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Immunological unresponsiveness induced in adult mice to synthetic polypeptides built on multichain polyproline and multichain polyalanine
hmmmochemi~tr~. 1974. Vol. I I, pp. 347 353.
Pergamon Press.
Printed in Great Britain
IMMUNOLOGICAL UNRESPONSIVENESS INDUCED IN ADULT MICE TO SYNTHETIC POLYPEPTIDES BUILT ON MULTICHAIN POLYPROLINE A N D MULTICHAIN POLYALANINE* BENJAMIN BONAVIDA,t EDNA MOZES, G. M. SHEARER:~ and M I C H A E L SELA§ From the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel (First received 19 March 1973; in revised form 20 July 1973)
Abstract--Tolerance to synthetic antigens was obtained by injecting them in soluble centrifuged form. Adult C3H.SW mice were injected with 2 mg of the soluble, multichain synthetic polypeptides poly (Tyr, Glu)-poly Pro-poly Lys, (T, G)-Pro~-L; poly (Tyr, Glu)-poly DL Ala-poly Lys, (T, G)-A-L; or poly-(Phe, Glu)-poly DL Ala poly Lys, (Phe, G)-A L, administered in one, two or seven aqueous injections and then immunized with the homologous antigens in complete Freund's adjuvant. All three schedules of soluble antigen injection induced efficiently tolerance to (T, G)-A-L and (Phe, G)-A-L, whereas for (T, G)-Pro-L, only the seven-injection protocol was highly effective. Unresponsiveness upon primary and secondary challenge of tolerant mice was also tested as a function of the dose of (T, G)-Pro-L (1 ng - 2 mg), administered in two injections. The intermediate range of 100 ng to 1 #g was most effective in inducing unresponsiveness in the primary response, whereas the upper (2 mg) and lower (1-10 ng) appeared to be more effective in the secondary response. Mice made tolerant to the Pro-L moiety of (T, G)-Prc~L elicited normal immune responses to the T. G determinant in {T. GFA L. Mice made tolerant to (T. GFPro L. and immunized ~ith {Phc. Gl-Pro L. elicited normal primary and secondary anti-(Phe. G) immune responscs but remained tolerant to the Pro L moiety. Since {T, G)-Pro L and {Phe, G)-Pro L arc cross reacting antigens and share 80 per cent of their structure. it is concluded that "'breaking of tolerance by cross reacting antigens" is not due to antibody formation to the determinants to which the animal is tolerant (in this case Pro-L) but to the formation of antibodies to a new determinant in the cross antigen.
INTRODUCTION
Numerous studies have dealt with the induction of immunological unresponsiveness (Mitchison, 1964; Weigle, 1967; Dresser and Mitchison, 1968; Landy and Braun, 1969; Weigle, 1971) and the apparent termination of tolerance by immunization with cross-reacting immunogens (Weigle, 1967; Benjamin and Weigle, 1970: Nachtigal. 1971 : Schechtcr et al., 1964). In many of these investigations complex macromolecular proteins have been used which contain poorly defined immunopotent regions and/or antigenic determinants. Only recently (Benjamin and Weigle, 1971) have attempts been made to ascertain immunochemically the antibody responses obtained after tolerance indue* Supported in part by the Thyssen Foundation. t Present address: Department of Medical Microbiology and Immunology, University of California at Los Angeles, Los Angeles, California 90024. :~Performed during tenure of American Cancer Society, Postdoctoral Fellowship PF-524. Present address: Immunology Branch, National Cancer Institutes of Health, Bethesda, Md. 20014. § Send correspondence to: Prof. Michael Sela, Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel. 347
tion following immunization with cross-reacting antigens. Serum albumins have been used extensively in studies on the termination of the unresponsive state (Weigle, 1967; Benjamin and Weigle, 1970; Nachtigal, 1971 ; Schechter et al., 1964). Conceivably an apparent state of termination may result if the tolerogen contains antigenic regions (capable of reacting with antibodies) which are neither immunopotent nor tolerogenie (Bonavida and Sercarz, 1971), and which are present in an immunopotent form in the cross-reacting immunogen used for tolerance abrogation. The use of the multichain synthetic polypeptide antigens offer an advantage in the elucidation of immunological tolerance, since these polymers possess defined chemical structure, in which the antigenic determinants are restricted (Sela et al., 1962; Fuchs and Sela, 1964; Jaton and Sela, 1968), and it is possible to construct crossreacting antigens in which the common parts as well as the different parts of the molecule are precisely known. For example, (T, G)-Pro-L and (Phe, G)-ProL share the P r o - L moiety which comprises about 80 per cent of the molecule and differ in the other regions, being either (T, G) or (Phe, G). Immunological tolerance, induced in rabbits toward synthetic antigens, has been previously described (Scla
348
B. BONAVIDA, E. MOZES, G. M. SHEARER and M. SELA
et al., 1963, 1971 ; Maurer et al., 1963, 1965; Bauminger and Sela, 1969). In the experiments described in this report, tolerance induction against various synthetic polypeptides built on multichain poly L proline and on multichain poly DL alanine has been successfully achieved in adult mice. Since genetic differences have been observed in the ability of different mouse strains to respond to synthetic polypeptides (McDevitt and Benacerraf, 1969; Mozes and Shearer, 1972), the C3H.SW strain, which is a good responder to all the above described immunogens (McDevitt and Chinitz, 1969; Mozes et al., 196%), was used in the present study. Mice were made tolerant to the synthetic polypeptide (T, G)-Pro-L (which contains a single immunopotent region, Pro-L) and later immunized with a related polypeptide (Phe, G ) - P r ~ L (which contains a second immunopotent region (Phe, G) in addition to the Pro-L region found in the tolerogen). The present report demonstrates lack of termination of the unresponsive state following immunization with a cross-reacting antigen, although concomitant antibody formation was obtained for the determinant contained in the immunogen only. MATERIALS AND METHODS
Animals The mice used in these experiments were 8-12 weeks old C3H.SW animals of both sexes, obtained from the Experimental Animal Unit, The Weizmann Institute of Science. Antigens The multichain synthetic polypeptides used as tolerogens, immunogens and assaying antigens were poly (LTyr, LGlu)poly L Pro-poly L Lys, denoted (T, G)-Pro-L (Jaton and Sela, 1968); poly (LPhe, LGlu)-poly L Pro-Poly-L Lys, (Phe, G)-Pro-L (Jaton and Sela, 1968); poly (LPhe, LGlu)-poly DL Ala-poly L Lys, (Phe, G)-A-L (Fuchs and Sela, 1964); and poly (LTyr, LGlu)-poly OL Ala-poly L Lys, denoted (T, G)A-L (Sela et al., 1962). The description, synthesis, characterization and immunogenicity of these branched chain polymers have been published elsewhere (Jaton and Sela, 1968; Mozes et al., 1969a; Fuchs and Sela, 1964; Sela et al., 1962; McDevitt and Chinitz, 1969). Tolerance induction and immunization Unresponsiveness was produced in the mice by injecting soluble, centrifuged antigens. The antigens were dissolved in phosphate-buffered saline (0'15 N NaCI, 0.01 M phosphate buffer, pH 7) (PBS) and centrifuged at 15,000 g for 30 min. Only the upper 2/3 of the supernatant was used for injection. The dissolved, centrifuged antigens were injected intraperitoneally in a volume of 0.2 ml. They were administered in a total dose of 2.0 mg, injected as one, two or seven injections. The two injections were spaced 1 week apart and the seven injections were given on alternate days over a period of 2 weeks. Lower doses of(T, G)-Pro-L (ranging from 1 ng to 2 mg) were also given as tolerogen, using the two-injections protocol. The immunogens (10 pg/mouse) were injected intradermally in the hind foot pads in complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, Michigan) 2 weeks after a single administration of tolerogen, or 1 week after the two
