Immunology, 1969, 17, 819.

The Effect of the Carrier Protein on the Immune Responseand on the Induction of Tolerance in Mice to the

2,4-Dinitrophenyl Determinant

H. F. HAVAS

Department of Microbiology, Temple University Health Sciences Center,Philadelphia, Pennsylvania, U.S.A.

(Received 2nd December 1968)

Summary. The role of the protein carrier on the primary and anamnesticresponse to the 2,4-dinitrophenyl (DNP) determinant was studied in random-bred ICR and in an inbred BALB/c AnNIcr strain of mice. It was found that thelevel of the response was related to the immunogenicity of the carrier molecule.Mouse y-globulin (MGG) was the most effective carrier for inducing tolerance toDNP regardless of the route of administration, while only occasional tolerancewas achieved with the highly immunogenic haemocyanin. When human y-globulin (HGG) served as a carrier, hyporesponsiveness to the DNP group wasinduced in the majority of neonate and adult mice when injected intraperitoneally,while an immune response ensued when the antigen was incorporated in adjuvantand injected into the footpads. Conversely, the most effective carrier for immuniza-tion against the DNP determinant was haemocyanin, while little immunityagainst either DNP or the protein carrier resulted from immunization withDNP-MGG. The antibody response of the mice when measured by the passivetanned haemagglutinating test was directed mostly against the hapten carriercomplex and the protein carrier rather than the DNP-determinant alone, regard-less of the initial route of administration. In contrast to the results obtained invitro, the antibody responsible for the PCA reaction in mice is directed exclusivelyagainst the hapten and the hapten-protein conjugate but not against the proteincarrier.

INTRODUCTIONIn order to study the role of individual determinants in the induction of the immune

response or oftolerance, antigens should be used with which antibody response to an individ-ual determinant can be dissociated from the overall response to the whole molecule, e.g.conjugates of proteins coupled to chemically defined haptens. The coupling of definedhaptenic groups to 'tolerogenic' antigens should thus provide a useful experimentalapproach to the study of the specificity of immune unresponsiveness (Nachtigal andFeldman, 1964; Weigle, 1965).However, since the antigenic determinant may involve both the hapten and the carrier,

819

the carrier to which a hapten is coupled may influence the immunological specificity of theanti-hapten response (Fronstein, Sage and Vazquez, 1967).

Hapten-carrier specificity has been involved in the secondary response in rabbits(Ovary and Benacerraf, 1963; Eisen and Siskind, 1964), in delayed hypersensitivity insensitized guinea-pigs (Benacerraf and Levine, 1962; Siskind, Paul and Benacerraf,1966; Weigle, 1967) and in the antibody response of mice (Dietrich, 1966; Brownstone,Mitchison and Pitt-Rivers, 1966; Maurer and Pinchuck, 1967). Tolerance to haptens hasbeen induced in rabbits (Cinader and Pearce, 1958; Yoschimura and Cinader, 1966;Weigle, 1967), guinea-pigs (Battisto and Chase, 1963; Coe and Salvin, 1963) and mice(Dietrich, 1966; Brownstone et al., 1966; Maurer and Pinchuck, 1967).

It was found that tolerance as well as immunity is directed to specific antigenic deter-minants rather than to the antigen as a whole (Smith, 1961; Weigle, 1967), and that newantigenic determinants, which are created when a hapten is coupled to a protein carrierprovide a new steric configuration which incorporates parts of both the hapten and thecarrier molecules. Although extensive immunological studies involving 2,4-dinitrophenyl(DNP) substituted antigens have been made in rabbits (Ovary and Benacerraf, 1963;Eisen and Siskind, 1964), mice (Fronstein et al., 1967; Pinchuck and Maurer, 1968)and guinea-pigs (Paul, Siskind and Benacerraf, 1966), no study has been reported of theinduction of tolerance to DNP in mice and of the role of the carrier in this process.

In a previous paper, tolerance to human y-globulin in ICR random bred mice wasinvestigated (Havas and Senff, 1967). These studies have now been extended to the studyof the immune response and the induction of unresponsivensss to the 2,4-dinitrophenyldeterminant in ICR albino and an inbred strain of BALB/c AnNIcr (BALB/c) mice.The effect of the carrier on enhancing or suppressing the immunogenicity of the DNP

group was investigated in neonatally injected as well as in adult mice. The immuneresponse to the hapten, the hapten-protein conjugate and to the protein was compared inmice initially injected with soluble antigen to those injected with antigen incorporatedinto Freund's adjuvant.

