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Biological effects of anti-idiotypic antibodies on lymphocyte function
CELLULAR
50, 369-378 (1980)
IMMUNOLOGY
Biological
Effects of Anti-ldiotypic Antibodies Lymphocyte Function
I. Analysis of the Effects on B Lymphocytes of Combining Framework-Directed Anti-T-15 ldiotypic Antibodies’ HIROSHI
YAMAMOTO~
AND DAVID
on Site and
H. KATZ
Anti-idiotypic antibodies against TEPC-15 myeloma protein (BALBlc origin) were raised in allogeneic animals by immunization of A/J mice with the myeloma protein. The antibody activities were fractionated into two specificities by TEPC-15 immunoadsorbent affinity columns by elution with free hapten (phosphorylcholine, PC), followed by elution with acidic buffer (glycine-HCI, pH 2.3). Idiotype binding analysis indicated that the fraction eluted with hapten could be inhibited in its binding to TEPC-15 by free hapten (i.e.. binding site-directed anti-idiotypic antibody), whereas the acid-eluted fraction could not (i.e., framework-directed anti-idiotypic antibody). When analyzed for their biological activities on PC-specific B lymphocytes producing T-15 idiotype-bearing antibodies, both anti-idiotypic antibody fractions had similar suppressive effects on the in tifro production of antiphosphorylcholine antibody in culture.
INTRODUCTION The V-region structure of antibody molecules possesses unique antigenic determinants which have been termed idiotypes (Id) (1,2). Idiotypic determinants are present on two distinguishable areas of the antibody molecule: One such area is the combining site of the antibody molecule itself (hereafter referred to as “binding-site” idiotypes), whereas the other is located in one or more noncombining site areas of the V-region (hereafter referred to as “framework” idiotypes) (reviewed in (3)). Immunization with idiotypes obtained from homogeneous antibodies or myeloma proteins induces production of anti-idiotypic antibodies in xenogeneic, allogeneic, and even in syngeneic animals. Immunochemical analysis of idiotypic determinants by specific anti-idiotypic antibodies has been used as a sophisticated tool for analyzing the immunogenetics of V-region genes and the diversification of such genes (reviewed in (4)). During the past few years the biological effects of anti-idiotypic antibodies have received increasing attention due to (i) the postulated role idiotype-anti-idiotype responses may play in both regulation and diversification of the immune system, as ’ This is publication No. 91 from the Department of Cellular and Developmental Immunology and publication no. 1694from the Immunology Departments, Scripps Clinic and Research Foundation, La Jolla, Calif. This work was supported by United States Public Health Service Grant AI-13781. 2 Supported by a Fellowship from the Cancer Research Institute, Inc., New York, N.Y. 369 0008-8749/80/040369-10$02.00/O Copyright 8 1980by Academic Press.Inc. All rights of reproductionin any form reserved.
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AND KATZ
fostered by Jerne (5), and (ii) the possible clues such anti-idiotypic reactivities might provide to the ultimate clarification and elucidation of the mystery concerning the molecular nature of T-cell receptors (reviewed in (6, 7)). Several well-known and thoroughly characterized idiotypic specificities have been particularly attractive for studies of this type. The T- 15 idiotypic marker defines the unique determinants on the BALB/c myeloma protein, TEPC-15 (IgA, K), a paraprotein which has specificity for phosphorylcholine (PC)” (8,9); PC is the major antigenic determinant of the cell wall of pneumococci strain R36A. The T-15 idiotype exists in significant amounts in normal BALB/c serum (10). Cosenza and Kohler (11) first reported that PC-specific plaques developed with R36A-coupled or azophenylphosphorylcholine-coupled sheep erythrocytes could be prevented by the addition of anti-T- 15 anti-idiotypic serum obtained from properly sensitized A/J mice. Subsequently, these investigators demonstrated the inhibitory effects of anti-T-15 antibodies on development of PC-specific primary in vitro antibody responses; these suppressive effects were shown to be independent of IgG subclass (12) or of the strain of mice in which such antibodies were raised (13). Contrasting effects of anti-idiotypic antibodies, namely those in which stimulation of responses could be observed following administration of such antibodies, have also been described. Thus, Eichmann (14) and Rajewsky and Eichmann (15) reported that anti-idiotypic antibodies of the IgG, class raised in guinea pigs sensitized with mouse antibodies specific for the group-specific streptococcal wall carbohydrate (A-CHO) could sensitize A-CHO-specific helper T as well as B lymphocytes when administered to mice of the A/J strain. On the other hand, such anti-A5A-idiotypic antibodies of the IgGz class were shown to be potent in inducing a subpopulation of specific suppressor T lymphocytes in these mice. Subsequently, it was shown that even among the IgG, anti-A5A-idiotypic antibodies, a further segregation could be demonstrated in which only certain of this subclass of antibodies were effective in inducing helper T cells while virtually all of this subclass of antibodies could sensitize A-CHO-specific B lymphocytes (7). These interesting contrasting effects of anti-idiotypic antibodies, namely enhancing in some instances while suppressive in others, have not as yet been fully explained. Certain obvious possibilities, such as the species in which anti-idiotypic antibodies are raised in relation to the species of origin of the idiotypic determinants as well as the species in which such antibodies are tested, do not provide any conclusive answers to this problem. Upon scrutinizing the published reports concerning biological effects of anti-idiotypic antibodies, we were struck by the fact that as yet no comparative analysis had been made of homologous anti-idiotypic antibodies segregated on the basis of their respective reactivities with determinants present in either the binding-site(s) or framework residues of the antibody molecule. It is conceivable that in any preparation of anti-idiotypic antibodies the biological activities observed could reflect the net sum of the effects of these independent specificities. In other words, while antibodies directed to binding site determinants might exert one biological effect, those directed to framework residues could have somewhat different, even opposite, biological effects; hence, the overall activity observed with a given anti-idiotypic antiserum could be ’ Abbreviations used: PC, phosphorylcholine; DNP, 2,4-dinitrophenyl; hemocyanin; BSA, bovine serum albumin; MyG, mouse y-globulin.
