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Induced recovery of a suppressed idiotype by immunization with anti-idiotype
CELLULAR
Induced
IMMUNOLOGY
52, 458-463 (1980)
Recovery of a Suppressed ldiotype Anti-ldiotypel HEINZ K~HLER,JOHN
La Rabida-University
by Immunization
FUNG,~ANDSUE
SMYK
of Chicago Institute and the Department of Pathology, Chicago, Illinois 60649 Received November
with
University
of Chicago,
30, 1979
Neonatal Balb/c mice were suppressed for the HI/T15 idiotype by injection of homologous anti-H8 (D. S. Strayer, D. A. Rowley, and H. Kohler, et alJ. Zmmunol. 114,722, 1975. Six to eight weeks later groups of these suppressed mice were immunized up to three times with isologous anti-H8 raised in Balb/c. After a rest of 4 weeks each group was challenged with R36a vaccine and bleedings were obtained before and after this challenge. All sera were assayed for total anti-PC and H8 idiotype amounts by solid-phase radioimmunoassay. After two-preimmunizations with isologous anti-H8 no increase of the H8 levels was observed though these mice responded to R36a immunization with an increase of total anti-PC antibodies. After the third preimmunization, however, the H8 idiotype was increased in sera taken before and after challenge with R36a. These findings demonstrate that the state of neonatal idiotype suppression can be broken by immunization with complementary anti-idiotype.
INTRODUCTION The response of Balb/c mice to phosphorylcholine (PC)3 is characterized by the dominance of the T15 idiotype (1). The T15 idiotype is not the only idiotype the Balb/c mouse possesses. If the T15 clone is chronically suppressed by neonatal anti-idiotype application (2) other, non-T15 clones respond to immunization with PC (3). Furthermore, analysis of the neonatal precursor repertoire has shown that neonatal liver contains mainly PC precursors which are not of the dominant T15 idiotype (4). The dominance of the T15 idiotype in the Balb/c response to PC appears late in ontogeny and is not seen before Day 5 after birth (5). During the time of the first week after birth we (6) and others (3) have observed a small amount of auto-anti-idiotype in the serum of normal Balb/c neonates. This finding led us to speculate that auto-anti-idiotypic antibody stimulates the expression of the complementary idiotype (6). In the present study we wantedto test this hypothesis in a model system in which the dominant T15 idiotype had been chronically suppressed. This kind of adult 1 Supported by USHPS Grant AI-1 1080. * Supported by Training Grant T32-GM07281 3 Abbreviations used: PC, phosphorylcholine; T15 and H8, purified PC-binding myeloma proteins from TEPC-15 and HOPC-8 Balb/c tumors, respectively; RIA, radioimmunoassay. 458 0008-8749/80/080458-06$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
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animal mimics the early neonate with respect to the lack of T15 dominance. Neonatally suppressed Balb/c were preimmunized with purified Balb/c anti-T15 antibody before challenging with PC antigen. Only after repeated anti-idiotype immunizations the T15 idiotype appeared dominant in the anti-PC response. MATERIALS
AND METHODS
Animals. Six- to eight-week female Balb/c mice were purchased from Cumberland Farms (Clinton, Tenn.) and female A/He of similar age from Jackson Laboratories (Bar Harbor, Maine). The HOPC-8 tumor line was a gift from M. Potter (NCI) and was maintained in our animal facilities by serial transfer in Balb/c mice. Purification of myeloma protein and anti-idiotypic antibody: HOPC-8 protein was purified on affinity chromatography by elution with 10e2M PC according to Chesebro and Metzger (7). Balb/c anti-H8 sera were raised in neonatally suppressed mice as described previously (8). The anti-idiotypic antibodies were purified as described (9). Briefly, 1 ml of Balb/c anti-H8 serum was diluted with T&-saline and adsorbed on an equal volume of HS-Sepharose. After incubation of 37°C for 30 min the Sepharose was extensively washed and eluted with 2 ml of 1 M acetic acid. The eluted antibody was immediately neutralized by adding 10x Tris buffer, pH 7.8, dialyzed against PPS, and concentrated. The antibody activity of purified anti-H8 was compared to a standard anti-H8 serum in RIA. The protein concentration was determined by amino acid