Eur. J. Immunol. 1990. 20: 1311-1316
Ulrich Theopold and Georges Kohler Max-Planck-Institut fur Immunbiologie, Freiburg
Partial tolerance in p-galactosidase-transgenicmice
1311
Partial tolerance in p-galactosidase-transgenic mice A transgenic mouse line was produced which allowed the expression of E . coli P-galactosidase (P-Gal) under the regulatory elements of the immunoglobulin heavy chain locus. Expression of the transgene is found in spleen and bone marrow. Upon immunization of the transgenic mice with P-Gal, a reduced but clearly detectable antibody response was obtained. Affinity purification with sera from immunized transgenic mice suggests that they contain lower affinity antibodies as compared to normal littermates. Transgenic and nontransgenic mice immunized with bovine serum albumin (BSA) alone or as a mixture with @-Galgave comparable anti-BSA responses. Immunization with a chemically cross-linked (Gal-BSA)-protein, however, showed a 10- to 30-fold difference in the anti-BSA response. Partial unresponsiveness to P-Gal in the transgenic mice is best explained by a dominant, peripheral suppression mechanism linked to the antigen-presenting potential of B cells.
1 Introduction In recent years, transgenic mice have been used as model systems to study the development of tolerance towards the transgenic product (TGP) as a model autoantigen. Some of the hypotheses about tolerance have been supported by these investigations. Clonal deletion of autoreactive T cells in the thymus [l] as well as the inactivation [2] or elimination [3] of autoreactive B cells have been shown t o occur in these model systems. The importance of the time point of expression of theTGP has also been confirmed [4]. Beside these systems, where tolerance towards the TGP was clearly observable, there are other transgenic mouse lines, which show no tolerance towards the TGP For example a transgenic mouse line expressing a soluble form of an MHC class I gene product at a level of 200 ng/ml and early enough during ontogeny to induce tolerance showed no tolerance upon immunization and in MLR [5]. The question arises if the autoantibodies observed in these non-tolerant mice after immunization are comparable with natural autoantibodies, which have also been reported in healthy individuals [6-81. In the present study, a prokaryotic protein ( E . coli P-galactosidase; P-Gal) was expressed as a novel B cell protein in transgenic mice. Since the response to the TGP was only partially reduced in the transgenic mice, this reduction and the residual activity could be studied.
an M13 sequencing vector (m13mp8; [9]) and an expression vector for P-Gal fusion proteins (ptrRBO; [lo]). After manipulations of the 5' and 3' end of the lacZ gene by oligonucleotides which delivered the desired restriction sites for further cloning steps in the correct reading frame, the lac Z gene was introduced into a vector containing the regulatory elements of the Ig heavy chain locus cloned from the hybridoma line Sp6 [ ll] . A Hind 111 fragment containing the exons coding for the transmembrane region of IgM together with the polyadenylation site downstream of those exons was introduced in a last step to produce the plasmid pC5-6 (see Fig. 1). 2.2 Animals
The lacZ gene was introduced into the germ line by injecting the gel-purified large Sma I-Bam HI fragment of plasmid pC5-6 into SJL x C57BL/6 zygotes. For the immunizations, mice from the third and fourth backcross of the founder mouse to C57BL/6 background were used.
2.3 Analysis of the transgenic mice for expression of the TGP
2.1 Construction of the eukaryotic P-Gal expression vector
Mouse organs were homogenized on ice in an extraction buffer (PBS containing 1% NP40 and 5 mM PMSF) using a teflon pestel. The lysates were immediately cleared by centrifugation at 10000 x g for 5 min. After being separated on an SDS-polyacrylamide gel, the fractions of the lysates were analyzed by the Western blot technique using a commercially available anti-@-Gal mAb (Promega Biochtech, Madison, WI).
For the construction of an appropriate expression vector, the lac Z gene was assembled via an internal Aoc I site from
2.4 Cross-linking of BSA to P-Gal
2 Materials and methods
[I 81371 Correspondence: Georges Kohler, Max-Planck-Institute for Immunobiology, Postfach 1169, D-7800 Freiburg, FRG Abbreviations: P-Gal: P-Galactosidase TGP: Transgenic product 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
For cross-linking P-Gal (Boehringer Mannheim, Mannheim, FRG) with BSA, 1 mg of P-Gal was first incubated for 2 h at room temperature in 1 ml PBS containing 2% glutaraldehyde. The P-Gal was separated from glutaraldehyde on an HPLC column (Dupont, Wilmington, DE; GF-250 XL) run with 200mM potassium phosphate, p H 6.5, at 1 mumin. It was then incubated for 2 h at room OO14-2980/YO/0606-1311$02.50/0
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Eur. J. Immunol. 1990.20: 1311-1316
U. Theopold and G. Kohler
temperature with 1 mg of BSA in a total volume of 1 ml. The reaction was stopped by adding glycine to a final concentration of 1%. The high-molecular-weight fraction was purified again on an HPLC and concentrated afterwards for immunization. Each mouse was immunized with one tenth of this fraction.This fraction was also analyzed in a Western blot and shown to contain both P-Gal and BSA molecules. The exact ratio of P-Gal to BSA and the total amount of each of the components was not determined. 2.5 Immunization Mice were immunized i.p. with 100 pg of the respective antigen in CFA except for the cross-linked protein (see above). Ten days after each immunization, mice were bled and specific antibodies were detected in an ELISA using the antigen(s) as solid phase reagent, the mAb against P-Gal as a standard and a commerciallyavailable (Southern Biotechnology, Birmingham, AL) mouse x light chain-specific antiserum produced in goat as second-step antibody. Titers of Ag-specific Ab were determined from the standard and were expressed as pg/ml for P-Gal-specific Ab and in U/ml for BSA-specific Ab. The sera used for the affinity purification were obtained in the following way. Mice were back-crossed once to BALB/c and immunized twice with the P-Gal-expressing transfectant C5-6 [ 121. Since only low P-Gal-specific titers were observed, the mice were boosted with 100 pg of E. coli P-Gal in 1FA.Tendays later, sera were collected and tested individually (not shown). Their titers resembled titers found in primary responses to E. coli P-Gal. The sera were pooled and analyzed in detail (see Sect. 2.7 and 2.8). 2.6 Affinity purification of anti-P-Gal Ab The coupling of P-Gal to CNBr-activated Sepharose as well as the purification of the antisera were performed as recommended by the supplier (Pharmacia, Freiburg, FRG). The pooled and 10-fold diluted antisera from transgenic and normal mice were passed three times over two similar columns which were washed with 20 times the EcoRI.Smal.BamH1.S.ll.
