Clin. exp. Immunol. (1979) 38, 158-165.

Antibody-antigen complex stimulated lysis of non-sensitized sheep red cells by human lymphocytes I. REQUIREMENT FOR IgG COMPLEXES E.W. FUSON, M.W. SHAW, R.A. HUBBARD & E.W. LAMON Department of Medical Biology, The University of Tennessee Memorial Research Center, Center for the Health Sciences, Knoxville, Tennessee, the Veterans Administration Hospital and the Department ofSurgery and Cancer Research and Training Center, University of Alabama in Birmingham, School of Medicine, Birmingham, Alabama, USA

(Accepted fir publication 20 March 1979) SUMMARY

IgG antibody-antigen complexes stimulated lysis of non-sensitized sheep erythrocytes (SRBC) by normal human peripheral blood lymphocytes (PBL). Heat-aggregated human IgG, rabbit IgG-ovalbumin complexes and rabbit IgG-sensitized ox erythrocytes (ORBC) were effective in the induction of SRBC lysis by PBL. However, IgM-sensitized ORBC and IgM-complementsensitized ORBC were ineffective. As only SRBC and not ORBC or chicken erythrocytes (CRBC) were lysed under identical experimental conditions, it is conceivable that the SRBC receptor present on the T cell is involved. Furthermore, 4500 inhibition of lysis was obtained by pretreating the effector cells with anti-human thymocyte globulin (ATG) and complete inhibition was obtained by adding SRBC stroma to the reaction mixture. The requirement for the inclusion of IgG complexes and the absence of specific anti-target cell antibody distinguish this reaction from natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Immune killer T cells would not appear to be responsible as eight different donors were used and none of these were cytotoxic to SRBC in the absence of IgG complexes. The induction of this cytotoxic reaction appears to require the recognition and interaction by the effector cells of two separate molecular entities, i.e. the SRBC membrane by the T cell and the IgG Fc region by an IgG-Fc receptor-bearing cell.

INTRODUCTION Antibody-antigen complexes have been implicated in a variety of cell-mediated reactions both in vitro and in vivo. When antibody is complexed to cell surface antigens, normal lymphoid cells are stimulated to lyse the antibody-sensitized cell (Pearson, 1978; Cerotinni & Brunner, 1974). This phenomenon has been designated antibody-dependent cell-mediated cytotoxicity (ADCC). In vitro ADCC can be inhibited by free complexes (MacLennan, 1972; Perlmann, Perlmann & Wigzell, 1972a), but under the appropriate experimental conditions complexes (Perlmann, Perlmann & Biberfeld, 1972b) and certain subclasses of IgG (Spiegelberg, Perlmann & Perlmann, 1976) (without target cell specificity) have been shown to stimulate lysis of unsensitized target cells. The lymphocytes involved as effector cells against antibody-sensitized target cells have been functionally designated as K cells. Many studies have indicated that the most efficient K cells are found in a minor subpopulation of lymphocytes possessing no easily detectable T or B cell markers (Wisl0ff, Fr0land & Michaelsen, 1974); however, a number of recent reports have indicated that T cells may Correspondence: Dr E.W. Fuson, Department of Medical Biology, The University of Tennessee Memorial Research Center, Center for the Health Sciences, 1924 Alcoa Highway, Knoxville, Tennessee 37920, USA. 0099-9104/79/1000-0158$02.00 ©) 1979 Blackwell Scientific Publications