MATERIALS AND METHODS

AntigensHuman y-globulin (HGG) (Lots Nos. C-839 and C-906) was donated by Dr Franklin

M. Phillips (Lederle Laboratories, Pearl River, New Jersey); haemocyanin (Hy) fromLimulus polythymus by Dr Karush (University of Pennsylvania Medical School); Boviney-globulin (BGG), bovine serum albumin (BSA) and horse serum albumin (HSA) wereobtained commercially from Pentex. Mouse y-globulin (MGG) was prepared in ourlaboratory by repeated (NH4)2SO4 precipitation from mouse ascites fluid (Havas et al.,1967). From the immunoelectrophoretic pattern it could be seen that MGG contained allthe globulins and was essentially free ofalbumin. The 2,4-dinitrophenyl protein conjugateswere prepared with varying degrees of substitution according to the method of Eisen(1964). The number of groups per protein molecule were calculated by determining theoptical density (OD) of the derivatized proteins in 0-1 N NaOH at 280 and 360 my on aHitashi spectrophotometer and by protein determination by the Micro-Kjeldahl method.The number of DNP groups per protein molecule of the DNP conjugates is indicated bythe numbers, DNP56BGG, DNP40HGG, DNP35MGG, DNP9Hy and DNP31Hy (Hyper molecular weight 100,000).

820 H. F. Havas

Immune Response of Mice to Dinitrophenylated Proteins

AnimalsThe original breeders for the ICR albino and BALB/c mice were kindly donated by

Dr Wolf of the Institute for Cancer Research (Philadelphia). ICR albino mice wererandomly mated while BALB/c mice were propagated by brother-sister mating. Adultrabbits were obtained commercially. All animals were given food and water ad libitum.

Immunization and induction of toleranceThe procedure of Dietrich and Weigle (1964) was followed for the induction of both

the immune and unresponsive state in neonate as well as adult mice. However, because ofthe toxicity of the DNP derivatized proteins to newborn mice and the ensuing highmortality, most of the studies were carried out in adult mice.Mice were immunized with antigens emulsified in Freund's incomplete adjuvant

(Difco). A total volume of 0-05 ml of the antigen adjuvant mixture containing 0.1 mgof antigen was injected into three footpads.To induce unresponsiveness, mice were given a single intraperitoneal injection of anti-

gen in saline. The antigen dosage for adult mice was [ 0 mg in 0 1 ml and for neonatemice 1[2-1 4 mg in 005 ml of saline.

Rabbit antiseraRabbit antisera for testing the sensitized cells were obtained by immunizing rabbits by a

single injection of 5 mg of protein antigens or of DNP derivatized protein followed bymonthly intravenous booster injections of 2 mg of the antigen as described by Eisen(1964).

Sources of antibody (mice)Blood was obtained from the retro-orbital plexus or by heart puncture for the final

bleeding.

In vitro testfor antibodyThe immune response to both the carrier and the hapten was ascertained, following

antigenic challenge, by testing the sera by the passive tanned cell haemagglutination test,essentially as described by Stavitsky (1954). The concentration of the DNP-conjugatedproteins used to sensitize the cells was 0-0048-00096 per cent, and the concentration of theprotein antigens was 0s 15 per cent. All dilutions were made in 1 per cent heat inactivated,SRBC absorbed, normal rabbit serum in pH 7-2 phosphate buffer (NRS).

Standard anti-DNP rabbit antisera were titrated against each batch of freshly tannedsensitized cells and used only in the assay if titres fell within a two tube dilution range.Normal mouse serum also was tested each time for non-specific haemagglutination of theantigen-coated cells. Serial dilutions of serum were started at 1: 25 in a volume of 0 5 mlof NRS to which were added 0 05 ml of the sensitized cells. All tests were performed induplicate against the appropriate antigens: the homologous proteins and DNP coupledto the homologous and heterologous proteins. The geometric mean (GM) of the serumtitres was calculated as previously described (Havas and Senr, 1967) and the reciprocalof the geometric mean (RGM) titres are presented in the tables.