PIlI.H8ndlY
volume of the column and eluted with 3.5 M MgC12. The concentration of total Ig and of P-Gal-specific Ab was determined in all fractions in an ELISA technique. 2.7 LD of B cells
A dilution series of spleen cells was incubated for 8 days with 25 pglml of LPS (kindly provided by Dr. C. Galanos, Freiburg, FRG). The number of wells containing P-Gal-specificAb was then determined in an ELISA. From the number of positive wells, the precursor frequency was determined with the help of a computer program (kindly provided by Dr. I. Melchers, Freiburg).
3 Results For the expression of E. coli 6-Gal in transgenic mice, a plasmid was constructed which contains the lac Z gene coding for E. coli P-Gal and sequences coding for the leader exon, including the promoter and enhancer region and the transmembrane region of the Ig p chain (see Fig. 1). The plasmid was tested by transfection into a mutant hybridoma cell line igm 662 [111 as described elsewhere [121. Briefly, the plasmid leads to the expression of a protein of the expected size, a part of which can be detected on the surface of the transfected hybridoma called C5-6. Other plasmids were constructed which lead to the expression of intracellular and secreted versions of @-Gal [12]. The plasmid pC5-6 was used to create transgenic mouse lines by injection into fertilized oocytes. The mouse lines which contained the transgene as assayed by dot blot analysis of tail DNA were examined for the expression of the transgenic product by Western blot analysis and cell staining of spleen cells, LPS-stimulated spleen cells and of hybridomas derived from the mice. One out of four lines of transgenic mice turned out to express the transgene. It contained approximately 10 copies of the transgene and was analyzed in more detail. Fig. 2a, b shows a Western blot analysis of some organs derived from a
8amHI.Sall. PSII,Hlnd Ill
Lac 2
linker BH 1
G418'
Figure I. Construction of the plasmid pC5-6. For details see Sect. 2.1. lac i = lac repressor, lac Po = lac promoter and operator, lac Z = gene for 6-Gal, IgE = enhancer of Ig heavy chains, IgPo=promoter of Ig heavy chain, L = leader of Ig heavy chain, TK = thymidine kinase, p(A) = polyadenylation site, Amp', G418' = genes for resistance to Ampicillin and Neomycin, T K Pr = promoter of the TK gene, C5, C6 = exons coding for p transmembrane region, BHI = BamHI.
Eur. J. Immunol. 1990.20: 1311-1316
Partial tolerance in P-galactosidase-transgenicmice
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Figure 2. Tissue specificity and amount of P-Gal expression by the transgenic mouse line CGa156. Protein (200 pg) of organs from a transgenic (+) and a normal (-) mouse (a, b) were analyzed by PAGE and a Western blot with an anti-P-Gal mAb (see Sect. 2.3).The position of E. coli P-Gal is indicated by the arrow head.The hybrid protein expressed in the transgenic mice is larger due to the presence of the y transmembrane region. (br = brain, liv = liver, ki = kidney, thy = thymus, spl = spleen, spl LPS = spleen cells stimulated for 5 days with LPS, b.m. = bone marrow. A29 = an Abelson-murine-leukemia virus (A-MuLV)-transformed line obtained from the mouse line CGa156. Ay = a control A-MuLV line obtained from an IgM transgenic mouse line). Fig. 2c shows an estimation of the amount of P-Gal expressed in 200 yg protein from the spleen of different individual mice from the line CGa156 and a normal control littermate compared with different amounts of E. coli P-Gal.
transgenic mouse and a non-transgenic littermate. A band of the expected size (121 kDa) is seen in the spleen and in the BM as well as in LPS-stimulated spleen cells of transgenic mice and not in those of the littermate. Fig. 2c shows that the amount of the TGP detected in different individual mice is clearly below 625 pg/200 Fg total protein and was estimated to be 20 ng/spleen.
Table 1. Concentration of anti-P-Gal Ab before and after passage over a P-Gal affinity columna)
Serum before Flow through Affinity-purified purification (cI&hnl) fraction (cl%/ml) (Irg/ml) If.ansgeniC
Transgenic and normal mice were immunized repeatedly with P-Gal from E. coli to assay for their capacity to mount an immune response to the TGF! One of these experiments is shown in Fig. 3a. The transgenic mice (filled circles) produced a significant titer of P-Gal-specific Ab which was, however, 3-10-fold lower than that of non-transgenic littermates (open circles). Transgenic mice immunized with BSA (Fig. 3c) or with a mixture of BSA and P-Gal (Fig. 3b) showed a normal response towards BSA and again a reduced response towards @-Galcompared with normal control littermates. When mice were immunized with P-Gal which was crosslinked to BSA by glutaraldehyde, the response to P-Gal was 5-7-fold lower. Also the response to BSA was between 10and 30-fold lower in transgenic than in non-transgenic littermates (Fig. 3d). Since the amount of BSA present in the P-Gal-BSA complex was not determined, it is not clear, whether the low anti-BSA response in the transgenicmice is due to insufficient stimulation with BSA antigen or due to suppression imposed onto the BSA response by the covalently linked P-Gal molecule (see Sect. 4). The frequency of LPS-reactive P-Gal-specific B cells was determined to be about 11200 for both the transgenic and the littermate control mice. Thus, the bulk of anti-pGal-producing B cells were unaffected by the expression of the transgene. This is surprising in view of the low anti-@-Gal response in the transgenic mice which may, however, be due to the lack of B cells with high-affinity Ig receptors for P-Gal.
