their re-expression in tissue culture (Gormus et al., 1978). These observations, and observations by others (Hallberg, 1974; Wisl0ff et al., 1974; Dickler, 1974) indicating that ADCC can be almost totally inhibited by pre-treatment of effector peripheral blood lymphocytes (PBL) with aggG, raisedquestions regarding the identity of Fc receptors required for ADCC. Thus, in the present report, studies were undertaken to compare the abilities of native (untreated) and of stripped PBL (having markedly altered abilities to bind aggG and EA) to mediate ADCC before and after aggG pre-treatment. Since our previous observations (Gormus et al., 1978) suggested that binding of human Rh-positive, type 0 erythrocytes (HRBC-A) sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley) was similar to binding of chicken erythrocytes (CRBC) sensitized with rabbit anti-CRBC IgG (CRBC-A), experiments were conducted to test the suggestion of others that HRBC-ARipley complexes have PBL-binding specificity different from other EA complexes (Fr0land, Natvig & Michaelson, 1974a, Fr0land, Wisloff & Michaelson, 1974b; Kurnick & Grey, 1975; Dickler, 1976).
MATERIALS AND METHODS Methods for isolation of human PBL free of monocytes, detection of membrane receptors, proteolytic stripping of PBL with pronase and papain, and preparation of Fab'2 fragments of rabbit anti-CRBC IgG have been described in an accompanying report (Gormus et al., 1978). Preparation of rabbit anti-CRBC serum. Rabbits were immunized by intravenous injection of 1 ml of a 10% washed CRBC suspension three times weekly for 3 weeks. Antisera were harvested from animals bled 1 week after the third injection. 5 1Cr-labelling ofCRBC. Blood was collected aseptically in modified Alsever's solution from female white Leghorn chickens, stored at 4°C, and used within 5 days. An aliquot of blood containing 1 x 108 CRBC was removed and washed three times in RPMI-1640. The washed cell pellet was resuspended in 100 pCi of Na51CrO4 (1 mCi/ml, New England Nuclear Corp., Boston, Massachusetts) and incubated for 60 min at 37°C. The labelled cells were washed three times in RPMI-1640 to remove unbound 51Cr and resuspended in 5 ml RPMI containing 6% heat-inactivated foetal calf serum (FCS) that had been absorbed previously with CRBC. After counting, 2 x 106 cells were removed and diluted to 12-5 ml with RPMI-6% FCS. ADCC. The assay was performed as described by Perlmann & Perlmann (1971). Briefly, 8x 105 lymphocytes in 0-2 ml RPMI-6% FCS were mixed with 0-2 ml 51Cr-labelled CRBC suspension (3-2 x 104 CRBC in RPMI-6% FCS) and 0-4 ml of a 1:50,000 dilution of rabbit anti-CRBC serum added. This dilution of anti-CRBC serum had been determined to give optimal cytotoxicity. Various controls were included: (1) a 1: 50,000 dilution of normal rabbit serum (NRS) instead of antiCRBC; (2) graded concentrations of Fab'2 fragments of anti-CRBC IgG above, below, and including 1:50,000 instead of anti-CRBC serum; (3) RPMI-6% FCS instead of anti-CRBC serum; and (4) 8 x 105 unlabelled CRBC instead of lymphocytes. All cells and reagents were prepared in RPMI-6y/ FCS and each tube was set up in duplicate or triplicate. Tubes were incubated for 4 hours at 37°C in 5% C02-air. Thereafter, they were chilled in ice and centrifuged (150 x g) for 10 min at 4°C. Supernatant fluids were separated from pelleted cells and both were counted in a Packard gamma spectrometer. Cytotoxic indices (CI) were calculated as follows: CI= cpm (anti-CRBC)- cpm (NRS) X 100. cpm (Total)- cpm (NRS)
Total radioactivity/tube was determined by counting a diluted aliquot of the 5'Cr-labelled CRBC suspension. Pre-treatment of lymphocytes with aggG. Normal and pronase-, or papain-stripped lymphocytes (1 x 106/mi) from normal donors were incubated with 1 mg/ml of aggG in 0-05M PBS, pH 8-0 at room temperature for 30 min as described by Dickler (1974). One mg/ml of aggG was determined to be excess of the optimal concentration required to give maximal inhibition of ADCC and EA rosetting. Control cells (stripped and unstripped) were treated with buffer alone. PBL were then washed thrice in cold RPMI with centrifugation at 150g for 10 min at 4°C. Thereafter, lymphocytes were resuspended to the appropriate concentrations in RPMI-6% FCS, and studied for their ability to mediate ADCC. Depletion oflymphocytes adhering to EA monolayers. The method of Kedar, Ortiz de Landazuri & Bonavida (1974a) was used. Sheep erythrocytes (SRBC), CRBC, or HRBC were permitted to form monolayers by adherence to poly-L-lysine (PLL) coated, 60 x 15 mm polystyrene tissue-culture dishes (Falcon Plastics, Oxnard, California). After washing to remove excess
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B. J. Gormus, Mildred Woodson
M. E. Kaplan
RBC, the monolayers were sensitized with the IgG fraction of rabbit anti-SRBC antiserum (Cappel Laboratories, Downington, PA.), rabbit anti-CRBC, or Ripley anti-Rh serum, respectively. Control plates were treated with NRS that had been absorbed with appropriate RBC. All sera were heat-inactivated and centrifuged at 150,000g just prior to use, to remove aggG. Aliquots of 2-5 ml containing 107 PBL/ml were added to each dish and the lymphocytes allowed to adhere to the EA monolayers using the centrifugation procedure as described by Kedar et al. (1974a). After incubation, non-adherent lymphocytes were removed, washed twice and immediately studied for surface markers and assayed for cytolytic activity in ADCC.
