is there really a “lack of natural tolerance to allotypic γ-globulins in rabbits”?

Eur. J. Immunol. 1991. 21: 2543-2551 Response to maternally derived incompatible Ig allotypes 2543

Daniel Meier and Is there really a “lack of natural tolerance to allotypic y-globulins in rabbits” ?

Andrew S. Kelus

Basel Institute for Immunology+, Basel

We present data of extended studies on the possibility of maternally derived allotype Ig inducing a state of natural immunological tolerance to a non-inherited allotype in the offspring. Rabbits homozygous at the a locus, encoding allotypes in the variable region of immunoglobulin heavy chains, and rabbits homozygous at the unliked b locus, encoding allotypes of the constant region of x l light chains, were immunized at the age of 2 months against the non-inherited allotype of their heterozygous mothers to which they had been exposed in utero and in early life. As control, we immunized rabbits of the same Ig phenotype but born to homozygous mothers, and therefore not exposed to that allotype. Immunization was done in a3/a3 offspring of either al/a3 or a3/a3 mothers, by injecting a1 IgG, and in b6/b6 offspring of b4/b6 or b6h6 mothers, by injecting b4 1gG.The IgG was injected either in a soluble form or emulsified in adjuvant. Injection of soluble IgG elicited only a low response, if any, revealing no differences between the various groups. All rabbits responded upon immunization with IgG in adjuvant. We have not found any good evidence for natural tolerance to a non-inherited allotype, although progeny of al/a3 mothers had slightly decreased responses to al. On the contrary, progeny of b4h6 mothers responded even better than offspring of b6h6 mothers, upon such immunization with b4. To induce tolerance experimentally, we injected newborn rabbits, either from heterozygous al/a3 or from homozygous a3/a3 mothers, with a1 serum or IgG. Newborn of heterozygous b4/b6 or of homozygous b6/b6 mothers were injected with b4 serum or IgG in the same way. Such treatment resulted in partial tolerance to each allotype. In an attempt to amplify the tolerizing effect of the maternal a1 Ig, we injected newborn rabbits of al/a3 mothers with the serum of their mother. The response upon subsequent immunization with a1 allotype of another individual did not differ significantly from the response of control rabbits. The response was much poorer when rabbits were injected with nonmaternal tolerogen at birth, and when the same Ig preparation was used as immunogen. In a control experiment, neonatal injection of xenogeneic proteins, human IgG or bovine serum albumin, clearly resulted in tolerance. We speculate that tolerance to allotypes is established in theT cell repertoire only but bypassed by recognition of idiotypic determinants on antigen molecules by helper T cells, which trigger anti-allotype antibody formation by allotype-specific B cells. The end result of it is a lack of natural tolerance.

1 Introduction

The capacity to discriminate between self and non-self is generated during embryonic and neonatal development of the immune system and specific immunological tolerance to autologous antigens is acquired [1-6].Tolerance in theT cell repertoire is achieved during development in the thymus by negative selection, i. e., deletion of potentially harmful cells

[I 96361

+ The B a d Institute for Immunology was founded and is supported by F. Hoffmam-La Roche and Co., Ltd., Basel, Switzerland.

Correspondence: Andrew Kelus, Basel Institute for Immunology, Postfach, CH-4005 Basel, Switzerland

Abbreviation: HIgG: Human IgG

with receptors for self antigens plus self MHC. The T cells bearing receptors for foreign antigens plus self MHC are positively selected for further development [7, 81. Anti- body diversity in the B cell compartment is generated in two phases: one occurs by gene rearrangement of immature B cells in the primary lymphoid organs, while the other one takes place in peripheral lymphoid organs by somatic hypermutation upon antigen contact [9, 101. Induction of tolerance in the B cell repertoire can occur at both phases of diversification, and self-reactive B cells can be either eliminated, a process called clonal deletion, or functionally inactivated, a process termed clonal anergy [l, 41. Self tolerance in B cells is not absolute but since the active participation of Th cells is required in most B cell responses, tolerance in the Tcell repertoire usually prevents the production of autoantibsdies.

Tolerance can be induced experimentally by injecting potential antigen (tolerogen) into newborn animals [ll-141. Adult animals can be made unresponsive, if the

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 oO14-2980/91/1010-2543$3 .50 + .25/0

2544 D. Meier and A. S. Kelus Eur. J. Immunol. 1991.21: 2543-2551

antigen is either presented in a soluble form free of aggregates [15] or given at very high or very low doses, a phenomenon known as high-zone and low-zone paralysis [16, 17l.Tolerance is induced inTcells and B cells, or in the Tcell repertoire only, depending on the dose of antigen injected [18, 191.

Ig of the rabbit expresses genetic markers called allotypes. They are found in the CH and, so far uniquely, in the rabbit, in theVH region of Ig chains. Allotypes of theVH region as well as the allotypes of the CL x l chain are found in all classes of rabbit Ig [20-22].Two or more alleles exist at each locus and are inherited in a simple Mendelian fashion.

