MHC class-I transgenic mice.

Rev. Immunol. 1991. 9:297-322 Copyright © 1991 by Annual Reviews Inc. All rights reserved

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MHC CLASS-I TRANSGENIC MICE Bernd Arnold and Gunter J. Hammerling Institute for Immunology and Genetics, German Cancer Research Center, 1m Neuenheimer Feld 280, 6900 Heidelberg, Germany . KEY WORDS:

structure-function correlation of MHC antigens, tissue specific expression, peripheral tolerance, HLA transgenic mice

Abstract The introduction of cloned genes into the germline of mice has been proven to be a powerful tool to investigate the role of the respective gene products within the immune system. Here we summarize the transgenic mouse models that have been established with major histocompatibility complex (MHC) class-I genes. Foreign class-I alleles can be expressed in transgenic mice according to their normal expression patterns as authentic self mol ecules and can function in T-cell responses in the same way as endogenous class-I molecules. Since this is also true for most of the introduced human HLA class-I alleles, there is great interest in establishing mouse models for HLA-linked diseases. A new field of experimental approaches concerning self-tolerance has been opened by tissue specific expression of MHC anti gens under specific promoters. Besides negative selection in the thymus, peripheral mechanisms could be identified that induce and maintain self tolerance. INTRODUCTION

Since T lymphocytes are involved in most antigen-specific immune responses, a fundamental question has always been, how do T cells recog nize antigen and discriminate between self and nonself? Our knowledge about T-cell antigen recognition has advanced tremendously due to cloning of the molecular components participating in this process ( l , 2) and to 297 0732-0582/91/041 0--0297$02.00


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verifying their function by gene transfers into tissue culture. cells (3-6). It is now generally accepted that major histocompatibility complex (MHC) encoded molecules bind and present protein fragments (7, 8) and that this complex is recognized by T cells via their IX, f3 T-cell receptors (TCR) (5, 6). This antigen-specific interaction can, however, only result in a functional consequence if there is support by other adhesion events like the binding of CD4 or CD8 molecules on T cells to class-II or class-I MHC molecules on stimulator/target cells (9, 10). The possibility of introducing genes into the germline of mice (11) has now created the opportunity to study the role of well-characterized single components participating in T-cell antigen recognition and stimulation. In this review we mainly concentrate on MHC c1ass-I transgenic mice. Such mice have been used (a) to study the expression and function of various class-I alleles encoded by different loci; (b) to investigate thymic and extrathymic mechanisms leading to a T-cell repertoire selection, which assures self-tolerance; and (c) to establish mouse models for HLA-linked diseases. EXPRESSION OF CLASS-I GENES UNDER THEIR OWN REGULATORY SEQUENCES

The Transgene Products Function As Transplantation Antigens and Restriction Elements Transgenic mice have been produced with cloned MHC genes from mouse, miniature swine, and human (see Table 1) to find out whether the intro duced gene can be expressed in a way similar to that of the endogenous counterparts or according to the expression in the respective species, and also whether the transgene product can fulfill its function. Expression and tissue distribution of the murine H-2Dd (12, 13) and H-2Kb (14), of the porcine PD l (15, 16), and of human HLA protein (discussed later in detail) showed the expected pattern: high class-I synthesis in lymphoid tissues and moderate in others. Expression was inducible in response to interferon and suppressible by transformation with the human adenovirus 12 (13). Introduction of the murine Tld'-3 gene in transgenic mice led to expression only on thymocytes, as it is known for TL antigens (14). If, however, the T3b coding sequence was expressed under the regulatory elements of H2 Kb, the T3b expression pattern paralleled the one observed for H-2Kb in the H-2Kb transgenic mice (14). In all cases the transgenic animals were tolerant of the transgene product.On the other hand, the transgenic MHC molecules could act as transplantation antigens because skin grafts from the transgenic mice were rejected from normal mice of the same inbred