and seven injection schedules of the tolerogen. Two weeks after primary immunization, the mice were bled from the retro-orbital plexus and boosted 1 week later with 10 #g of immunogen in PBS. The animals were bled for secondary responses 10 days later. Passive hemagglutination and plaque assays Sheep erythrocytes were formalinized, tanned (Herbert, 1967) and coated either with (T, G)-Pro~L or with (Phe, G)A-L as previously described (Shearer et al., 1971). Passive microhemagglutination tests were performed using disposable microtiter plates (Cooke Engineering Co., Alexandria, Va.) by two-fold serial dilutions of antisera in phosphatebuffered saline containing 0"1~o bovine plasma albumin (crystallized, Armour Pharmaceutical Co., Chicago, Ill.) (Kabat, 1968). The plates were incubated at 20°C and read 2 hr later. Hemolytic plaque-forming cell assays were made for the (T, G)-Pro-L immune response. The antigen was coupled to fresh sheep erythrocytes (SRBC) by incubating equal volumes of washed SRBC, (T, G)-Pro-L (10 mg/ml in PBS) and chromium chloride (10 mg/ml in PBS) for 3 rain at 20°C (Perucca et al., 1969). The SRBC were washed three times in PBS and diluted to a final concentration of 10% in PBS. Base layers were prepared by adding 1.5 ml of an isotonic solution of 1.5% agarose (L'Industrie Biologique Francaise S.A., Gennevilliers, France) in Eagle's Minimum Essential Medium (Grand Island Biological Co., Grand Island, N.Y.) to 60 mm dia plastic disposable petri dishes. An overlay of 0'7~o agarose in Minimum Essential Medium was kept in a 47°C water bath until the cells were plated. Spleen cell suspensions from the immunized mice were prepared as described (Shearer et al., 1968). The antigen-coated SRBC (0.1 ml) and spleen cells (0.2 ml) were added to 0.6 ml of the top agarose, and the mixture was quickly poured onto the base layers. The plates were incubated at 37°C for 1.5 hr without complement and an additional 1.5 hr with I ml per plate of 1 : 10 diluted lyophilized guinea pig complement (Grand Island Biological Co., Grand Island, N.Y.) (Jerne et al., 1963). RESULTS
Immunological unresponsiveness to (T, G)-Pro-L compared by passive hemagglutination and~ hemolytic plaque-forming cell assays Three groups of mice were injected with a total of 0.2, 1 or 2 mg of soluble centrifuged (T, G ) - P r ~ L administered in seven injections, spaced at 2-day intervals. One week after the final injection, the animals were immunized with 10 #g (T, G)-Pro-L and boosted 3 weeks later. Immune responses specific for (T, G)Pro-L were assayed 10 days after the second immunization by passive hemagglutination of the sera and by a modified hemolytic plaque-forming cell assay. The results obtained from individual mice are shown in Table 1. A reduction was obtained in the immune responses assayed by both methods with all three doses of soluble (T, G)-Pro-L. With one exception, all animals showing a titer of less than 1:4 (considered in this study to be tolerant) also exhibited less than 400 anti-(T, G)-Pro-L P F C per spleen, Immunized mice not given solutle (T, G)-Pro-L showed titers of 1 : 32-1 : 64 and P F C responses of 660-2000 per spleen. The P F C responses in mice injected with 2 mg
Tolerance of Mice to Synthetic Polypeptides
349
Table 1. Comparison of passive hemagglutination titers and (T, G)-Pro-L hemolytic plaque-forming cells per spleen in C3H.SW mice made unresponsive to different doses of (T, G)-Pro-L and immunized with (T, G)-Pr6-L 2.0 Mouse number
HA
PFC
1 2 3 4 5 6 7
< 1:4 <1:4 <1:4 < 1:4 < 1:4 <1:4 < 1:4
10 20 70 40 40 10 70
Total tolerogen dose (mg/mouse)• 1.0 0.2 HA PFC HA PFC < 1:4 < 1:4 < 1:4 < 1:4 <1:4 <1:4 < 1:4
60 1020 60 360 80 130 90
< 1:4 < 1:4 < 1:4 1:4 < 1:4 <1:4 < 1:4
0
20 140 40 240 290 80 100
HA
PFC
1:64 1:32 1:32 1:32 1:64
660 670 1500 2000 1100
' The total tolerogen dose was administered by seven injections spaced at 2-day intervals. The mice were immunized with 10/~g (T, G)-Pro-L 7 days after the last injections of tolerogen and boosted 3 weeks later. HA--Hemagglutination titers; PFC---(T, G)-Pro-L plaque-forming cells/spleen.
PRIMARY RESPONSE
h-
<1:4
I;4
h8
mw 8 f
if
1:16
1:32
1:64
SECONDARY RESPONSE
<1:4
1:4
1:8
1:16
1:32
1:64
HEMAGGLUTINATION TITERSWITH(T,G)-PRO--L
Fig. l. Primary and secondary hemagglutination titers of sera from C3H.SW mice immunized with 10/~g(T, G)-Pro-L in CFA after injection of 2 mg of soluble, centrifuged (T, G)-Pro-L administered in one ([]), two or seven (ll) injections. Titers of immunized mice not given the tolerogen are shown by (1). soluble (T, G)-Pro-L were lower than those detected in mice which received a total of 1 mg or 0-2 mg of the tolerogen, suggesting that 2 mg is a better tolerogenic dose. No differences were observed in the hemagglutination titers among the different doses of tolerogen used, since a dilution of 1:4 was the lowest titer assayed. Passive hemagglutination titers of 1:4 were occasionally obtained in sera from unimmunized mice. Thus, sera giving titers of 1:4 were considered to be negative. Since tolerance was verified by two independent techniques, only hemagglutination assays were used in subsequent experiments. Tolerance induced by 2 m# of soluble centrifuged polypeptides administered in one, two or seven injections The induction.of tolerance to the multichain synthetic polypeptides (T, G)-Pro-L, fl', G)-A-L, and (Phe, G)-A-L was investigated by administering 2 mg of the polypeptides in one injection, in two injections
spaced 1 week apart, or in seven injections spaced at 2-day intervals. The three groups of mice were immunized with 10 ~tg of antigen in CFA 2 weeks after tolerogen for the single injection group, and 1 week after the second or seventh tolerogen injections for the other two groups. All animals were bled 14 days after primary immunization, boosted with 10/~g of antigen in PBS 3 weeks after primary immunization, and bled for secondary immune responses 10 days after boosting. The hemagglutination titers obtained in the primary and secondary responses to (T, G)-Pro-L, (T, G)A - L and (Phe, G)-A-L for the three schedules of tolerogen are shown in Figs. 1-3, respectively. For (T, G)-Pro-L (see Fig. 1), unresponsiveness was more pronounced in the secondary than in the primary response. Administration of the tolerogen in two injections appeared to be least effective in the primary response, whereas a schedule of seven injections was most effective in the secondary response. When (T, G)-A-L was used as the tolerogen and immunogen (see Fig. 2), no differences were detected in the primary and secondary responses for one, two or
'!F
~o
•
PRIMARY RESPONSE
1:4
1:8
1
1:16
1:32
1:64
<1:4 1:4 118 hi6 1:32 I;64 HEMMAGLUTINATION TITERS WITH (T,G)-A--L
1:128
1:128
Fig. 2. Primary and secondary hemagglutination titers of sera from C3H.SW mice immunized with 10 #g (T, G)-A-L in CFA after injection of 2 mg of soluble, centrifuged (T, G)-A-L administered in one ([]), two ( I or seven ( I ) injections. Titers of immunized mice not given the tolerogen are shown by (I).