The IgG and IgM molecules, which provide a newborn rabbit with passive immunity, are of maternal origin. They are transmitted to the fetus via the yolk sac splanchnopleur [23]. Thus maternal antibodies protect the newborn during the first several weeks of life. The acquirement of Ig via colostrum and milk does not significantly influence this passive immunity received in utero. In our experience, a 2-month-old rabbit is immunologically mature.

Allotypes provide useful genetic markers which allow one to distinguish between maternal and progeny Ig, and hence enable us to study the influence of products of the maternal immune system on the developing immune system of the offspring. Allotypically homozygous newborn of mothers, heterozygous at one or several allotype loci, contain large quantities of circulating Ig of the non-inherited allotype, which may be considered as foreign antigen. This would seem to be an ideal situation to establish tolerance to the allotype. Surprisingly, as shown by Gel1 and Kelus, in Lack of natural tolerance to allotypic y-globulins in rabbits [24], such animals could be readily immunized against non- inherited maternal allotypes. It was even possible to demonstrate spontaneous formation of low-level antibody responses to the maternal allotypes in such rabbits [25-271.

We present here data from extended studies on this topic and confirm the inability of rabbits to establish a state of natural tolerance to non-inherited allotypes, to which they had been exposed in utero and during the first few weeks of life. Nevertheless, we could demonstrate that if rabbits are injected with serum or IgG of the appropriate allotype neonatally, they do show partial tolerance when immunized at the age of 2 months with the same antigen. In control experiments tolerance to BSA or to human IgG (HIgG) was induced by injecting rabbits with those antigens shortly after birth.

2 Materials and methods

2.1 Animals

Rabbits identical by descent at the Ig loci were bred and maintained at the animal colony of the Basel Institute for Immunology (RodersdorfISO, Switzerland). Females of allotype a31a3 b4h4 or al/a3 b4/b4 were mated to a31a3 b4h4 males, and females of allotype al/al b6h6 or al/al b4h6 were mated to al/al b6h6 males. Some of the resulting litters were injected within 24 h after birth with a1 b4 serum or IgG. The rabbits received between one and five

(given every 2days) injections of the tolerogen. At 2 months of age, the homozygous progeny (a3/a3 b4h4 and al/al b6/b6) was immunized with IgG (a1 b4) either as soluble Ag in PBS or emulsified in CFA, in order to produce either anti-a1 or anti-b4 antibodies.

The serum or IgG used for neonatal injection, as well as the IgG employed for immunization, derived from one individual rabbit which was immunized against Proteus vulgaris X19. Other groups of rabbits, some of which had been injected after birth with the Ag, were immunized in the same way with HIgG (Sandoglobulin, Sandoz AG, Basel, Switzerland) or BSA (Sigma, St. Louis, MO).

2.2 Auotyping

Glass plates were coated with agarose gel (1.5% agarose, 2% PEG 6000 in PBS, pH 7.2). Six wells arranged around a centrally placed well were filled with undiluted test sera. The center well was filled with undiluted anti-allotype antiserum. The plates were incubated at 4°C and the precipitation lines read after 24 h and 48 h. All measurements were performed using two antisera of the same specificity [21, 281.

2.3 Preparation of anti-allotype antisera

2.3.1 Immunization using IgG complexed with Proteus

Precipitating anti-allotype antisera used for double diffu- sion tests were obtained by injecting rabbits i.v., with a few courses of 1 mg heat-killed, lyophilized Proteus vulgaris X19, a non-motile strain, complexed with anti-Proteus IgG which differed from the recipient’s haplotype only in the allotype towards which the antiserum would be directed [21]. Usually three to four courses consisting of three injectionslweek, with a break of about a month between each course, were sufficient to raise strong antisera. Anti- Proteus antisera were produced in the same way by injecting the bacillus alone. The anti-Proteus IgG was isolated on protein A-Sepharose CL-4B (Pharmacia, Upp- sala, Sweden) [29]. The rabbits were bled from the central ear artery about 10 days after the last injection of the Ag, and tested for specificity against a panel of allotypically defined sera.

2.3.2 Immunization with soluble or CFA-emulsified IgG

Immunization of homozygous a31a3 and b6h6 rabbits under study was done by injecting 5 mg/ml a1 b4 IgG in PBS i.v. Every rabbit got three injections with a break of 2 weeks between the first and second and one of a month between the second and third injection. Blood samples were col- lected 2 weeks after the injections. Another set of a3/a3 and b6/b6 rabbits was immunized by injecting 1 mg of a1 b4 IgG in PBS, in 2 ml of a 1 : 1 emulsion in CFA, applied at several sites, S.C. and i.m. After 4 weeks a blood sample was taken followed by a second course of immunization, and another bleed was taken 4 weeks later. In a control experiment, rabbits of various allotypes were injected in the same way either with soluble HIgG or BSA or with these Ag emulsified in CFA.