MHC CLASS-I TRANSGENIC MICE

Table 1

Mouse and miniature swine class I transgenic mice Reference

H-2 gene

Recipient strain

Dd

C57Bl/6

12

Kb

C3H/He

14

CBA/Ca B6.C _H _2bml B6.C_H_2bml

20

Kba 2 Kbm l 5 ml55 Kb

Kbml56


299

l B6.C_H_2bm

20 20

T1aa-3

C3H/He

14

T3b

C3H/He

14

Modified H-2 gene Kd/Kk

C3H/He

19

Db/Q9/Db Kk/Q7/Ld

CBA/Ca

24

CBA/Ca

24

Db/Q9

CBAjCa

Kk/P2m

C57B1/6

Dd/QIO

24 x

DBA/2

C57B1/6

46 48

SLA gene PDI

BlO

15

PD7

BlO

1 27

" A. Mellor, London, personal communication.

strain ( 1 3, 1 5). In addition, the H-2Dd transgenic mice generated antigen specific H-2Dd-restricted cytotoxic T lymphocytes (CTL), a fact indicating that the H-2Dd molecule could also function as a restriction element for antigen recognition ( 1 3). Taken together, class-I proteins can be expressed in transgenic mice according to their normal expression patterns and can function in T-cell responses in the same way as do endogenous class-I molecules. This holds also true for chimeric class-I molecules. It has been shown that replacement of the rx 1 domain of the H-2Kk protein by the rx 1 domain of the H-2Kd allele results in a total abrogation of the function of the hybridantigen as a restriction element for H_2Kd _ and H-2Kk-restricted T cells during virus infection ( 1 7). In contrast, new class-I determinants are formed ( 1 8). Transgenic mice expressing this hybrid antigen can respond to influenza A virus with virus-specific CTL restricted to hybrid class-I determinants not present on either H-2Kk or H-2Kd ( 19). These results raise the additional possibility that the exchange of even larger fragments of DNA between homologous H-2 genes could contribute to the poly morphism among MHC antigens ( 19).

300

ARNOLD & HAMMERLING


Alteration in the T-Cell Repertoire by Introduction of Mutant Class-I Antigens Even the exchange of a single amino acid in class-I molecule can alter the ability of the respective transgenic mouse to mount virus-specific CTL responses (20). The B6.C_H_2hml mutant mouse expresses an H-2Kb mol ecule with three amino acid substitutions in positions 152, 155, and 156 (21). This alteration causes CTL nonresponsiveness to Sendai virus (22). To assess the effect of each amino acid substitution, the H-2Kb gene was mutated at the individual positions and the three resulting mutant genes were introduced into B6.C-H_2hml (20). Immunization of the transgenic mice with Sendai virus led to a virus-specific response in H_2Khm152 mice, which was restricted to either H_2Kbm152 or H-2Kb but did not raise virus specific CTL in H_2Kbm155 transgenic mice. This is very interesting because fibroblasts transfected with the H_2Kbm155 gene and infected with Sendai virus can constitute the target structure recognized by Sendai virus-spe cific, H-2Kb-restricted CTL. In other words, the H_2Kbrn155 molecule could apparently bind and present the relevant Sendai virus peptide (20). These preliminary data suggest that a single amino acid exchange in the H-2Kb protein (position 155 Arg -+ Tyr) can alter the T-cell repertoire, causing CTL nonresponsiveness to Sendai virus. One could speculate that this may be due to differential binding of self-peptides by H-2Kb and H_2Kbm155 molecules in the thymus, which could influence the set of the positively selected T-cell repertoire, as suggested recently (23).

Glycophosphatidyl Inositol-Anchored Class-I Proteins Transmit a Signal for T-Cell Proliferation In another approach to correlate structure and function of class-I proteins, the Qa-2 antigen was studied (24). A number of cell surface proteins are anchored in the cell membrane by glycophosphatidyl inositol (GPI) linkages instead of by hydrophobic protein domains. Treatment of mouse T lymphocytes with antibodies specific for such proteins, such as Thy-I, Ly-6, and Qa-2, could induce proliferation (24-26). To determine whether the GPI-anchor is important for this T cell-activation pathway, transgenic mice were produced that expressed either the normal Qa-2 protein or Qa2 molecules with the membrane spanning domain of H-2Db (24). In both cases the coding sequences were expressed under the H-2Db promoter. Only lymphocytes from transgenic mice carrying GPT-anchored forms of Qa-2 could be stimulated to proliferate by Qa-2 specific antibodies. In addition, T cells of transgenic mice expressing a GPI-anchored form of H2Db could also be activated by anti-H-2Db antibodies. These results point out the importance of the GPI-anchor for this T cell-activation pathway (24).