In vivo antibody testPassive cutaneous anaphylaxis (PCA): the specificity of antibody contained in the as-

cites fluid which fixes to mouse skin was tested according to the procedure of Ovary

821

(1964) with minor modifications. ICR albino mice were injected intradermally on foursites on the back with 0 05 ml of increasing dilutions in saline diluent of mouse anti-DNP-Hy ascites fluid. They were challenged j hour later with either DNP-HGG,DNP-Hy or Hy by intravenous injection (into the tail vein) of the antigen (0.1 mlantigen/0 1 ml of a 1 per cent Evans blue solution in saline). The mice were killed andskinned 15 minutes later and the area of blue coloration measured. Only those mice whichgave a skin reaction of 1 mm2 or greater were considered positive.

RESULTS

ROLE OF CARRIER IN IMMUNE RESPONSE TO DNP

The effect of different protein carriers on the immunogenicity of the 2,4-dinitrophenylgroup and the immune responses of ICR and BALB/c mice were compared (Table 1).It can be seen that no antibody solely directed against DNP could be detected in miceimmunized with DNP-MGG (Group 2, Table 1) when tested with DNP-BSA sensitizedcells. However, immunization with DNP-Hy resulted in high anti-DNP titres (Groups3 and 4).

Sera ofmice immunized with DNP-HGG (Group 1) and even with DNP-MGG (Group2) had high titres against DNP when tested against DNP coupled to the homologousprotein. Antibody levels against the protein carrier were low or negative in all groups,except in Group 1.

It can be seen from these results that Hy is superior to HGG which in turn is superiorto MGG in enhancing the antigenicity of DNP.When the anti-DNP response of ICR and BALB/c was compared (Groups 3 and 4),

the immune response of the BALB/c mice was greater (RGM 274 and 1054, Groups 3 and4, respectively) (also unpublished observations). Because of the obvious advantages ofusinginbred strains, only BALB/c mice were used in most subsequent experiments.

TABLE 1ANTIBODY RESPONSE TO DNP AND THE PROTEIN CARRIER IN BALB/C AND ICR ALBINO MICE IMMUNIZED WITH DNP-

PROTEIN CONJUGATES

Days of Strain and Reciprocal of HA* titres on days:Groups Immunizing Test immuniza- No. of

antigent antigen tion mice 28 54-57 70-73

1 DNP-HGG DNP-BSA 0, 59 BALB/c 0, 0/9 88, 4/7$HGG 9 11, 3/9 226, 8/9

2 DNP-MGG DNP-BSA 0, 59 BALB/c 0, 0/13 6, 1/11§MGG 13 36, 7/13 0, 0/13

3 DNP-Hyll DNP-BGG 0, 21, 42 ICR 274, 8/12 373¶Hy 51 ND ND

4 DNP-Hyll DNP-BGG 0, 21, 42 BALB/c 1054, 12/12 152¶Hy 47 0, 0/12 ND

ND, None done.* Reciprocal of geometric means; number of mice with titres of 1: 25 or greater/number tested.t Injected with 0 1 mg DNP-conjugate in 0 05 ml Freund's incomplete adjuvant into three footpads.t HA titre against DNP-HGG was 2702 with eight out of eight responders.§ HA titre against DNP-MGG was 790 with three out of three responders.Injected with 0- 1 mg DNP-conjugate in 0 05 ml complete adjuvant.

¶ HA titres are RGM ofHA titres of pooled sera from mice.

822 H. F. Havas

Immune Response of Mice to Dinitrophenylated Proteins

IMMUNE RESPONSE TO DNP OF MICE INJECTED NEONATALLY WITH DNP-PROTEIN CONJUGATES

When mice were injected at birth with DNP-Hy and were challenged 78 days laterwith DNP-Hy in Freund's adjuvant, seven of eight mice responded (Group 1, Table 2)compared to three of six of their littermate controls not injected at birth (Group 2). Inaddition, the titres of the mice injected at birth (Group 1) were higher than those of thecontrols. It thus appears that the neonates were sensitized rather than paralysed by theinitial injection ofDNP-Hy at birth. When rechallenged at 139 days all the mice respondedin both groups, but at that time the anti-DNP antibody titres of the controls were approxi-mately twice as high as those of the neonatally injected mice (RGM 1193 in Group 2,compared with 616 in Group 1), possibly indicating a delayed depression of the immuneresponse against DNP. When DNP-HGG was injected at birth (Groups 3 and 4) animalswere essentially unresponsive against the DNP hapten 2 weeks after the first challenge,but after the second rechallenge at 176 days with DNP-Hy (Groups 3 and 4), all mice inboth groups gave an immune response to the hapten.