2.4 Purification of anti-allotype antibodies

Immunosorbent columns were prepared by covalently linking IgG of appropriate allotype to CNBr-activated Sepharose 4B (Pharmacia) [30]. After passage of antiserum in PBS, the adsorbed Ab was eluted with 0.56% acetic acid in saline (pH 3.0) followed by dialysis against PBS. A sheep anti-rabbit Fcy was absorbed on a column with covalently linked HIgG to remove cross-reacting antibodies. In this case the non-adsorbed eluent was used for further preparations.

2.5 Coupling of p-galactosidase to antibody

Affinity-purified anti-allotype Ab or anti-allotype IgG purified on protein A-Sepharose, as well as anti-HIgG or anti-BSA IgG were conjugated to 0-galactosidase (5 mg Ig/lO mg enzyme; Boehringer-Mannheim, Mannheim, FRG) using glutaraldehyde (Sigma) [31].

2.6 ELISA

Concentrations of allotypes and titers of antisera were measured by ELISA [32,33]. Briefly, nonsterile microtitra- tion plates (Flow Laboratories, McLean,VA) were coated with Ag (a1 b5 IgG to test anti-a1 antisera, a3 b41gG to test anti-b4 antisera; HIgG or BSAwere used for coating to test anti-HIgG and anti-BSA antisera, respectively). A lOO-vl quantity of sodium carbonate (0.1 M, pH 9.6) containing 5 pg/ml Ag was added to every well. After incubation at 4°C overnight, the plates were washed four times by flicking the contents and rinsing with PBS containing 0.1% Tween 20 (T-20; Merck-Schuchardt, Hohenbrunn, FRG). Serial twofold dilutions of the assay samples in PBS/T-20 containing 0.25% gelatin (PBS/T-2O/G) were added (100 @/well).

The titration of a reference sample, to check variation from plate to plate, and blank wells containing PBS/T-20IG only, were included in every plate. All measurements were done in duplicate. After 1 h at 37"C, followed by four washings, 100 pl PBS/T-20/G containing 2.5 pg/ml Ab conjugated to 0-galactosidase was added to evey well.To measure the titer of anti-a1 antisera (produced in a3 b4), a conjugate of purified anti-b4 antibodies was used, and to measure anti-b4 titer (produced in a1 b6), a conjugate of purified anti-a1 antibodies was employed. Titers of anti-HIgG and anti-BSA antisera were measured with conjugated sheep IgG anti-rabbit Fc (HIgG absorbed). After 1 h at 37"C, the plates were washed again four times, 100 yl of the substrate o-nitrophenyl-0-D-galactopyranoside (Sigma) was added [0.8 mg/ml in 0.1 M phosphate buffer, pH 7.0, containing 0.002 M Mg-Titriplex (Merck-Schuchardt), 0.0002 M MnS04 and 0.001 M MgS041 and the plates were incubated at room temperature for 30-60 min. The reaction was stopped by adding 25 pl of 2 M NaZC03, and the rq405 ,,,,, was measured in a Titeflek Multiskan MCC/340 (Eflab, Hel- sinki, Finland).The specificity of the assay was checked by a variety of negative control sera. Measurements which seemed to be doubtful were repeated. In spite of slight variations of the titer, the differences were not significant. Antisera of higher titer gave more reproducible results.

The same method was used to test remaining maternal or injected Ig in the offspring. Plates were coated with affinity-purified Ab directed towards an allotype of the maternal or injected Ig, but not present in the genome of the rabbit tested. The same Ab used to coat the plates was employed as the enzyme-Ab conjugate. IgG of known concentration of a homozygous allal b4/b4 rabbit was titrated as standard in every plate. A similar procedure was applied to test HIgG or BSA using protein A-purified rabbit IgG containing the specific Ab.

2.7 Calculations

A reference antiserum of high titer was selected for every specificity.The titers of the test samples were determined by plotting the absorbance vs the log of the dilution and comparison of the graph with that of the reference antiserum. A point was defined at the lower part of the reference curve (around &.I) and the titer of the test samples were calculated by selecting the dilution which gave the absorbance value closest to the defined one. The reference samples, as well as blank wells, were included in every plate as controls for the reliability of the tests. Since most of the pre-inoculation samples, due to unspecific binding at low dilutions, did not give reliable curves, the + and - signs (Tables 1 and 2) indicate only where samples differed clearly from the background. All calculations were performed on Macintosh IIcx computer, using the Excel program (Microsoft, Bellevue,WA). The significance of the differences of the means of the Ab titers in the various groups was determined by Student's t-test. Therefore, the numbers were transformed into logarithmic form and the test was performed using the StatViewTM program (Abacus Concepts, Berkeley, CA).

3 Results

We tested if rabbits can be tolerized to an allotype,which is not a product of their genome, by exposure to this allotype in utero and in early life, either through maternally derived Ig, or through neonatal injection of Ig. Responses to allotypes of the VH alocus and the CL b locus were measured. The results obtained from rabbits which have been immunized with soluble IgG did not allow comparison of the various groups (data not tabulated).The responses of these rabbits, measured in ELISA, differed only slightly, if at all, from the background. It seems therefore that soluble IgG is rather a poor immunogen. Tables 1 and 2 show the results and the statistical significance of the differences between various groups immunized with IgG in CFA.