KLH,
keyhole
limpet
ANTI-IDIOTYPIC
ANTIBODY
REGULATION
I
371
conditioned by the relative proportions of one specificity versus the other in such preparations. In the present study, we have used highly purified preparations of anti-T-15 idiotypic antibodies, segregated for binding-site versus framework residue reactivities, and analyzed the activities of these respective antibodies on PC-specific B lymphocytes in vitro. MATERIALS
AND METHODS
Animals BALB/c mice of both sexes and male A/J mice aged 8- 12weeks from the Scripps breeding colony and New Zealand red rabbits (Triple R Rabbitry, Manasquan, N.J.) were used throughout this study. Protein Antigens and Chemicals Keyhole limpet hemocyanin (KLH) and bovine serum albumin (Cohn fraction V, BSA) were purchased from Pacific Bio-Marine Supply Company, Venice, California, and Miles Laboratories, Inc., Kankakee, Illinois, respectively. Diazonium phenyl phosphorylcholine (DPPC) was synthesized by the method of Chesebro and Metzger (16) and conjugated to KLH (PC,-KLH) and BSA (PC,,-BSA). Phosphorylcholine chloride was obtained from Sigma Chemical Company, St. Louis, MO. Dinitrophenyl (DNP)-KLH and BSA were prepared as previously described (17). Myeloma Proteins Myeloma tumors TEPC-15, HOPC-8, MOPC-167, MOPC-511, and W3207, all of which are IgA, K, and have binding affinity for PC, were maintained in BALB/c mice and ascites were collected. The ascites fluids were processed for mild reduction and alkylation (16) and myeloma proteins were purified by passing through PC-BSA-conjugated Sepharose 4B (CNBr-activated Sepharose 4B from Pharmacia Fine Chemicals, Uppsala, Sweden) immunoadsorbent column and eluted with lo-:’ M phosphorylcholine chloride in borate-buffered saline (pH 8.0). After extensive dialysis, affinity-purified myeloma proteins were used throughout this study. Anti-idiotypic
Antisera
(a) Preparation. Anti-idiotypic antisera were raised in A/J mice and rabbits. In mice, TEPC-15 myeloma protein was given according to the methods of Potter and Leiberman (9). Seventy-five micrograms per 0.15 ml of TEPC-15 was emulsified in equal volume of complete Freund’s adjuvant (CFA, Difco Laboratories, Detroit, Mich.) and injected into six sites, two hind foot pads, two axillary areas, one intraperitoneally. and one hind trunk area. At 4-day intervals, animals were boosted with the same amount of TEPC-15 into the same sites, once with incomplete Freund’s adjuvant (IFA, Difco) and twice in saline. Four weeks after primary immunization, weekly bleedings were started. Rabbits were injected intravenously with deaggregated a-globulin from normal A/J mice as a means for inducing tolerance to major immunoglobulin (Ig) determinants. Twenty-four hours later, they were immunized with 100 pg of
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AND KATZ
TEPC-15 myeloma proteins in CFA into two hind foot pads. Two weeks later they were boosted with 50 kg in IFA into the same sites and immune sera were obtained 2 weeks thereafter. (b) Purijication. Antisera were absorbed by passing through MOPC- 167myeloma protein-coupled Sepharose 4B to remove rabbit anti-mouse Ig or rabbit and mouse antiallotypic antibodies. Anti-idiotypic antibodies from such absorbed immune sera were purified on TEPC- 15coupled Sepharose 4B immunoadsorbent columns. After extensive washing with borate-buffered saline, adsorbed antibodies were eluted from the column bed with lo-” M phosphorylcholine chloride in saline followed by 0.1 M glycine-HCl buffer (pH 2.3). Figure 1 illustrates the elution profile of A/J anti-TEPC-15 antibodies obtained as a pool of three bleedings on Days 28, 35, and 42 after primary immunization. Two antibody fractions were obtained in this way, one of which could be eluted with free hapten (termed hereafter B, for binding-site, fraction-see under Results), while the second was eluted by acid (termed F, for framework, fraction-see under Results). Rabbit anti-TEPC-15 antibodies, on the other hand, did not show elution with phosphorylcholine chloride but only with acidic buffer (data not shown). After extensive dialysis against phosphate-buffered saline (PBS, pH 7.2) the recovered fractions were concentrated over PM 10 Diaflo membrane filter (Amicon Corp., Lexington, Mass.) and used for radioimmunoassay or dialyzed again against Mishell-Dutton culture medium for use in in vitro antibody responses. In Vitro Antibody Responses. Spleen cells from BALB/c mice which were preimmunized with 20 pg of KLH in CFA intraperitoneally were cultured in Mishell-Dutton medium (18) supplemented with lo-” M of 2-mercaptoethanol. The 2.5 x lo6 cells were cultured in plastic tubes (Falcon 2057, Falcon, Oxnard, Calif.) in 1.Oml volume with lo-” pg of PC-KLH or DNP-KLH and with or without normal sera, anti-idiotypic antisera, or purified antibodies from such sera for certain periods (see RESULTS for details). The cells were washed and recultured in Falcon microculture wells (Falcon 3040) at 0.5 x lo6 cells per well in 0.2 ml for 5 to 7 days with daily feeding of 10 ~1 of 30% fetal calf serum-containing Mishell-Dutton
0.4/
B Fraction
1
F Fraction
F
Q 0.3-
%
OO t
20 30 10 TUBE NO. / Iml/t&J t
6C-,d3M
6,f MGly-WI
40
(pH 2.3)
FIG. 1. Elution profile of anti-idiotypic antibodies from TEPC-I5 myeloma protein-conjugated Sepharose 4B. The antibodies were eluted from immunoadsorbent column consecutively with 25 ml of lo-’ A4 of phosphorylcholine chloride in saline and 0.1 M glycine-HC1 buffer, pH 2.3. Effluents were collected and optical densities at 280 nm were measured.
ANTI-IDIOTYPIC
ANTIBODY
REGULATION
medium. Anti-PC, anti-DNP and TEPC-I5 idiotype-positive antibodies in culture supernatants were measured. Solid-Phase
373
I
(T-15) anti-PC
Radioimmunoassay
Anti-idiotypic specificities and antibodies in culture supernatants were analyzed in a solid-phase radioimmunoassay system originally described by Klinman et al. (19). Briefly, polyvinyl plates (Microtiter, Cooke Engineering Co., Alexandria, Va.) were exposed to 100 ~1 of 30-50 ,ug/ml of affinity-purified anti-idiotypic antibodies or 100~1 of PC-BSA (or DNP-BSA) at concentrations of 1 mgiml for 4 hr at room termperature; the plates were then washed with tap water and quenched with 10 mgiml of BSA in PBS overnight at 4°C. After washing out BSA, 50 ~1 of ‘z”I-labeled TEPC-15 with inhibitors was added to antibody-coated plates, or 25-50 ~1 of culture supernatants were added to respective antigen-coated plates and incubated for 4 hr or more at room temperature or overnight at 4°C. Wells were washed with water followed with 0.5 M sodium chloride-containing phosphate buffer (pH 7.2) and washed again with water. For anti-idiotypic analysis, wells were cut and counted. Antibody measurements were done by exposing the wells with ““I-labeled rabbit anti-mouse Fab (generously supplied by Dr. Norman Klinman of our Department) for anti-PC (or anti-DNP) antibody responses and with *2ZI-labeled affinity-purified rabbit anti-T-15 anti-idiotypic antibody diluted in 10% fetal calf serum-containing PBS for T-15 idiotypic anti-PC antibody responses. Statistical
Analysis
of Data
Antibody levels of culture supernatant in nanograms/milliliter were logarithmically transformed and then geometric means and standard errors were calculated. The sensitivity limit of our radioimmunoassay system was 2-8 ngiml and samples below this were arbitrarily assigned the limit amount in each individual experiment. COffCEN?-RA~ONOf PHOSPHORYLCHOLINE CHLORIDEf/W) 10-6to-5 10.4 10-J 10-210-6 m-5 10-4 iti3 icy 10-6o5 10-410’3 10-2
t~~~~~
10.’ 100 IO’ 10’ 103 10-f roe IO’ 402 f03 to” 100 IO’ 102 103 CONCENTRATIQVOF MYELOMAPROTEINSf//p/m/) FIG. 2. Specificities of anti-idiotypic (T-15) antibodies detected in competitive inhibition assay. Polyvinyl plates were coated with (A) Rabbit anti-TEPC-15 antibodies eluted with acidic buffer; (B) A/J anti-TEPC-15 eluted with phosphorylcholine chloride, and(C) with acidic buffer. Percentage binding of I-labeled TEPC-15 myeloma protein in the presence of unlabeled TEPC-15 (O), HOPC-8 (A), and other myeloma proteins such as MOPC- 167, MOPC-5 1I, and W3207 (shaded area) and phosphorylcholine chloride (0) at various concentrations is illustrated.