Normal
24.7 109.2
11.5 11.0
1.3 (6.5) 58.0 (72.8)
a) For the purified fraction, the amount of total Ig eluted from the column is indicated in parentheses.The amount of total Ig in the serum before purification was similar in transgenic and normal mice (5.7 vs. 4.2 mg/ml).The anti-P-Gal Ab in the flow-through fraction were enriched for IgM and in the purified fraction for IgG as compared to the unpurified serum.
In order to determine the quality of the P-Gal-specificAb in the transgenic mice, pooled sera from transgenic and normal mice were separated on a P-Gal affinity column. As shown inTable 1, two fractions of antibodies which bind to P-Gal in an ELISA were obtained: the first which did not bind to the affinity column and which was present in the same amount in both types of sera and a second one which binds to the affinity column and was present in the sera of normal mice in much higher quantities than in those of transgenic mice.The transgenic mouse lacks around 90% of the high-avidity Ab able to bind to the P-Gal column.
4 Discussion In the present study, mice transgenic for E. coli @-Galwere analyzed for their capability of mounting an immune response towards the TGF! As expected for a construct with the regulatory elements of the p heavy chain locus, expression of the transgene was restricted to B cells in BM
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1
Eur. J. Immunol. 1990. 20: 1311-1316
U. Theopold and G. Kohler
drn'
Ga I
(d) bplm,
-
2 10
0
8
1
10
0
0
0 10
10.1
.. so
-Ipb 20
3O
i:
-
2
3O
Figure 3. Analysis of immune responses. Serum titers of transgenic ( 0 )and normal (0)mice immunized with P-Gal (a), BSA (c), a mixture (b) and a BSA-P-Gal protein, cross-linked by glutaraldehyde (d) are shown. Mice were immunized as described in Sect. 2.6. For the different immunizations the age of the mice was (a) 6-14 weeks, (b) 8-10 weeks, (c) 4 months, (d) 9-12 weeks; 0" = pre-immune serum, 1°,20,30= sera obtained from primary, secondary and tertiary responses.Titers of specific Ab were determined in pg/ml for P-Gal and in U/ml for BSA-specific antibodies as described in Sect. 2.4.
and spleen. In contrast to the hen egg lysozyme-transgenic qualitative difference of the immunizing vs. the transgenic mice, where the TGP was expressed under the control of protein. E. coli f3-Gal, in contrast to the TGP, is not the metallothionein promoter [2], the mice described here glycosylated and lacks the transmembrane region of IgM. show no absolute tolerance. Although being reduced by a This is reminiscent of the transgenic mice expressing a factor of 3 to 10, the antibody response to the TGP is secreted version of an MHC molecule [5], which showed readily detectable. The reduction of the titer of f3-Gal- both a normal humoral and cellular imune response upon specific Ab is more striking among the IgGl Ab than among sensitization with a membrane-bound version of the same the IgM Ab (data not shown). This result is in agreement molecule. In our case, the Ab generated in the transgenic with the finding that autoantibodies are more frequently mouse bind with the same titer difference to E. coli- and found among the IgM class [8].The induction of antigen- myeloma-derived p-Gal and detect the TGP in Western specific Ab upon immunization of (3-Gal-transgenic mice blots (not shown). Therefore autoantibodies have been with E. coli P-Gal might at least in part be due to a generated in the transgenic mouse. We were unable to
Eur. J. Immunol. 1990. 20: 1311-1316
detect surface expression of P-Gal in unstimulated spleen cells. Surface expression has, however, been observed in myeloma cells transfected with the construct [12] and might occur at low levels in the transgenic mouse. As our mice show no signs of an autoimmune disease, even after immunization with P-Gal, they offer the possibility to gain insight into the nature of natural, non-harmful Ab with specificity for autoantigens. The finding that such Ab belong to the repertoire of normal, healthy individuals has for years been a puzzling observation [7, 81. Our findings indicate, that compared to those of normal mice, P-Gal-specific Ab in transgenic mice have a lower affinity for the TGP Low-affinity Ab have also been obtained in hen egg lysozyme-transgenic mice, after their immunization with a carrier hen egg lysozyme complex [2]. There are several possibilities to explain the partial tolerance observed in our system.Tcel1 tolerance in the thymus of p-Gal-transgenic mice could deplete them of /%Galspecific T cell help. According to this hypothesis, early expression and presentation of antigen in the thymus, would lead to the elimination of antigen-specific T cells [ 13-16]. That P-Gal could potentially be expressed in Tcells of the thymus is indicated by previous findings that Ig promoter and enhancer-driven genes are often expressed in thymocytes and also in peripheral Tcells [17, 181. In our transgenic mice, P-Gal was not detectable in the thymus by the Western blot method. The signal obtained from unstimulated spleen cells was weak (Fig. 2). A two- to fourfold reduction of the amount of the TGP in the thymus could easily give rise t o a negative result. In addition, B cells residing in the thymus could present P-Gal to the developingT cell repertoire and delete specific clones. Deletion of P-Gal-specificT cells would then result in partial unresponsiveness to the P-Gal antigen due t o suboptimal T cell help. However, it is difficult to envisage how this mechanism could influence the