We immunized homozygous a3Ia3 rabbits, the progeny of al/a3 or a3Ia3 mothers mated to a3/a3 fathers, with a1 IgG in CFA. Some of these rabbits have therefore been exposed to a1 in utero. No maternal Ig was found in these offspring when tested at 2 months of age. Comparison of the anti-a1 titers in those two groups shows that rabbits exposed in utero to a1 reacted slightly weaker, than rabbits not exposed to a1 in utero (Table 1; A vs C). We found a low anti-a1 titer in serum samples taken before immunization (Table 1; rabbits no. T-24 and T-28).

2546 D. Meier and A. S. Kelus Eur. J. Immunol. 1991. 21: 2543-2551

Table 1. Rabbits of a3 allotype immunized with ala)

Offspring of aM13 b 4 M mothers Treatment after Rabhit antibody titer birth no.

Pre Course 1 Course I1

(A) No treatment T- I - T- 2 - T- 3 - T- 4 - T- 5 - T- 6 - T- 7 - T- X - T- 9 - T-10 - T-I1 - T-12 - T-I3 -

(B) 3 x 10mg a1 igG T-I4 - T-lS - T-16 - T-17 - T-18 -

5 x 2 ml a l seruni T-I9 - T-20 - T-21 - T-22 - T-23 -

a) Note: t-test

aYa3 mothers 1’s a l h 3 mothers: no treatment

a333 mothers L ’ S a3/a3 mothers: progeny injccted after birth with aUal alla3 mothers cs alh13 mothers; progeny injccted after birth with a l h l al/a3 mothers vs a h 3 mothers; progeny injected after birth with miitcrnal serum al/a3 mothers v.s al/a3 mothers: progeny injected after birth with a3h3 serum al/a3 mothers vs aIki3m others, suppressed for a1

;13/a3 mothcrs V S ;rl/a3 mothers. suppressed for a1

Treatment after birth

(C) N o treatment

(D) I x 2 ml al serum

(E) 5 x 2 ml maternal serum

(F) S x 2 ml a3 serum

(G) Mother al-suppressed. no treatment after birth

Offspring of alla3 bllbj mothers

no. Rabbit antibody titer

Pre CourscI CourscII

T-24 T-25 T-26 T-27 T-28 T-29 T-30 T-3 1 T-32 T-33 T-34 T-35 T-36 T-37 T-3s T-39 T-40 T-4 I T-42 T-43 T-4-4 T-15 T-46 T-17 T-76 T-77 T-78 T-79 T-80 T-8 1 T-82 T-83 T-84 T-85 T-86 T-87

+

+

+ -

ND ND ND ND - -

ND

Groups Course I I Geometric mean of

titers

A 1’s c I 8/13 i m / soi A t1.S c II w13 s s n ~ 2 3 7 7

c v s ~ I x/ 7 mi/ x8

A cs 8 I 13/10 1330/ 80 A vs B I1 13/10 SSOW 226

C vs D I1 8/ 7 2377/ 131 C vs E I 8/ S 501/ 211

C v s F I 8/ 4 SOl/ 423

c v . s ~ I 8/12 sol/ ~ 1 7 C vs G I1 8/12 23771h039 A vs G I 13/12 1330/ 8.17 A vs G I1 13/12 5SW6039

DF I

I Y 2.20 19 2.87 21 7.82 21 10.76 13 2.93 13 5.83 11 1.43

400 4 ow 2000 loo0 4ooo 4 0 1 0 2 OM)

0 20

320 80

160 320 10

ND ND ND ND ND ND ND ND ND 3 200 6 XK) 6 900 6 j o 6300 3 2nn h 40 3 200

12 800 12 NK) 3 200

12 NIO

10 0.23

18 1.0.1 18 355 23 1.19 23 0.38

P

0.82 I2

Eur. J. Immunol. 1991. 21: 2543-2551

Table 2. Rabbits of b6 allotype immunized with Ma)

Response to maternally derived incompatible Ig allotypes 2547

Offspring of allal b6/b6 mothers Treatment after Rabbit antibody titer birth no.

Pre Course1 Coursc I1

(A) No treatment T-48 - T-49 - T-50 - T-51 - T-52 - T-53 - T-54 - T-55 - T-56 - T-57 - T-58 - T-59 -

(B) 3 x 10 mg b4 IgG T-60 + T-61 + T-62 -

1600 1600 1600 400

1600 80 800

1600 400

1 6 0 00

1 0

200 200 100

3 200 6 1 0 6 1 0

Joo 3 200 1 6 0 1600 3 200 3 200 3 200 6400 3 200

200 800 400

5 x 2ml b4 serum T-63 - 800 1600 T-63 + Joo 80

a) Note: t-test

Offspring o f al/al INh6 mothers Treatment after Rabbit antibody titcr birth no.