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YAMAMOTO
AND KATZ
RESULTS AND DISCUSSION Fine Specificity of Anti-Zdiotypic (T-15) Antibodies Employed in These Studies In order to determine the fine specificity of the anti-idiotypic antibodies tested in the subsequent functional assays, lz51-labeled TEPC- 15 myeloma protein was mixed with varying concentrations of unlabeled myeloma proteins (TEPC-15, HOPC-8, MOPC-167, MOPC-511, and W3207) or phosphorylcholine chloride and the mixtures then incubated in anti-idiotypic (T-15) antibody-coated polyvinyl plates. The relevant data from this competitive inhibition assay is summarized in Fig. 2. Rabbit anti-idiotypic antibodies were not inhibited by free hapten, but were inhibited to a comparable extent by both TEPC-15 and HOPC-8 (Fig. 2A). None of the other myeloma proteins employed manifested any detectable inhibition of binding of TEPC-15 (shaded areas). These results are comparable to previous observations that the TEPC-15 and HOPC-8 myeloma proteins share identical idiotypic determinants, whereas others employed in the present study do not (8,9). Our findings disagree, however, with those of Sakato and Eisen (20) who found cross-reactivity between TEPC-15 and MOPC-511. Analysis of the mouse anti-idiotypic antibodies, purified by affinity chromatography, revealed that the B and F fractions, respectively, manifested different inhibition patterns. Thus, the B fraction was clearly inhibited by free hapten (Fig. 2B), whereas the F fraction was not so inhibited (Fig. 2C). These findings indicate obvious differences in the respective anti-idiotypic specificities of these two fractions in that the B fraction is directed to the antigen-combining site-specific idiotypic determinants while the F fraction reacts to the nonbinding site-specific determinants (i.e., framework residues). TEPC- 15 binding to anti-T- 15 idiotypic antibodies was also inhibited by HOPC-8 (Figs. 2A andC), although the extent of Antigen -
Serum Cont. (%I -
PC- KLH
O.ii
DNP-KLH
0.11
Exp.I
Exp. 2
i
C C I
1
I
I
I
I
0
20
40
60
60
400
I
ANTI- PC /ANTf -DNPI ANTIBODY IN CULTLM SUPERNATANTfng/mll FIG. 3. Suppression of in vitro PC-specific antibody responses by anti-idiotypic (T-15) antiserum. KLH-primed spleen cells were cultured in the presence of A/J anti-TEPC-15 antiserum (W) or normal A/J serum @Id).Specificity ofthis antiserum was also shown in Experiment 2 with stimulation by DNP-KLH.
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ANTIBODY
REGULATION
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inhibition of these affinity-purified mouse anti-idiotypic antibodies differed slightly from the inhibition of rabbit anti-T-15 idiotype by HOPC-8 (Fig. 2A). Effects of Antiddiotypic
Antibodies
on B Cell Responses.
(a) Suppression of in vitro PC-specific antibody responses by anti-idiotypic (T-15) antiserum. The 2.5 x lo6 KLH-primed BALB/c spleen cells were cultures (1
ml/tube) with various concentrations of either normal A/J serum of A/J anti-idiotypic antiserum. Twenty-four hours later, a second dose of the same quantities of normal or anti-idiotypic serum was added to each culture together with IO-” pg of PC-KLH. After a subsequent 24 hr, the cells in each culture tube were thoroughly washed to remove serum and antigen and then dispensed into microculture wells at a concentration of 0.5 x lOYO.2 ml/well calculated from the original cell concentrations. Seven days after the addition of antigen, culture supernatants were harvested and the PC-specific antibody concentrations were determined. Preliminary studies demonstrated that the cell concentrations employed and the day of harvest of culture supernatants were optimal in this system (data not shown). The results from such studies are summarized in Fig. 3. In Experiment 1, (top panel) it can be seen that cells exposed to PC-KLH in the absence of any added serum developed readily detectable PC-specific antibody responses. The addition of anti-idiotypic antiserum at concentrations of 0.04 and 0.11% strongly suppressed PC-specific antibody responses; normal serum at these concentrations did not significantly affect synthesis of anti-PC antibody. At higher concentrations (0.33 and 1.0%) nonspecific suppression due to normal serum was also observed. Seventy to eighty percent of the PC-specific antibodies produced in all cultures were found to be T- 15idiotype-positive. In experiment 2 (bottom panel), the antigen specificity of the suppressive effects of anti-T-15 antiserum is illustrated. Thus, in the presence of 0.11% anti-T-15 antiserum, there is a substantial suppression of the PC-specific responses but no detectable suppression of anti-DNP antibody responses elicited with DNP-KLH. This is quite similar to the observations of Cosenza and Kohler (1 l), though the experimental systems are different. (b) Suppression of T-15 idiotypic anti-PC antibody responses in vitro by af$nity-purijed anti-idiotypic antibodies. Spleen cells from KLH-primed BALBic
mice were cultured with either normal A/J y-globulin (purified by DEAE-cellulose column chromatography) or with the B or F fractions of affinity-purified anti-idiotypic antibodies in various concentrations for 24 hr. Twenty-four hours later, the same quantities of normal A/J mouse y-globulin (MyG) or anti-idiotypic antibodies, together with lo-” pg of PC-KLH were added to each culture tube, After an additional 24 hr, all cells were washed and then placed in culture for an additional 6 days. Culture supernatants were harvested and the quantities of anti-PC antibodies determined. As shown in Fig. 4, both fractions of anti-idiotypic antibodies suppressed the T- 15 idiotypic anti-PC antibody responses at concentrations of 1.1 pg and higher, The results with B fraction are identical to the data of Kohler et al. (21) who used heterologous anti-idiotypic antibodies. In contrast, no significant suppression was observed with normal MyG. At the highest concentration employed (10 pg), the F fraction suppressed responses significantly more than that observed with the B
376
YAMAMOTOANDKATZ
fraction of anti-idiotypic antibodies. Elution of antibodies by acidic buffer might have a stronger denaturing effect on antibodies. Though we have no data of quantitative analysis based on weight and titer of individual antibody preparations, in no instances were any enhancing activities of either B or F fractions on such in vitro responses observed. (c) Kinetics of anti-idiotypic antibody-mediated suppression of in vitro T-15 idiotypic anti-PC antibody production. Five micrograms of either normal MyG or
the B or F fractions of affinity-purified anti-T-15 anti-idiotypic antibodies were added at various times prior to the addition of either lo-” pg of PC-KLH or uncoupled KLH to cultures of KLH-primed BALB/c spleen cells. Twenty-four hours after the addition of antigen, all cultures were washed thoroughly and then replated for an additional 6 days at which time culture supernatants were harvested and assayed for anti-PC antibodies. The results in this experiment were expressed as percentage responsiveness of T-15 idiotypic anti-PC antibody responses as compared with cultures exposed to normal MyG. As shown in Fig. 5, both antibody fractions behaved precisely the same, illustrating that no specificity differences exist in their biological effects on anti-PC antibody responses. Optimal suppression was observed when anti-idiotypic antibody fractions were added either 3 hr before or at the same time as antigen. The apparent enhancement of responses when such antibodies were added 12 hr after antigen was not significantly different from the responses observed in cultures of cells exposed to normal MyG at the same time. The present studies have been directed to questions concerning comparative biological effects of anti-idiotypic antibodies segregated in terms of their determinant binding specificities on the basis of affinity chromatography Culture I
AntibodyAdded None
I
/
1
1
1
0
ioo
200
300
400
T- t5 /D/OTYP/C ANT/-PC ANTIBODY IN CL/LEWE SUERNA TANT fng/ml / FIG. 4. Suppression of T-15 idiotype positive anti-PC antibody responses by affinity-purified anti-idiotypic antibodies. KLH-primed spleen cells were cultured with normal A/J y-globulin (R), B fraction (El), and F fraction (0). see text.