response to an unrelated antigen such as BSAwhen covalently linked to P-Gal. On the other hand, it cannot be excluded, that the amount of BSA covalently coupled to P-Gal was too low to induce an optimal anti-BSA response. In such a situation, the P-Gal moiety might act as a carrier and help to mount the immune response to BSA in normal mice but would be unable to do so in transgenic mice, where P-Gal-specific help is compromised. For systems where elimination of self-reactive T lymphocytes in the thymus could not explain tolerance, peripheral inactivation or suppression mechanisms have been postulated [19-211. The increase in P-Gal expression resulting from antigenic stimulation (as mimicked by LPS treatment of splenic B cells, see Fig. 2) might raise the amount of P-Gal to a level where such a failsafe mechanism of tolerance induction is required. One possibility, which has received some attention, is the two-signal hypothesis of tolerance induction [20]. T or B cells which recognize antigen and do not receive at the same time a second signal (e.g. the appropriate interleukin) are turned into a nonresponsive (anergic) state. Such a mechanism has been put forward to explain the nonresponsive state of Tcells in transgenic mice expressing alloreactive MHC class I antigens extrathymically on their pancreatic islet cells [21]. It also might explain the anergic state
Partial tolerance in P-galactosidase-transgenicmice
1315
of B cells observed in the hen egg lysozyme-transgenic mouse [2]. Alternatively, in our particular case, co-expression of P-Gal and Ab with specificity for P-Gal in the endoplasmic reticulum could lead to internal antigen-antibody complexes formation and to self-destruction of the B cells, perhaps from a certain affinity threshold on (a mechanism recently hypothesized for T cells [22]). T!., alltlgen crosslinking experiment (see Fig. 3d) argues against deletion or anergy of P-Gal-specific B cells as a mert L pnenomenon, since BSA-specific B cells should not be affected by the expression of P-Gal from the transgene. Another hypothesis according to which peripheral B cell tolerance is explained by a dominant suppression mechanism conveyed by T cells [23-271 is more compatible with our results. BSA-specific B cells will present 6-Gal fragments on MHC class I1 only when P-Gal is covalently linked to BSA. The BSA-specific B cells could then be recognized and suppressed by P-Gal-specificT effector cells of both the CD8 or CD4 phenotype, possibly via cytolytic mechanisms. A dominant suppressive mechanism for the induction of peripheral tolerance is also more suitable to explain the preferential loss of antibodies with high affinity for the autoantigen. If one assumes that the affinity determines the amount of presentation of the antigen and thereby the density of the complexes between antigen-derived peptides and the MHC molecules, high-affinity B cells should attract T cells more easily than lower-affinity B cells. If these B cells are suppressor cells, an elimination of only the high-affinity pool of B cells is imaginable. In contrast, if they are helper T cells, they should lead to an expansion of these B cells. Thus the existence of suppressor cells which recognize self-peptides presented by MHC class I1 molecules may be a failsafe mechanism to avoid the production of high-affinity autoantibodies. The antigen presentation potential of B cells is essential for this kind of regulation of the B cell response. We thank I! Renard, R. Lamers and A . Iglesias for the generation of the transgenic mouse lines, U. Fritzsche for the generation of the Abelson lines, U. Birsner, B. Croetschel and C. Jacot for technical assistance, R. Lamers, G. McMaster, I! Nielsen and M. M . Simon for critically reading the manuscript and for useful discussions, S. Kleinhans for art work and G. Prosch for typing the manuscript.
Received November 27, 1989; in revised form February 22, 1990.
5 References Kisielow, I?, Bliithmann, H . , Staerz, U., Steinmetz, M. and Von Boehmer, H . , Nature 1988. 333: 742. Goodnow, C. C., Crosbie, J . , Adelstein, S., Lavoie, T. B., Smith-Gill, S. J., Brink, R. A . , Pritchard-Briscoe, H.,Wotherspoon, J. S., Loblay, R. H., Raphael, K., Trent, R. J. and Basten, A., Nature 1988. 334: 676. Nemazee, D. A . and Biirki, K . , Nature 1988. 337: 562. Adams,T. E., Alpert, S. and Hanahan, D., Nature 1987. 325: 223. Arnold, B., Dill, O., Kiiblbeck, G . , Jatsch, L . , Simon, M. M . , Tucker, J. and Hammerling, G. J., Proc. Natl. Acad. Sci. USA 1988. 85: 2269. Holmberg, D. and Coutinho, A . , Immunol. Today 1985. 6: 356.
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7 Payelle-Brogard, B., Ternyck, T., Guilbert, B. and Avrameas, S., Mol. Immunol. 1989. 26: 121. 8 McHyzer-Williams,M. and Nossal, G. J.V., J. Immunol. 1988. 141: 4118. 9 Messing, J., Crea, R. and Seeburg, F! H., Proc. Natl. Acad. Sci. USA 1977. 74:3642. 10 Burglin, T. R. and De Robertis, E., EMBO J. 1987. 6: 2617. 11 Kohler, G., Potash, M. J., Lehrach, H. and Shulman, M. J., EMBO J. 1982. 1: 555. 12 Rammensee, H.-G., Schild, H. and Theopold, U., Immunogenetics 1989. 30: 296. 13 Van Ewijk, W., Ron,Y., Monaco, J., Kappler, J., Marrack, P., LeMeur, M., Gerlinger,l?, Durand, B., Benoist, C. and Mathis, D., Cell 1988. 53: 357. 14 MacDonald, H. R., Schneider, R., Lees, R. K., Howe, R. C., Acha-Orbea, H., Festenstein, H., Zinkernagel, R. M. and Hengartner, H., Nature 1988. 332: 40. 15 Kappler, J.W., Staerz, U.,White, J. and Marrack, €? C., Nature 1988. 332: 35.