Prc Chrsc I Coursc I 1

(C) No treatment T-65 + T-66 - T-67 + T-f4 - T-69 + T-70 +

(D) 3 x 10 ml b4 IgG T-71 - T-72 - T-73 + T-74 + T-75 +

(E) Mother b4-sup- T-88 - prcssed. no treat- T-8Y - mcnt aftcr birth T-90 -

T-91 - T-92 - T-93 - T-94 - T-95 - T-96 - T-97 - T-98 - T-9y -

Groups Course

b6M mothers vs b4/b6 mothers; no treatment AvsC 1 A vs C I1

b6/b6 mothers vs b6h6 mothers; progeny injected after birth A us B I with Mh4 A vs B I1 Wb6 mothers vs b4M mothers; progeny injected after birth C vs D 1 with bJ/bJ C vs D I1 bJlb6 mothers vs b4/b6 mothers. suppressed for b4 C vs E I

C v s E I1 b6/b6 mothers vs b4h6 mothers, suppressed for b4 A v s E I

A vs E 11

When a3la3 offspring of a31a3 mothers were injected with a1 serum or IgG neonatally, in the attempt to induce tolerance experimentally, a significantly lower response upon an Ag challenge with a1 was noted (Table 1; A vs B). We found traces of neonatally injected a1 in sera of 2-month-old rabbits, taken before immunization (Table 1; rabbits n0.T-19 and T-20: 5 pgh1l~T-22: 10 pgld and T-23: 1 Pgld).

Offspring of alIa3 mothers had lower titers of anti-al, when ived injections of a1 after birth, than offspring

which were exposed to a1 in utero only (Table 1; C vs D).

n

l Z 4 1 2 6 12i 5 l Y 5 41 5 6 / 5 4/12 6/12

12112 12112

Geometric mean of

titers

lMYI2692 285 1/3589 lM9l 264 28511 607 269U 459 3589l1393 26Y2/ 283 3589/1600 1069/ 283 2851/1600

DF t

14 2.53 16 0.56 15 4.22 1s 3.74 7 3.25 9 1.61

I4 4.32 16 1.31 22 4.33 22 1.28

P

0.0240 0.5818 0.0007 0.0020 0.0 140 0.1413 0.0007 0.20" 0.0m3 0.2128

This shows that such treatment induces partial tolerance to a1 allotype. Interestingly, no statistically significant difference in response to a single injection of a1 Ag was found between control rabbits and rabbits treated after birth, when the neonatally injected serum was of maternal (al/a3) origin (Table 1; C vs E). As expected, injection of a3Ia3 serum, performed as a control experiment, had no influence and the response of these rabbits did not differ from untreated ones (Table 1; C vs F).

In another experiment, we tested the response to a1 in offspring of a1 suppressed alla3 mothers. These mothers

2548

Table 3. Rabbits immunized with HIgGa)

D. Meier and A. S. Kelus

Trcatmcnt after birth

Rabbit no.

(B) 1 x 1 mg HIgG

Sx I mg HIgG

5 x 1 0 mg HIgG

( A ) No trcatmcnt T- 1(W) T- 10 1 T- I02 T- 103 T-104 T-105 T-106 T- 107 T- I 08 T-IOY T-110

a) Note: t-test

Anti body titer

Course II

Control rabbits vs rabbits injected aftcr birth with HlgG

Table 4. Rabbits immunized with BSAa)

Trcatmcnt :tfter birth Rabbit no.

( A ) N o trcatmcnt

(B) l x 1mgBSA 3 x 10mgBSA

3 x I00 nig BSA

a) Note: t-test

T-I 1 I T-I12 T-I13 T-114 T-115 T-I16 T-117 T-I18 T-I 19 T- 120 T-121 T-122 T- 123 T- I24 T- 125 T-126 T-127 T- I28

Eur. J. Immunol. 1991. 21: 2543-2551

Groups Course n Geometric DF t P mean of

titers

A V S B 11 5/15 319890f2735 9 13.30 O.ooO1

Antibody titer Course I Course I 1

Control r;ihhits I'S rabbits injected aftcr birth with BSA

32 OOO 128 00 128000 128000 32 O00 32 000 4 0 0

16 0 32 OOO 2000 4 ouo 8 000 2000 1OOO 320 160 320 320

Groups Course 18 Geometric DF r P mean of

titcrs

A vs B I 5N 34W318 12 4.21 0.0012 II 9N 3732Y957 16 6.24 0.OOOI


were the progeny of anti-al-producing a3/a3 females and al/al males. They lacked the a1 Ig in circulation when tested at 2 months of age, and allotype suppression was maintained by repeated injections of anti-a1 antiserum. Mating of these females to a3/a3 males resulted in offspring which got only little maternal al.The concentration of a1 in the mothers at the time of delivery was between 10 and 50 pg/ml (- 5 mg/ml in normal heterozygous rabbits). The response upon immunization with a1 of the a3/a3 offspring of these allotype-suppressed females was comparable to the response of progeny of a3/a3 mothers (Table 1; A vs G).