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also function as K cells (Biberfeld et al., 1975; Perlmann et al., 1975; Van Oers, Zeijlemaker & Schellekens, 1977; Wahlin, Perlmann & Perlmann, 1976). Human T cells form spontaneous rosettes (E-rosettes) with sheep red blood cells (SRBC). In addition to an SRBC receptor, T cells also possess IgM (Moretta et al., 1975) and IgG (Ferrarini et at., 1975) Fc receptors. We have recently compared the activity of IgM and IgG antibody in the induction of ADCC against erythrocyte target cells (Fuson et al., 1978). Both classes of antibody induced lysis, but IgM antibody was 500-1000 times more efficient than IgG on a molar basis under optimal conditions. The molecular structures involved in the binding of lymphocytes to sensitized target cells with subsequent lysis appears to be the IgM (Fuson & Lamon, 1977) and IgG (Pearson, 1978) Fc receptors on the effector cells. In those experiments we employed SRBC as target cells in the ADCC assay, and because T cells possess an SRBC receptor, we conducted the following experiments to determine the possible role of the SRBC receptor in lymphocyte-mediated lysis of SRBC. In this study we evaluate the ability of IgG antibodyantigen complexes and IgG aggregates to stimulate cell-mediated lysis of unsensitized erythrocyte target cells, including SRBC. MATERIALS AND METHODS Preparation of IgG and IgM anti-ORBC. IgG was prepared from serum obtained from rabbits hyperimmune to ox erythrocytes (ORBC) by DEAE-cellulose chromatography with a 0-0175 M P04 (Na+) buffer, pH 6 5. The IgG was found to be free of other antibody classes when analysed by immunodiffusion against class-specific antisera (Microbiological Associates). The IgM was prepared by pooling the leading half of the first protein peak produced by two separate G-200 fractionations (2-6 x 90 cm column with PBS) of serum obtained from rabbits injected 5 days previously with ORBC. The 19S fraction was concentrated and passed over a Staphylococcus aureus protein A coupled to Sepharose CL-4B column (Pharmacia Fine Chemicals, Uppsala, Sweden) to remove possible trace contaminants of IgG. Haemolytic endpoint titres were determined in microplates (Cooke Microplates, Alexandria, Virginia) with serial dilutions of antibody and guinea-pig sera as a source of complement. In some experiments, before use in the assay, the antibody was adsorbed for 1 hr on ice with either sheep erythrocytes (SRBC) or chicken erythrocytes (CRBC) to remove any cross-reactive antibody. Indicator cells. SRBC, ORBC or CRBC were labelled with 51Cr (Na25'CrO4, New England Nuclear, Boston, Massachusetts; 250 pCi/1 5 x 0I cells) by incubating for 1 hr at 37°C. The "ICr labelled erythrocytes were washed in phosphatebuffered saline (PBS) and suspended in medium 199 containing 0-1 mg/ml gentamicin (Schering Corp., Port Reading, New Jersey) and 10% heat-inactivated (60 min, 560C) foetal calf serum (complete medium). Sensitization of ORBC. ORBC were washed in PBS and suspended at 2 x 106/ml in an appropriate dilution of IgG or IgM anti-ORBC in complete medium and placed on a tube rotator for 30 min at room temperature. The antibody-sensitized ORBC were then washed and resuspended in complete medium before use in the assay. Erythrocyte-antibody-complement complexes (EoXAMC) were prepared by incubating IgM-sensitized ORBC (EOXAM) in a 1:4 dilution of whole A/J mouse serum at 370C for 15 min. The EOKAMC interacted with human complement receptor lymphocytes (CRL) to form EAC rosettes when PBL were pelleted with the EAC complexes and incubated 1 hr at 250C. Heat-aggregated IgG. Human IgG from the sera of normal, healthy donors was prepared by DEAE-cellulose chromatography (0-0175 M P04 pH 6 5). A 10 mg/ml solution of the IgG in PBS was incubated for 30 min at 630C. The heat-aggregated IgG was then diluted for use in the assay. IgG-ovalbumin complexes. The IgG fraction of hyperimmune rabbit anti-ovalbumin was prepared by DEAE-cellulose chromatography (0.0175 M P04, pH 6.5). The IgG preparation was mixed with an equal molar concentration of ovalbumin and incubated at 10°C overnight. The resulting precipitate was washed three times in cold PBS and then dialysed against a sodium acetate buffer (pH 4.0) and fractionated on G-200 with the same acetate buffer. The first protein peak was concentrated to 1 mg/ml after dialysis against PBS and 2 mg/ml of ovalbumin were added to form soluble complexes. Effector cells. PBL from the blood of healthy donors were isolated by the method of Boyum (1964). Adherent cells were removed by overnight incubation at 37°C on plastic. The non-adherent cells were washed and suspended at 1 x 107/ml in complete media. An aliquot of the lymphocyte preparation was tested for the number of phagocytic cells by incubating for 1 hr with latex beads. The mean percentage of phagocytic cells was determined to be < 3%. ATG treatment of PBL. Anti-human thymocyte globulin (ATG) prepared in horses was obtained from Dr A.G. Diethelm, Department of Surgery, University of Alabama in Birmingham. The preparation of this reagent and its specificity for human T cells has been described previously (Diethelm et al., 1974; 1976; Balch et al., 1977). PBL at l x 107/ml were suspended in a 1/12,000 dilution of ATG with an agglutination titre of 6000 and incubated at room temperature for 30 min. The PBL were then washed and used as effector cells in the assay. Cytotoxicity assay. 1 X 106 effector cells were added to triplicate tubes with 2 x 10'5" Cr-labelled SRBC, ORBC or CRBC. The JgG complex was provided by adding various concentrations of aggregated human IgG, rabbit-IgG-ovalbumin complexes or rabbit IgG-sensitized unlabelled ORBC to the tubes. Controls consisted of effector cells mixed with 5'Cr-labelled SRBC, ORBC or CRBC only and "Cr-labelled erythrocytes without lymphocytes to determine spontaneous lysis. This

