[30] IgG-binding factors

[30]

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403

[30] I g G - B i n d i n g F a c t o r s By W. H. F R I D M A N , M. J. GELABERT, M. DACRON, J. MONCUIT, I. LOwv, J. TH~ZE, and C. N~AUPORT-SAUTI~S

Immunoglobulin-binding factors (IBF) 1 are a group of substances that regulate immunoglobulin (Ig) isotype production. IBF specific for IgG (IgG-BF), la,2 IgE (IgE-BF), 3 and IgA (IgA-BF) 4 have been reported. These factors share common properties: (1) they can be induced by the interaction of T cells with a given Ig isotype, (2) they bind to the Fc portion of the Ig isotype that they regulate, and (3) in general, they suppress the production of the isotype to which they bind. In the case of IgEBF, in addition to suppressor IgE-BF, a potentiating factor with affinity for IgE has been described. 5 Subclass-specific mouse IgG-BF, suppressing IgGj, and IgG2 antibody production are induced by incubating T cells with murine monoclonal IgG~ and IgG2, respectively. 6 They are isolated by affinity chromatography on IgG. They can be detected by a rosette inhibition assay. Their immunoregulatory activities are evaluated on in vitro secondary antibody responses. Their biochemical characteristics are analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE), by chromatofocusing, and by chromatography on Blue Sepharose.

Abbreviations: IBF, immunoglobulin-binding factors; Ig, immunoglobulin; ATC, alloantigen-activated T cells; BSS, balanced salt solution; SRBC, sheep red blood cells; PBS, phosphate buffered saline; FCS, fetal calf serum; HAT, Hypoxanthine-aminopterinthymidine medium; PEG, polyethylene glycol; DNP-OVA, dinitrophenyl-ovalbumin conjugate; PFC, plaque-forming cells; TNP-OVA, trinitrophenol-ovalbumin conjugate; S DSPAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ta W. H. Fridman and P. Golstein, Cell. Immunol. 11, 442 (1974). 2 R. H. Gisler and W. H. Fridman, Cell. lmmunol. 23, 99 (1976). 3 j. Yodoi and K. Ishizaka, J. lmmunol. 124, 1322 (1980). 4 j. Yodoi, M. Adachi, K. Teshigawara, M. Myamainaba, T. Masuda, and W. H. Fridman, J. ImmunoL 131, 303 (1983). 5 j. Yodoi, M. Hirashima, and K. Ishizaka, J. lmmunol. 125, 1436 (1980). 6 I. LOwy, C. Brezin, C. N6auport-Saut~s, J. Th6ze, and W. H. Fridman, Proc. Natl. Acad. Sci. U.S.A. 86, 2323 (1983).

METHODS IN ENZYMOLOGY, VOL. 116

Copyright ~ 1985 by Academic Press, Inc. All righls of reproduction in any form reserved.

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IgG-BF-Producing Cells Alloantigen-activated T cells (ATC) TMand T cell hybridomas7 are used for the production of IgG-BF. Several lines of evidence suggest that IgGBF-producing cells are part of the subset of T cells expressing receptors for the Fc portion of IgG(FcvR). 8 Thus, preparations of ATC containing more than 15% of Fc~R positive cells as detected by rosette formation are used for the production of IgG-BF.

Preparation of lgG-BF-Producing Cells Preparation of ATC. Thymuses from 3- to 4-week-old mice are dissected free of parathymic lymph nodes. Cell suspensions are prepared in balanced salt solution (BSS) by passage through a nylon mesh (125 mesh, Fyltis, Lyon, France). The cells are washed twice and resuspended in BSS at a density of 2 x 10 6 trypan blue-excluding cells/ml. Allogeneic mice (2-3 months old) are irradiated with 850 R and injected intravenously with 0.5 ml of the cell suspension. The mice are sacrificed 5 days later, and a suspension of spleen cells is prepared, washed once, and adjusted to 2 x 106 viable cells/ml in BSS. These cells are more than 95% T lymphocytes. The percentage of cells expressing Fcr receptors is determined by rosette formation with sheep red blood cells (SRBC) sensitized with rabbit IgG. The rosette-forming cells should be between 30 and 50%. IgG-BF production is not affected by the genetic background of the mice. Critical to good IgG-BF production is the number of FcrR (+) T cells recovered. If fewer than 15% of T cell express such receptors, the preparation is not suitable for the production of IgG-BF. Preparation and Selection of T Cell Hybridomas. Clones of T cell hybridomas, obtained by fusion of the BW-5147 T lymphoma from AKR mice with activated T cells and selected on the basis of FcrR expression, represent a homogeneous and reliable cell source for the production and induction of IgG-BF. IgG-BF-producing cells used to prepare T cell hybridomas are either alloantigen-activated T cells (ATC) or spleen cells from mice injected intraperitoneally 5 days before with 108 SRBC. BW-5147 T lymphoma cells sensitive to hypoxanthine, aminopterin, and thymidine (HAT) are used as a fusion partner and grown in RPMI