ANTI-IDIOTYPIC
ANTIBODY
REGULATION
I
377
purification procedures and differential elution of the antibody-binding activities by free hapten, on the one hand, and acidic buffer, on the other. Claflin and Davie (22) reported that although rabbit anti-HOPC 8 idiotypic antibodies exhibited specificity exclusively for binding-site determinants, only marginal quantities of binding site-specific antibodies could be detected in A/J mouse anti-HOPC 8 serum. In contrast, we could purify significant amounts of antibinding site, as well as antiframework, idiotypic antibodies from antiserum raised in A/J mice against TEPC-15, but no detectable binding site-specific antibodies in rabbit anti-T- 15 antiserum. It is important to emphasize the fact that the present studies demonstrate the feasibility of inducing anti-idiotypic antibodies of distinct specificities in the same species of animals since this will provide a useful tool for analysis of regulatory effects of heterogeneous anti-idiotypic antibodies on immune responses of members of the same species. Our original purpose was to investigate the immunoregulatory activities of anti-idiotypic antibodies which have different specificities. Specifically, we were interested in determining whether the respective activities obtained by these methods-namely, those antibodies binding to combining site determinants versus those specific for framework residues-exerted different, perhaps even opposing biological effects on the capacity of idiotype-positive B lymphocytes to secrete their antibody products. These studies demonstrate unequivocally, however, that no such differences in biological effects exist. These studies indicate, therefore, that the previously described contrasting effects of anti-idiotypic antibodies on lymphocyte function reported by Eichmann (14) and Rajewsky and Eichmann (15), which in some cases are enhancing and in other cases suppressive, clearly do not reflect differences in determinant specificities of the anti-idiotypic antibodies employed, at least insofar as the T-15
ANTIBODES ADDED AJ: fhfd FIG. 5. Effects of anti-idiotypic antibodies on the production of T-15 idiotype positive anti-PC antibodies at different timing. Antibodies were added at various times indicated and IO-” pg of PC-KLH was added at 0 hr. B fraction (O), F fraction (0). Data of normal mouse y-globulin at respective times were 59, 86, 78, 61, 44, and 44 ngiml. Nonadded control was 75 ngiml.
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YAMAMOTO
AND KATZ
idiotype and the in vitro system described here are concerned. Studies currently underway in our laboratory which are directed to making similar analyses of these different anti-idiotypic antibodies on PC-specific T-cell functions may provide better clarification of these questions. ACKNOWLEDGMENTS The authors are grateful to Drs. T. Hamaoka and Barry J. Skidmore for helpful discussions during the early phase of this work. Drs. Erik Lindh and Fu-Tong Liu and Mr. Robert M. Gordon provided discussions and excellent technical assistance and we thank Anthea Hugus and Keith Dunn for assistance in preparation of the manuscript. Dr. Norman Klinman critically reviewed the paper and commented thereon in a most helpful way.
REFERENCES 1. Kunkel, H. G., Mannik, M., and Williams, R. C., Jr., Science 140, 1218, 1963. 2. Oudin, J., and Michael, M., C. R. Acad. Sci. (Paris) 257, 805, 1963. 3. Nisonoff, A., Hopper, J. E., and Spring, S. B., In “The Antibody Molecule,” p. 444. Academic Press, New York, 1975. 4. Kunkel, H. G., and Kindt, T., In “Immunogenetics and Immunodeficiency,” MTP Press, Lancaster, England, p. 55, 1975. 5. Jerne, N. K., Ann. lmmunol. (Inst. Pasteur) 12X, 373, 1974. 6. Binz, H., and Wigzell, H., Cold Spring Harbor Symp. Quant. Biol. 41, 275, 1976. 7. Krawinkel, U., Cramer, M., Berek, C. Hammerling, G., Black, S. J., Rajewski, K., and Eichmann, K., Cold Spring Harbor Symp. Quant. Biol. 41, 285, 1976. 8. Cohn, M., Notani, G., and Rice, S. A., Immunochemistry 6, 111, 1969. 9. Potter, M., and Lieberman, R., J. Exp. Med. 132, 737, 1970. 10. Lieberman, R., Potter, M., Mushinski, E. B., Humphrey, W., Jr., and Rudikoff, S., J. Exp. Med. 139, 983, 1975.
11. 12. 13. 14. 15. 16. 17. 18. 19.
Cosenza, H., and Kohler, H., Science 176, 1027, 1972. Kohler, H., Richardson, B. C., Rowley, D. A., and Smyk, S., J. Immunol. 119, 1977. Kohler, H., Richardson, B. C., and Smyk, S., J. Immunol. 120, 233, 1978. Eichmann, K., Eur. J. Immunol. 5, 511, 1975. Rajewsky, K., and Eichmann, K., In “Contemporary Topics in Immunobiology” 7, 69, 1977. Chesebro, B., and Metzger, H. Biochemistry 5, 766, 1972. Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B. J. Exp. Med. 132, 261, 1970. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. Klinman, N. R., Pickard, A. R., Sigal, N. H., Gearhart, P. J., Metcalf, E. S., and Pierce, S. K.,Ann. Immunol.
(Inst. Pasteur)
127C, 489, 1976.
20. Sakato, N., and Eisen, H. N., 1. Exp. Med. 141, 1411, 1975. 21. Kohler, H., Smyk, S., and Kluskens, L., Cell. Immunol. 17, 295, 1975. 22. Claflin, J. L., and Davie, J. M. J. Immunol. 114, 70, 1975.