TABLE 2ANTIBODY RESPONSE OF NEONATE TOLERANT MICE TO DNP-PROTEIN CONJUGATES

Reciprocal haemagglutination titres§ againstAntigent DNP-BGG sensitized tanned SRBC

Group* injected Challenge Days ofat birth antigent challenge 89-91 112-125 138 152 166-175 185-189

1 DNP-Hyll DNP-HyT 78, 139 168 0 6167/8 0/8 8/8

2 - DNP-HyT 78, 139 33 0 11933/6 0/6 6/6

3 DNP-HGG DNP-Hyll 101, 176 0 0 0 8000/5 0/5 0/5 5/5

4 DNP-HGG DNP-Hyll¶ 101, 176 0 56 0 4300/6 4/6 0/6 5/5

* 1 and 2, and 3 and 4 were littermates.t Initial dose of antigen 1-2 mg of DNP-Hy; 1-4 mg DNP-HGG.I Challenged with 0 1 mg antigen in Freund's incomplete adjuvant injected into three footpads.§ Reciprocal of geometric means; number of mice with titre of 1: 25 or greater/number tested.DNP-Hy thirty-one groups/100,000 Hy.

¶DNP-Hy nine groups/100,000 Hy.

ANTIBODY RESPONSE OF MICE IMMUNE OR 'TOLERANT TO DNP-HGG

Immune or 'tolerant' mice injected with DNP-HGG (Groups 1 and 2, Table 3) willon rechallenge with the homologous conjugate produce antibody against both DNP-HGG and HGG, although the 'tolerant' group at lower levels. The greater antibody responseofboth groups was directed against DNP-HGG. No antibody (< 1: 25) was detectable withDNP-BSA or DNP-HSA sensitized cells. In the 'tolerant' mice of Group 1 a split immuneresponse occurs with only four of ten mice making anti-HGG antibody, while all tenproduced antibody against DNP-HGG: in contrast in the immune mice of Group 2, fiveof six mice made antibody against both HGG and DNP-HGG. When DNP-HGGimmune or 'tolerant' mice were challenged with HGG instead (Groups 3 and 4) anti-HGGtitres were considerably higher. However, anti-DNP-HGG titres were comparable in theimmune mice of Groups 2 and 4, but not in the tolerant mice of Groups 1 and 3. We again

823

824 H. F. Havasobserved a split immunity in the 'tolerant' mice only, two of eight in Group 3 producedanti-DNP antibodies, but all were immune to HGG.When mice initially injected with DNP-HGG were challenged with DNP-Hy, a highly

immunogenic carrier, the antibody response of both 'tolerant' and immune mice was lowagainst DNP-HGG and HGG (Groups 7 and 8) but rose to 229 4 weeks after challengein the immune mice (Group 7). The titres ofthe anti-DNP-HGG controls (Group 8) werecomparable to those of the tolerant mice of Group 1, 2 weeks after injection of DNP-HGG but were ten-fold higher after 4 weeks (RGM 925 compared to 93, see footnote toTable 3).

TABLE 3ANTIBODY RESPONSE OF DNP-HGG INJECTED TOLERANT OR IMMUNE MICE FOLLOWING CHALLENGE WITH HGG OR DNP-

CONJUGATES

Reciprocal ofHA* titres 2 weeksAntigen injected post-challenge against

GroupsInitial Challenget DNP-HGG HGG

1 DNP-HGGJ DNP-HGG 115jJ 34Tolerant 10/10 4/10

2 DNP-HGG§ DNP-HGG 446 188Control 5/6 5/6

3 DNP-HGG+ HGG 12 417Tolerant 2/8 8/8

4 DNP-HGG§ HGG 659 674Control 5/6 6/6

5 HGGt DNP-HGG 0 32Tolerant 0/9 6/12

6 DNP-HGG$ DNP-Hy 13 9Tolerant 3/12 2/12

7 DNP-HGG§ DNP-Hy 25¶ 8Control 3/6 1/6

8 - DNP-HGG 82** 8Control 6/7 1/7

Pre-challenge titres were uniformly low with only an occasional responder. Sera tested with DNP-BSA or DNP-HSA sensitized cells were < 1: 25 throughout the experiment. Some pertinent 4-week post-challenge titres are listed.