7 C. N6auport-Saut6s, C. Rabourdin-Combe, and W. H. Fridman, Nature (London) 277, 656 (1979). W. H. Fridman, D. Fradelizi, A. Guimezanes, C. Platter, and J. C. Leclerc, Eur. J. lmmunol. 7, 549 (1977).

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1640-10% fetal calf serum (FCS). They are available at the American Type Culture Collection, Rockville, MD.

Media and Reagents RPMI 1640 (Gibco) containing 1 mM sodium pyruvate, 100 U/ml penicillin and streptomycin, and 2 mM glutamine. FCS: heat inactivated (30 min at 56°) and screened for best cloning efficiency. Solution of polyethylene glycol (PEG) [50% (w/v)] 1550 (Serva, Heidelberg, Germany) in RPMI 1640. To prepare this solution, RPMI is added to PEG while warm (about 37°) after sterilization. Stock solution (50 x) of HAT prepared by mixing one volume of 100 × stock solution of hypoxanthine (136 mg/100 ml) and thymidine (38 mg/100 ml) with one volume of 100× stock solution of aminopterin (i.76 mg/100 ml adjusted to pH 7 with 0.1 N NaOH). Stock solutions are kept frozen in aliquots. Preparation and Cloning of Hybridomas. All reagents used for fusion are warmed at 37° before use. Fusion is performed in a water bath at 37°; 108 spleen cells and 107 BW-5147 T lymphoma cells are mixed and centrifuged at 24° for 10 min at 200 g. One milliliter of 50% PEG is slowly added to the pellet with mixing over 1-min. The suspension is slowly diluted to 10 ml (in 5 min) with RPMI 1640 and centrifuged. The pellet is gently resuspended with a pipette in 10 ml RPMI 1640-10% FCS, and 0.2 ml of the cell suspension (106 cells) is distributed in wells of microplates (Falcon No. 3040). Cells are grown in selective HAT medium (RPMI 1640 supplemented with 10% FCS and HAT) in a CO2 incubator (5% CO2-95% air). Two weeks later, the selective medium is removed and replaced by RPMI 1640-10% FCS. Hybridomas are selected for the presence of membrane FcrR by a rosette technique using sheep erythrocytes sensitized with rabbit IgG anti-Forssman antibodies (see below). Clones from FcrR positive hybrids, obtained by distribution of individual cells in microplates or by limiting dilution, 9 are grown in RPMI 1640-FCS in microplates (Falcon 3040) over feeder layers of C3H (H2 k) thymocytes (2 × 105 cells/well). 9 Resulting hybridomas are described in Ref 7. The T2D4 cell line is commonly used as a source of IgG-BF. It is prepared by the fusion of the BW5147 lymphoma cell line with alloantigen-activated spleen T cells (H2k, THY 1.2 + and 40% Fc~/receptor positive) from B10.BR mice. The alloantigen-activated cells are obtained by injecting thymocytes from 3week-old BALB/c mice into B 10.BR mice (Jackson Laboratory, Bar Harbor, ME) irradiated 5 days before with 850 rads. 9 V. T. Oi and L. A. Herzenberg, in "Selected Methods in Cellular Immunology" (B. B. MisheU and S. M. Shiigi, eds.), p. 351. Freeman and Co, San Francisco, 1980.