50, 369-378 (1980)
IMMUNOLOGY
Biological
Effects of Anti-ldiotypic Antibodies Lymphocyte Function
I. Analysis of the Effects on B Lymphocytes of Combining Framework-Directed Anti-T-15 ldiotypic Antibodies’ HIROSHI
YAMAMOTO~
AND DAVID
on Site and
H. KATZ
Anti-idiotypic antibodies against TEPC-15 myeloma protein (BALBlc origin) were raised in allogeneic animals by immunization of A/J mice with the myeloma protein. The antibody activities were fractionated into two specificities by TEPC-15 immunoadsorbent affinity columns by elution with free hapten (phosphorylcholine, PC), followed by elution with acidic buffer (glycine-HCI, pH 2.3). Idiotype binding analysis indicated that the fraction eluted with hapten could be inhibited in its binding to TEPC-15 by free hapten (i.e.. binding site-directed anti-idiotypic antibody), whereas the acid-eluted fraction could not (i.e., framework-directed anti-idiotypic antibody). When analyzed for their biological activities on PC-specific B lymphocytes producing T-15 idiotype-bearing antibodies, both anti-idiotypic antibody fractions had similar suppressive effects on the in tifro production of antiphosphorylcholine antibody in culture.
INTRODUCTION The V-region structure of antibody molecules possesses unique antigenic determinants which have been termed idiotypes (Id) (1,2). Idiotypic determinants are present on two distinguishable areas of the antibody molecule: One such area is the combining site of the antibody molecule itself (hereafter referred to as “binding-site” idiotypes), whereas the other is located in one or more noncombining site areas of the V-region (hereafter referred to as “framework” idiotypes) (reviewed in (3)). Immunization with idiotypes obtained from homogeneous antibodies or myeloma proteins induces production of anti-idiotypic antibodies in xenogeneic, allogeneic, and even in syngeneic animals. Immunochemical analysis of idiotypic determinants by specific anti-idiotypic antibodies has been used as a sophisticated tool for analyzing the immunogenetics of V-region genes and the diversification of such genes (reviewed in (4)). During the past few years the biological effects of anti-idiotypic antibodies have received increasing attention due to (i) the postulated role idiotype-anti-idiotype responses may play in both regulation and diversification of the immune system, as ’ This is publication No. 91 from the Department of Cellular and Developmental Immunology and publication no. 1694from the Immunology Departments, Scripps Clinic and Research Foundation, La Jolla, Calif. This work was supported by United States Public Health Service Grant AI-13781. 2 Supported by a Fellowship from the Cancer Research Institute, Inc., New York, N.Y. 369 0008-8749/80/040369-10$02.00/O Copyright 8 1980by Academic Press.Inc. All rights of reproductionin any form reserved.
370
YAMAMOTO
AND KATZ
fostered by Jerne (5), and (ii) the possible clues such anti-idiotypic reactivities might provide to the ultimate clarification and elucidation of the mystery concerning the molecular nature of T-cell receptors (reviewed in (6, 7)). Several well-known and thoroughly characterized idiotypic specificities have been particularly attractive for studies of this type. The T- 15 idiotypic marker defines the unique determinants on the BALB/c myeloma protein, TEPC-15 (IgA, K), a paraprotein which has specificity for phosphorylcholine (PC)” (8,9); PC is the major antigenic determinant of the cell wall of pneumococci strain R36A. The T-15 idiotype exists in significant amounts in normal BALB/c serum (10). Cosenza and Kohler (11) first reported that PC-specific plaques developed with R36A-coupled or azophenylphosphorylcholine-coupled sheep erythrocytes could be prevented by the addition of anti-T- 15 anti-idiotypic serum obtained from properly sensitized A/J mice. Subsequently, these investigators demonstrated the inhibitory effects of anti-T-15 antibodies on development of PC-specific primary in vitro antibody responses; these suppressive effects were shown to be independent of IgG subclass (12) or of the strain of mice in which such antibodies were raised (13). Contrasting effects of anti-idiotypic antibodies, namely those in which stimulation of responses could be observed following administration of such antibodies, have also been described. Thus, Eichmann (14) and Rajewsky and Eichmann (15) reported that anti-idiotypic antibodies of the IgG, class raised in guinea pigs sensitized with mouse antibodies specific for the group-specific streptococcal wall carbohydrate (A-CHO) could sensitize A-CHO-specific helper T as well as B lymphocytes when administered to mice of the A/J strain. On the other hand, such anti-A5A-idiotypic antibodies of the IgGz class were shown to be potent in inducing a subpopulation of specific suppressor T lymphocytes in these mice. Subsequently, it was shown that even among the IgG, anti-A5A-idiotypic antibodies, a further segregation could be demonstrated in which only certain of this subclass of antibodies were effective in inducing helper T cells while virtually all of this subclass of antibodies could sensitize A-CHO-specific B lymphocytes (7). These interesting contrasting effects of anti-idiotypic antibodies, namely enhancing in some instances while suppressive in others, have not as yet been fully explained. Certain obvious possibilities, such as the species in which anti-idiotypic antibodies are raised in relation to the species of origin of the idiotypic determinants as well as the species in which such antibodies are tested, do not provide any conclusive answers to this problem. Upon scrutinizing the published reports concerning biological effects of anti-idiotypic antibodies, we were struck by the fact that as yet no comparative analysis had been made of homologous anti-idiotypic antibodies segregated on the basis of their respective reactivities with determinants present in either the binding-site(s) or framework residues of the antibody molecule. It is conceivable that in any preparation of anti-idiotypic antibodies the biological activities observed could reflect the net sum of the effects of these independent specificities. In other words, while antibodies directed to binding site determinants might exert one biological effect, those directed to framework residues could have somewhat different, even opposite, biological effects; hence, the overall activity observed with a given anti-idiotypic antiserum could be ’ Abbreviations used: PC, phosphorylcholine; DNP, 2,4-dinitrophenyl; hemocyanin; BSA, bovine serum albumin; MyG, mouse y-globulin.