Eur. J. Immunol. 1990. 20: 1311-1316 16 Sha, W. C., Nelson, C. A., Newberry, R. D., Kranz, D. M., Russell, J. H. and Loh, D. Y., Nature 1988. 336: 73. 17 Storb, U., Annu. Rev. Immunol. 1987. 5: 151. 18 Lamers, M. C.,Vakil, M., Kearney, J. F., Langhorne, J., Paige, C. J., Julius, M. H., Mossmann, H., Carsetti, R. and Kohler, G., Eur. J. lmmunol.1989. 19: 459. 19 Zamoyska, R., Waldman, H. and Matzinger, P., Eur. J. Immunol. 1989. 19: 111. 20 Bretscher, F! and Cohn, M., Science 1970. 169: 1042. 21 Morahan, G., Allison, J. and Miller, J. F. A. F,! Nature 1989. 339: 622. 22 Miller, J. F. A. F! and Watson, J. D., Scand. J. Immunol. 1988. 28: 389. 23 Tite, J. F! and Janeway, L. A,, Eur. J. Immunol. 1984. 14: 878. 24 Shinohara, N., Watanabe, M., Sachs, D. H. and Hozumi, N., Nature 1988. 336: 481. 25 Lanzavecchia, A., Immunol. Today 1989. 10: 157. 26 Fleischer, B., Nature 1984. 308: 365. 27 Simpson, E., Nature 1988. 336: 426.
Ulrich Theopold and Georges Kohler Max-Planck-Institut fur Immunbiologie, Freiburg
Partial tolerance in p-galactosidase-transgenicmice
1311
Partial tolerance in p-galactosidase-transgenic mice A transgenic mouse line was produced which allowed the expression of E . coli P-galactosidase (P-Gal) under the regulatory elements of the immunoglobulin heavy chain locus. Expression of the transgene is found in spleen and bone marrow. Upon immunization of the transgenic mice with P-Gal, a reduced but clearly detectable antibody response was obtained. Affinity purification with sera from immunized transgenic mice suggests that they contain lower affinity antibodies as compared to normal littermates. Transgenic and nontransgenic mice immunized with bovine serum albumin (BSA) alone or as a mixture with @-Galgave comparable anti-BSA responses. Immunization with a chemically cross-linked (Gal-BSA)-protein, however, showed a 10- to 30-fold difference in the anti-BSA response. Partial unresponsiveness to P-Gal in the transgenic mice is best explained by a dominant, peripheral suppression mechanism linked to the antigen-presenting potential of B cells.
1 Introduction In recent years, transgenic mice have been used as model systems to study the development of tolerance towards the transgenic product (TGP) as a model autoantigen. Some of the hypotheses about tolerance have been supported by these investigations. Clonal deletion of autoreactive T cells in the thymus [l] as well as the inactivation [2] or elimination [3] of autoreactive B cells have been shown t o occur in these model systems. The importance of the time point of expression of theTGP has also been confirmed [4]. Beside these systems, where tolerance towards the TGP was clearly observable, there are other transgenic mouse lines, which show no tolerance towards the TGP For example a transgenic mouse line expressing a soluble form of an MHC class I gene product at a level of 200 ng/ml and early enough during ontogeny to induce tolerance showed no tolerance upon immunization and in MLR [5]. The question arises if the autoantibodies observed in these non-tolerant mice after immunization are comparable with natural autoantibodies, which have also been reported in healthy individuals [6-81. In the present study, a prokaryotic protein ( E . coli P-galactosidase; P-Gal) was expressed as a novel B cell protein in transgenic mice. Since the response to the TGP was only partially reduced in the transgenic mice, this reduction and the residual activity could be studied.
an M13 sequencing vector (m13mp8; [9]) and an expression vector for P-Gal fusion proteins (ptrRBO; [lo]). After manipulations of the 5' and 3' end of the lacZ gene by oligonucleotides which delivered the desired restriction sites for further cloning steps in the correct reading frame, the lac Z gene was introduced into a vector containing the regulatory elements of the Ig heavy chain locus cloned from the hybridoma line Sp6 [ ll] . A Hind 111 fragment containing the exons coding for the transmembrane region of IgM together with the polyadenylation site downstream of those exons was introduced in a last step to produce the plasmid pC5-6 (see Fig. 1). 2.2 Animals
The lacZ gene was introduced into the germ line by injecting the gel-purified large Sma I-Bam HI fragment of plasmid pC5-6 into SJL x C57BL/6 zygotes. For the immunizations, mice from the third and fourth backcross of the founder mouse to C57BL/6 background were used.
2.3 Analysis of the transgenic mice for expression of the TGP
2.1 Construction of the eukaryotic P-Gal expression vector
Mouse organs were homogenized on ice in an extraction buffer (PBS containing 1% NP40 and 5 mM PMSF) using a teflon pestel. The lysates were immediately cleared by centrifugation at 10000 x g for 5 min. After being separated on an SDS-polyacrylamide gel, the fractions of the lysates were analyzed by the Western blot technique using a commercially available anti-@-Gal mAb (Promega Biochtech, Madison, WI).