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Detection and Characterization of Fc~R by Rosette Formation Cell membrane FcrR are detected by the binding of IgG labeled with a marker that can be visualized when bound to cells. Among commonly used markers are radioisotopes, fluorescent molecules, and erythrocytes. Erythrocytes are convenient because, once bound to nucleated cells, they form rosettes that are readily recognized under the microscope. IgG can be bound to erythrocytes chemically, by means of coupling reagents, or immunologically, as antigen-antibody complexes. In the latter case, Fc portions are oriented on erythrocytes in such a way that they can interact optimally with Fc~R. Antibodies directed toward erythrocytes antigens, such as anti-Forssman antibodies, are suitable reagents. These are prepared by gel filtration of ammonium sulfate-precipitated immunoglobulins from the immune serum of rabbits immunized with sheep erythrocyte stroma. ~° Alternatively, anti-hapten antibodies can be used for coating hapten-modified erythrocytes. In order to study receptors for subclasses of IgG, monoclonal antibodies of defined isotypes, produced by hybridoma or myeloma cells, are usually preferred. However, one has to check that the monoclonal antibodies used have not undergone mutation(s) that might alter cytophilic properties. These reagents are sold by New England Nuclear (Boston, MA) and Beckton Dickinson (Mountain View, CA). Anti-SRBC hybridoma cells can be purchased at the American Type Culture Collection (Rockville, MD). In practice, washed SRBC or ox red blood cells, unmodified or hapten-modified [for instance with trinitrophenol (TNP), according to the method of Rittenberg and Pratt~q suspended at 1% (v/v) in BSS, are incubated with dilutions of antibodies in the same medium for 30 min at 37°, then for another 30 min at 0°, with manual shaking every 15 min. Preliminary determination of the subagglutinating concentration of antibodies may provide useful information on the dilutions to be used. However, since experimental conditions for hemagglutination and rosette formation are not identical, differences may be found between the first nonhemagglutinating dilution and the optimal sensitizing dilution. It is, therefore, preferable to test first every antibody to be used at several dilutions (Fig. 1). After incubation with antibodies, RBC are washed at least three times in BSS and resuspended at 1% in the same medium. Indicator cells should be stored at 0° and used within 48 hr. Cells to be assayed are washed at least twice in BSS and resuspended at 5 x 106/ml in BSS supplemented with 5-10% FCS. Twenty microliters to M. M. Mayer, in "Kabat and Mayer's Experimental Immunochemistry," pp. 33, 240. Thomas, Springfield, Illinois, 1971. ~l M. B. Rittenberg and K. L. Pratt, Proc. Soc. Exp. Biol. Med. 132, 575 (1969).

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FIG. 1. Titration of monoclonal antibodies for coating indicator cells used to detect rosette formation by T2D4 cells. Sheep erythrocytes (SRBC), resuspended at 1% (v/v) in BSS are coated as described in the text with dilutions of ascitic fluids containing monoclonal anti-SRBC antibodies of the IgG~ (U.56.162), IgG2~ (4.88.2.3), or IgG3 (P.35.RU.2) subclasses. T2D4 cells harvested from 72-hr culture are then assayed for rosette formation with each preparation of indicator cells.

of this cell suspension is mixed with 20/zl of indicator cells (1% in BSS) in 5 ml clear plastic tubes. The mixture is centrifuged at 200 g for 2 min at 4° and pellets are incubated at 0° for 30-60 min. Cells are resuspended by gently rotating the tubes until an even suspension is obtained. One volume is taken out and gently mixed with one volume of 0.1% toluidine blue (in PBS 10% FCS). With this procedure, only nucleated cells are stained, live cells in light blue and dead cells in dark blue. One drop of the cell suspension is introduced in a counting chamber and examined under the microscope. Live cells with 3 or more attached erythrocytes are scored as rosettes. The percentage of rosetted cells is determined by counting at least 300 cells per sample. One usually finds that a given cell type forms rosettes with various indicator cells. For instance, T2D4 cells form high proportions of rosettes with erythrocytes coated with IgG1, IgG2b, or IgG3 (Fig. 1). In order to define the receptors involved in the formation of these various types of rosettes, the specificity of rosette formation must be studied in competition experiments between sensitized indicator cells and an excess of free monoclonal immunoglobulin of every isotype, purified preferably by affinity chromatography. Immunoglobulins should not react with erythrocyte antigens or erythrocyte-bound hapten. Also, it must be checked that their cytophilic properties are not impaired by establishing that they are capable of inhibiting rosettes of the same isotype. Competition experiments are performed by preincubating the cells to be rosetted for 30 min at 0°, with various concentrations of purified monoclonal IgG of different sub-