KLH,
keyhole
limpet
ANTI-IDIOTYPIC
ANTIBODY
REGULATION
I
371
conditioned by the relative proportions of one specificity versus the other in such preparations. In the present study, we have used highly purified preparations of anti-T-15 idiotypic antibodies, segregated for binding-site versus framework residue reactivities, and analyzed the activities of these respective antibodies on PC-specific B lymphocytes in vitro. MATERIALS
AND METHODS
Animals BALB/c mice of both sexes and male A/J mice aged 8- 12weeks from the Scripps breeding colony and New Zealand red rabbits (Triple R Rabbitry, Manasquan, N.J.) were used throughout this study. Protein Antigens and Chemicals Keyhole limpet hemocyanin (KLH) and bovine serum albumin (Cohn fraction V, BSA) were purchased from Pacific Bio-Marine Supply Company, Venice, California, and Miles Laboratories, Inc., Kankakee, Illinois, respectively. Diazonium phenyl phosphorylcholine (DPPC) was synthesized by the method of Chesebro and Metzger (16) and conjugated to KLH (PC,-KLH) and BSA (PC,,-BSA). Phosphorylcholine chloride was obtained from Sigma Chemical Company, St. Louis, MO. Dinitrophenyl (DNP)-KLH and BSA were prepared as previously described (17). Myeloma Proteins Myeloma tumors TEPC-15, HOPC-8, MOPC-167, MOPC-511, and W3207, all of which are IgA, K, and have binding affinity for PC, were maintained in BALB/c mice and ascites were collected. The ascites fluids were processed for mild reduction and alkylation (16) and myeloma proteins were purified by passing through PC-BSA-conjugated Sepharose 4B (CNBr-activated Sepharose 4B from Pharmacia Fine Chemicals, Uppsala, Sweden) immunoadsorbent column and eluted with lo-:’ M phosphorylcholine chloride in borate-buffered saline (pH 8.0). After extensive dialysis, affinity-purified myeloma proteins were used throughout this study. Anti-idiotypic
Antisera
(a) Preparation. Anti-idiotypic antisera were raised in A/J mice and rabbits. In mice, TEPC-15 myeloma protein was given according to the methods of Potter and Leiberman (9). Seventy-five micrograms per 0.15 ml of TEPC-15 was emulsified in equal volume of complete Freund’s adjuvant (CFA, Difco Laboratories, Detroit, Mich.) and injected into six sites, two hind foot pads, two axillary areas, one intraperitoneally. and one hind trunk area. At 4-day intervals, animals were boosted with the same amount of TEPC-15 into the same sites, once with incomplete Freund’s adjuvant (IFA, Difco) and twice in saline. Four weeks after primary immunization, weekly bleedings were started. Rabbits were injected intravenously with deaggregated a-globulin from normal A/J mice as a means for inducing tolerance to major immunoglobulin (Ig) determinants. Twenty-four hours later, they were immunized with 100 pg of
372
YAMAMOTO
AND KATZ
TEPC-15 myeloma proteins in CFA into two hind foot pads. Two weeks later they were boosted with 50 kg in IFA into the same sites and immune sera were obtained 2 weeks thereafter. (b) Purijication. Antisera were absorbed by passing through MOPC- 167myeloma protein-coupled Sepharose 4B to remove rabbit anti-mouse Ig or rabbit and mouse antiallotypic antibodies. Anti-idiotypic antibodies from such absorbed immune sera were purified on TEPC- 15coupled Sepharose 4B immunoadsorbent columns. After extensive washing with borate-buffered saline, adsorbed antibodies were eluted from the column bed with lo-” M phosphorylcholine chloride in saline followed by 0.1 M glycine-HCl buffer (pH 2.3). Figure 1 illustrates the elution profile of A/J anti-TEPC-15 antibodies obtained as a pool of three bleedings on Days 28, 35, and 42 after primary immunization. Two antibody fractions were obtained in this way, one of which could be eluted with free hapten (termed hereafter B, for binding-site, fraction-see under Results), while the second was eluted by acid (termed F, for framework, fraction-see under Results). Rabbit anti-TEPC-15 antibodies, on the other hand, did not show elution with phosphorylcholine chloride but only with acidic buffer (data not shown). After extensive dialysis against phosphate-buffered saline (PBS, pH 7.2) the recovered fractions were concentrated over PM 10 Diaflo membrane filter (Amicon Corp., Lexington, Mass.) and used for radioimmunoassay or dialyzed again against Mishell-Dutton culture medium for use in in vitro antibody responses. In Vitro Antibody Responses. Spleen cells from BALB/c mice which were preimmunized with 20 pg of KLH in CFA intraperitoneally were cultured in Mishell-Dutton medium (18) supplemented with lo-” M of 2-mercaptoethanol. The 2.5 x lo6 cells were cultured in plastic tubes (Falcon 2057, Falcon, Oxnard, Calif.) in 1.Oml volume with lo-” pg of PC-KLH or DNP-KLH and with or without normal sera, anti-idiotypic antisera, or purified antibodies from such sera for certain periods (see RESULTS for details). The cells were washed and recultured in Falcon microculture wells (Falcon 3040) at 0.5 x lo6 cells per well in 0.2 ml for 5 to 7 days with daily feeding of 10 ~1 of 30% fetal calf serum-containing Mishell-Dutton
0.4/
B Fraction
1
F Fraction
F
Q 0.3-
%
OO t
20 30 10 TUBE NO. / Iml/t&J t
6C-,d3M
6,f MGly-WI
40
(pH 2.3)
FIG. 1. Elution profile of anti-idiotypic antibodies from TEPC-I5 myeloma protein-conjugated Sepharose 4B. The antibodies were eluted from immunoadsorbent column consecutively with 25 ml of lo-’ A4 of phosphorylcholine chloride in saline and 0.1 M glycine-HC1 buffer, pH 2.3. Effluents were collected and optical densities at 280 nm were measured.
ANTI-IDIOTYPIC
ANTIBODY
REGULATION
medium. Anti-PC, anti-DNP and TEPC-I5 idiotype-positive antibodies in culture supernatants were measured. Solid-Phase
373
I
(T-15) anti-PC
Radioimmunoassay
Anti-idiotypic specificities and antibodies in culture supernatants were analyzed in a solid-phase radioimmunoassay system originally described by Klinman et al. (19). Briefly, polyvinyl plates (Microtiter, Cooke Engineering Co., Alexandria, Va.) were exposed to 100 ~1 of 30-50 ,ug/ml of affinity-purified anti-idiotypic antibodies or 100~1 of PC-BSA (or DNP-BSA) at concentrations of 1 mgiml for 4 hr at room termperature; the plates were then washed with tap water and quenched with 10 mgiml of BSA in PBS overnight at 4°C. After washing out BSA, 50 ~1 of ‘z”I-labeled TEPC-15 with inhibitors was added to antibody-coated plates, or 25-50 ~1 of culture supernatants were added to respective antigen-coated plates and incubated for 4 hr or more at room temperature or overnight at 4°C. Wells were washed with water followed with 0.5 M sodium chloride-containing phosphate buffer (pH 7.2) and washed again with water. For anti-idiotypic analysis, wells were cut and counted. Antibody measurements were done by exposing the wells with ““I-labeled rabbit anti-mouse Fab (generously supplied by Dr. Norman Klinman of our Department) for anti-PC (or anti-DNP) antibody responses and with *2ZI-labeled affinity-purified rabbit anti-T-15 anti-idiotypic antibody diluted in 10% fetal calf serum-containing PBS for T-15 idiotypic anti-PC antibody responses. Statistical
Analysis
of Data
Antibody levels of culture supernatant in nanograms/milliliter were logarithmically transformed and then geometric means and standard errors were calculated. The sensitivity limit of our radioimmunoassay system was 2-8 ngiml and samples below this were arbitrarily assigned the limit amount in each individual experiment. COffCEN?-RA~ONOf PHOSPHORYLCHOLINE CHLORIDEf/W) 10-6to-5 10.4 10-J 10-210-6 m-5 10-4 iti3 icy 10-6o5 10-410’3 10-2
t~~~~~
10.’ 100 IO’ 10’ 103 10-f roe IO’ 402 f03 to” 100 IO’ 102 103 CONCENTRATIQVOF MYELOMAPROTEINSf//p/m/) FIG. 2. Specificities of anti-idiotypic (T-15) antibodies detected in competitive inhibition assay. Polyvinyl plates were coated with (A) Rabbit anti-TEPC-15 antibodies eluted with acidic buffer; (B) A/J anti-TEPC-15 eluted with phosphorylcholine chloride, and(C) with acidic buffer. Percentage binding of I-labeled TEPC-15 myeloma protein in the presence of unlabeled TEPC-15 (O), HOPC-8 (A), and other myeloma proteins such as MOPC- 167, MOPC-5 1I, and W3207 (shaded area) and phosphorylcholine chloride (0) at various concentrations is illustrated.