350
B. BONAVIDA, E. MOZES, G. M. SHEARER and M. SELA
I!f PRIMARYRESPONSE
LL
<1:4
114
1:8
1:t6
1:32
1:16
1:~2
1:64
1:128
Ol~(.i SECONDARYRESPONSE
'
<1:4
t:4
1:8
l_J
1:64
1:~28
HEMAGGLUTINAT~ON T~TERS WITH (~HE,G)-Z--L
Fig. 3. Primary and secondary hemagglutination titers of sera from C3H.SW mice immunized with 10 #g (Phe, G)A-L in CFA after injection of 2 mg of soluble, centrifuged (Phe, G)-A-L administered in one (E]), two ( ) or seven ([]) injections. Titers of immunized mice not given the tolerogen are shown by (11). seven injections, except that titers of 1:8 and 1:16 were detected in the secondary responses of two animals given a single injection of tolerogen. The results obtained using (Phe, G)-A-L as tolerogen and immunogen are summarized in Fig. 3. A greater degree of unresponsiveness was seen after secondary than after primary immunization for all three schedules of tolerogen administration. The primary responses of normal control mice immunized with (Phe, G)-A-L were lower than those observed for (T, G)-Pro-L and (T, G)-A-L, and were not higher than the groups of mice injected with the tolerogen. huluction o[ tolerance as a [m+ction 0[" the amount 0[ soluble (T, G)-Pr~-L il!iected Six groups of mice (10 15 mice per group), were injected with varying amounts of soluble, centrifuged (T, G)-Pro-L ranging from 1 ng to 2 nag. Tolerogen was administered in two injections spaced 1 week apart. Primary and secondary immunizations were made as described above. The percentage of mice with positive hemagglutination titers after primary or secondary immunization are shown in Fig. 4. 1130 ng and 1 #g appeared to be the most effective doses for inhibition of the primary response (less than 20 per cent of the mice giving a detectable response), whereas the extreme doses of I ng and 2 mg were the least effective (50 per cent or more of the mice giving significant titers). In contrast, for the secondary response the most effective tolerogen dose used was 1 ng, whereas 1 #g appeared to be the least effective (42 per cent of the mice exhibited serum titers ranging from 1 : 16 to 1:64). Specificity requirements of the tolerogen and the immunogen Immunization of mice with (T, G)-Pro-L results in an immune response specific for the Pro-L moiety only (Mozes et al., 1969a). From this observation it would appear that (T, G) on polyprolyl side chains is not immunogenic. |n contrast, immunization with (T,
G)-A-L results mainly in antibodies specific for the sequences of tyrosine and glutamic acid (McDevitt and Sela, 1965). Therefore, it was of interest to investigate whether it is possible to induce tolerance to (T, G) by using (T, G)-Pro-L as the tolerogen and (T, G)-A-L as the immunogen. One group of mice made tolerant to (T, G)-Pro-L and a second group of untreated control mice were immunized with (T, G ) - A - L The results shown in Table 2 demonstrate that no reduction in the immune response to (T, G)-A-L was detected in the mice tolerant to (T, G)-Pro--L (T, G)-Pro-L and (Phe, G)-Pro-L share the Pro-L immunopotent region, and (Phe, G)-Pro-L contains the additional (Phe, G) immunopotent region (Mozes et al., 1969a). Thus, it was possible to investigate whether induction of tolerance to (T, G)-Pro-L followed by immunization with (Phe, G)-Pro-L would result in the production of antibodies specific for (Phe, G) only, the production of antibodies specific for both (Phe, G)-and Pro-L, or whether no immune response would be detected to either of these specificities, Mice were injected with a total of 2 mg of soluble (T, G)-Pro-L in one, two or seven injections. The animals were then immunized and boosted as described above with 10 pg (Phe, G)-Pro-L. The percentage of sera showing specific anti-(Phe, G)-A-L and anti(T, G) Pro-L titers are shown in Fig. 5. Tolerance was obtained in the primary response for the Pro-L specificity with all three schedules of injection. No significant degree of tolerance was seen in the primary response for the (Phe, G) specificity. Although only 50 per cent of the tolerant animals given seven injections of tolerogen responded to (Phe, G), in the control also only 67 per cent of the mice (not given tolerogen) gave a detectable primary response. L, O nr" W F~-
I00
,~ 80
~ 6o ~ 40
~ zo
t~ n
I ng
I0 ng I00 ng
I~g 200/~
2 mg
DOSE OF (T,GI-PRO--L TO'LEROGEN
Fig. 4. Percentage of sera giving anti-(T, G)-Pro--L titers greater than 1:4 as a function of the dose of soluble, centrifuged (T, G)-Pro--L (administered in two injections). Primary response denoted by (IS]); secondary response denoted by (U).
Tolerance of Mice to Synthetic Polypeptides Table 2. Passive hemagglutination titers in mice made tolerant" to (T. G)-Pro L and immunizedb with (T, G)-A L Secondary anti-(T, G)-A-L titer (T, G)-Pr~L No tolerogen tolerogen
Mouse number
1:32 1:64 1:64 1:64 1:64 1:64
1
2 3 4 5 6
1:128 1:128 1:64 1:64 1:128 1:64
"Two mg soluble (T. G)-Pro-L given in seven injections spaced at 2-day intervals. b Ten /~g (T, G)-A-L given intraperitoneally in CFA I week after last injection of tolerogen. Secondary immunization was 10 #g (T, G)-A-L in PBS 3 weeks after primary immunization.
i 0 0 PRIMARY RESPONSE
~
60
~ 4o ~
2o
o
0
I
'°°r
BOI
2 R
7
0
NS
6o
,=, 4 o
zo o
I 2 7 0 NUMBER OF TOLEROGEN INJECTIONS (TOTAL OF Z rng (T,G)-PRO--L)
Fig. 5. Percentage of sera giving anti-(Phe, G)-A L (D) or anti-(T, G)- Pro--L (11) titers greater than 1:4 in mice immunized with 10/~g (Phe, G)-(Pro--L after injection of 2 mg soluble, centrifuged (T, G)-Pro-L in one, two or seven injections. In the secondary response, distinctions were observed in the induction of tolerance to Pro--L as a function of the schedule of tolerogen injection. Tolerance to P r o - L was terminated when 2 mg of soluble (T, G)-Pro-L was administered in one injection. In contrast, 50 and 18 per cent of the animals showed positive titers when the tolerogen was given in two or seven injections, respectively. No tolerance was observed however in the secondary response to the (Phe, G) specificity for any of the tolerogen schedules used. Thus, these results indicate that the immune response obtained was specific for an immunopotent region of the immunogen (Phe, G) which was not contained in the tolerogen.