408

ANTIGEN NONSPECIF|C LYMPHOKINES

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classes. (They can be purchased at Litton Bionetics, Kensington, MD.) After incubation, the rosette assay is performed as described above, without washing the cells. Competition between immunoglobulins and erythrocyte-bound antibodies is favored by using indicator cells sensitized with suboptimal concentrations of antibodies. Maximal inhibition occurs when 50% cells form rosettes in the absence of inhibitor. In the case reported in Fig. I, both IgGl and IgG2b rosettes are inhibited by IgGl and IgG2b but not by IgG2a or IgG3, while IgG3 rosettes are inhibited by IgG3 only. This defines two separate receptors: one shared by IgG1 and IgG2b and one specific for IgG3.12 Production and Preparation of IgG-BF

Production and Preparation of Constitutive IgG-BF Preparation of Cell Supernatants. ATC or T cell hybrids (2 x 106) are incubated in 1 ml BSS in 5 ml plastic tubes (Falcon No. 2053) for 2 hr at 37 °. Cells are removed by centrifugation at 200 g for 10 min. IgG-BFcontaining supernatants are collected, pooled, and centrifuged at 1500 g for 10 rain. This incubation in serum-free medium results in a decrease in Fc~R expression on ATC or T cell hybrids as determined by rosette formation with rabbit IgG-sensitized erythrocytes. Preparation of Constitutive IgG-BF by Affinity Chromatography on Rabbit IgG-Sepharose. Based on the affinity of the factor for IgG, affinity chromatography is used to prepare IgG-BF. Rabbit IgG is commonly used for the purification of "constitutive IgG-BF."

Preparation of lmmunoadsorbents Reagents CNBr-activated Sepharose 4B (Pharmacia) Rabbit IgG at 10 mg/ml in bicarbonate buffer (Miles Laboratories) Bicarbonate buffer, 0.1 M NaHCO3 0.5 M NaCI, pH 8.0 0.001 M HCI 1 M ethanolamine, pH 8.5 Acetate buffer, 0.1 M sodium acetate, 0.5 M NaCI, pH 4.0 Borate buffer, 0.1 M sodium borate, 0.5 M NaCI, pH 8.0 Phosphate buffer, 0.02 M NazHPO4/NaH2PO4, pH 7.0 Glycine buffer, 0.2 M glycine-HCl, pH 2.8 Procedure. Activated sepharose is washed with 0.001 M HCI on a glass filter (200 ml HC1/g sepharose) and then equilibrated with bicarbonate buffer pH 8.0. One volume of activated sepharose is incubated with 12 M. Da6ron, J. Yodoi, C. N6auport-Saut6s, J. Moncuit, and W. H. Fridman, Eur. J. I m m u n o l . (1985), in press.

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two volumes of rabbit IgG (10 mg/ml) in bicarbonate buffer for 2 hr at 22° or 14 hr at 4 ° with gentle agitation. At the end of the incubation, the sepharose is washed with bicarbonate buffer until the OD at 280 nm of the effluents is less than 0.02. The coupling yield should be greater than 95% as determined by the OD at 280 nm of the supernatant. The sepharose beads are then incubated with 1 M ethanolamine pH 8.5 for 2 hr at 22° in order to saturate the remaining free NH2 groups. The immunoadsorbent is then washed (on a glass filter or by centrifugation at 1500 g for 10 min.) with acetate buffer pH 4.0, followed by borate buffer pH 8.0. This procedure is repeated three times, after which the material is washed once with glycine buffer pH 2.8 and equilibrated in 0.02 M phosphate buffer pH 7.0 with 0.02% sodium azide and kept at 4°. Affinity Chromatography. A batch procedure is commonly used. IgGBF-containing supernatants are concentrated 4-fold on Amicon (YM 10) filters, dialyzed against 0.02 M phosphate buffer, pH 7.0 and centrifuged at 1500 g for 10 min. The coupled sepharose beads are washed in the same buffer. Two volumes of concentrated supernatants are mixed with one volume of immunoadsorbent and incubated either for 1 hr at room temperature followed by 1 hr at 4° or overnight at 4°. The mixture is centrifuged at 1500 g for 10 min to recover the effluent. The beads are then washed in 0.02 M phosphate buffer pH 7.0 until the OD at 280 nm is less than 0.02. To elute bound material, the beads are incubated with an equivalent volume of glycine buffer pH 2.8 for 10 min at 4° with shaking and then centrifuged at 1500 g for 10 min. The same procedure is repeated twice. The eluates are pooled and filtered through a 0.45-/zm Millipore filter to remove residual beads and neutralized to pH 7.5 with 1 M KzHPO4. The three eluates are then concentrated to 1/10 of the original volume of the supernatant on YM 10 membranes. Finally they are dialyzed against an appropriate buffer. When used for the measurement of suppressive activity, the material needs to be sterilized on 0.22-~m Millipore filters.