* Reciprocal of geometric means; number of titres of 1: 25 or greater/number tested.t Challenged with 0 1 mg antigen in Freund's incomplete adjuvant into footpads 42-45 days after initial injection.t Injected i.p. with 1 0 mg antigen in 0 I ml of saline on day 0.§ Injected with 0 I mg of DNP-HGG in 0-05 ml Freund's incomplete adjuvant into three footpads on day 0.1j 4 weeks post-challenge the RGM against DNP-HGG was 93 with eight of ten responders.¶ 4 weeks post-challenge the RGM against DNP-HGG was 229 with five of five responders.** 4 weeks post-challenge the RGM against DNP-HGG was 925 with seven of seven responders.

EFFECT OF THE MGG CARRIER ON THE IMMUNE RESPONSE TO DNP

When DNP is coupled to the carrier MGG, antibodies directed exclusively against thehapten or carrier are only rarely produced regardless of the initial route and dosage ofinjection (Table 4) and regardless of the subsequent challenge antigen. Only one ofthirteen mice in control Group 4 produced anti-hapten antibodies (against DNP-HGGsensitized cells). There was a difference between the 'tolerant' and control mice of Groups3 and 4. None formed anti-DNP-MGG antibodies in the former compared to three ofthree in the controls. In addition, after the initial injection seven of thirteen of the controlmice of Group 4 produced anti-MGG antibodies, compared to none in the tolerant Group3 (see footnote to Table 4).

Immune Response of Mice to Dinitrophenylated ProteinsTABLE 4

EFFECr OF MGG CARRIER ON THE ANTI-DNP RESPONSE

Reciprocal of HA* titre 2 weeks post-Antigen injected challenge against:

Groups Initial Challenget DNP-MGG MGG DNP-HGG

1 DNP-MGG§ DNP-Hy 11 11 0Tolerant i.p. 1/9 1/9 0/9

2 DNP-MGGt DNP-Hy 10 0 9Control f.p. 0/6 0/6 1/3

3 DNP-MGG§ DNP-MGG 8 011 0Tolerant i.p. 1/4 0/12 0/11

4 DNP-MGGt DNP-MGG 790 0¶ 6Control f.p. 3/3 0/13 1/13

* Reciprocal of geometric means; number of mice with titres of 1:25 or greater/number tested.t Challenged with 0-1 mg antigen in Freunds incomplete adjuvant into three footpads 42 days after

initial injection.t Injected into three footpads on day 0 with 0- 1 mg antigen/0-05 ml Freund's incomplete adjuvant.§ Injected i.p. with 10 ng DNP-MGG in 0-1 ml saline on day 0.¶ Before re-challenge seven of thirteen mice in Group 4 had anti-MGG antibodies but none in the

tolerant Group 3.

PASSIVE CUTANEOUS ANAPHYLAXIS (PCA)

To measure the skin sensitizing activity of mouse anti-hapten antisera, increasing dilu-tions of anti-DNP-Hy serum were injected intradermally on the back of ICR mice untilan end point was obtained. When the mice were subsequently challenged with the homo-logous protein, DNP-heterologous protein and DNP-homologous protein, highest titreswere obtained on challenge with the completely homologous antigen (except for two often sera from donor mice 2 and 8). There was a complete absence of reaction on challengewith the homologous protein although the sera's anti-haemocyanin titres ranged up to1: 400 (Table 5).The PCA titres of all sera tested (except donor mouse 8) were lower than those obtained

in the haemagglutination titration test with DNP-BGG. Although it would have beenpreferable to have used the same antigen for both the HA and PCA tests, DNP-HGG

TABLE 5PCA REACTIONS ELICITED IN ICR MICE WITH ICR ANTi-DNP-Hy SERUM

Reciprocal of HA titre* PCA response reciprocal of titre of serum givingagainst cells sensitized with: distinct PCA reactions on challenge with:

Donor mouseDNP-BGG Hy Hyt DNP-Hyj DNP-HGGt

1 300 150 0 190(3) 802 800 75 0 90(3) 160§3 800 37 0 160(4) 80§4 200 50 0 45(3) 405 1200 - 0 320(4) 1606 1200 100 0 160(3) 807 800 50 0 240(3) -8 12 400 0 40(2) 160§9 75 + 0 30(3) 2010 200 50 0 80(3) 80

* Average HA titre of duplicate tests.t One mouse per test.t No. of mice per test in parentheses.§ No endpoint was reached.

825

and DNP-BGG were comparable as test antigens in the haemagglutination test. Thestrong PCA activity is indicative that the yl-globulin was responsible for the anti-haptenskin sensitizing activity (Barth and Fahey, 1965).