For the construction of an appropriate expression vector, the lac Z gene was assembled via an internal Aoc I site from
2.4 Cross-linking of BSA to P-Gal
2 Materials and methods
[I 81371 Correspondence: Georges Kohler, Max-Planck-Institute for Immunobiology, Postfach 1169, D-7800 Freiburg, FRG Abbreviations: P-Gal: P-Galactosidase TGP: Transgenic product 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
For cross-linking P-Gal (Boehringer Mannheim, Mannheim, FRG) with BSA, 1 mg of P-Gal was first incubated for 2 h at room temperature in 1 ml PBS containing 2% glutaraldehyde. The P-Gal was separated from glutaraldehyde on an HPLC column (Dupont, Wilmington, DE; GF-250 XL) run with 200mM potassium phosphate, p H 6.5, at 1 mumin. It was then incubated for 2 h at room OO14-2980/YO/0606-1311$02.50/0
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Eur. J. Immunol. 1990.20: 1311-1316
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temperature with 1 mg of BSA in a total volume of 1 ml. The reaction was stopped by adding glycine to a final concentration of 1%. The high-molecular-weight fraction was purified again on an HPLC and concentrated afterwards for immunization. Each mouse was immunized with one tenth of this fraction.This fraction was also analyzed in a Western blot and shown to contain both P-Gal and BSA molecules. The exact ratio of P-Gal to BSA and the total amount of each of the components was not determined. 2.5 Immunization Mice were immunized i.p. with 100 pg of the respective antigen in CFA except for the cross-linked protein (see above). Ten days after each immunization, mice were bled and specific antibodies were detected in an ELISA using the antigen(s) as solid phase reagent, the mAb against P-Gal as a standard and a commerciallyavailable (Southern Biotechnology, Birmingham, AL) mouse x light chain-specific antiserum produced in goat as second-step antibody. Titers of Ag-specific Ab were determined from the standard and were expressed as pg/ml for P-Gal-specific Ab and in U/ml for BSA-specific Ab. The sera used for the affinity purification were obtained in the following way. Mice were back-crossed once to BALB/c and immunized twice with the P-Gal-expressing transfectant C5-6 [ 121. Since only low P-Gal-specific titers were observed, the mice were boosted with 100 pg of E. coli P-Gal in 1FA.Tendays later, sera were collected and tested individually (not shown). Their titers resembled titers found in primary responses to E. coli P-Gal. The sera were pooled and analyzed in detail (see Sect. 2.7 and 2.8). 2.6 Affinity purification of anti-P-Gal Ab The coupling of P-Gal to CNBr-activated Sepharose as well as the purification of the antisera were performed as recommended by the supplier (Pharmacia, Freiburg, FRG). The pooled and 10-fold diluted antisera from transgenic and normal mice were passed three times over two similar columns which were washed with 20 times the EcoRI.Smal.BamH1.S.ll.
PIlI.H8ndlY
volume of the column and eluted with 3.5 M MgC12. The concentration of total Ig and of P-Gal-specific Ab was determined in all fractions in an ELISA technique. 2.7 LD of B cells
A dilution series of spleen cells was incubated for 8 days with 25 pglml of LPS (kindly provided by Dr. C. Galanos, Freiburg, FRG). The number of wells containing P-Gal-specificAb was then determined in an ELISA. From the number of positive wells, the precursor frequency was determined with the help of a computer program (kindly provided by Dr. I. Melchers, Freiburg).
3 Results For the expression of E. coli 6-Gal in transgenic mice, a plasmid was constructed which contains the lac Z gene coding for E. coli P-Gal and sequences coding for the leader exon, including the promoter and enhancer region and the transmembrane region of the Ig p chain (see Fig. 1). The plasmid was tested by transfection into a mutant hybridoma cell line igm 662 [111 as described elsewhere [121. Briefly, the plasmid leads to the expression of a protein of the expected size, a part of which can be detected on the surface of the transfected hybridoma called C5-6. Other plasmids were constructed which lead to the expression of intracellular and secreted versions of @-Gal [12]. The plasmid pC5-6 was used to create transgenic mouse lines by injection into fertilized oocytes. The mouse lines which contained the transgene as assayed by dot blot analysis of tail DNA were examined for the expression of the transgenic product by Western blot analysis and cell staining of spleen cells, LPS-stimulated spleen cells and of hybridomas derived from the mice. One out of four lines of transgenic mice turned out to express the transgene. It contained approximately 10 copies of the transgene and was analyzed in more detail. Fig. 2a, b shows a Western blot analysis of some organs derived from a
8amHI.Sall. PSII,Hlnd Ill
Lac 2
linker BH 1
G418'
Figure I. Construction of the plasmid pC5-6. For details see Sect. 2.1. lac i = lac repressor, lac Po = lac promoter and operator, lac Z = gene for 6-Gal, IgE = enhancer of Ig heavy chains, IgPo=promoter of Ig heavy chain, L = leader of Ig heavy chain, TK = thymidine kinase, p(A) = polyadenylation site, Amp', G418' = genes for resistance to Ampicillin and Neomycin, T K Pr = promoter of the TK gene, C5, C6 = exons coding for p transmembrane region, BHI = BamHI.