analysis. Immunizations. Each experimental group of mice consisted of 8-10 animals. Neonatal Balb/c mice were suppressed by injection of 50 ~1 of 1:5 diluted A/He anti-H8 serum within 24-48 hr after birth (2). At the age of 6-8 weeks these and normal litter mates were used in different experimental protocols. One group of neonatally suppressed mice was preimmunized with 10 pg of purified isologous anti-H8 in complete Freund’s adjuvant one time; another group was preimmunized twice at monthly intervals and a third group three times at biweekly intervals; the second and third preimmunizations were in incomplete Freund’s adjuvant. Then all groups were rested for 1 month before they were immunized with R36a intravenously. Bleedings from all animals were taken before the first preimmunization, after the last preimmunization, and 5 days after the immunization with R36a. Radioimmunoassays. H8 idiotype and anti-PC antibody were measured by a modification of Klinman’s solid-phase method (10). For measuring anti-PC antibodies polyvinyl titer plates were first coated with 100~1 of PC-BSA (10 p.g/ml) and the remaining sites were saturated with BSA (1% in PBS). Standard amounts of purified H8 or dilutions of sera were added to the wells followed by addition of rabbit XIti-K (Bionetics, Kensington, Md.), and 1251-iodinated goat anti-rabbit (5 x lo4 cpmwell). Maximum binding was between 25 and 40%. Idiotype amounts were measured using titer wells coated with 100~1 H8 solution of (0.1 pg/ml). The binding of 1251-iodinated(Fab’), of H8 by A/He anti-H8 (2 x lo4 cpm/well) was inhibited by standard amounts of T15 or dilutions of sera. The binding in the absence of inhibitors was 30-42%. The amount of H8 or anti-PC in sera was calculated using known amounts of T15 from linear regression of the binding or inhibition. The radioactivity was measured in a 4/600 micrometics gamma counter.
SHORT COMMUNICATIONS
RESULTS Effects of Immunization Suppressed Mice
with Anti-H8 on the Expression of H8 in Neonatally
The expression of the H8 idiotype in conventional Balb/c mice is substantial even without immunization with PC (11). Therefore normal Balb/c are not a good choice for studying the effects of anti-idiotype immunization on the level of the H8 idiotype. In contrast, Balb/c which were suppressed as neonates for the H8 idiotype (2) have only small amounts of circulating H8 idiotype and thus are better animals for stimulating the expression of H8 by anti-idiotype. Anti-H8 antibodies can be induced in normal Balb/c mice only with difficulties, but are readily induced in neonatally suppressed Balblc (8). Anti-H8 antibodies were purified from hyperimmunized, neonatally suppressed Balb/c by elution from HB-Sepharose immunoabsorbents. Groups of neonatally suppressed Balb/c were immunized with 10pg of purified isologous anti-H8 up to three times. Sera were obtained from these groups before and after these immunizations; then the mice were rested for 4 weeks, challenged with R36a, and bled 5 days later. The amounts of H8 and of total anti-PC antibodies were measured by RIA; the data are shown in Table 1. Neonatally suppressed mice have five times less circulating H8 than normal but equal amounts of total anti-PC antibodies. Upon immunization with R36a the antiPC antibodies increase while the H8 remains the same. The same pattern of response is observed in the two other groups of neonatally suppressed Balb/c which had received one or two preimmunizations with anti-H8. The amount of H8 in these TABLE 1 Idiotype Expression in Neonatally Suppressed Balb/c Anti-Idiotype immunizationb
PC immunizationc
HS k.dml)
Normal
-
+
25.3 48.8
33.8 55.3
Neonatally suppressed
-
+
5.6 5.7
33.8 109.3
Neonatally suppressed
lx lx
+
4.1 4.6
44.9 80.6
Neonatally suppressed
2x 2x
+
7.8 6.8
44.7 82.3
Neonatally suppressed
3x 3x
+
13.4 24.7
5.3 48.4
Animals”
Anti-PCd (dml)
(1Groups of normal or neonatally suppressed Balb/c mice, 6-8 weeks of age, were used. b Neonatally suppressed mice were preimmunized either one time with 10 pg purified Balb/c anti-HI, or twice at monthly intervals, or three times at biweekly intervals. c After the last preimmunization all groups were rested for 1 month and then immunized with R36a and bled 5 days later. d The serum levels of anti-PC and H8 of pooled sera from the different groups were determined by solid-phase RIA.