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mixture was centrifuged at 400 g for 2 min and incubated at 370C in 5% CO2 for 1 hr. After the incubation period the cell pellets and supernatants were assayed for 51Cr content in a well-type gamma counter. Maximum releasable counts were determined by adding 1 ml of water to tubes containing "Cr-labelled erythrocytes only. The percentage of lysis was determined by the following formula: percentage total (experimental)-percentage total (spontaneous) x 100. percentage total (maximum)-percentage total (spontaneous) Antibody-dependent cell-mediated cytotoxicity (ADCC) was determined by pelleting 2 x 105 5"Cr-labelled, antibodysensitized erythrocytes with 1 x 106 PBL in triplicate tubes with complete medium (Fuson et al., 1978). The tubes were incubated at 370C for 1 hr, the 5 1Cr content of the pellet and supernatant was counted and the percentage lysis determined using the above formula. Percentage lysis

=

RESULTS Lysis of non-sensitized SRBC by non-immune PBL 51Cr-labelled SRBC (2 x 105 cells) were mixed with varying numbers of IgG-sensitized unlabelled ORBC and 1 x 106 PBL in a total volume of 0 3 ml of complete medium. The cell mixture was pelleted and incubated for 1 hr at 370C and the percentage lysis of SRBC ascertained. Fig. 1 illustrates the results from three separate experiments. This cytotoxic reaction produced 430 lysis in 1 hr when 2 x 106 IgG sensitized ORBC were used. The decrease in cytotoxicity was linear with two-fold decreases in the number of IgG-sensitized ORBC. Fig. 2 illustrates the SRBC lysis induced by IgG-sensitized ORBC when the total number of cells was constant (2 x 105/tube) but sensitized with two-fold dilutions of the IgG. 50 _ 40 30

20 -

10

0-5 0-25 0-125 2 Number of IqG-ORBC/tube x 105

FIG. 1. Titration of IgG-ORBC in the induction of SRBC lysis by IgG-ORBC stimulated PBL. Each point represents triplicate tubes from three separate experiments. Tubes contained 2x 105 "Cr-labelled SRBC, 1 X 106 PBL and IgG-ORBC. The cells were pelleted and incubated 1 hr at 37TC before determining the '"Cr release. The vertical lines represent standard deviation.

IgG complexes that stimulate SRBC Iysis by non-immune PBL 2 x 105 IgG-sensitized ORBC, 3 mg of a final concentration of aggregated human IgG or rabbit IgG-ovalbumin complexes were added to triplicate tubes containing 2 x 105 5tCr-labelled SRBC and 1 x 106 PBL. In each case, as shown in Table 1, lysis of the SRBC was induced.