351 DISCUSSION
The experiments described in this report demonstrate that tolerance can be induced in adult mice to multichain synthetic polypeptides of both the 'A-L' and 'Pro-L' series. (T, G)-A-L and (Phe, G)-A L appear to be better tolerogens in C3H.SW mice than (T, G)-Pro--L. These two polypeptides of the A-L series are also better immunogens than (T, G)-Pro-L in this mouse strain (McDevitt and Benacerraf, 1969; Mozes et al., 1969a). In the present example there would appear to be a direct correlation between immunogenicity and the ability to induce unresponsiveness. This agrees with the findings of some investigations (Rajewsky and Rottl~inder, 1967; Collotti and Leskowitz, 1969). However, it is not a universal observation, since a lot of evidence has been presented that immunogenicity is not a necessary criterion for tolerogenicity (Janeway and Humphrey, 1968; Parish and Ada, 1969; Roelants and Goodman, 1970). An unresponsive state has been obtained to proteins by injection of high or very low doses of antigen. These phenomena have been respectively referred to as high and low zones of tolerance (Dresser and Mitchison, 1968; Mitchison, 1964), and a considerable amount of attention has been focused on the selective inactivation of different cell populations by high. and low zone tolerance (Mitchison, 1971). A significant reduction in the immune responses to (T, G)-Pro-L was obtained for different doses oftolerogen injected (see Fig. 4). The most effective dose range of soluble (T, G)-Pro-L for the primary response was 100 ng to 1/~g, and the least effective were the extreme doses of 1 ng and 2 mg. In contrast, for the secondary response 1 #g was the least effective tolerogen dose, whereas the 1 10 ng range were most effective. The reason for this difference between the primary and secondary response is unknown, although it is possible that different functional cell populations might be selectively affected in the primary and secondary response as a function of tolerogen dose. It also may be that the production of different classes of antibodies is differentially affected by primary and secondary responses after tolerance induction with the different doses (Weber and Kt~lsch, 1972). It should be noted that the boost consisted of 10 ~g soluble aggregated antigen, some of which is in a tolerogenic form. This material might have induced additional tolerance. In mice the immune response to (T, G ) - P r ~ L is directed mainly to the ProwL portion of the molecule (Mozes et al., 1969a), whereas immunization with (T, G)-A-L primarily generates antibodies specific for the short sequences of tyrosine and glutamic acid (McDevitt and Sela, 1965). The injection of soluble (T, G)Pro-L as tolerogen followed by immunization with (T, G)-A-L resulted in a normal response to the immunogen (Table 2). Thus, the observation that a normal response was obtained to (T, G)-A-L in mice made tolerant to (T, G)-Pro-L agrees with the earlier observation (discussed above) that a weakly or non-immunogenic-determinant may also be a poor tolerogen.
352
B. BONAVIDA. E. MOZES, G. M. SHEARER and M. SELA
However, the possibility also exists that the mice were tolerant to the short sequences of tyrosine and glutamic acid on Pro-L, but that breakage of tolerance to (T, G) occurred as a result of immunization with (T, G) A-L. Immunization with (Phe, G)-Pro-L results in populations of antibadies specific for the short terminal sequences of (Phe, G), as well as for the polyproline side chains (Mozes et al., 1969a, b). In contrast, immunization with (T, G)-Pro-L results in an immune response specific for Pro-L only. Thus, tolerance induction to (T, G)-Pro-L followed by immunization with (Phe, G)-Pro-L provides an experimental situation in which the tolerogen contains a single, repeating immunopotent region, whereas the immunogen contains a new immunopotent region as well as the one in common with the tolerogen. Neither the primary nor the secondary anti-(Phe, G) responses were significantly below those obtained in the normal immunized control mice (see Fig. 5). Thus, a normal immune response was obtained for a new determinant on an immunogen which also possessed the determinants in common with the tolerogen. In contrast, the anti-Pro-L primary response was considerably below the control group (20-32 per cent vs 100 per cent) for all three schedules of tolerogen injection. A difference was seen in the secondary response to Pro-L as a function of the tolerogen injection schedule. A normal secondary anti-ProL response was seen when the tolerogen was administered in a single injection. Two mg of soluble (T, G)Pro-L in two injections resulted in 50 per cent response, whereas less than 20 per cent of the mice given 2 mg of tolerogen in seven injections responded to the Pro-L specificity. A possibility not tested in these experiments is that the animals given a single injection of tolerogen may have escaped the unresponsive state after immunization and boosting more readily than those animals given the same amount of tolerogen in multiple injections. Intramolecular competition between (Phe, G) and Pro-L was found to occur in mice immunized with (Phe, G)-Pro-L (Taussig et al., 1972). In this case P r o L was the dominant antigen and (Phe, G) the suppressed one. The results described in this paper that mice rendered tolerant to Pro-L respond to (Phe, G), agree with other observations that antigenic competition does not take place in an animal which is tolerant to an otherwise dominant antigen (Taussig, 1973). The results described in this report differ from those previously reported by Weigle and colleagues (Weigle, 1967; Benjamin and Weigle, 1970), who demonstrated termination of immunological unresponsiveness in rabbits made tolerant to bovine serum albumin (BSA) and immunized with related albumins such as those of human, pig, horse and guinea pig. However, sheep serum albumin did not terminate tolerance to BSA and did not induce antibodies specific to the sheep albumin determinants not present on BSA (Weigle, 1961). It was concluded from these studies that termination is obtained if the tolerogen and immunogen exhibit
major structural differences (Benjamin and Weigle, 1971). The results of immunization of mice unresponsive to (T, G)-Pro-L and injected with (Phe, G)-Pro--L were not similar to the above, since tolerance to Pro-L was not terminated by immunization with (Phe, G)Pro-L. The combination of synthetic polypeptides as tolerogens and immunogens can be useful in investigating the cellular aspects of antigen-induced unresponsiveness.
REFERENCES
Bauminger S., Schechter I. and Sela M. (1967) lmmunochemistry 4, 169. Bauminger S. and Sela M. (1969) Israel J. reed. Sci. 5, 177. Benjamin D. C. and Weigle W. O. (1970) J. exp. Med. 132, 66. Benjamin D. C. and Weigle W. O. (1971) lmmunochemistry 8, 1087. Bonavida B. and Sercarz E. (1971) Eur. J. Immun. 1, 166. Collotti C. and Leskowitz S. (1969) Nature, Lond. 222, 97. Dresser D. W. and Mitchison N. A. (1968) Advan. Immunol. 8, 129. Fuchs S. and Sela M. (1964) Biochem. J. 93, 566. Gill T. J., Kunz H. W. and Papermaster D. S. (1967) J. biol. Chem. 242, 3308. Herbert W. J. (1967) Handbook of Experimental Immunology (Edited by Weir D. M.), p. 720. Blackwell Scientific Publications, Ltd., Oxford. Janeway C. A. and Humphrey J. H. (1968) Immunology 14, 225. Janeway C. A, and Sela M. (1967) Immunology 13, 29. Jaton J.-C. and Sela M. (1968) d. biol. Chem. 243, 5616. Jerne N. K., Nordin A. A. and Henry C. (1963) Cell Bound Antibodies (Edited by Amos B. and Koprowski H.), p. 109. The Wistar Institute Press, Philadelphia. Kabat E. A. (1968) Structural Concepts in Immunolooy and lmmunochemistry, p. 32. Holt, Rinehart and Winston, New York. Landy M. and Braun W. (Eds.) (1969) 1mmunolooical Tolerance. A Reassessment of Mechanisms of the Immune Response. Academic Press, New York. Maurer P. H., Lowy R. and Kierney C. (1963) Science 139,
1061. Maurer P. H., Pinchuk P. and Gerulat B. F. (1965) Proc. Soc. exp. Biol. 118, 1113. McDevitt H. O. and Benacerraf B. (1969) Advan. 1mmunol. 11, 31. McDevitt H. O. and Chinitz A. (1969) Science 163, 1207, McDevitt H. O. and Sela M. (1965) J. exp. Med. 122, 517. Mitchison N. A. 0964) Proe. R. Soc. B 161,275. Mitchison N. A. ( 1971) Cell Interactions and R ecel,tor A ntibodies in hnmune Responses (Edited by M/ikel~i O., Cross A. and Kosunen T. U.), p. 249. Academic Press, New York, London. Mozes E., McDevitt H. O., Jaton J.-C. and Sela M. (1969a) J. exp. Med. 130, 493. Mozes E., McDevitt H. O., Jaton J.-C. and Sela M. (1969b) J. exp. Meal. 130, 1263. Mozes E. and Shearer G. M. (1972) Curt. topics Microbiol. hnmunol. 59, 167. Nachtigal D. (1971) hn,mnology 20, 719. Parish C. R. and Ada G, L. (1969) hnmunoloqy 17, 153.