SHORTCOMMUNICATIONS
461
sera is only 5-8% of the total anti-PC antibodies. However, after the third preimmunization with anti-H8 the relative and absolute amount of HS idiotype has increased. In pooled sera from such mice more H8 idiotype than anti-PC is present. After challenge with R36a both titers increase, and the amount of H8 represents about half of the total anti-PC activity. When normal Balb/c mice were subjected to an identical schedule of three preimmunizations with anti-H8 and challenged with R36a, the H8 level prior to R36a challenge was within the range of normal untreated mice and after immunization with R36a anti-PC and H8 increased in parallel fashion like in normal immunized Balb/c (data not shown). H8 and Anti-PC
Expression
in Individual
Mice
It was observed that the amount of total anti-PC antibodies in individual neonatally suppressed mice in the experimental groups shown in Table 1 varies over a wide range. In contrast the H8 idiotype levels in individual mice did not vary much. Since these findings are not evident from determinations on pooled sera Fig. 1 is presented to demonstrate this point. The H8 and total anti-PC antibodies in individual mice of an experimental group which had been primed twice with Balb/c anti-H8 and challenged with R36a are shown. The levels before and after challenge with R36a are shown. The individual variation in the anti-PC titers is unexpectedly large while the H8 levels are more or less uniform. This seems to indicate that there is no apparent linkage between the state of specific idiotype suppression and the total anti-PC antibody amount. It is also evident that double priming with anti-H8 did not increase the H8 expression in any of the individually tested mice. Similar findings were seen with mice after single priming with anti-H8 (data not shown& DISCUSSION The data in this report are relevant to two previous experimental findings: neonatal T15 idiotype suppression (2) and induction of an idiotype by anti-idiotype (12, 13). Neonatal application of even very small amounts of anti-T15 anti-idiotype serum (K. Gleason and H. Kohler, unpublished observation) induces a long-lasting state of idiotype suppression. If these mice are challenged with PC antigen they respond with non-T15 anti-PC antibodies. Evidence has been presented that this state is actively maintained by a cellular mechanism (14). This mechanism could prevent the reemergence of idiotype-committed precursors after an initial reduction of idiotype precursors at the time of anti-idiotype injection, since the number of idiotype-positive cells in spleens of neonatally suppressed mice is drastically reduced in fluorescence studies (15). Assuming that anti-idiotype can induce the expression of idiotypes (13, 14, 16)it is somewhat surprising to find that it takes three injections of anti-idiotype to induce recovery of the suppressed idiotype. This relative resistance to stimulation by anti-idiotype is compatible with the notion that an active mechanism is operating in neonatally suppressed mice which tends to maintain the state of suppression. To further support this hypothesis germ-free mice in which the natural H8 is low were used in attempts to induce the idiotype through anti-idiotype; it was observed that already small doses of antiidiotype induced the expression of H8 (J. Fung, unpublished observations).
462
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MICE
FIG. 1. H8 idiotype (upper panel) and anti-PC amounts (lower panel) from individual neonatally suppressed Balb/c. The corresponding bars in both panels give the amounts of H8 and anti-PC from the same mouse. (A) Bleedings from neonatally suppressed I-week-old Balb/c; (B) bleedings from the same mice after two preimmunizations with anti-H8; (C) bleedings from the same mice 5 days after immunizations with R36a.