Production and Preparation of Induced IgG-BF Induction of Fc~R and IgG-BF. Both Fc~R and IgG-BF formation are induced by treating T cells with IgG. After a short preincubation with inducer immunoglobulins, the target cells are washed, resuspended in fresh culture medium, and incubated overnight. The cells are then separated from the supernatant by centrifugation and Fc~R are detected on cell surface by rosette formation with the appropriate indicator cells. Biological activities are determined in cell-free supernatants by studying their effect on a secondary in vitro antibody response.

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In practice, 48- to 72-hr-old cultures are used as a source of hybridoma cells. These are washed twice in BSS, resuspended at 2 to 3 x l06 ml in RPMI 1640 with 10% FCS, 4 mM L-glutamine, 150 U/ml penicillin, and 100 /zg/ml streptomycin and incubated for 2-3 hr at 37° with purified monoclonal IgGt or IgGz (250/zg/ml final concentration). (They can be purchased at Litton Bionetics, Kensington, MD.) At the end of the incubation, the cells are washed in sterile BSS, resuspended at 2-3 x 106/ml in fresh culture medium and placed at 37 ° in a humidified atmosphere of 5% CO2-95% air. Culture supernatants, harvested after 18 hr incubation, contain isotype-specific IgG-BF. The cells show an enhanced expression of Fc~R that can be detected by rosette formation with indicator cells coated with IgG of the same subclass as that of inducer immunoglobulins. Depending on the cell and/or the isotype studied, however, receptor induction may not be observed. For instance, on T2D4 cells, IgG2, induces the transient expression of Fc~R that can be detected by rosette formation with IgG2a-coated erythrocytes between the sixth and the twelfth hour only, after initial contact with IgG2a. By contrast, IgG~, IgG2b, and IgG3 induce FcvR that remain expressed at the membrane, j3

Preparation of Induced IgG-BF by Affinity Chromatography on Mouse lgG-Sepharose. Immunoadsorbents are prepared with murine monoclonal immunoglobulins. The coupling procedure is similar to that used for rabbit IgG, except that mouse immunoglobulin is coupled at a concentration of 3-5 mg/ml. Subclass-specific induced IgG-BF is then prepared from immunoglobulin-induced supernatants by affinity chromatography using the same procedures as for constitutive IgG-BF, except that supernatants are not concentrated before immunoadsorption. Biological Activities of IgG-BF

Rosette Inhibition Functionally defined by its affinity for IgG, IgG-BF can be not only purified but also detected through this biological property. One of the most convenient test system proved to be a rosette inhibition assay. It consists in allowing IgG-BF to bind to IgG molecules, at the surface of appropriate indicator cells, thus preventing the subsequent binding of these indicator cells to FcrR(+) cells. Indicator cells are antibody-coated erythrocytes prepared as previously described, so that about 50% test

13 M. Da6ron, C. N6auport-Saut6s, J. Yodoi, J. Moncuit, and W. H. Fridman, Eur. J. lmmunol. (1985), in press.

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cells form rosettes. Antibodies can be either polyclonal rabbit IgG or monoclonal mouse IgG of various subclasses. F%R(+) test cells can be either tumor cell lines maintained in culture or fresh spleen lymphocytes. Normal lymphocytes have the advantage of avoiding variations in FcR expression seen in cultured cells. They however, may not express FcR for the studied isotype. In practice, 50-tzl aliquots of IgG-coated erythrocytes suspended at 1% in BSS are added to 500/zl 0.1% BSA dissolved in PBS and pelleted by centrifugation in polypropylene 5 ml tubes (Falcon ref 2063). Supernatants are carefully aspirated and replaced by 50 /zl IgG-BF containing solution, diluted in 0.1% BSA-PBS. The mixtures are incubated at 4° for at least 3 hr (overnight incubation is convenient) on a rotator, so that erythrocytes are continuously maintained in suspension. At the end of incubation, the tubes are immediately vortexed to recover any red cells remaining on cell walls; 20 tzl of the suspensions are added to 20/zl test cells resuspended at 5 x 106/ml in 10% FCS containing BSA and allowed to form rosettes as previously described. Rosette inhibition, observed with indicator cells incubated with IgG-BF, is expressed using, as 100%, the percentage of rosettes formed by the same test cells with indicator cells incubated under the same conditions with PBS-BSA.