Tolerance
of Mice to Synthetic
Perucca P. J., Faulk W. P. and Fudenberg H. H. (1969) J. Inumm 102, 8 12. Rajewsky K. and RottlHnder E. (1967) Cold Spring Hark Symp. quant. Biol. 32, 541. Roelants G. E. and Goodman J. W. (19701 Nuture. Lund. 221, 175. Schechter I., Bauminger S., Sela M., Nachtigal D. and Feldman M. (1964) Immunochemistry 1, 249. Sela M., Fuchs S. and Arnon R. (1962) Biochrm. J. 85, 223. Sela M., Fuchs S. and Feldman M. (1963) Science 139, 342. Sela M., Fuchs S.. Maron R. and Gertner B. (1971) Eur. J. Immun. 1, 36. Shearer G. M., Cudkowicz G., Connell M. S. J. and Priore R. L. (1968) J. <‘.Y,‘.Mrtl. 128, 437.
Polypeptides
353
Shearer G. M., Mazes E. and Sela M. (1971) J. exp. Med. 133, 216. Taussig M. J. (1973) Curr. Topics Microhiol. Immunol. 60, 125. Taussig M. J., Mazes E., Shearer G. M. and Sela M. (1972) Eur. J. lmmun. 2. 448. Weber G. and KGlsch E. (1972) Eur. J. Immun. 2, 191. Weiele W. 0. (1961) J. exe. Mod. 114. 111. Weigle W. 0.‘(196$ Natural and A&wired Immunoloyical Unresponsiveness. World Publishing Co., Cleveland, Ohio. Weigle W. 0. (197 I ) Cliu. cxp. /mrmr~~ol.9, 437.
Pergamon Press.
Printed in Great Britain
IMMUNOLOGICAL UNRESPONSIVENESS INDUCED IN ADULT MICE TO SYNTHETIC POLYPEPTIDES BUILT ON MULTICHAIN POLYPROLINE A N D MULTICHAIN POLYALANINE* BENJAMIN BONAVIDA,t EDNA MOZES, G. M. SHEARER:~ and M I C H A E L SELA§ From the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel (First received 19 March 1973; in revised form 20 July 1973)
Abstract--Tolerance to synthetic antigens was obtained by injecting them in soluble centrifuged form. Adult C3H.SW mice were injected with 2 mg of the soluble, multichain synthetic polypeptides poly (Tyr, Glu)-poly Pro-poly Lys, (T, G)-Pro~-L; poly (Tyr, Glu)-poly DL Ala-poly Lys, (T, G)-A-L; or poly-(Phe, Glu)-poly DL Ala poly Lys, (Phe, G)-A L, administered in one, two or seven aqueous injections and then immunized with the homologous antigens in complete Freund's adjuvant. All three schedules of soluble antigen injection induced efficiently tolerance to (T, G)-A-L and (Phe, G)-A-L, whereas for (T, G)-Pro-L, only the seven-injection protocol was highly effective. Unresponsiveness upon primary and secondary challenge of tolerant mice was also tested as a function of the dose of (T, G)-Pro-L (1 ng - 2 mg), administered in two injections. The intermediate range of 100 ng to 1 #g was most effective in inducing unresponsiveness in the primary response, whereas the upper (2 mg) and lower (1-10 ng) appeared to be more effective in the secondary response. Mice made tolerant to the Pro-L moiety of (T, G)-Prc~L elicited normal immune responses to the T. G determinant in {T. GFA L. Mice made tolerant to (T. GFPro L. and immunized ~ith {Phc. Gl-Pro L. elicited normal primary and secondary anti-(Phe. G) immune responscs but remained tolerant to the Pro L moiety. Since {T, G)-Pro L and {Phe, G)-Pro L arc cross reacting antigens and share 80 per cent of their structure. it is concluded that "'breaking of tolerance by cross reacting antigens" is not due to antibody formation to the determinants to which the animal is tolerant (in this case Pro-L) but to the formation of antibodies to a new determinant in the cross antigen.
INTRODUCTION
Numerous studies have dealt with the induction of immunological unresponsiveness (Mitchison, 1964; Weigle, 1967; Dresser and Mitchison, 1968; Landy and Braun, 1969; Weigle, 1971) and the apparent termination of tolerance by immunization with cross-reacting immunogens (Weigle, 1967; Benjamin and Weigle, 1970: Nachtigal. 1971 : Schechtcr et al., 1964). In many of these investigations complex macromolecular proteins have been used which contain poorly defined immunopotent regions and/or antigenic determinants. Only recently (Benjamin and Weigle, 1971) have attempts been made to ascertain immunochemically the antibody responses obtained after tolerance indue* Supported in part by the Thyssen Foundation. t Present address: Department of Medical Microbiology and Immunology, University of California at Los Angeles, Los Angeles, California 90024. :~Performed during tenure of American Cancer Society, Postdoctoral Fellowship PF-524. Present address: Immunology Branch, National Cancer Institutes of Health, Bethesda, Md. 20014. § Send correspondence to: Prof. Michael Sela, Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel. 347
tion following immunization with cross-reacting antigens. Serum albumins have been used extensively in studies on the termination of the unresponsive state (Weigle, 1967; Benjamin and Weigle, 1970; Nachtigal, 1971 ; Schechter et al., 1964). Conceivably an apparent state of termination may result if the tolerogen contains antigenic regions (capable of reacting with antibodies) which are neither immunopotent nor tolerogenie (Bonavida and Sercarz, 1971), and which are present in an immunopotent form in the cross-reacting immunogen used for tolerance abrogation. The use of the multichain synthetic polypeptide antigens offer an advantage in the elucidation of immunological tolerance, since these polymers possess defined chemical structure, in which the antigenic determinants are restricted (Sela et al., 1962; Fuchs and Sela, 1964; Jaton and Sela, 1968), and it is possible to construct crossreacting antigens in which the common parts as well as the different parts of the molecule are precisely known. For example, (T, G)-Pro-L and (Phe, G)-ProL share the P r o - L moiety which comprises about 80 per cent of the molecule and differ in the other regions, being either (T, G) or (Phe, G). Immunological tolerance, induced in rabbits toward synthetic antigens, has been previously described (Scla
348
B. BONAVIDA, E. MOZES, G. M. SHEARER and M. SELA
et al., 1963, 1971 ; Maurer et al., 1963, 1965; Bauminger and Sela, 1969). In the experiments described in this report, tolerance induction against various synthetic polypeptides built on multichain poly L proline and on multichain poly DL alanine has been successfully achieved in adult mice. Since genetic differences have been observed in the ability of different mouse strains to respond to synthetic polypeptides (McDevitt and Benacerraf, 1969; Mozes and Shearer, 1972), the C3H.SW strain, which is a good responder to all the above described immunogens (McDevitt and Chinitz, 1969; Mozes et al., 196%), was used in the present study. Mice were made tolerant to the synthetic polypeptide (T, G)-Pro-L (which contains a single immunopotent region, Pro-L) and later immunized with a related polypeptide (Phe, G ) - P r ~ L (which contains a second immunopotent region (Phe, G) in addition to the Pro-L region found in the tolerogen). The present report demonstrates lack of termination of the unresponsive state following immunization with a cross-reacting antigen, although concomitant antibody formation was obtained for the determinant contained in the immunogen only. MATERIALS AND METHODS