DISCUSSION

In order to study the effect of the protein carrier on the DNP-ligand, a study was under-taken of the immune response of random bred (ICR) and an inbred (BALB/c) strain ofmice. The antibody response to the hapten, the DNP-protein conjugate and the proteincarrier were compared, following two different routes and dosages of antigen administra-tion; injection in soluble form by the intraperitoneal route and antigen suspended inwater oil emulsion injected into the footpads. The former mode of injection resulted intolerance and the latter in immunity to HGG (Dietrich and Weigle, 1964; Havas andSenfl, 1967). However, following the injection of the DNP-protein conjugate the responseto the DNP group was often only marginal, even with adjuvant, and most of the antibodywas directed against the hapten-protein conjugate determinants and against the homolo-gous protein. This is in agreement with the findings of Weigle (1965) that the antibodyresponse is directed to areas on the conjugates larger than the hapten, i.e. areas adjacentto the hapten altered as a result of conjugation.

In order to study the antibody response of mice against the hapten and protein carrier,a sensitive test using minimal amounts of sera was needed. Pinchuck and Maurer (1968)found the passive haemagglutination test to be as sensitive and reliable as a techniqueinvolving specific binding by tritiated antigen. In addition, the small amounts of serumrequired per test allows one to follow the immune response at different time intervalsagainst three to four test antigens. The HA test was thus considered a suitable test toanswer the questions of the specificity of 'tolerance' and of the immune response followingthe injection of DNP coupled to different carrier proteins.When the sera of the mice were tested for anti-DNP antibodies with DNP-BSA or

DNP-HSA coated cells, few positive HA titres were observed although antibody againstDNP-HGG or DNP-MGG (Groups 1 and 2, Table 1) was present. Either the amino acidneighbours of the haptens linkage on the BSA molecule differ sufficiently from those onHGG or MGG, or the anti-DNP antibody specificity was directed not only againstthe hapten but also against the carrier part covalently linked to the hapten. Presumablya DNP-conjugate of a heterologous globulin would be a more sensitive, but a less specifictest of anti-DNP antibody. The dearth of sera obtained by eye or heart bleedings unfor-tunately precluded testing all sera against additional DNP-conjugates.When the contribution of the different protein carriers to the hapten specific antibody

response was compared, it was found that the most immunogenic carrier, haemocyanin,also produced the greatest anti-DNP response (Table 1). These data support the conceptsuggested by others (Siskind et al., 1966; Fronstein et al., 1967) that a direct relationshipexists between the immunogenicity of a protein molecule and its effectiveness as a haptencarrier.At the lower end of the scale of immunogenicity for mice is the carrier MGG (prepared

from the ascites fluid from the homologous BALB/c strain) which induced anti-DNPantibody only in two of sixteen mice (Groups 2 and 4, Table 4), and a transient antibodyresponse to MGG in seven of thirteen mice in Group 4. The results differ from thoseobtained by Fronstein et al. (1967) in a different strain ofmice who found that DNP-MGG

826 H. F. Havas

Immune Response of Mice to Dinitrophenvlated Proteins

elicited no detectable antibodies directed against the carrier, but significant levels of anti-DNP antibody. However, Fronstein also obtained a correlation between the 'foreignness'of the carrier protein and its ability to elicit an anti-hapten response: the carrier proteinfrom an animal most distinct phylogenetically from the recipient elicited the highest anti-body response.

Results obtained after the injection of DNP-protein conjugates into neonatal miceindicated that no antibodies were detectable up to 175 days if the carrier was HGG(Groups 3 and 4, Table 2), but that an immune response resulted if haemocyanin servedas the protein carrier (Group 1). Because of high mortality in neonatal mice due to DNPtoxicity, and since use of neonates is no longer considered obligatory for the production ofthe tolerant state, all subsequent experiments were carried out in adult BALB/c mice 8-14weeks of age.When mice were initially injected intraperitoneally with DNP-HGG (Table 3, Group

1), the greatest subsequent antibody response was always directed against DNP-HGG,the hapten carrier complex, some against HGG, and none against the hapten alone. Asimilar but greater response is given by the immune mice in Group 2. It thus appears thatregardless of the initial mode of administration after challenge with DNP-HGG, antibodyspecificity is directed against the hapten carrier complex rather than the hapten alone.