Isotype Nonspecific Suppression of Secondary Anti-SRBC Antibody Response by Constitutive IgG-BF Constitutive IgG-BF suppresses IgG synthesis regardless of the subclasses produced. 2,6 It is tested by enumerating IgG-secreting cells in cultures with and without IgG-BF. Mice (usually B6D2F1, Jackson Laboratory) are injected intraperitoneally (ip) with 108 SRBC. Eight to ten days later the animals are sacrificed. A single-cell suspension is prepared from the spleens. The cells are centrifuged and the pellet is resuspended in culture medium containing RPMI 1640, 10% FCS, 1% horse serum, 3% sodium bicarbonate, 200 mM glutamine, penicillin, and streptomycin (500 units/ml of each). These steps are carried out in the cold. The cells (8 x 106/ml) are then placed in 12 × 75 mm Falcon Plastic tubes (Falcon, Oxnard, California) in a final volume of 1 ml. One drop of SRBC (1% suspension in BSS) is added to each tube, except for the controls. The tubes are incubated for 5 days at 37° in an atmosphere of 5% CO2 and 95% air. Various substances may be added to the culture for testing. The final volume, however, should be about 1 ml. Each sample should be run at least in duplicate. The duration of the procedure, from sacrifice of the mice to the start of the culture, should not exceed 60 min. IgG-BF is added at the initiation of the culture and IgG plaque-forming cells are measured at day 5.

412

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lsotype-Specific Suppression of Secondary Anti-DNP Antibody Response by Induced IgG-BF The secondary anti-SRBC response in mice is a convenient and reproducible model for the study of IgG production. It is not suitable, however, for measuring IgG subclass production since in this system, the majority of the IgG produced is of the IgGj isotype. Another assay is therefore necessary for the determination of subclass-specific IgG-BF. In this assay, the in vitro secondary IgG production to dinitrophenylated ovalbumin (DNP-OVA) (see Ref. 14 for preparation) of BALB/c mice is measured. Mice are injected ip with 100 /xg of DNP-OVA in Maalox (Laboratoire Rorer, Paris, France) and pertussis vaccine (Institut Pasteur Production, Garches, France); 6-8 weeks later they are boosted with one ip injection of 100 p.g of DNP-OVA in saline. The spleens are used 4-5 days later as a source of DNP-OVA-primed cells. Spleen cells (106) a r e cultured in a final volume of 100/xl in each well of a 96-well microtiter plate. Culture medium is composed of Eagle's MEM supplemented with 2 mM glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, 50 U/ml streptomycin, 50 U/ml penicillin, 20 mM Hepes, 10/zg/ml nucleotides (adenosine, uridine, cytosine, and guanosine), 0.05 mM 2-mercaptoethanol, and 5% FCS.~5 IgG-BF-containing samples are added at various dilutions at the initiation of the cultures. Cultures are fed at day 1 with 50 ~1 per well of nutritional cocktail. Nutritional cocktail is made of Eagle's MEM culture medium supplemented with 5× nonessential amino acids, 10 mM glutamine, 1% sodium bicarbonate, 1% glucose, and 40 txg/ ml nucleotides. Cultures are incubated at 37 ° in 7.5% CO2 and humidified atmosphere. After 5 days of culture, the cells are washed in BSS and assayed for plaque formation as described elsewhere. 16 Since a direct plaque assay detects only IgM-producing cells, IgG antibodies must be detected with an indirect plaque assay. For this purpose, the following components are mixed together in wells of a microtitration plate: 100/xl of cell suspension usually at 106 cells/ml; 25 /zl of guinea pig serum (as a source of complement) previously absorbed with SRBC to remove heterologous antibodies; 25/xl of SRBC or TNP-SRBC (20% in BSS) prepared using the technique of Rittenberg and PrattH; 25/zl of one of the following antibodies in BSS: anti-IgG, anti-IgG1, anti-IgGza, or anti-IgG2b. They can be purchased at New England Nuclear (Boston, MA), Beckton Dickinson (Mountain View, CA), or Litton Bionetics (Kensington, MD). 14 H. N. Eisen, Methods Med. Res. 10, 94 (1964). 15 I. Lefkovits, Eur. J. lmmunol. 2, 360 (1972). 16 W. H. Fridman and E. Falcoff, this series, Vol. 79, p. 510.