RESULTS Stripping of lymphocytes with either pronase or papain failed to significantly alter their cytotoxic activities, despite significant removal or inactivation of aggG receptors and pronounced changes in the percentages of cells bearing EA receptors (Table 1). These results indicated that isolated PBL can, TABLE 1. Effects of pronase and papain treatment on PBL receptors and ADCC % Receptor-Positive cells
Lymphocytes
Control Pronase-stripped Papain-stripped
AggG
HRBC-A Ripley
CRBC-A
CI (%)*
13.8+0.9 5 5±0 9t 1 9+ 0-5t
1141+ F0 245+±2-5t 5 6+ 10t
11-4+1-1 17-1+ It 5-9+ 1-6t
41-6+2-0 43-8+4-9 36-2+ 2-7
* Each valve represents the mean+ 1 s.e. of at least twelve observations (see Gormus et al., 1978). t Experimental results that differ significantly (P< 0-005) from control values
A
(Student's t-test).
function normally in ADCC after essentially total removal, or inactivation of, their aggG receptors and after significant increases (pronase-induced) or decreases (papain-induced) in the percentages of cells bearing EA receptors. In studies utilizing Fab'2 fragments of rabbit anti-CRBC IgG for sensitization, no rosetting and little ADCC activity was observed before and after proteolytic treatment of PBL. Normal and stripped PBL failed to bind unsensitized RBC. To further test the efficiency of aggG-receptor removal and to study the relationship between aggG receptors and ADCC, ADCC activities were measured after normal and stripped lymphocytes were exposed to aggG. Pre-treatment of control lymphocytes with aggG resulted in 90%0 inhibition of ADCC, whereas, stripped lymphocytes were significantly more refractory to aggG inhibition (57%° and 28% inhibition of pronase- and papain-stripped lymphocytes, respectively) (Table 2). After aggG pre-treatment of pronase-stripped cells residual cytotoxicity (19.4%) was significantly less than that of pronase-stripped cells not treated with aggG (43-80%) (Table 3). However, residual cytoTABLE 2. Reduction in ADCC by aggG treatment of control and stripped lymphocytes
Effector cells
Control Pronase-stripped Papain-stripped
CI (% change)*
Pt
-903+ 2-6
-
- 57 0+ 5-7
< 0 0005 < 0 0005
-27-8+ 6-1
* Each value represents the mean+ 1 s.e. of at least twelve observations. 1 Significance of difference between stripped and control lymphocytes (Student's t-test).
277 Heterogeneity of human lymphocyte Fc receptors. II. toxicity remaining after aggG pre-treatment of papain-stripped cells (26 6%) differed only slightly from papain-stripped cells not treated with aggG (36.2%), but differed markedly from unstripped, aggGtreated control cells (4 5%4). Thus, papain treatment of PBL almost totally abolished their ability to bind aggG (Table 1) and almost completely prevented aggG inhibition of ADCC induced by these cells. Pronase treatment reduced the number of aggG-binding cells by only 60% (Table 1), and ADCC mediated by these cells remained partially inhibitable by aggG (Tables 2 and 3). However, this degree of inhibition was significantly less than that observed with unstripped, aggG-treated cells. TABLE 3. ADCC with and without aggG treatment of control and stripped lymphocytes
(CI, %)* - aggG
Lymphocytes
41V6+2-0 Control Pronase-stripped 438+ 4.9 Papain-stripped 36-2+ 2-7
+aggG
Pt
4-5+±06
< 00005 < 0-0005 < 0 025
19-4+ 3-3 26-6+ 3.4
* Each value represents the mean+ 1 s.e. of at least twelve observations. t Significance of differences between aggG-treated and the corresponding aggG-nontreated samples (Student's ttest).