Animals The mice used in these experiments were 8-12 weeks old C3H.SW animals of both sexes, obtained from the Experimental Animal Unit, The Weizmann Institute of Science. Antigens The multichain synthetic polypeptides used as tolerogens, immunogens and assaying antigens were poly (LTyr, LGlu)poly L Pro-poly L Lys, denoted (T, G)-Pro-L (Jaton and Sela, 1968); poly (LPhe, LGlu)-poly L Pro-Poly-L Lys, (Phe, G)-Pro-L (Jaton and Sela, 1968); poly (LPhe, LGlu)-poly DL Ala-poly L Lys, (Phe, G)-A-L (Fuchs and Sela, 1964); and poly (LTyr, LGlu)-poly OL Ala-poly L Lys, denoted (T, G)A-L (Sela et al., 1962). The description, synthesis, characterization and immunogenicity of these branched chain polymers have been published elsewhere (Jaton and Sela, 1968; Mozes et al., 1969a; Fuchs and Sela, 1964; Sela et al., 1962; McDevitt and Chinitz, 1969). Tolerance induction and immunization Unresponsiveness was produced in the mice by injecting soluble, centrifuged antigens. The antigens were dissolved in phosphate-buffered saline (0'15 N NaCI, 0.01 M phosphate buffer, pH 7) (PBS) and centrifuged at 15,000 g for 30 min. Only the upper 2/3 of the supernatant was used for injection. The dissolved, centrifuged antigens were injected intraperitoneally in a volume of 0.2 ml. They were administered in a total dose of 2.0 mg, injected as one, two or seven injections. The two injections were spaced 1 week apart and the seven injections were given on alternate days over a period of 2 weeks. Lower doses of(T, G)-Pro-L (ranging from 1 ng to 2 mg) were also given as tolerogen, using the two-injections protocol. The immunogens (10 pg/mouse) were injected intradermally in the hind foot pads in complete Freund's adjuvant (CFA) (Difco Laboratories, Detroit, Michigan) 2 weeks after a single administration of tolerogen, or 1 week after the two
and seven injection schedules of the tolerogen. Two weeks after primary immunization, the mice were bled from the retro-orbital plexus and boosted 1 week later with 10 #g of immunogen in PBS. The animals were bled for secondary responses 10 days later. Passive hemagglutination and plaque assays Sheep erythrocytes were formalinized, tanned (Herbert, 1967) and coated either with (T, G)-Pro~L or with (Phe, G)A-L as previously described (Shearer et al., 1971). Passive microhemagglutination tests were performed using disposable microtiter plates (Cooke Engineering Co., Alexandria, Va.) by two-fold serial dilutions of antisera in phosphatebuffered saline containing 0"1~o bovine plasma albumin (crystallized, Armour Pharmaceutical Co., Chicago, Ill.) (Kabat, 1968). The plates were incubated at 20°C and read 2 hr later. Hemolytic plaque-forming cell assays were made for the (T, G)-Pro-L immune response. The antigen was coupled to fresh sheep erythrocytes (SRBC) by incubating equal volumes of washed SRBC, (T, G)-Pro-L (10 mg/ml in PBS) and chromium chloride (10 mg/ml in PBS) for 3 rain at 20°C (Perucca et al., 1969). The SRBC were washed three times in PBS and diluted to a final concentration of 10% in PBS. Base layers were prepared by adding 1.5 ml of an isotonic solution of 1.5% agarose (L'Industrie Biologique Francaise S.A., Gennevilliers, France) in Eagle's Minimum Essential Medium (Grand Island Biological Co., Grand Island, N.Y.) to 60 mm dia plastic disposable petri dishes. An overlay of 0'7~o agarose in Minimum Essential Medium was kept in a 47°C water bath until the cells were plated. Spleen cell suspensions from the immunized mice were prepared as described (Shearer et al., 1968). The antigen-coated SRBC (0.1 ml) and spleen cells (0.2 ml) were added to 0.6 ml of the top agarose, and the mixture was quickly poured onto the base layers. The plates were incubated at 37°C for 1.5 hr without complement and an additional 1.5 hr with I ml per plate of 1 : 10 diluted lyophilized guinea pig complement (Grand Island Biological Co., Grand Island, N.Y.) (Jerne et al., 1963). RESULTS
Immunological unresponsiveness to (T, G)-Pro-L compared by passive hemagglutination and~ hemolytic plaque-forming cell assays Three groups of mice were injected with a total of 0.2, 1 or 2 mg of soluble centrifuged (T, G ) - P r ~ L administered in seven injections, spaced at 2-day intervals. One week after the final injection, the animals were immunized with 10 #g (T, G)-Pro-L and boosted 3 weeks later. Immune responses specific for (T, G)Pro-L were assayed 10 days after the second immunization by passive hemagglutination of the sera and by a modified hemolytic plaque-forming cell assay. The results obtained from individual mice are shown in Table 1. A reduction was obtained in the immune responses assayed by both methods with all three doses of soluble (T, G)-Pro-L. With one exception, all animals showing a titer of less than 1:4 (considered in this study to be tolerant) also exhibited less than 400 anti-(T, G)-Pro-L P F C per spleen, Immunized mice not given solutle (T, G)-Pro-L showed titers of 1 : 32-1 : 64 and P F C responses of 660-2000 per spleen. The P F C responses in mice injected with 2 mg
Tolerance of Mice to Synthetic Polypeptides
349
Table 1. Comparison of passive hemagglutination titers and (T, G)-Pro-L hemolytic plaque-forming cells per spleen in C3H.SW mice made unresponsive to different doses of (T, G)-Pro-L and immunized with (T, G)-Pr6-L 2.0 Mouse number
HA
PFC
1 2 3 4 5 6 7
< 1:4 <1:4 <1:4 < 1:4 < 1:4 <1:4 < 1:4
10 20 70 40 40 10 70
Total tolerogen dose (mg/mouse)• 1.0 0.2 HA PFC HA PFC < 1:4 < 1:4 < 1:4 < 1:4 <1:4 <1:4 < 1:4
60 1020 60 360 80 130 90
< 1:4 < 1:4 < 1:4 1:4 < 1:4 <1:4 < 1:4
0
20 140 40 240 290 80 100
HA
PFC
1:64 1:32 1:32 1:32 1:64
660 670 1500 2000 1100
' The total tolerogen dose was administered by seven injections spaced at 2-day intervals. The mice were immunized with 10/~g (T, G)-Pro-L 7 days after the last injections of tolerogen and boosted 3 weeks later. HA--Hemagglutination titers; PFC---(T, G)-Pro-L plaque-forming cells/spleen.