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Seventy five microliters of the mixture of these components is introduced into chambers made by sealing two slides with double-faced Scotch tape. The chambers are closed with melted paraffin and incubated for 1 hr at 37°. Plaque forming cells (PFC) are then counted with a magnifying lens under dark field illumination and expressed in PFC/106 cells. The antibodies used must be isotype-specific. Monoclonal antibodies reacting with isotypic or allotypic determinants are the best for this use. If polyclonal reagents are used, their specificity should be determined after proper absorbtion with various isotypes. The dilution of the antibodies should be determined in preliminary assays. The IgM response can be determined in a parallel direct PFC assay, and the number of PFC obtained subtracted from the indirect PFC, or it can be inhibited with antiIgM antibodies which are added to the mixture.

Biological Activity of lgG-BF on Isotype Production Constitutive IgG-BF produced by T2D4 cells and purified by affinity chromatography on rabbit IgG suppresses IgG production to SRBC as well as production of IgGj, IgG~a, and IgGzb antibodies to D N P - O V A . 6 The effect is detectable up to a 1 : 200 dilution of IgG-BF. Supernatants of T2D4 cells induced to produce IgG1-BF by incubation with IgG~ under the conditions described above, inhibit IgG1 anti-DNP response at a 1 : 2000 dilution without significantly affecting at dilutions higher than 1 : 200, the synthesis of the other isotypes tested. This effect is due to IgG1-BF since IgGj suppressor activity is recovered in the acid eluates of IgG~ immunoadsorbents. Supernatants of T2D4 cells induced by IgGza suppress both IgG2a and IgGzb but not IgG~ anti-DNP responses at a ! : 2000 dilution. Conversely, supernatants of T2D4 cells induced with IgGzb inhibit both IgG2b and IgGza, but not IgG~ antibody production. IgGz-BF can be purified on both IgG2a and IgGzb immunoadsorbents. 6 Physicochemical Characterization of IgG-BF IgG-BF is a glycoprotein. ~7 It can be stored for 24 hr at 4°, freezethawed once, and heated for 10 min at 70° in the presence of 0.1% SDS without loss of biological activity. Treatment with proteolytic enzymes but not with neuraminidase, prevents its binding to IgG.~a 17C. N6auport-Saut6s and W. H. Fridman, J. l m m u n o l . 119, 1269 (1977). 18M. Joskowicz, C. Rabourdin-Combe, C. N6auport-Saut6s, and W. H. Fridman, J. Immunol. 121, 777 (1978).

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Molecular Size o f lgG-BF Estimated by One-Dimensional SDS-PAGE Standard conditions for electrophoresis using a discontinuous Trisglycine buffered S D S - P A G E system are essentially as described by Laemmli. Slab gel (1.2 mm thick), with a 5% acrylamide stacking layer containing I0 sample wells, and a 10 cm high 12.5% acrylamide running layer, are poured. Ten-fold concentrated IgG-BF (90/.d) (Centriflow CF 25 membranes; Amicon, Danvers, MA) is mixed with 10/xl of I% SDS and incubated for 10 min at 70 °. Ten microliters of glycerol is then added and the mixture is applied in the sample well. When phenol red has reached the bottom of the gel, migration is stopped. Vertical slots corresponding to wells containing IgG-BF are cut and sliced into 0.45 cm slices. Following homogeneization each slice is incubated for 16 hr at 0° in the presence of 0.5 ml RPMI supplemented with 10% FCS. After centrifugation, the medium is recovered and sterilized through 0.22-/.~m Millipore filters. Slots corresponding to well containing markers are cut similarly and the radioactivity is determined in a 3' counter. Samples eluted from the gels are added at day 0 to secondary in vitro anti-SRBC cultures. Indirect PFC are measured at day 5. Results of a typical experiment using constitutive IgG-BF from T2D4 cells, and expressed as percentage inhibition of the indirect PFC responses of control cultures, are illustrated in Fig. 2. Under these conditions (which do not significantly alter biological activity of IgG-BF), suppressive activity is present in two peaks of 42,000 and 20,000 molecular weight. The suppressive activity found in the first two slices of the gel corresponds to protein(s) of molecular weight 75,000 as estimated on 10% acrylamide gels. The 20,000 peak is absent when IgG-BF is purified in the presence of protein synthesis inhibitors. The same results are obtained by using constitutive IgG-BF produced by ATC or induced IgG-BF.19