Specificity of SRBC lysis When 51Cr-labelled ORBC or CRBC were used in lieu of the "Cr-labelled SRBC, no lysis was observed under identical experimental conditions. Table 1 illustrates the specificity of this lytic reaction for SRBC with IgG-ORBC, heat-aggregated IgG or IgG-ovalbumin complexes added to induce lysis. These results suggest that the SRBC receptor on the T cell may be involved. We therefore attempted

IgG complex stimulated lysis of SRBC

161

40 0

30

20

if10 _ Cr

O

\

8

o6 0~~~~~

.0~~~~~ \ 2

16

Dilution-'

32 64 128 256 512 of IgG used to sensitize ORBC

FIG. 2. Titration of antibody in the induction of SRBC lysis by IgG-ORBC stimulated PBL. Each point Cr-labelled SRBC, Ix 106 PBL and 2 x 105 IgG-ORBC. The ORBC were sensitized with rabbit IgG anti-ORBC at the dilutions indicated. The haemolytic endpoint titre of the IgG was 128. The cells were pelleted and incubated 1 hr at 370C before determining the "Cr release. represents triplicate tubes. Tubes contained 2 x 105

TABLE 1. Specificity of the cytotoxic reaction for SRBC

Indicator cell*

IgG stimulus

Percentage lysis

SRBC ORBC CRBC SRBC ORBC CRBC SRBC ORBC CRBC

IgG-ORBCt IgG-ORBC IgG-ORBC Aggregated IgG$ Aggregated IgG Aggregated IgG IgG-ovalbumin§ IgG-ovalbumin IgG-ovalbumin

43 0 0 42 0 0 30 0 0

* Indicator cells were labelled with 5'Cr and dispensed 2 x 105 per tube. t DEAE-cellulose purified rabbit anti-ORBC IgG with a haemolytic titre of 128 used at a 1:64 dilution. I Heat-aggregated IgG (30 min, 630C) purified from healthy individuals used at 3 mg/ml. § DEAE-cellulose purified rabbit anti-ovalbumin. Complex prepared as described in the Materials and Methods section and used at 3 mg/ml of IgG.

to inhibit the lysis of SRBC by pre-treating the PBL with a horse ATG, which has been shown to inhibit E-rosette formation, and by adding SRBC stroma to the mixture to hinder SRBC-T cell interaction (Table 2). The ATG treatment of PBL produced a 45%0 inhibition of lysis. When SRBC stroma were added to the cell mixture, lysis was completely inhibited. 1.

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E. W. Fuson et al. TABLE 2. Inhibition of SRBC lysis

Inhibitor

IgG stimulus

Percentage lysis (±+ s.d.)

Percentage inhibition

IgG-0RBCj

22+ 1-2

-

ATG*

IgG-ORBC

12+2d1

45

IgG-ORBC IgG-ORBC

29+0 5

SRBC stromat

-

0

100

* PBL were suspended in a 1: 12,000 dilution of horse anti-human thymocyte globulin with an agglutination titre of 6000. The PBL were incubated with the ATG for 30 min at room temperature, washed and used as effector cells. t 5 x 109 SRBC were lysed by suspension in distilled water. The stroma was pelleted in a high speed centrifuge, washed with PBS, resuspended in 1 ml of complete medium and 0-1 ml was added to 0 3 ml of containing cells. $ Tubes contained lx 106 PBL, 2x 105 51Cr-SRBC and 2x 105 IgGsensitized ORBC in 0 3 ml volume.

SRBC lysis requires IgG complexes IgG-sensitized ORBC were efficient in the induction of SRBC lysis. However, as can be seen in Table 3, IgM sensitized ORBC (EoxAM) did not induce lysis. Furthermore, C3 on the surface of the ORBC (EOXAMC) was also ineffective. Comparison of SRBC lysis induced by IgG complexes and ADCC That the lysis of SRBC by non-immune PBL observed in the presence of IgG complexes might be a function of IgG-induced ADCC must be considered. To test the possibility that the SRBC lysis might be ADCC-induced by undetectable amounts of anti-target cell IgG, we added IgG immune complexes or heat-aggregated IgG to tubes containing IgG-sensitized 51Cr-labelled SRBC and 1 x 106 PBL or non-sensitized "Cr-labelled SRBC under identical experimental conditions (Table 4). In tubes to which no aggregate or complex were added, 43%O lysis of the IgG-sensitized SRBC and 0%/O lysis of non-sensitized SRBC was produced. When IgG complexes or aggregated IgG were added to the tubes containing the IgG-sensitized SRBC, the percentage lysis was inhibited by 58% and 56%, respectively. On the other hand, addition to tubes containing non-sensitized SRBC resulted in the induction of lysis of 29% and 30°/,,, respectively. TABLE 3. Requirement for IgG complexes Immune complex