374
YAMAMOTO
AND KATZ
RESULTS AND DISCUSSION Fine Specificity of Anti-Zdiotypic (T-15) Antibodies Employed in These Studies In order to determine the fine specificity of the anti-idiotypic antibodies tested in the subsequent functional assays, lz51-labeled TEPC- 15 myeloma protein was mixed with varying concentrations of unlabeled myeloma proteins (TEPC-15, HOPC-8, MOPC-167, MOPC-511, and W3207) or phosphorylcholine chloride and the mixtures then incubated in anti-idiotypic (T-15) antibody-coated polyvinyl plates. The relevant data from this competitive inhibition assay is summarized in Fig. 2. Rabbit anti-idiotypic antibodies were not inhibited by free hapten, but were inhibited to a comparable extent by both TEPC-15 and HOPC-8 (Fig. 2A). None of the other myeloma proteins employed manifested any detectable inhibition of binding of TEPC-15 (shaded areas). These results are comparable to previous observations that the TEPC-15 and HOPC-8 myeloma proteins share identical idiotypic determinants, whereas others employed in the present study do not (8,9). Our findings disagree, however, with those of Sakato and Eisen (20) who found cross-reactivity between TEPC-15 and MOPC-511. Analysis of the mouse anti-idiotypic antibodies, purified by affinity chromatography, revealed that the B and F fractions, respectively, manifested different inhibition patterns. Thus, the B fraction was clearly inhibited by free hapten (Fig. 2B), whereas the F fraction was not so inhibited (Fig. 2C). These findings indicate obvious differences in the respective anti-idiotypic specificities of these two fractions in that the B fraction is directed to the antigen-combining site-specific idiotypic determinants while the F fraction reacts to the nonbinding site-specific determinants (i.e., framework residues). TEPC- 15 binding to anti-T- 15 idiotypic antibodies was also inhibited by HOPC-8 (Figs. 2A andC), although the extent of Antigen -
Serum Cont. (%I -
PC- KLH
O.ii
DNP-KLH
0.11
Exp.I
Exp. 2
i
C C I
1
I
I
I
I
0
20
40
60
60
400
I
ANTI- PC /ANTf -DNPI ANTIBODY IN CULTLM SUPERNATANTfng/mll FIG. 3. Suppression of in vitro PC-specific antibody responses by anti-idiotypic (T-15) antiserum. KLH-primed spleen cells were cultured in the presence of A/J anti-TEPC-15 antiserum (W) or normal A/J serum @Id).Specificity ofthis antiserum was also shown in Experiment 2 with stimulation by DNP-KLH.
ANTI-IDIOTYPIC
ANTIBODY
REGULATION
I
375
inhibition of these affinity-purified mouse anti-idiotypic antibodies differed slightly from the inhibition of rabbit anti-T-15 idiotype by HOPC-8 (Fig. 2A). Effects of Antiddiotypic
Antibodies
on B Cell Responses.
(a) Suppression of in vitro PC-specific antibody responses by anti-idiotypic (T-15) antiserum. The 2.5 x lo6 KLH-primed BALB/c spleen cells were cultures (1
ml/tube) with various concentrations of either normal A/J serum of A/J anti-idiotypic antiserum. Twenty-four hours later, a second dose of the same quantities of normal or anti-idiotypic serum was added to each culture together with IO-” pg of PC-KLH. After a subsequent 24 hr, the cells in each culture tube were thoroughly washed to remove serum and antigen and then dispensed into microculture wells at a concentration of 0.5 x lOYO.2 ml/well calculated from the original cell concentrations. Seven days after the addition of antigen, culture supernatants were harvested and the PC-specific antibody concentrations were determined. Preliminary studies demonstrated that the cell concentrations employed and the day of harvest of culture supernatants were optimal in this system (data not shown). The results from such studies are summarized in Fig. 3. In Experiment 1, (top panel) it can be seen that cells exposed to PC-KLH in the absence of any added serum developed readily detectable PC-specific antibody responses. The addition of anti-idiotypic antiserum at concentrations of 0.04 and 0.11% strongly suppressed PC-specific antibody responses; normal serum at these concentrations did not significantly affect synthesis of anti-PC antibody. At higher concentrations (0.33 and 1.0%) nonspecific suppression due to normal serum was also observed. Seventy to eighty percent of the PC-specific antibodies produced in all cultures were found to be T- 15idiotype-positive. In experiment 2 (bottom panel), the antigen specificity of the suppressive effects of anti-T-15 antiserum is illustrated. Thus, in the presence of 0.11% anti-T-15 antiserum, there is a substantial suppression of the PC-specific responses but no detectable suppression of anti-DNP antibody responses elicited with DNP-KLH. This is quite similar to the observations of Cosenza and Kohler (1 l), though the experimental systems are different. (b) Suppression of T-15 idiotypic anti-PC antibody responses in vitro by af$nity-purijed anti-idiotypic antibodies. Spleen cells from KLH-primed BALBic
mice were cultured with either normal A/J y-globulin (purified by DEAE-cellulose column chromatography) or with the B or F fractions of affinity-purified anti-idiotypic antibodies in various concentrations for 24 hr. Twenty-four hours later, the same quantities of normal A/J mouse y-globulin (MyG) or anti-idiotypic antibodies, together with lo-” pg of PC-KLH were added to each culture tube, After an additional 24 hr, all cells were washed and then placed in culture for an additional 6 days. Culture supernatants were harvested and the quantities of anti-PC antibodies determined. As shown in Fig. 4, both fractions of anti-idiotypic antibodies suppressed the T- 15 idiotypic anti-PC antibody responses at concentrations of 1.1 pg and higher, The results with B fraction are identical to the data of Kohler et al. (21) who used heterologous anti-idiotypic antibodies. In contrast, no significant suppression was observed with normal MyG. At the highest concentration employed (10 pg), the F fraction suppressed responses significantly more than that observed with the B
376
YAMAMOTOANDKATZ
fraction of anti-idiotypic antibodies. Elution of antibodies by acidic buffer might have a stronger denaturing effect on antibodies. Though we have no data of quantitative analysis based on weight and titer of individual antibody preparations, in no instances were any enhancing activities of either B or F fractions on such in vitro responses observed. (c) Kinetics of anti-idiotypic antibody-mediated suppression of in vitro T-15 idiotypic anti-PC antibody production. Five micrograms of either normal MyG or
the B or F fractions of affinity-purified anti-T-15 anti-idiotypic antibodies were added at various times prior to the addition of either lo-” pg of PC-KLH or uncoupled KLH to cultures of KLH-primed BALB/c spleen cells. Twenty-four hours after the addition of antigen, all cultures were washed thoroughly and then replated for an additional 6 days at which time culture supernatants were harvested and assayed for anti-PC antibodies. The results in this experiment were expressed as percentage responsiveness of T-15 idiotypic anti-PC antibody responses as compared with cultures exposed to normal MyG. As shown in Fig. 5, both antibody fractions behaved precisely the same, illustrating that no specificity differences exist in their biological effects on anti-PC antibody responses. Optimal suppression was observed when anti-idiotypic antibody fractions were added either 3 hr before or at the same time as antigen. The apparent enhancement of responses when such antibodies were added 12 hr after antigen was not significantly different from the responses observed in cultures of cells exposed to normal MyG at the same time. The present studies have been directed to questions concerning comparative biological effects of anti-idiotypic antibodies segregated in terms of their determinant binding specificities on the basis of affinity chromatography Culture I
AntibodyAdded None
I
/
1
1
1
0
ioo
200
300
400
T- t5 /D/OTYP/C ANT/-PC ANTIBODY IN CL/LEWE SUERNA TANT fng/ml / FIG. 4. Suppression of T-15 idiotype positive anti-PC antibody responses by affinity-purified anti-idiotypic antibodies. KLH-primed spleen cells were cultured with normal A/J y-globulin (R), B fraction (El), and F fraction (0). see text.