Eur. J. Immunol. 1990.20: 1311-1316
Partial tolerance in P-galactosidase-transgenicmice
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Figure 2. Tissue specificity and amount of P-Gal expression by the transgenic mouse line CGa156. Protein (200 pg) of organs from a transgenic (+) and a normal (-) mouse (a, b) were analyzed by PAGE and a Western blot with an anti-P-Gal mAb (see Sect. 2.3).The position of E. coli P-Gal is indicated by the arrow head.The hybrid protein expressed in the transgenic mice is larger due to the presence of the y transmembrane region. (br = brain, liv = liver, ki = kidney, thy = thymus, spl = spleen, spl LPS = spleen cells stimulated for 5 days with LPS, b.m. = bone marrow. A29 = an Abelson-murine-leukemia virus (A-MuLV)-transformed line obtained from the mouse line CGa156. Ay = a control A-MuLV line obtained from an IgM transgenic mouse line). Fig. 2c shows an estimation of the amount of P-Gal expressed in 200 yg protein from the spleen of different individual mice from the line CGa156 and a normal control littermate compared with different amounts of E. coli P-Gal.
transgenic mouse and a non-transgenic littermate. A band of the expected size (121 kDa) is seen in the spleen and in the BM as well as in LPS-stimulated spleen cells of transgenic mice and not in those of the littermate. Fig. 2c shows that the amount of the TGP detected in different individual mice is clearly below 625 pg/200 Fg total protein and was estimated to be 20 ng/spleen.
Table 1. Concentration of anti-P-Gal Ab before and after passage over a P-Gal affinity columna)
Serum before Flow through Affinity-purified purification (cI&hnl) fraction (cl%/ml) (Irg/ml) If.ansgeniC
Transgenic and normal mice were immunized repeatedly with P-Gal from E. coli to assay for their capacity to mount an immune response to the TGF! One of these experiments is shown in Fig. 3a. The transgenic mice (filled circles) produced a significant titer of P-Gal-specific Ab which was, however, 3-10-fold lower than that of non-transgenic littermates (open circles). Transgenic mice immunized with BSA (Fig. 3c) or with a mixture of BSA and P-Gal (Fig. 3b) showed a normal response towards BSA and again a reduced response towards @-Galcompared with normal control littermates. When mice were immunized with P-Gal which was crosslinked to BSA by glutaraldehyde, the response to P-Gal was 5-7-fold lower. Also the response to BSA was between 10and 30-fold lower in transgenic than in non-transgenic littermates (Fig. 3d). Since the amount of BSA present in the P-Gal-BSA complex was not determined, it is not clear, whether the low anti-BSA response in the transgenicmice is due to insufficient stimulation with BSA antigen or due to suppression imposed onto the BSA response by the covalently linked P-Gal molecule (see Sect. 4). The frequency of LPS-reactive P-Gal-specific B cells was determined to be about 11200 for both the transgenic and the littermate control mice. Thus, the bulk of anti-pGal-producing B cells were unaffected by the expression of the transgene. This is surprising in view of the low anti-@-Gal response in the transgenic mice which may, however, be due to the lack of B cells with high-affinity Ig receptors for P-Gal.
Normal
24.7 109.2
11.5 11.0
1.3 (6.5) 58.0 (72.8)
a) For the purified fraction, the amount of total Ig eluted from the column is indicated in parentheses.The amount of total Ig in the serum before purification was similar in transgenic and normal mice (5.7 vs. 4.2 mg/ml).The anti-P-Gal Ab in the flow-through fraction were enriched for IgM and in the purified fraction for IgG as compared to the unpurified serum.
In order to determine the quality of the P-Gal-specificAb in the transgenic mice, pooled sera from transgenic and normal mice were separated on a P-Gal affinity column. As shown inTable 1, two fractions of antibodies which bind to P-Gal in an ELISA were obtained: the first which did not bind to the affinity column and which was present in the same amount in both types of sera and a second one which binds to the affinity column and was present in the sera of normal mice in much higher quantities than in those of transgenic mice.The transgenic mouse lacks around 90% of the high-avidity Ab able to bind to the P-Gal column.
4 Discussion In the present study, mice transgenic for E. coli @-Galwere analyzed for their capability of mounting an immune response towards the TGF! As expected for a construct with the regulatory elements of the p heavy chain locus, expression of the transgene was restricted to B cells in BM
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1
Eur. J. Immunol. 1990. 20: 1311-1316
U. Theopold and G. Kohler
drn'
Ga I
(d) bplm,
-
2 10
0
8
1
10
0
0
0 10
10.1
.. so
-Ipb 20
3O
i:
-
2
3O
Figure 3. Analysis of immune responses. Serum titers of transgenic ( 0 )and normal (0)mice immunized with P-Gal (a), BSA (c), a mixture (b) and a BSA-P-Gal protein, cross-linked by glutaraldehyde (d) are shown. Mice were immunized as described in Sect. 2.6. For the different immunizations the age of the mice was (a) 6-14 weeks, (b) 8-10 weeks, (c) 4 months, (d) 9-12 weeks; 0" = pre-immune serum, 1°,20,30= sera obtained from primary, secondary and tertiary responses.Titers of specific Ab were determined in pg/ml for P-Gal and in U/ml for BSA-specific antibodies as described in Sect. 2.4.