TABL 4. Effects of depletion of PBL by adherence to CRBC-A monolayers
%Y of Cells binding* Lymphocytes
CRBC-A
HRBC-A Ripley
Unfractionated Controlt Non-adherent to CRBC-A
10-2+ 2-3 10 0+ 4 0 18+ 1-I
14-0+ 2-2 9.8+ 15 1 0+ 1 0T
aggG
CI (%O)*
11-1+0-9 67-5+ 3-1 10-7+ 24 59-6+ 6-0 1-9+ 0-7t 7 4± 19t
* Each value represents the mean+ 1 s.e. of three observations. t PBL non-adherent to E-NRS. I Experimental values differing significantly (P< 0.025) from corresponding controls (Student's t-test).
TABLE 5. Effects of depletion of PBL by adherence to HRBC-A Ripley monolayers
% of Cells binding* Lymphocytes
Unfractionated Controlt Non-adherent to HRBC-A Ripley
aggG
CI (%)*
9-7+ 2-7 9-7+ 3-8
11-3+0-7 8-0+ 2-6
49 7+ 5 2 36-7+ 6-4
1-5±12t
2-3+0-9t 14-3+4-41:
HRBC-A Ripley CRBC-A 9-0+ 3-1 10-3+ 5-5
1-3+0-3t
* Each value represents the mean+ 1 s.e. of three observations. t PBL non-adherent to E-NRS. I Experimental values differing significantly (P< 0 025) from corresponding controls (Student's t-test).
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B. J. Gormus, Mildred Woodson & M. E. Kaplan
Depletion of EA-binding lymphocytes on PLL-EA (CRBC-anti-CRBC) monolayers essentially abolished ADCC and aggG binding by the resultant non-adherent fraction, and totally removed cells that rosetted with CRBC-A or with HRBC-A Ripley (Table 4). Essentially identical results were obtained by depletion on PLL-SRBC-A monolayers (data not shown). In similar experiments, when PBL were depleted of cells binding to HRBC-A Ripley-PLL monolayers, non-adherent cells showed significantly diminished aggG binding and ADCC activity, and failed to rosette with CRBC-A or HRBC-A Ripley (Table 5). In control experiments, lymphocytes exposed to CRBC, SRBC or HRBC monolayers treated with normal sera failed to show significantly diminished ADCC activity, rosetted normally with CRBC-A and HRBC-A Ripley, and bound aggG normally (Tables 4 and 5).
DISCUSSION ADCC is an extremely sensitive index of K-cell Fc-receptor activity (Moller & Svehag, 1972). The blocking of K-cell Fc receptors by aggG would be expected to result in significant inhibition of ADCC if the receptors for aggG and EA (CRBC-A) are identical. However, as we have demonstrated in an accompanying paper (Gormus et al., 1978), these receptors differ in their susceptibility to proteolytic digestion by pronase and papain. Stripping of PBL with either of these two enzymes resulted in marked diminution in aggG-receptor sites; concomitantly, papain induced a significant decrease in EA-receptor activity, whereas pronase digestion markedly enhanced EA-rosette formation. If there are different receptors for aggG and EA on individual peripheral blood K cells, it would be of interest to determine which, if either, receptor is involved in ADCC. The data indicate that (1) proteolysis leading to significant diminution in aggG receptors on PBL does not alter the ability of these cells to mediate ADCC (Table 1); (2) pre-treatment of PBL with aggG almost totally abrogates ADCC by normal lymphocytes, but inhibits ADCC by enzyme-stripped lymphocytes to a much lesser degree (Table 2). From these data we conclude that aggG receptors are not required for ADCC. However, removal of aggG receptors resulted in significant decreases in the susceptibility to ADCC inhibition by aggG. This observation suggests that aggG receptors almost certainly are present on normal K cells in close association with the Fc receptors involved in ADCC. These findings imply steric inhibition or modulation of Fc receptors that are involved in ADCC by membrane-bound aggG. If a reasonable assumption is made that Fc receptors of the EA type are required for ADCC (since the target cell itself is an EA, i.e. CRBC-A), then it is necessary to explain the poor correlation between the percentages of cells binding EA and the degree of ADCC induced by a given lymphocyte population. Thus, papain-stripping results in significant diminution in the percentages of PBL rosetting with EA but does not induce a parallel decrease in ADCC activity. Conversely, pronase induces marked increases in the percentages of PBL rosetting without a concomitant increase in ADCC activity. There are several possible explanations for these apparently conflicting findings: (1) EA receptors detected by the rosetting methodologies employed may be different from those that induce ADCC. However, the studies demonstrating depletion of K cells by EA monolayers provide strong evidence against this hypothesis; (2) it is possible that receptors that induce ADCC by stripped PBL are different (e.g., not Fc receptors) from those that mediate ADCC by untreated lymphocytes. However, the studies demonstrating lack of cytotoxic activity against unsensitized CRBC or CRBC sensitized with Fab'2 antibody by untreated, as well as by proteolytically stripped, lymphocytes strongly suggest that Fc receptors are involved; and (3) heterogeneity may exist among EA receptors, papain removing these receptors from non-K cells and pronase exposing EA receptors on a yet unidentified subpopulation of lymphocytes that are incapable of supporting ADCC. At present, the last hypothesis (number 3) offers the most reasonable explanation for the results obtained. Indeed, other investigators, in particular Cordier, Samarut & Revillard (1976), and Revillard et al. (1975) have published data strongly supporting the concept of EA-receptor heterogeneity. Thus, Cordier et al. (1976) have shown that PBL depleted of lymphoid cells having very high avidity EA receptors (1-2X% of total PBL) byrosetting, totally inhibited ADCC activity; the residual fraction of PBL having less avid Fc receptors were incapable of mediating ADCC. Revillard etal. (1975),
Heterogeneity ofhuman lymphocyte Fc receptors. II.