When finally recovery of the suppressed H8 is observed after repeated application of anti-H8 the recovery is not accompanied by an increase of total antiPC antibody level. As seen in Fig. 1 the amount of anti-PC in individual animals is highly variable and some mice have less anti-PC antibodies than HS idiotype. This indicates that those animals responded to preimmunization with H8 with a selective increase of H8. Such specific stimulation of an idiotype without the usually associated antigen specificity has also been observed in rabbits by Urbain (13) and Cazenave (12). The bleedings of the group of mice which had been preimmunized three times with anti-H8 were assayed for anti-PC and H8 levels after the pooling of sera. Therefore the data in Table 1 do not allow us to distinguish between two possibilities accounting for the increase in H8 idiotype observed in the group preimmunized three times. Either a few animals had responded selectively with a
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very large increase of H8 or all animals had responded selectively in the group with a moderate H8 increase. In either case the same conclusion can be drawn, namely, that three preimmunizations with isologous anti-idiotypic antibodies are required to induce a recovery from idiotype suppression. The dissociation of idiotype and antigen specificity becomes a challenging argument in the discussion of idiotypic networks (1, 17, 18). In the case of the H8 idiotype, which in the normal Balb/c mouse represents more than 95% of the total anti-PC response, the construction of idiotypic circuits regulating the response becomes even more complex. If the H8 idiotype is the target for idiotype-specific regulation (19,20) and if this regulation is responsible for the H8 dominance (6,21) the independent behavior of H8 levels and anti-PC levels observed in this study is not easily explained by a simple idiotype-anti-idiotype circuitry. REFERENCES Kohler, H., 7’ransplanf.Rev. 27, 29, 1975. Strayer, D. S., Rowley, D. A., and Kohler H., J. Immunol. 114, 728, 1975. Augustin, A., and Cosenza, H., Eur. .I. Immunol. 6, 497, 1976. Fung, J., Gleason, K., Ward, R., and Kohler, H., In “Membranes, Receptors, and the Immune Response” (E. P. Cohen and H. Kohler, Eds.), Liss, New York, in press. 5. Sigal, N. H., Gearhart, P. J., Press, J. L., and Klinman, N. R., Nature (London) 259, 51, 1976. 6. Kohler, H., Kaplan, D., Kaplan, R., Fung .I., and Quintans, J., In “Cells of Immunoglobulin Synthesis” (B. Pernis and H. Vogel, Eds.), p. 357. Academic Press, New York, 1979. 7. Chesebro, B., and Metzger, H., Biochemistry 11, 766, 1972. 8. Kohler, H., Richardson, B. C., and Smyk, S., J. Immunol. 120, 233, 1978. 9. Kohler, H., Richardson, B. C., Rowley, D. A., and Smyk, S., J. Zmmunol. 119, 1979, 1977. 10. Klinman, N. R., Pickard, A. R., Sigal,N. H., Gearhart, P. J., Metcalf, E. S., and Pierce, S. K., Ann. Immunol. 127C, L, 89, 1976. Il. Lieberman, R., Potter, M., Mushinski, E. B., Humphrey, W., and Rudikoff, S.,J. Exp. Med. 139, 1. 2. 3. 4.
983, 1974.
12. Cazenave, P. A., Proc. Nat. Acad. Sci. USA 74, 5122, 1977. 13. Urbain, J., Wikler, M., Franssen, J. P., and Collingnon, T., Proc. Nat. Acad. Sci. USA 74, 5126, 1977. 14. Du Clos, T. W., and Kim, B. S., J. Zmmunol. 119, 1769, 1977. 15. Kohler, H., Strayer, D. S., and Kaplan, D. R., Science 186, 643, 1979. 16. Kohler, H., In “Genetic Control of Autoimmune Disease” (N. R. Rose, P. E. Bigazzi, and N. L. Warner, Eds.), p. 343. Elsevier/North-Holland, Amsterdam, 1978. 17. Jeme, N. K., Ann. Inst. Pasteur 125C, 373, 1979. 18. Rowley, D. A., Kohler, H., and Cowan, J. D., In “Contemporary Topics in Immunobiology” (J. Marchalonis, Ed.), Plenum, New York, in press. 19. Kluskens, L., and Kohler, H., Proc. Nut. Acad. Sci. USA 71, 5083, 1974. 20. Rowley, D. A., Kohler, H., Schreiber, H., Kay, S. T., and Lorbach, I. J. Exp. Med. 144, 946, 1976. 21. Kohler, H., Rowley, D. A., Du Clos, T., and Richardson, B. C. Fed. Proc. 36, 221, 1977.