PRIMARY RESPONSE
h-
<1:4
I;4
h8
mw 8 f
if
1:16
1:32
1:64
SECONDARY RESPONSE
<1:4
1:4
1:8
1:16
1:32
1:64
HEMAGGLUTINATION TITERSWITH(T,G)-PRO--L
Fig. l. Primary and secondary hemagglutination titers of sera from C3H.SW mice immunized with 10/~g(T, G)-Pro-L in CFA after injection of 2 mg of soluble, centrifuged (T, G)-Pro-L administered in one ([]), two or seven (ll) injections. Titers of immunized mice not given the tolerogen are shown by (1). soluble (T, G)-Pro-L were lower than those detected in mice which received a total of 1 mg or 0-2 mg of the tolerogen, suggesting that 2 mg is a better tolerogenic dose. No differences were observed in the hemagglutination titers among the different doses of tolerogen used, since a dilution of 1:4 was the lowest titer assayed. Passive hemagglutination titers of 1:4 were occasionally obtained in sera from unimmunized mice. Thus, sera giving titers of 1:4 were considered to be negative. Since tolerance was verified by two independent techniques, only hemagglutination assays were used in subsequent experiments. Tolerance induced by 2 m# of soluble centrifuged polypeptides administered in one, two or seven injections The induction.of tolerance to the multichain synthetic polypeptides (T, G)-Pro-L, fl', G)-A-L, and (Phe, G)-A-L was investigated by administering 2 mg of the polypeptides in one injection, in two injections
spaced 1 week apart, or in seven injections spaced at 2-day intervals. The three groups of mice were immunized with 10 ~tg of antigen in CFA 2 weeks after tolerogen for the single injection group, and 1 week after the second or seventh tolerogen injections for the other two groups. All animals were bled 14 days after primary immunization, boosted with 10/~g of antigen in PBS 3 weeks after primary immunization, and bled for secondary immune responses 10 days after boosting. The hemagglutination titers obtained in the primary and secondary responses to (T, G)-Pro-L, (T, G)A - L and (Phe, G)-A-L for the three schedules of tolerogen are shown in Figs. 1-3, respectively. For (T, G)-Pro-L (see Fig. 1), unresponsiveness was more pronounced in the secondary than in the primary response. Administration of the tolerogen in two injections appeared to be least effective in the primary response, whereas a schedule of seven injections was most effective in the secondary response. When (T, G)-A-L was used as the tolerogen and immunogen (see Fig. 2), no differences were detected in the primary and secondary responses for one, two or
'!F
~o
•
PRIMARY RESPONSE
1:4
1:8
1
1:16
1:32
1:64
<1:4 1:4 118 hi6 1:32 I;64 HEMMAGLUTINATION TITERS WITH (T,G)-A--L
1:128
1:128
Fig. 2. Primary and secondary hemagglutination titers of sera from C3H.SW mice immunized with 10 #g (T, G)-A-L in CFA after injection of 2 mg of soluble, centrifuged (T, G)-A-L administered in one ([]), two ( I or seven ( I ) injections. Titers of immunized mice not given the tolerogen are shown by (I).
350
B. BONAVIDA, E. MOZES, G. M. SHEARER and M. SELA
I!f PRIMARYRESPONSE
LL
<1:4
114
1:8
1:t6
1:32
1:16
1:~2
1:64
1:128
Ol~(.i SECONDARYRESPONSE
'
<1:4
t:4
1:8
l_J
1:64
1:~28
HEMAGGLUTINAT~ON T~TERS WITH (~HE,G)-Z--L
Fig. 3. Primary and secondary hemagglutination titers of sera from C3H.SW mice immunized with 10 #g (Phe, G)A-L in CFA after injection of 2 mg of soluble, centrifuged (Phe, G)-A-L administered in one (E]), two ( ) or seven ([]) injections. Titers of immunized mice not given the tolerogen are shown by (11). seven injections, except that titers of 1:8 and 1:16 were detected in the secondary responses of two animals given a single injection of tolerogen. The results obtained using (Phe, G)-A-L as tolerogen and immunogen are summarized in Fig. 3. A greater degree of unresponsiveness was seen after secondary than after primary immunization for all three schedules of tolerogen administration. The primary responses of normal control mice immunized with (Phe, G)-A-L were lower than those observed for (T, G)-Pro-L and (T, G)-A-L, and were not higher than the groups of mice injected with the tolerogen. huluction o[ tolerance as a [m+ction 0[" the amount 0[ soluble (T, G)-Pr~-L il!iected Six groups of mice (10 15 mice per group), were injected with varying amounts of soluble, centrifuged (T, G)-Pro-L ranging from 1 ng to 2 nag. Tolerogen was administered in two injections spaced 1 week apart. Primary and secondary immunizations were made as described above. The percentage of mice with positive hemagglutination titers after primary or secondary immunization are shown in Fig. 4. 1130 ng and 1 #g appeared to be the most effective doses for inhibition of the primary response (less than 20 per cent of the mice giving a detectable response), whereas the extreme doses of I ng and 2 mg were the least effective (50 per cent or more of the mice giving significant titers). In contrast, for the secondary response the most effective tolerogen dose used was 1 ng, whereas 1 #g appeared to be the least effective (42 per cent of the mice exhibited serum titers ranging from 1 : 16 to 1:64). Specificity requirements of the tolerogen and the immunogen Immunization of mice with (T, G)-Pro-L results in an immune response specific for the Pro-L moiety only (Mozes et al., 1969a). From this observation it would appear that (T, G) on polyprolyl side chains is not immunogenic. |n contrast, immunization with (T,
G)-A-L results mainly in antibodies specific for the sequences of tyrosine and glutamic acid (McDevitt and Sela, 1965). Therefore, it was of interest to investigate whether it is possible to induce tolerance to (T, G) by using (T, G)-Pro-L as the tolerogen and (T, G)-A-L as the immunogen. One group of mice made tolerant to (T, G)-Pro-L and a second group of untreated control mice were immunized with (T, G ) - A - L The results shown in Table 2 demonstrate that no reduction in the immune response to (T, G)-A-L was detected in the mice tolerant to (T, G)-Pro--L (T, G)-Pro-L and (Phe, G)-Pro-L share the Pro-L immunopotent region, and (Phe, G)-Pro-L contains the additional (Phe, G) immunopotent region (Mozes et al., 1969a). Thus, it was possible to investigate whether induction of tolerance to (T, G)-Pro-L followed by immunization with (Phe, G)-Pro-L would result in the production of antibodies specific for (Phe, G) only, the production of antibodies specific for both (Phe, G)-and Pro-L, or whether no immune response would be detected to either of these specificities, Mice were injected with a total of 2 mg of soluble (T, G)-Pro-L in one, two or seven injections. The animals were then immunized and boosted as described above with 10 pg (Phe, G)-Pro-L. The percentage of sera showing specific anti-(Phe, G)-A-L and anti(T, G) Pro-L titers are shown in Fig. 5. Tolerance was obtained in the primary response for the Pro-L specificity with all three schedules of injection. No significant degree of tolerance was seen in the primary response for the (Phe, G) specificity. Although only 50 per cent of the tolerant animals given seven injections of tolerogen responded to (Phe, G), in the control also only 67 per cent of the mice (not given tolerogen) gave a detectable primary response. L, O nr" W F~-
I00
,~ 80
~ 6o ~ 40
~ zo
t~ n
I ng
I0 ng I00 ng
I~g 200/~
2 mg
DOSE OF (T,GI-PRO--L TO'LEROGEN
Fig. 4. Percentage of sera giving anti-(T, G)-Pro--L titers greater than 1:4 as a function of the dose of soluble, centrifuged (T, G)-Pro--L (administered in two injections). Primary response denoted by (IS]); secondary response denoted by (U).