279
observed decreases in EA rosetting PBL with no change in ADCC activity after treatment with papain (1975). Perlmann et al. (1975), utilizing an ADCC plaque assay technique, found that approximately 1% of PBL are capable of mediating ADCC, whereas much larger percentages of PBL have demonstrable EA receptors by conventional rosetting techniques (Fr0land & Natvig, 1973; Revillard et al., 1975; Cordier et al., 1976). Thus, it appears from these and our data (1) that not all cells that have EA receptors are capable of inducing ADCC; (2) that EA receptors that are inactive in ADCC are more readily removed by papain digestion; and (3) the cryptic EA receptors exposed by pronase digestion are inactive in ADCC. Kedar, Ortiz de Landazuri & Fahey (1974b) utilizing rat-spleen lymphocytes, reported that EAreceptor modulation, induced by proteolysis with various concentrations of pronase and papain, resulted in generally poor correlations between ADCC and the percentages of EA-rosette-forming cells. Our data confirm these observations (Table 1). However, in contrast to the results herein reported, Kedar observed (1) moderate decreases in percentages of EA-rosetting cells and modest diminution in ADCC after lymphocytes were treated with pronase (or trypsin); (2) increased percentages of EA-rosetting cells and somewhat enhanced ADCC activities after treatment with papain (Kedar et al., 1974b). These results, which are totally different from ours, may be due to differences in experimental design and methods (e.g. use of rat-spleen lymphocytes rather than human PBL, differences in EA-rosetting methods, differences in enzyme concentrations or length ofproteolytic digestion, etc.). Indeed, Fr0land et al. (1974b) observed increases in EA rosetting by human PBL after trypsin treatment and Cordier et al. (1976) reported decreases in EA-rosette formation by human PBL after papain treatment. It has been suggested that HRBC-A Ripley rosette with a small subset of human PBL that do not rosette with other EA used to detect Fc receptors (Dickler, 1976). In studies designed to explore this possibility, we have shown that PBL incubated with SRBC-A or CRBC-A monolayers resulted in depletion of (1) cells rosetting with CRBC-A and HRBC-A Ripley; (2) cells binding aggG; and (3) K cells (Table 4). Conversely, exposure of PBL to HRBC-A Ripley monolayers depleted cells that (1) rosetted with HRBC-A Ripley and CRBC-A; (2) bound aggG; and (3) killed target cells in ADCC (Table 5). These data strongly suggest that the population of PBL binding HRBC-A Ripley is essentially identical to the population that binds other types of EA. Moreover, the same PBL appear to bind aggG (Tables 4 and 5). In summary, the data herein presented strongly suggest that: (1) PBL K cells have receptors for aggG and for EA; (2) PBL deficient in functional aggG receptors (enzymatically removed or inactivated) are capable of inducing normal levels of ADCC; (3) aggG and EA receptors appear to be closely associated topographically on K-cell membranes; (4) there is no clear-cut relationship in a given lymphocyte population between the presence ofeither aggG or EA receptors and ADCC activity; and (5) populations of PBL binding HRBC-A Ripley overlap with, and may be identical to, those binding aggG and other types of EA complexes, viz., CRBC-A and SRBC-A. This work was supported by a Research Grant from the Veterans Administration. We wish to express our appreciation Ms Linda B. Good and Juanita Olson for the typing of this manuscript.
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