We also mated b4h6 or b6h6 females to b6h6 males and immunized the homozygous progeny with b4 IgG. In contrast to the situation of a3/a3 rabbits, which have been exposed in utero to a l , we found that perinatal exposure to maternal b4 enhanced slightly the response to the Ag after a single injection, when compared to rabbits which were not exposed to b4 in utero (Table 2; A vs C). But the differences disappeared after a boost with the same Ag. Four out of six offspring, which have been exposed to b4 in utero, even showed weak anti-b4 Ab in pre-immune sera (Table2; rabbits no. T-65, T-67, T-69 and T-70).

Progeny of b6h6 mothers, neonatally injected with b4, produced lower titers of anti-b4 than untreated controls (Table 2; A vs B) showing that a partial tolerance can be induced experimentally, although three of these rabbits had anti-b4 Ab of low titer in their pre-immune sera (Table 2; rabbits no. T-60,T-61 and T-64). The response of progeny of b4h6 mothers, injected with b4 after birth, and of progeny exposed to maternal b4 solely, differed statistically only after the first antigenic injection but not after the second (Table 2; C vs D). Low anti-b4 titers were also found in the pre-immune sera of some of these rabbits (Table 2; rabbits no. T-73, T-74 and T-75). Traces of b4 Ig (10 pg/ml) were found in the pre-immune sample of rabbit no. T-71.

An unexpected observation was made in b6/b6 offspring of b4 allotype suppressed b4h6 mothers. These mothers were the progeny of anti-b4 producing b6h6 mothers and b4h4 fathers, and they were repeatedly injected with strong anti-b4 antiserum to maintain suppression. Offspring of these mothers responded very poorly after a single injection of b4 Ag. This differed clearly from the response of rabbits exposed to b4 in utero, and from rabbits not exposed to maternal b4. There was no significant difference after a second Ag injection (Table 2; C vs E and A vs E).

In a control experiment, we injected newborn rabbits within 24 h after birth and for various times thereafter with HIgG or BSA to induce tolerance to these Ag. Immunization at 2 months of age with soluble HIgG or BSA did not elicit responses which allowed comparison of pretreated and untreated animals (data not tabulated). The titers measured by ELISA were very low, and in most cases not different from the background.

The anti-HIgG and anti-B Ab titers of pretreated and not pretreated rabbits, afte munization using CFA, were significantly different (Tables 3 and 4). As little as 1 mg HIgG or BSA injected into neonates could induce tolerance in the animals. Traces of HIgG (< 1 pg/ml) were found in the pre-immune sample of some rabbits (Table 3; rabbits no. T-105, T-108, T-109 and T-110).

4 Discussion

It has been demonstrated using hen egg lysozyme ( E L ) transgenic mice as a model that these animals recognize the secreted new Ag as self and develop tolerance to it [34]. Mating of HEL transgenic mice to another strain, which expressed rearranged H and L chain transgenes encoding high-affinity anti-HEL antibodies, resulted in double transgenic HEL-tolerant mice. HEL-specific B cells were found to be present in their peripheral lymphoid tissue but failed to respond efficiently to the Ag even in the presence of Tcell help [35].Depending on the concentration of lysozyme secreted into the plasma of those animals, tolerance was found in the T and B cell repertoires or in T cells only [19]. The situation, where a rabbit is exposed to a non- inherited maternal allotype in utero is somewhat similar: a “foreign” Ag is present during the whole period of development of the immu,ne system. Although the Ag is not produced by the host, as in the HEL-transgenic mouse model, and therefore disappears gradually, it could be recognized as self, and tolerance could be induced. We examined the ability of the VH a locus and CL b locus allotypes a1 and b4, respectively, to induce a state of tolerance in animals which did not possess the allotype but which were exposed to it in utero and early life.

The responses, after injection of the Ag in a soluble form, were very weak and did not reveal any differences between the various groups of rabbits. Therefore Ag applied in that form seems to be only poorly immunogenic, if at all. But upon immunization with Ag in CFA, all the rabbits responded quite well. Anti-HIgG and anti-BSA Ab titers of rabbits which have been injected neonatally with Ag of the same batch as used for immunization, differed significantly from titers of control rabbits not treated after birth. They had established tolerance to these Ag.

The mean anti-a1 Ab titer after one and after two courses of immunization of rabbits,which were exposed to a1 in utero, was about 2.5-fold lower than the mean titer of rabbits not exposed in utero to al. Titers of progeny of al-suppressed al/a3 mothers resemble those of offspring of a3/a3 mothers. Although the differences showed statistical significance, we do not think that these results provide strong evidence for the existence of tolerance to this allotype. We found a low anti-a1 Ab titer in the pre-immune serum of some rabbits showing that maternally transmitted a1 can even induce antibody formation (Table 1).