EoxAr,* EOXAMt

EO.AMC+

Indicator cell

Percentage lysis

SCr-SRBC 51Cr-SRBC 5lCr-SRBC

41 0 0

* ORBC sensitized with a 1:1000 dilution of rabbit anti-ORBC IgG with a haemolytic titre of 2000.

t ORBC sensitized with a 1:256 dilution of rabbit anti-ORBC IgM with a haemolytic titre of 1024. + ORBC sensitized with a 1:1024 dilution of rabbit anti-ORBC IgM with a haemolytic titre of 1024. The EOXAM were then incubated for 15 min at 370C in a 1:4 dilution of whole AIJ mouse serum and washed before use.

163

IgG complex stimulated lysis ofSRBC TABLE 4. The effect of IgG-antigen complexes and heat-aggregated IgG on lymphocyte cytotoxicity in IgG-ADCC*i and.non-sensitized SRBC lysist'

IgG Percentage

Cytotoxic reaction ADCC ADCC ADCC SRBC lysis SRBC lysis SRBC lysis

Complexes

Aggregates§

lysis

+ + -

+ +

43 18 19 0 30 29

* Target cells in ADCC were IgG-sensitized, .5Cr-labelled SRBC. Effector cells were PBL. Effector-target ratio 5:1. t Target cells were non-sensitized, 5'Cr-labelled SRBC. Effector cells were PBL. Effector-target cell ratio = 5:1. + Complexes were IgG-ovalbumin at a final conicentration of 3 mg/ml. 5 Aggregates were heat-aggregated IgG at a final, qncentration of 3 mg/ml.

DISCUSSION We have presented data which indicates that soluble and insoluble IgG immune complexes and heataggregated IgG can induce normal human lymphocytes to be cytotoxic against SRBC. This reaction is apparently specific for SRBC as CRBC and ORBC are not lysed under the same experimental conditions. Human T cells have receptors for SRBC but not CRBC or ORBC; it appears, therefore, that the T cell may be involved in this reaction. Human T cells have also been shown to possess Fc receptors for IgG (Ferrarini et al., 1975) and IgM (Moretta et al., 1975). These experiments do not determine whether a single T cell possessing both SRBC receptors and IgG Fc receptors or more than one cell type reacting synergistically is responsible for the observed cytotoxicity. This question is the subject of a continuing investigation. An SRBC induction of non-specific lymphocyte cytotoxicity has been reported by Mackler, O'Neill & Meistrich (1977). In their experiments lymphocytes were pelleted with SRBC and incubated for 1 hr at 4°C. During the incubation period, a soluble factor(s) was released that could stimulate non-T cells to be non-specifically cytotoxic to allogeneic and autologous target cells. However, at 37°C the factor was not produced. Lymphoid cells from normal individuals which apparently are not immune to the pertinent cell surface membrane antigens, may produce significant levels of cytotoxicity against certain allogeneic tumour cells (Takasugi, Mickey & Terasaki, 1973). In this spontaneous or natural cellmediated cytotoxicity the effector cells are present in the human PBL population and the assay is carried out at 37°C. There is evidence that the natural killer cells (NK cells) possess receptors for SRBC and Fc receptors for IgG (Kay et al., 1977). The lymphocyte cytotoxic reaction reported here appears to have a different mechanism to natural cell-mediated cytotoxicity as it requires interaction with added IgG aggregates or complexes. Fernandez, Rajaraman & MacSween (1976) have reported a spontaneous cell-mediated cytotoxic reaction by human PBL against SRBC where experiments were carried out at room temperature with maximum cytotoxicity occurring at 5 hr. However, we have been unable to obtain lysis of non-sensitized SRBC by PBL at the same E: T ratios at 37°C after incubation for as long as 72 hr (unpublished observations). A variety of target cells, sensitized with antibodies against their surface antigens, can be lysed in vitro by lymphoid cells from normal, healthy individuals who have not been previously sensitized to the pertinent target cell antigen. This phenomenon has been designated as antibody-dependent cell-mediated