Tolerance of Mice to Synthetic Polypeptides Table 2. Passive hemagglutination titers in mice made tolerant" to (T. G)-Pro L and immunizedb with (T, G)-A L Secondary anti-(T, G)-A-L titer (T, G)-Pr~L No tolerogen tolerogen
Mouse number
1:32 1:64 1:64 1:64 1:64 1:64
1
2 3 4 5 6
1:128 1:128 1:64 1:64 1:128 1:64
"Two mg soluble (T. G)-Pro-L given in seven injections spaced at 2-day intervals. b Ten /~g (T, G)-A-L given intraperitoneally in CFA I week after last injection of tolerogen. Secondary immunization was 10 #g (T, G)-A-L in PBS 3 weeks after primary immunization.
i 0 0 PRIMARY RESPONSE
~
60
~ 4o ~
2o
o
0
I
'°°r
BOI
2 R
7
0
NS
6o
,=, 4 o
zo o
I 2 7 0 NUMBER OF TOLEROGEN INJECTIONS (TOTAL OF Z rng (T,G)-PRO--L)
Fig. 5. Percentage of sera giving anti-(Phe, G)-A L (D) or anti-(T, G)- Pro--L (11) titers greater than 1:4 in mice immunized with 10/~g (Phe, G)-(Pro--L after injection of 2 mg soluble, centrifuged (T, G)-Pro-L in one, two or seven injections. In the secondary response, distinctions were observed in the induction of tolerance to Pro--L as a function of the schedule of tolerogen injection. Tolerance to P r o - L was terminated when 2 mg of soluble (T, G)-Pro-L was administered in one injection. In contrast, 50 and 18 per cent of the animals showed positive titers when the tolerogen was given in two or seven injections, respectively. No tolerance was observed however in the secondary response to the (Phe, G) specificity for any of the tolerogen schedules used. Thus, these results indicate that the immune response obtained was specific for an immunopotent region of the immunogen (Phe, G) which was not contained in the tolerogen.
351 DISCUSSION
The experiments described in this report demonstrate that tolerance can be induced in adult mice to multichain synthetic polypeptides of both the 'A-L' and 'Pro-L' series. (T, G)-A-L and (Phe, G)-A L appear to be better tolerogens in C3H.SW mice than (T, G)-Pro--L. These two polypeptides of the A-L series are also better immunogens than (T, G)-Pro-L in this mouse strain (McDevitt and Benacerraf, 1969; Mozes et al., 1969a). In the present example there would appear to be a direct correlation between immunogenicity and the ability to induce unresponsiveness. This agrees with the findings of some investigations (Rajewsky and Rottl~inder, 1967; Collotti and Leskowitz, 1969). However, it is not a universal observation, since a lot of evidence has been presented that immunogenicity is not a necessary criterion for tolerogenicity (Janeway and Humphrey, 1968; Parish and Ada, 1969; Roelants and Goodman, 1970). An unresponsive state has been obtained to proteins by injection of high or very low doses of antigen. These phenomena have been respectively referred to as high and low zones of tolerance (Dresser and Mitchison, 1968; Mitchison, 1964), and a considerable amount of attention has been focused on the selective inactivation of different cell populations by high. and low zone tolerance (Mitchison, 1971). A significant reduction in the immune responses to (T, G)-Pro-L was obtained for different doses oftolerogen injected (see Fig. 4). The most effective dose range of soluble (T, G)-Pro-L for the primary response was 100 ng to 1/~g, and the least effective were the extreme doses of 1 ng and 2 mg. In contrast, for the secondary response 1 #g was the least effective tolerogen dose, whereas the 1 10 ng range were most effective. The reason for this difference between the primary and secondary response is unknown, although it is possible that different functional cell populations might be selectively affected in the primary and secondary response as a function of tolerogen dose. It also may be that the production of different classes of antibodies is differentially affected by primary and secondary responses after tolerance induction with the different doses (Weber and Kt~lsch, 1972). It should be noted that the boost consisted of 10 ~g soluble aggregated antigen, some of which is in a tolerogenic form. This material might have induced additional tolerance. In mice the immune response to (T, G ) - P r ~ L is directed mainly to the ProwL portion of the molecule (Mozes et al., 1969a), whereas immunization with (T, G)-A-L primarily generates antibodies specific for the short sequences of tyrosine and glutamic acid (McDevitt and Sela, 1965). The injection of soluble (T, G)Pro-L as tolerogen followed by immunization with (T, G)-A-L resulted in a normal response to the immunogen (Table 2). Thus, the observation that a normal response was obtained to (T, G)-A-L in mice made tolerant to (T, G)-Pro-L agrees with the earlier observation (discussed above) that a weakly or non-immunogenic-determinant may also be a poor tolerogen.
352
B. BONAVIDA. E. MOZES, G. M. SHEARER and M. SELA
However, the possibility also exists that the mice were tolerant to the short sequences of tyrosine and glutamic acid on Pro-L, but that breakage of tolerance to (T, G) occurred as a result of immunization with (T, G) A-L. Immunization with (Phe, G)-Pro-L results in populations of antibadies specific for the short terminal sequences of (Phe, G), as well as for the polyproline side chains (Mozes et al., 1969a, b). In contrast, immunization with (T, G)-Pro-L results in an immune response specific for Pro-L only. Thus, tolerance induction to (T, G)-Pro-L followed by immunization with (Phe, G)-Pro-L provides an experimental situation in which the tolerogen contains a single, repeating immunopotent region, whereas the immunogen contains a new immunopotent region as well as the one in common with the tolerogen. Neither the primary nor the secondary anti-(Phe, G) responses were significantly below those obtained in the normal immunized control mice (see Fig. 5). Thus, a normal immune response was obtained for a new determinant on an immunogen which also possessed the determinants in common with the tolerogen. In contrast, the anti-Pro-L primary response was considerably below the control group (20-32 per cent vs 100 per cent) for all three schedules of tolerogen injection. A difference was seen in the secondary response to Pro-L as a function of the tolerogen injection schedule. A normal secondary anti-ProL response was seen when the tolerogen was administered in a single injection. Two mg of soluble (T, G)Pro-L in two injections resulted in 50 per cent response, whereas less than 20 per cent of the mice given 2 mg of tolerogen in seven injections responded to the Pro-L specificity. A possibility not tested in these experiments is that the animals given a single injection of tolerogen may have escaped the unresponsive state after immunization and boosting more readily than those animals given the same amount of tolerogen in multiple injections. Intramolecular competition between (Phe, G) and Pro-L was found to occur in mice immunized with (Phe, G)-Pro-L (Taussig et al., 1972). In this case P r o L was the dominant antigen and (Phe, G) the suppressed one. The results described in this paper that mice rendered tolerant to Pro-L respond to (Phe, G), agree with other observations that antigenic competition does not take place in an animal which is tolerant to an otherwise dominant antigen (Taussig, 1973). The results described in this report differ from those previously reported by Weigle and colleagues (Weigle, 1967; Benjamin and Weigle, 1970), who demonstrated termination of immunological unresponsiveness in rabbits made tolerant to bovine serum albumin (BSA) and immunized with related albumins such as those of human, pig, horse and guinea pig. However, sheep serum albumin did not terminate tolerance to BSA and did not induce antibodies specific to the sheep albumin determinants not present on BSA (Weigle, 1961). It was concluded from these studies that termination is obtained if the tolerogen and immunogen exhibit
major structural differences (Benjamin and Weigle, 1971). The results of immunization of mice unresponsive to (T, G)-Pro-L and injected with (Phe, G)-Pro--L were not similar to the above, since tolerance to Pro-L was not terminated by immunization with (Phe, G)Pro-L. The combination of synthetic polypeptides as tolerogens and immunogens can be useful in investigating the cellular aspects of antigen-induced unresponsiveness.
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