These findings are in agreement with results obtained in other laboratories that animalsdo not make a unique antibody molecule against DNP upon exposure to DNP antigensirrespective of the environment of the DNP determinant, but that populations of antibodymolecules are formed which bind the DNP hapten more or less strongly depending on thecarrier molecule used for immunization (Siskind et al., 1966). An interesting observationwas the anamnestic response to HGG of mice (tolerant to DNP-HGG) in Group 3(Table 3) but not to DNP-HGG to which they had been made tolerant. However, whenmice tolerant to HGG (Group 5) were challenged with DNP-HGG only six of twelvemice in Group 5 lost their tolerance to HGG and none produced anti-DNP-HGG anti-bodies. It thus appears that while HGG is capable of evoking an immune response to theHGG carrier in DNP-HGG 'tolerant' mice, DNP-HGG did not abrogate 'tolerance'directed against HGG.When DNP-HGG 'tolerant' mice were challenged with the DNP ligand linked to a

heterologous carrier Hy (Group 6) only three of twelve mice had a transient antibodyresponse to DNP-HGG. However, 4 weeks post-challenge, none of the tolerant, but all ofthe control mice formed antibody against DNP-HGG. This difference in response indicatesa difference in recognition mechanism between the immune and hyporesponsive group ofmice: the former responds to the priming and challenge antigen (initial 'antigenic sin')while the latter responds initially only to the challenge antigen to which it has not beenmade hyporesponsive or unresponsive. On comparing the response of the DNP-HGGimmune mice in Group 2 challenged with DNP-HGG with those of Group 7 challengedwith DNP-Hy it is apparent that the response against the initial antigen is stronger in theformer group. This is in agreement with the findings of Ovary et al. (1963) that the anti-hapten anamnestic response is controlled in part by the 'immunological backbone' of thecarrier.When mice were injected with DNP-MGG (Table 4), it was apparent that the

homologous MGG influenced the response to the hapten and protein carrier: only two ofsixteen of the immune control mice (Groups 2 and 4, Table 4) gave a detectable antibodyresponse against the hapten and only three of nine against DNP-MGG. Only one of nine

827

828 H. F. Havasmice in the tolerant group had detectable antibodies against DNP-MGG and MGG andnone against the hapten alone. It is thus apparent that a hapten linked to the host proteincarrier does not prime the immunocompetent cells against DNP and, therefore, no sub-sequent secondary response to the hapten follows regardless of whether the hapten carriercomplex is injected in a high dosage (1 mg) in solution or in a low dosage (0.1 mg) ad-ministered with adjuvant.The interpretation of the data as to whether tolerance was achieved is made difficult

by the fact that in general two injections of antigens in adjuvant are needed to evoke ameasurable response against DNP. The immune control mice injected twice with DNPconjugate will thus give an antibody response, while the 'tolerant' mice challenged onlyonce with antigen in adjuvant (following the intraperitoneal injection) will at best giveonly a poor response or none at all.

In contrast to the in vitro tests, the in vivo tests indicate that the specificity of the skinfixing antibody was directed against the hapten and hapten carrier complex but notagainst the carrier protein, thus differing in their specificity from the haemagglutinatingantibodies. An explanation for the differing specificities may be the different classes ofantibody involved in the reactions: while y1 is involved in the homologous skin sensitiza-tion (Barth and Fahey, 1965), the antibody responsible for haemagglutination is pre-dominantly Y2 or in early bleedings IgM. From these results one can infer that differentclasses of antibody with differing specificities are produced: y1 with anti-DNP and anti-DNP-carrier specificity and Y2 (and in early bleedings presumably also IgM) with anti-DNP-carrier and anti-carrier specificity. Both classes of antibody thus show specificitywhich involve both DNP and the carrier molecule while Y2 shows anti-carrier specificityas well.

ACKNOWLEDGMENTS

The author is indebted to Kristin Senff and Merle Sey for competent technical assist-ance and to James Dunne for performing the PCA reactions. The author also wishes toexpress her appreciation to Dr Fred Karush for his helpful criticism of some phases of thiswork, and to Dr Fred Karush and Dr Paul Maurer for their helpful criticism of the manu-script.

This work was performed during the tenure of a United States Public Health ServiceSpecial Fellowship (5-F3-CA-19,409) and a United States Public Health Service ResearchCareer Development Award (1-K3-CA-19,409-01). This investigation was also supportedby Grant AI-06556 from the United States Public Health Service and by the GeneralSupport Grant for the Temple University Health Sciences Center.

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