ANTI-IDIOTYPIC
ANTIBODY
REGULATION
I
377
purification procedures and differential elution of the antibody-binding activities by free hapten, on the one hand, and acidic buffer, on the other. Claflin and Davie (22) reported that although rabbit anti-HOPC 8 idiotypic antibodies exhibited specificity exclusively for binding-site determinants, only marginal quantities of binding site-specific antibodies could be detected in A/J mouse anti-HOPC 8 serum. In contrast, we could purify significant amounts of antibinding site, as well as antiframework, idiotypic antibodies from antiserum raised in A/J mice against TEPC-15, but no detectable binding site-specific antibodies in rabbit anti-T- 15 antiserum. It is important to emphasize the fact that the present studies demonstrate the feasibility of inducing anti-idiotypic antibodies of distinct specificities in the same species of animals since this will provide a useful tool for analysis of regulatory effects of heterogeneous anti-idiotypic antibodies on immune responses of members of the same species. Our original purpose was to investigate the immunoregulatory activities of anti-idiotypic antibodies which have different specificities. Specifically, we were interested in determining whether the respective activities obtained by these methods-namely, those antibodies binding to combining site determinants versus those specific for framework residues-exerted different, perhaps even opposing biological effects on the capacity of idiotype-positive B lymphocytes to secrete their antibody products. These studies demonstrate unequivocally, however, that no such differences in biological effects exist. These studies indicate, therefore, that the previously described contrasting effects of anti-idiotypic antibodies on lymphocyte function reported by Eichmann (14) and Rajewsky and Eichmann (15), which in some cases are enhancing and in other cases suppressive, clearly do not reflect differences in determinant specificities of the anti-idiotypic antibodies employed, at least insofar as the T-15
ANTIBODES ADDED AJ: fhfd FIG. 5. Effects of anti-idiotypic antibodies on the production of T-15 idiotype positive anti-PC antibodies at different timing. Antibodies were added at various times indicated and IO-” pg of PC-KLH was added at 0 hr. B fraction (O), F fraction (0). Data of normal mouse y-globulin at respective times were 59, 86, 78, 61, 44, and 44 ngiml. Nonadded control was 75 ngiml.
378
YAMAMOTO
AND KATZ
idiotype and the in vitro system described here are concerned. Studies currently underway in our laboratory which are directed to making similar analyses of these different anti-idiotypic antibodies on PC-specific T-cell functions may provide better clarification of these questions. ACKNOWLEDGMENTS The authors are grateful to Drs. T. Hamaoka and Barry J. Skidmore for helpful discussions during the early phase of this work. Drs. Erik Lindh and Fu-Tong Liu and Mr. Robert M. Gordon provided discussions and excellent technical assistance and we thank Anthea Hugus and Keith Dunn for assistance in preparation of the manuscript. Dr. Norman Klinman critically reviewed the paper and commented thereon in a most helpful way.
REFERENCES 1. Kunkel, H. G., Mannik, M., and Williams, R. C., Jr., Science 140, 1218, 1963. 2. Oudin, J., and Michael, M., C. R. Acad. Sci. (Paris) 257, 805, 1963. 3. Nisonoff, A., Hopper, J. E., and Spring, S. B., In “The Antibody Molecule,” p. 444. Academic Press, New York, 1975. 4. Kunkel, H. G., and Kindt, T., In “Immunogenetics and Immunodeficiency,” MTP Press, Lancaster, England, p. 55, 1975. 5. Jerne, N. K., Ann. lmmunol. (Inst. Pasteur) 12X, 373, 1974. 6. Binz, H., and Wigzell, H., Cold Spring Harbor Symp. Quant. Biol. 41, 275, 1976. 7. Krawinkel, U., Cramer, M., Berek, C. Hammerling, G., Black, S. J., Rajewski, K., and Eichmann, K., Cold Spring Harbor Symp. Quant. Biol. 41, 285, 1976. 8. Cohn, M., Notani, G., and Rice, S. A., Immunochemistry 6, 111, 1969. 9. Potter, M., and Lieberman, R., J. Exp. Med. 132, 737, 1970. 10. Lieberman, R., Potter, M., Mushinski, E. B., Humphrey, W., Jr., and Rudikoff, S., J. Exp. Med. 139, 983, 1975.
11. 12. 13. 14. 15. 16. 17. 18. 19.
Cosenza, H., and Kohler, H., Science 176, 1027, 1972. Kohler, H., Richardson, B. C., Rowley, D. A., and Smyk, S., J. Immunol. 119, 1977. Kohler, H., Richardson, B. C., and Smyk, S., J. Immunol. 120, 233, 1978. Eichmann, K., Eur. J. Immunol. 5, 511, 1975. Rajewsky, K., and Eichmann, K., In “Contemporary Topics in Immunobiology” 7, 69, 1977. Chesebro, B., and Metzger, H. Biochemistry 5, 766, 1972. Katz, D. H., Paul, W. E., Goidl, E. A., and Benacerraf, B. J. Exp. Med. 132, 261, 1970. Mishell, R. I., and Dutton, R. W., J. Exp. Med. 126, 423, 1967. Klinman, N. R., Pickard, A. R., Sigal, N. H., Gearhart, P. J., Metcalf, E. S., and Pierce, S. K.,Ann. Immunol.
(Inst. Pasteur)
127C, 489, 1976.
20. Sakato, N., and Eisen, H. N., 1. Exp. Med. 141, 1411, 1975. 21. Kohler, H., Smyk, S., and Kluskens, L., Cell. Immunol. 17, 295, 1975. 22. Claflin, J. L., and Davie, J. M. J. Immunol. 114, 70, 1975.