The anti-b4 titers of rabbits, which had been exposed to b4 in utero, were 2.5-fold higher after a single injection when compared to offspring of b6M mothers, and 60% of rabbits from b4h6 mothers had anti-b4 antibodies present in their pre-immune samples, although their titer was low [25-271. This suggests that exposure to non-inherited maternal CL b locus allotypes might have had a slight priming effect and not a tolerating one. After two injections there was no difference between those groups (Table 2).

The low response of offspring of b4-suppressed b4h6 mothers after a single immunization with b4 was striking. We speculate that anti-b4 antibodies, repeatedly injected into the mothers to maintain suppression, reached the circulation of the offspring and caused this effect via network interactions [36].

2550 D. Meier and A. S. Kelus Eur. J. Immunol. 1991. 21: 2543-2551

Partial tolerance was found in newborn rabbits injected shortly after birth with serum or IgG, and immunized with the same IgG at 2 months of age. Anti-a1 titers of rabbits born to a3/a3 mothers and treated after birth with a1 serum or IgG were up to 25 times (!) lower than titers of untreated rabbits. Additional neonatal injection of a1 in offspring of al/a3 mothers decreased their titers compared to that of control animals almost 20 times. The difference became more obvious after two immunizations, which is probably due to the relatively weak immunogenicity of VH a locus allotypes (Table l).We did not find any convincing evidence for tolerance when maternal al/a3 serum was injected into the offspring. Since the a1 allotype in a heterozygous al/a3 rabbit is present in > 50% of the Ig molecules [37], we do not think that this slight reduction of the a1 dose, in comparison to injection of serum of a homozygous al/al donor, is responsible for this inability to induce tolerance.

Progeny of b6b6 mothers, injected shortly after birth with b4 IgG or serum, had anti-b4 titers which were 4 to 5 times lower than those found in untreated offspring. Offspring of b4b6 mothers, injected neonatally with b4, reacted 6 times weaker after a single injection and 2.5 times weaker after a second injection of the Ag.

Although the situation of rabbits, which are constantly exposed to the Ag, a1 or b4 respectively, in utero and for some time after birth,offers at first sight a better model for tolerance induction than injection of the Ag after birth, the results suggest that there is no natural tolerance to non- inherited allotypes but that partial tolerance can be induced by injection of the Ag shortly after birth.

Partial tolerance to a1 is better established upon neonatal injection (mean titer up to 25-fold lower after neonatal treatment) than to b4 (mean titer up to 6-fold lower after neonatal treatment). It is well known that the CL b locus allotypes are stronger immunogens than the VH a locus allotypes, and this might be the reason for an earlier escape from tolerance and induction of anti-b4 Ab formation.

To explain the lack of natural tolerance to maternal allotypes on the one hand, and the possibility of inducing tolerance to allotypic determinants experimentally on the other, we propose the existence of unresponsiveness to allotypes in the Tcell repertoire but not in the B cell repertoire. Tolerance in the T cell repertoire could be bypassed for instance through recognition of idiotypic determinants [38, 391 on the Ag by Th cells. Idiotypes could therefore have a carrier effect, and Ag-presenting allotype- specific B cells could be triggered through idiotype-specific Th Cells.

We used serum or IgG from one individual rabbit as tolerogen and as antigen throughout the experiments. Since this Ag was derived from a rabbit which had been immunized with Proteus, one can presume that this preparation contained one or a few major idiotypes, which were present at an increased concentration [39]. The partial tolerance found in rabbits, which were injected with this Ag shortly after birth, could then be explained by a decreased availability of Tcell help for allotype-specific B cells, i.e., due to tolerance to those major idiotypes in the Tcell repertoire. Other idiotypes, present at a concentration too

low to induce tolerance, could be recognized and would elicit a poor antibody response. Further support for our speculation comes from the inability of maternal Ig to induce tolerance when injected neonatally. Although the a1 of the maternal Ig and the a1 of the tolerogen were products of the same ancestral gene, the Ig molecules differed surely in their idiotypic composition.

The HIgG preparation, used in a control experiment, unlike the individual rabbit IgG, originated form a large pool of sera (several thousand people). Since the same preparation was used throughout the experiment, tolerance in the T cell repertoire could have been established to human isotypic, allotypic and idiotypic determinants. It is also feasible that individual idiotypes in this pool were present at a concentration too low to be recongized, and which would therefore be inefficient as carriers.

Another explanation for the failure to induce natural tolerance could be that the in utero transmitted tolerogen is available to the newborn in a different form than that which was injected neonatally. One could imagine that by the former route Ig molecules reach the circulation of the offspring in an aggregate-free form, whereas preparative procedures to which IgG or serum have been subjected might have caused some aggregation. Intensive receptor cross-linking might be responsible for tolerance induction in immature B cells [2,4, 151. Aggregated Ig would better perform such cross-linking of receptors with low affinity, which immature B cells normally have, than maternal Ig in a non-aggregated form. Since injection of maternal al/a3 serum which should therefore also contain such complexes did not provide evidence for tolerance induction to a l , we favor the idiotype hypothesis.