Induced
IMMUNOLOGY
52, 458-463 (1980)
Recovery of a Suppressed ldiotype Anti-ldiotypel HEINZ K~HLER,JOHN
La Rabida-University
by Immunization
FUNG,~ANDSUE
SMYK
of Chicago Institute and the Department of Pathology, Chicago, Illinois 60649 Received November
with
University
of Chicago,
30, 1979
Neonatal Balb/c mice were suppressed for the HI/T15 idiotype by injection of homologous anti-H8 (D. S. Strayer, D. A. Rowley, and H. Kohler, et alJ. Zmmunol. 114,722, 1975. Six to eight weeks later groups of these suppressed mice were immunized up to three times with isologous anti-H8 raised in Balb/c. After a rest of 4 weeks each group was challenged with R36a vaccine and bleedings were obtained before and after this challenge. All sera were assayed for total anti-PC and H8 idiotype amounts by solid-phase radioimmunoassay. After two-preimmunizations with isologous anti-H8 no increase of the H8 levels was observed though these mice responded to R36a immunization with an increase of total anti-PC antibodies. After the third preimmunization, however, the H8 idiotype was increased in sera taken before and after challenge with R36a. These findings demonstrate that the state of neonatal idiotype suppression can be broken by immunization with complementary anti-idiotype.
INTRODUCTION The response of Balb/c mice to phosphorylcholine (PC)3 is characterized by the dominance of the T15 idiotype (1). The T15 idiotype is not the only idiotype the Balb/c mouse possesses. If the T15 clone is chronically suppressed by neonatal anti-idiotype application (2) other, non-T15 clones respond to immunization with PC (3). Furthermore, analysis of the neonatal precursor repertoire has shown that neonatal liver contains mainly PC precursors which are not of the dominant T15 idiotype (4). The dominance of the T15 idiotype in the Balb/c response to PC appears late in ontogeny and is not seen before Day 5 after birth (5). During the time of the first week after birth we (6) and others (3) have observed a small amount of auto-anti-idiotype in the serum of normal Balb/c neonates. This finding led us to speculate that auto-anti-idiotypic antibody stimulates the expression of the complementary idiotype (6). In the present study we wantedto test this hypothesis in a model system in which the dominant T15 idiotype had been chronically suppressed. This kind of adult 1 Supported by USHPS Grant AI-1 1080. * Supported by Training Grant T32-GM07281 3 Abbreviations used: PC, phosphorylcholine; T15 and H8, purified PC-binding myeloma proteins from TEPC-15 and HOPC-8 Balb/c tumors, respectively; RIA, radioimmunoassay. 458 0008-8749/80/080458-06$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
459
animal mimics the early neonate with respect to the lack of T15 dominance. Neonatally suppressed Balb/c were preimmunized with purified Balb/c anti-T15 antibody before challenging with PC antigen. Only after repeated anti-idiotype immunizations the T15 idiotype appeared dominant in the anti-PC response. MATERIALS
AND METHODS
Animals. Six- to eight-week female Balb/c mice were purchased from Cumberland Farms (Clinton, Tenn.) and female A/He of similar age from Jackson Laboratories (Bar Harbor, Maine). The HOPC-8 tumor line was a gift from M. Potter (NCI) and was maintained in our animal facilities by serial transfer in Balb/c mice. Purification of myeloma protein and anti-idiotypic antibody: HOPC-8 protein was purified on affinity chromatography by elution with 10e2M PC according to Chesebro and Metzger (7). Balb/c anti-H8 sera were raised in neonatally suppressed mice as described previously (8). The anti-idiotypic antibodies were purified as described (9). Briefly, 1 ml of Balb/c anti-H8 serum was diluted with T&-saline and adsorbed on an equal volume of HS-Sepharose. After incubation of 37°C for 30 min the Sepharose was extensively washed and eluted with 2 ml of 1 M acetic acid. The eluted antibody was immediately neutralized by adding 10x Tris buffer, pH 7.8, dialyzed against PPS, and concentrated. The antibody activity of purified anti-H8 was compared to a standard anti-H8 serum in RIA. The protein concentration was determined by amino acid analysis. Immunizations. Each experimental group of mice consisted of 8-10 animals. Neonatal Balb/c