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cytotoxicity (ADCC). This reaction requires the presence of IgM or IgG antibodies on the surface of the target cell (Perlmann & Perlmann, 1970; Lamon et al., 1975). The addition of extraneous IgM or IgM (Fc) 5 yu fragments (Fuson & Lamon, 1977) or IgG, IgG complexes, heat-aggregated IgG or IgG-Fc fragments (Pearson, 1978; Perlmann et al., 1972a; Spiegelberg et al., 1976) inhibits the appropriate ADCC reaction, presumably by covering available membrane Fc receptors. We have demonstrated that the reaction described in the present report is distinct from previously described ADCC reactions: (1) in some experiments the rabbit anti-ORBC antibodies were adsorbed with SRBC before use, and antibody prepared in this way would not sensitize SRBC to function as target cells in ADCC; (2) concentrations of IgG (complexes or aggregates) that induce the SRBC lysis reported here, inhibit IgG-ADCC; (3) IgG sensitized ORBC did not stimulate the lysis of non-sensitized ORBC which indicates that antibody does not dissociate from the sensitized ORBC and bind to another ORBC or SRBC and thus produce ADCC. Recently, Koide, Kwok & Takasugi (1978) reported the lysis of non-sensitized 51Cr-labelled SRBC in the presence of IgG-sensitized unlabelled SRBC. They suggested that the cytotoxicity was due to IgG anti-SRBC antibody being transferred to "Cr-labelled SRBC or onto the Fc receptors of effector lymphocytes which then reacted with target SRBC. We also observed PBL lysis of5 Cr-labelled unsensitized SRBC in the presence of IgG sensitized SRBC; however, we have discounted the possibility of the transfer of IgG anti-SRBC antibody by experiments employing normal human IgG aggregates, IgG-ovalbumin aggregates and IgG-sensitized ORBC (Table 1). In the present experiments the cytotoxicity was produced by lymphocytes from eight different donors. Therefore, this reaction potential appears to be present in normal donors and is not a result of prior exposure to the antigen and the production of antibody or immune cells. In murine systems it has been suggested that two recognition sites must be present on the surface of the target cell before immune killer T cells are actively cytotoxic (Zinkernagel et al., 1978). The target cell must possess the specific immunizing antigen (viral or haptenic) and the same H-2, D or K antigens as those present on the cells which initiated the immune response. This dual recognition hypothesis is also supported by experiments with human cytotoxic T cells in which only target cells carrying both the HLA-A2 antigen and the non-HLA target determinant of the original sensitizing cell were lysed (Goulmy et al., 1977). Similarly, the lymphocyte cytotoxicity reported here appears to require the interaction of two molecular moieties with the effector cell, i.e. the SRBC receptor with the SRBC and the Fc receptor with the Fc region of IgG-antigen complexes. That SRBC-T cell interaction is required was further supported by the inhibition of cytotoxicity in this assay by the treatment of effector cells with the IgG fraction of horse ATG and the addition of SRBC stroma to the reaction. These data may have in vivo significance in immune complex disease. If normal lymphocytes possess receptors for syngeneic HLA antigens it is conceivable that high concentrations of immune complexes may provide a second stimulus that will produce cell-mediated tissue damage. This system may provide a convenient model for the in vitro study of such phenomena. This investigation was supported by Grant number CA-17273-02 from the National Cancer Institute, National Institutes of Health. Credit is also given to project No. 5132-01 from the Veterans Administration Hospital, Birmingham, Alabama and a Biomedical Research Support Grant number NIH FR 5541 of the National Institutes of Health.

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