We wish to thank Willy Hanggi who bred and immunized the animals for this study so skillfully. We are most grateful to Alexandra Livingstone and Ivan Lejkovits for reading the manuscript critically. Our special thanks go to Charley Steinberg for his help with the statistical analysis.

Received May 28, 1991; in revised form July 5, 1991.

5 References

1 Lederberg, J., Science 1959. 129: 1649. 2 Howard, J. G. and Mitchison, N. A., Prog. Allergy 1975. 18:

3 Nossal, G. J. V. and Pike, B. L. , J. Exp. Med. 1978. 148:

4 Nossal, G. J.V, Annu. Rev. Immunol. 1983. I : 33. 5 Nossal, G. J.V., Int. Rev. Immunol. 1987. 2: 321. 6 Burnet, E M., Stone, J. D. and Edney, M., Aust. J . Exp. Biol.

7 Von Boehmer, H.,Teh, H. S. and Kisielow, l?, Immunol. Today

8 Von Boehmer, H. and Kisielow, l?, Science 1990. 248: 1369. 9 Jerne, N. K., Eur. J. Immunol. 1971. I: 1.

43.

1161.

Med. Sci. 1950. 28: 291.

1989. 10: 57.

10 Tonegawa, S., Nature 1983. 302: 575. 11 Burnet, E M. and Fenner, F., Theproduction of antibodies, 2nd

12 Billiigham, R. E., Brent, L. and Medawar, P. B., Nature 1953.

13 Hanan, R. and Oyama, J., J. Immunol. 1954. 73: 49.

Edit., Macmillan and Co. Ltd., London 1949.

172: 603.


14 Smith, R. T. and Bridges, R. A., J. Exp. Med. 1958. 108:

15 Dresser, D. W., Immunology 1962. 5: 378. 16 Mitchison, N. A., Proc. R. SOC. London, Ser. B 1964. 161:

275. 17 Dresser, D. W. and Mitchison, N. A., Adv. Immunol. 1968.8:

129. 18 Harris, D. E., Cairns, L., Rosen, F. S. and Borel,Y, J. Exp.

Med. 1982. 156: 567. 19 Adelstein, S., Pritchard-Briscoe, H., Anderson,T. A., Crosbie,

J., Gammon, G., Loblay, R. H., Basten, A. and Goodnow, C. C., Science 1991. 251: 1223.

20 Dray, S., Dubiski, S., Kelus, A., Lennox, E. S. and Oudin, J., Nature 1962. 195: 785.

21 Kelus, A. S. and Gell, l? G. H., Prog. Allergy 1967. 11: 141.

22 Mage, R. G., in Weir, D. M. (Ed.), Handbookof Experimental Immunology, 4th Edit., vol. 3, Blackwell Scientific Publica- tions, Oxford 1986, chapter 99.1.

23 Brambell, F. W. R., in Neuberger,A. and Tatum, E. L. (Eds.), Frontiers of Biology, 18, North-Holland Publishing Co., Amsterdam 1970.

227.

24 Gell, l? G. H. and Kelus, A. S., Nature 1966. 211: 766. 25 Adler, E L. and Noelle, R. J., J. lmmunol. 1975. 115: 620. 26 Adler, F. L. and Adler, L. T., Ann. N X Acad. Sci. 1982.392:

266.

27 Rodkey, L. S. and Adler, F. L., J. Exp. Med. 1983. 157:

28 Kelus, A. S. and Weiss, S., Nature 1977. 265: 156. 29 Ey, l? L., Prowse, S. J. and Jenkin, C. R., Immunochemistry

30 Axen, R., Porath, J. and Ernback, S., Nature 1967. 214:

31 Avrameas, S. , Immunochemistry 1969. 6: 43. 32 Avrameas, S. and Guilbert, B., Eur. J. Immunol. 1971. I:

394. 33 Engvall, E. and Perlmann, €?, Immunochemistry 1971. 8:

871. 34 Goodnow, C. C., Crosbie, J., Adelstein, S., Lavoie, T. B..

Smith-Gill, S. J., Brink, R. A., Pritchard-Briscoe, H.,Wother- spoon, J. S., Loblay, R.H., Raphael, K., Trent, R. J. and Basten, A., Nature 1988. 334: 676.

35 Adams, E., Basten, A. and Goodnow, C. C., Proc. Natl. Acad. Sci. USA 1990. 87: 5687.

36 Jeme, N. K., Ann. Immunol. (Inut. Pasteur) 1974. 125C: 373.

37 Lofts, R. S., Jr. and Rodkey, L. S., Mol. Immunol. 1981. 18: 433.

38 Oudin, J. and Michel, M., C. R. Acad. Sci. (Paris) 1963. 257: 805.

39 Kelus, A. S. and Gell, €! G. H., J. Exp. Med. 1968. 127: 215.

1920.

1978. 15: 429.

1302.

is there really a “lack of natural tolerance to allotypic γ-globulins in rabbits”?

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