mice were suppressed by injection of 50 ~1 of 1:5 diluted A/He anti-H8 serum within 24-48 hr after birth (2). At the age of 6-8 weeks these and normal litter mates were used in different experimental protocols. One group of neonatally suppressed mice was preimmunized with 10 pg of purified isologous anti-H8 in complete Freund’s adjuvant one time; another group was preimmunized twice at monthly intervals and a third group three times at biweekly intervals; the second and third preimmunizations were in incomplete Freund’s adjuvant. Then all groups were rested for 1 month before they were immunized with R36a intravenously. Bleedings from all animals were taken before the first preimmunization, after the last preimmunization, and 5 days after the immunization with R36a. Radioimmunoassays. H8 idiotype and anti-PC antibody were measured by a modification of Klinman’s solid-phase method (10). For measuring anti-PC antibodies polyvinyl titer plates were first coated with 100~1 of PC-BSA (10 p.g/ml) and the remaining sites were saturated with BSA (1% in PBS). Standard amounts of purified H8 or dilutions of sera were added to the wells followed by addition of rabbit XIti-K (Bionetics, Kensington, Md.), and 1251-iodinated goat anti-rabbit (5 x lo4 cpmwell). Maximum binding was between 25 and 40%. Idiotype amounts were measured using titer wells coated with 100~1 H8 solution of (0.1 pg/ml). The binding of 1251-iodinated(Fab’), of H8 by A/He anti-H8 (2 x lo4 cpm/well) was inhibited by standard amounts of T15 or dilutions of sera. The binding in the absence of inhibitors was 30-42%. The amount of H8 or anti-PC in sera was calculated using known amounts of T15 from linear regression of the binding or inhibition. The radioactivity was measured in a 4/600 micrometics gamma counter.
SHORT COMMUNICATIONS
RESULTS Effects of Immunization Suppressed Mice
with Anti-H8 on the Expression of H8 in Neonatally
The expression of the H8 idiotype in conventional Balb/c mice is substantial even without immunization with PC (11). Therefore normal Balb/c are not a good choice for studying the effects of anti-idiotype immunization on the level of the H8 idiotype. In contrast, Balb/c which were suppressed as neonates for the H8 idiotype (2) have only small amounts of circulating H8 idiotype and thus are better animals for stimulating the expression of H8 by anti-idiotype. Anti-H8 antibodies can be induced in normal Balb/c mice only with difficulties, but are readily induced in neonatally suppressed Balblc (8). Anti-H8 antibodies were purified from hyperimmunized, neonatally suppressed Balb/c by elution from HB-Sepharose immunoabsorbents. Groups of neonatally suppressed Balb/c were immunized with 10pg of purified isologous anti-H8 up to three times. Sera were obtained from these groups before and after these immunizations; then the mice were rested for 4 weeks, challenged with R36a, and bled 5 days later. The amounts of H8 and of total anti-PC antibodies were measured by RIA; the data are shown in Table 1. Neonatally suppressed mice have five times less circulating H8 than normal but equal amounts of total anti-PC antibodies. Upon immunization with R36a the antiPC antibodies increase while the H8 remains the same. The same pattern of response is observed in the two other groups of neonatally suppressed Balb/c which had received one or two preimmunizations with anti-H8. The amount of H8 in these TABLE 1 Idiotype Expression in Neonatally Suppressed Balb/c Anti-Idiotype immunizationb
PC immunizationc
HS k.dml)
Normal
-
+
25.3 48.8
33.8 55.3
Neonatally suppressed
-
+
5.6 5.7
33.8 109.3
Neonatally suppressed
lx lx
+
4.1 4.6
44.9 80.6
Neonatally suppressed
2x 2x
+
7.8 6.8
44.7 82.3
Neonatally suppressed
3x 3x
+
13.4 24.7
5.3 48.4
Animals”
Anti-PCd (dml)
(1Groups of normal or neonatally suppressed Balb/c mice, 6-8 weeks of age, were used. b Neonatally suppressed mice were preimmunized either one time with 10 pg purified Balb/c anti-HI, or twice at monthly intervals, or three times at biweekly intervals. c After the last preimmunization all groups were rested for 1 month and then immunized with R36a and bled 5 days later. d The serum levels of anti-PC and H8 of pooled sera from the different groups were determined by solid-phase RIA.