and spleen. In contrast to the hen egg lysozyme-transgenic qualitative difference of the immunizing vs. the transgenic mice, where the TGP was expressed under the control of protein. E. coli f3-Gal, in contrast to the TGP, is not the metallothionein promoter [2], the mice described here glycosylated and lacks the transmembrane region of IgM. show no absolute tolerance. Although being reduced by a This is reminiscent of the transgenic mice expressing a factor of 3 to 10, the antibody response to the TGP is secreted version of an MHC molecule [5], which showed readily detectable. The reduction of the titer of f3-Gal- both a normal humoral and cellular imune response upon specific Ab is more striking among the IgGl Ab than among sensitization with a membrane-bound version of the same the IgM Ab (data not shown). This result is in agreement molecule. In our case, the Ab generated in the transgenic with the finding that autoantibodies are more frequently mouse bind with the same titer difference to E. coli- and found among the IgM class [8].The induction of antigen- myeloma-derived p-Gal and detect the TGP in Western specific Ab upon immunization of (3-Gal-transgenic mice blots (not shown). Therefore autoantibodies have been with E. coli P-Gal might at least in part be due to a generated in the transgenic mouse. We were unable to
Eur. J. Immunol. 1990. 20: 1311-1316
detect surface expression of P-Gal in unstimulated spleen cells. Surface expression has, however, been observed in myeloma cells transfected with the construct [12] and might occur at low levels in the transgenic mouse. As our mice show no signs of an autoimmune disease, even after immunization with P-Gal, they offer the possibility to gain insight into the nature of natural, non-harmful Ab with specificity for autoantigens. The finding that such Ab belong to the repertoire of normal, healthy individuals has for years been a puzzling observation [7, 81. Our findings indicate, that compared to those of normal mice, P-Gal-specific Ab in transgenic mice have a lower affinity for the TGP Low-affinity Ab have also been obtained in hen egg lysozyme-transgenic mice, after their immunization with a carrier hen egg lysozyme complex [2]. There are several possibilities to explain the partial tolerance observed in our system.Tcel1 tolerance in the thymus of p-Gal-transgenic mice could deplete them of /%Galspecific T cell help. According to this hypothesis, early expression and presentation of antigen in the thymus, would lead to the elimination of antigen-specific T cells [ 13-16]. That P-Gal could potentially be expressed in Tcells of the thymus is indicated by previous findings that Ig promoter and enhancer-driven genes are often expressed in thymocytes and also in peripheral Tcells [17, 181. In our transgenic mice, P-Gal was not detectable in the thymus by the Western blot method. The signal obtained from unstimulated spleen cells was weak (Fig. 2). A two- to fourfold reduction of the amount of the TGP in the thymus could easily give rise t o a negative result. In addition, B cells residing in the thymus could present P-Gal to the developingT cell repertoire and delete specific clones. Deletion of P-Gal-specificT cells would then result in partial unresponsiveness to the P-Gal antigen due t o suboptimal T cell help. However, it is difficult to envisage how this mechanism could influence the response to an unrelated antigen such as BSAwhen covalently linked to P-Gal. On the other hand, it cannot be excluded, that the amount of BSA covalently coupled to P-Gal was too low to induce an optimal anti-BSA response. In such a situation, the P-Gal moiety might act as a carrier and help to mount the immune response to BSA in normal mice but would be unable to do so in transgenic mice, where P-Gal-specific help is compromised. For systems where elimination of self-reactive T lymphocytes in the thymus could not explain tolerance, peripheral inactivation or suppression mechanisms have been postulated [19-211. The increase in P-Gal expression resulting from antigenic stimulation (as mimicked by LPS treatment of splenic B cells, see Fig. 2) might raise the amount of P-Gal to a level where such a failsafe mechanism of tolerance induction is required. One possibility, which has received some attention, is the two-signal hypothesis of tolerance induction [20]. T or B cells which recognize antigen and do not receive at the same time a second signal (e.g. the appropriate interleukin) are turned into a nonresponsive (anergic) state. Such a mechanism has been put forward to explain the nonresponsive state of Tcells in transgenic mice expressing alloreactive MHC class I antigens extrathymically on their pancreatic islet cells [21]. It also might explain the anergic state
Partial tolerance in P-galactosidase-transgenicmice
1315
of B cells observed in the hen egg lysozyme-transgenic mouse [2]. Alternatively, in our particular case, co-expression of P-Gal and Ab with specificity for P-Gal in the endoplasmic reticulum could lead to internal antigen-antibody complexes formation and to self-destruction of the B cells, perhaps from a certain affinity threshold on (a mechanism recently hypothesized for T cells [22]). T!., alltlgen crosslinking experiment (see Fig. 3d) argues against deletion or anergy of P-Gal-specific B cells as a mert L pnenomenon, since BSA-specific B cells should not be affected by the expression of P-Gal from the transgene. Another hypothesis according to which peripheral B cell tolerance is explained by a dominant suppression mechanism conveyed by T cells [23-271 is more compatible with our results. BSA-specific B cells will present 6-Gal fragments on MHC class I1 only when P-Gal is covalently linked to BSA. The BSA-specific B cells could then be recognized and suppressed by P-Gal-specificT effector cells of both the CD8 or CD4 phenotype, possibly via cytolytic mechanisms. A dominant suppressive mechanism for the induction of peripheral tolerance is also more suitable to explain the preferential loss of antibodies with high affinity for the autoantigen. If one assumes that the affinity determines the amount of presentation of the antigen and thereby the density of the complexes between antigen-derived peptides and the MHC molecules, high-affinity B cells should attract T cells more easily than lower-affinity B cells. If these B cells are suppressor cells, an elimination of only the high-affinity pool of B cells is imaginable. In contrast, if they are helper T cells, they should lead to an expansion of these B cells. Thus the existence of suppressor cells which recognize self-peptides presented by MHC class I1 molecules may be a failsafe mechanism to avoid the production of high-affinity autoantibodies. The antigen presentation potential of B cells is essential for this kind of regulation of the B cell response. We thank I! Renard, R. Lamers and A . Iglesias for the generation of the transgenic mouse lines, U. Fritzsche for the generation of the Abelson lines, U. Birsner, B. Croetschel and C. Jacot for technical assistance, R. Lamers, G. McMaster, I! Nielsen and M. M . Simon for critically reading the manuscript and for useful discussions, S. Kleinhans for art work and G. Prosch for typing the manuscript.
Received November 27, 1989; in revised form February 22, 1990.
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