Isoelectric Point of IgG-BF Estimated by Chromatofocusing Chromatofocusing of IgG-BF is carried out with Pharmacia Equipment according to the specifications of the manufacturer. Chromatography is performed at 4 ° with polybuffer exchanger PBE 94. The eluate obtained after passage on IgG immunoadsorbent, as described above is dialyzed against 0.025 M imidazole-HC1 pH 7.4 and applied to a K/15 column (15 ml bed volume) equilibrated with the same t9 C. N6auport-Saut~s, M. J. G61abert, J. Moncuit, and W. H. Fridman, Febs Lett. 171, 41 (1984).

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FIG. 2. Molecular size of constitutive IgG-BF examined by SDS-PAGE. IgG-BF eluted from gel slices is used at 1/20 final dilution in a secondary in vitro anti-SRBC response. Values indicate the percentage of inhibition of the indirect PFC response of duplicate cultures. Control response: 3200 _+ 108 PFC/106 cells. From W. H. Fridman et al., Mol. I m m u n o l . 21 (1984), with permission of Pergamon Press.

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FIG. 3. Isoelectric points of constitutive IgG-BF obtained by chromatofocusing. Fractions eluted from the column are used at 1/20 final dilution in a secondary in vitro anti-SRBC response. Values indicate the percentage of inhibition of the indirect PFC response of duplicate cultures (control response: 2880 + 100 PFC/106 cells.

416

ANTIGEN NONSPECIFICLYMPHOKINES

[31]

buffer. Elution is performed at a flow rate 30-40 cm/hr with polybuffer 74 diluted eight times and adjusted at pH 4 with 2 N HC1. Each fraction is dialyzed against PBS and tested for suppressive activity. Chromatofocusing of constitutive IgG-BF from ATC results in two peaks of inhibitory activity corresponding respectively to pI 6.3 and 4.9 (Fig. 3). By using pH gradient from 9 to 6 two other peaks can be characterized. They correspond to molecules of pI 7.7 and 8.4. Blue Sepharose Chromatography Constitutive IgG-BF (1 ml) (prepared as described above) equilibrated with sodium phosphate buffer 0.02 M pH 7.0 is applied at a flow rate 4 ml/ hr to a column (1.6 × 4 cm, bed volume 7 ml) of Blue Sepharose CL-6B equilibrated with the same buffer. The bulk of suppressive IgG-BF is eluted with the starting buffer. No IgG-BF is eluted at high salt concentration (0.02 M sodium phosphate buffer, 1.5 M NaC1, pH 7.0) even in the presence of 50% polyethylene glycol.

[31] A S o l u b l e T C e l l S u p p r e s s o r F a c t o r for t h e M i x e d Leukocyte Response (MLR-TsF) B y MARGARET B E C K W I T H , CHRIS ARHELGER, and SUSAN RICH

Exposure to cells bearing disparate major (H-2) or minor histocompatibility alloantigens in vivo activates a population of murine splenic Ts~; upon further interaction in vitro with priming alloantigens Ts release MLR-TsF that prohibits alloantigen-induced T cell proliferation in the MLR. la Induction of MLR-Ts is incompletely understood but clearly distinctive in certain respects from activation of other T cell subsets, including the requirement for stimulator cells bearing I-J and/or I-C H-2 associated determinants, 2 the efficacy of purified T cells as stimulators, i Abbreviations: Ts, suppressor T cells; MLR, mixed lymphocyte reaction; TsF, T cell suppressor factor; IL-2, interleukin 2; HBSS, Hanks' balanced salt solution; FCS, fetal calf serum; GAMIg, goat anti-mouse immunoglobulin; slg, surface immunoglobulin; MEM, Eagle's minimal essential medium; SEM, standard errors of the mean; 2ME, 2mercaptoethanol; Ts2, second order suppressor cells; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonylfluoride. la S. Rich and R. Rich, J. Exp. Med. 142, 1391 (1975). 2 S. Rich, J. Exp. Med. 158, 738 (1983).

METHODS IN ENZYMOLOGY, VOL. 116

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.