SHORTCOMMUNICATIONS
461
sera is only 5-8% of the total anti-PC antibodies. However, after the third preimmunization with anti-H8 the relative and absolute amount of HS idiotype has increased. In pooled sera from such mice more H8 idiotype than anti-PC is present. After challenge with R36a both titers increase, and the amount of H8 represents about half of the total anti-PC activity. When normal Balb/c mice were subjected to an identical schedule of three preimmunizations with anti-H8 and challenged with R36a, the H8 level prior to R36a challenge was within the range of normal untreated mice and after immunization with R36a anti-PC and H8 increased in parallel fashion like in normal immunized Balb/c (data not shown). H8 and Anti-PC
Expression
in Individual
Mice
It was observed that the amount of total anti-PC antibodies in individual neonatally suppressed mice in the experimental groups shown in Table 1 varies over a wide range. In contrast the H8 idiotype levels in individual mice did not vary much. Since these findings are not evident from determinations on pooled sera Fig. 1 is presented to demonstrate this point. The H8 and total anti-PC antibodies in individual mice of an experimental group which had been primed twice with Balb/c anti-H8 and challenged with R36a are shown. The levels before and after challenge with R36a are shown. The individual variation in the anti-PC titers is unexpectedly large while the H8 levels are more or less uniform. This seems to indicate that there is no apparent linkage between the state of specific idiotype suppression and the total anti-PC antibody amount. It is also evident that double priming with anti-H8 did not increase the H8 expression in any of the individually tested mice. Similar findings were seen with mice after single priming with anti-H8 (data not shown& DISCUSSION The data in this report are relevant to two previous experimental findings: neonatal T15 idiotype suppression (2) and induction of an idiotype by anti-idiotype (12, 13). Neonatal application of even very small amounts of anti-T15 anti-idiotype serum (K. Gleason and H. Kohler, unpublished observation) induces a long-lasting state of idiotype suppression. If these mice are challenged with PC antigen they respond with non-T15 anti-PC antibodies. Evidence has been presented that this state is actively maintained by a cellular mechanism (14). This mechanism could prevent the reemergence of idiotype-committed precursors after an initial reduction of idiotype precursors at the time of anti-idiotype injection, since the number of idiotype-positive cells in spleens of neonatally suppressed mice is drastically reduced in fluorescence studies (15). Assuming that anti-idiotype can induce the expression of idiotypes (13, 14, 16)it is somewhat surprising to find that it takes three injections of anti-idiotype to induce recovery of the suppressed idiotype. This relative resistance to stimulation by anti-idiotype is compatible with the notion that an active mechanism is operating in neonatally suppressed mice which tends to maintain the state of suppression. To further support this hypothesis germ-free mice in which the natural H8 is low were used in attempts to induce the idiotype through anti-idiotype; it was observed that already small doses of antiidiotype induced the expression of H8 (J. Fung, unpublished observations).
462
SHORT COMMUNICATIONS
INDIVIDUAL
MICE
FIG. 1. H8 idiotype (upper panel) and anti-PC amounts (lower panel) from individual neonatally suppressed Balb/c. The corresponding bars in both panels give the amounts of H8 and anti-PC from the same mouse. (A) Bleedings from neonatally suppressed I-week-old Balb/c; (B) bleedings from the same mice after two preimmunizations with anti-H8; (C) bleedings from the same mice 5 days after immunizations with R36a.
When finally recovery of the suppressed H8 is observed after repeated application of anti-H8 the recovery is not accompanied by an increase of total antiPC antibody level. As seen in Fig. 1 the amount of anti-PC in individual animals is highly variable and some mice have less anti-PC antibodies than HS idiotype. This indicates that those animals responded to preimmunization with H8 with a selective increase of H8. Such specific stimulation of an idiotype without the usually associated antigen specificity has also been observed in rabbits by Urbain (13) and Cazenave (12). The bleedings of the group of mice which had been preimmunized three times with anti-H8 were assayed for anti-PC and H8 levels after the pooling of sera. Therefore the data in Table 1 do not allow us to distinguish between two possibilities accounting for the increase in H8 idiotype observed in the group preimmunized three times. Either a few animals had responded selectively with a
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very large increase of H8 or all animals had responded selectively in the group with a moderate H8 increase. In either case the same conclusion can be drawn, namely, that three preimmunizations with isologous anti-idiotypic antibodies are required to induce a recovery from idiotype suppression. The dissociation of idiotype and antigen specificity becomes a challenging argument in the discussion of idiotypic networks (1, 17, 18). In the case of the H8 idiotype, which in the normal Balb/c mouse represents more than 95% of the total anti-PC response, the construction of idiotypic circuits regulating the response becomes even more complex. If the H8 idiotype is the target for idiotype-specific regulation (19,20) and if this regulation is responsible for the H8 dominance (6,21) the independent behavior of H8 levels and anti-PC levels observed in this study is not easily explained by a simple idiotype-anti-idiotype circuitry. REFERENCES Kohler, H., 7’ransplanf.Rev. 27, 29, 1975. Strayer, D. S., Rowley, D. A., and Kohler H., J. Immunol. 114, 728, 1975. Augustin, A., and Cosenza, H., Eur. .I. Immunol. 6, 497, 1976. Fung, J., Gleason, K., Ward, R., and Kohler, H., In “Membranes, Receptors, and the Immune Response” (E. P. Cohen and H. Kohler, Eds.), Liss, New York, in press. 5. Sigal, N. H., Gearhart, P. J., Press, J. L., and Klinman, N. R., Nature (London) 259, 51, 1976. 6. Kohler, H., Kaplan, D., Kaplan, R., Fung .I., and Quintans, J., In “Cells of Immunoglobulin Synthesis” (B. Pernis and H. Vogel, Eds.), p. 357. Academic Press, New York, 1979. 7. Chesebro, B., and Metzger, H., Biochemistry 11, 766, 1972. 8. Kohler, H., Richardson, B. C., and Smyk, S., J. Immunol. 120, 233, 1978. 9. Kohler, H., Richardson, B. C., Rowley, D. A., and Smyk, S., J. Zmmunol. 119, 1979, 1977. 10. Klinman, N. R., Pickard, A. R., Sigal,N. H., Gearhart, P. J., Metcalf, E. S., and Pierce, S. K., Ann. Immunol. 127C, L, 89, 1976. Il. Lieberman, R., Potter, M., Mushinski, E. B., Humphrey, W., and Rudikoff, S.,J. Exp. Med. 139, 1. 2. 3. 4.
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12. Cazenave, P. A., Proc. Nat. Acad. Sci. USA 74, 5122, 1977. 13. Urbain, J., Wikler, M., Franssen, J. P., and Collingnon, T., Proc. Nat. Acad. Sci. USA 74, 5126, 1977. 14. Du Clos, T. W., and Kim, B. S., J. Zmmunol. 119, 1769, 1977. 15. Kohler, H., Strayer, D. S., and Kaplan, D. R., Science 186, 643, 1979. 16. Kohler, H., In “Genetic Control of Autoimmune Disease” (N. R. Rose, P. E. Bigazzi, and N. L. Warner, Eds.), p. 343. Elsevier/North-Holland, Amsterdam, 1978. 17. Jeme, N. K., Ann. Inst. Pasteur 125C, 373, 1979. 18. Rowley, D. A., Kohler, H., and Cowan, J. D., In “Contemporary Topics in Immunobiology” (J. Marchalonis, Ed.), Plenum, New York, in press. 19. Kluskens, L., and Kohler, H., Proc. Nut. Acad. Sci. USA 71, 5083, 1974. 20. Rowley, D. A., Kohler, H., Schreiber, H., Kay, S. T., and Lorbach, I. J. Exp. Med. 144, 946, 1976. 21. Kohler, H., Rowley, D. A., Du Clos, T., and Richardson, B. C. Fed. Proc. 36, 221, 1977.