301


H-2Dd Protein Plays a Role in Hybrid Resistance The transgenic mouse models mentioned so far were established to verify known class-I functions and to study structure function relationships. With the following transgenic system, a new function could be found for class-I proteins. The phenomenon of F I hybrid resistance is defined as the rejection of transplanted hematopoietic tissue or tumors of parental origin by F I hybrids of two inbred strains (27). This resistance has been linked to the MHC locus, but it was still unclear whether H-2 genes or other closely linked genes are responsible for the phenomenon (2S). The earlier mentioned H-2Dd transgenic CS7Blj6 mice, CS (12), were used to analyze the genetic control of host resistance to tumors (29) and bone marrow grafts (30). Tumor growth of the RBL-5 lymphoma (H-2b) was observed in C57BI/6 (H-2b), but could not be seen in (C57Blj6 x BIOD2)F I (H2b x H_2d) nor in DS (H_2b, H-2Dd) mice. H-2Dd positive (CS7Blj6 x DS)F I mice also rejected the RBL-S lymphoma cells. Tumor resistance segregated with H-2Dd in (C57Bl/6 x DS)F I x CS7Bl/6 backcross mice. It could be abrogated by treatment with anti-asialo GM 1 antiserum or with anti-NK 1 .1 monoclonal antibodies, indicating a role for NK cells (29). Similar results were obtained with bone marrow grafts in irradiated mice, showing again a direct involvement of the H-2Dd antigen in F I hybrid resistance (30). Since this NK cell-mediated reaction is relevant in bone marrow transplantations and graft-versus-host disease, this transgenic mouse model may prove to be very valuable in clarifying the conditions under which NK cell-dependent nonresponsiveness is switched off. CELL TYPE AND TISSUE-SPECIFIC EXPRESSION OF MHC ANTIGENS

A new field of experimental approaches concerning self-tolerance has been opened by thc possibility to express genes under foreign regulatory elements that direct the expression of the gene products to certain cell types or tissues in transgenic mice (see Table 2).

Expression in Subsets of Thymic Cells and Their Influence on Selection Processes The repertoire of immunocompetent T lymphocytes capable of dis criminating self and nonself is generated by two selection processes within the thymus (31). Immature thymic lymphocytes, which have randomly rearranged and expressed their T cell-receptor genes, are positively selected for their capacity to bind self-MHC molecules (32-34). This event is

302 Table 2

ARNOLD & HAMMERLING

Transgenic mice expressing class I genes under foreign promoters Promoter/enhancer

Recipient

Reference

Db

Q9

C3H/He

Kb

T3b

C3H/He

14

QIO

Q I O/Ld

C3H/He

65

Rat insulin

Kb Kka

C57BI/6

Immunoglobulin heavy chain

Kb

Metallothionein

Kb

Rat insulin

Metallothionein


Class- I gene

Myelin basic protein Myelin basic protein IX

lactalbumin

Keratin IV Keratin IV CO2

Glial fibrillary acidic protei n

24

x

SJL

C57Bl/6

x

SJL

43

C57Bl/6

x

SJL

66

Kb c hh

C57Bl/6

x

SJL

Kbd Kb Kbe Kbe

CBA/Ca C57Bl/6

x

DBA/2

C57Bl/6

x

DBA/2

C57BI/6

x

DBA/2

C57BI/6

x

DBA/2

Dd b

53

K

K be

Personal communications: aGo Kohler, Freiburg, d A. Mellor, London, 'the authors lahoratory.

hG. Jay,

Rockville, 'J. F. A. P. Miller, Melbourne,

the basis for the finding that mature T cells recognize antigen (peptides) presented only by self-MHC molecules (MHC restriction) (35). T cells with high affinity to self-MHC molecules plus self-peptides are clonally deleted in a second thymic selection process (36-38). By this negative selection, self-tolerance is achieved. Although both processes have been demonstrated, there is still a debate about the view that only epithelial cells in the cortex are responsible for positive selection and that only macrophagesjdendritic cells transmit the signals for negative selection. This problem could be approached by expression of MHC molecules only in defined subsets of thymic cells of transgenic mice. For example, the issue of positive selection has been reinvestigatcd with transgenic mice which expressed the I E class-II molecule either in the cortex or in the medulla or in both thymic compartments, depending on deletions in the lEa promoter region (39). The fate of the anti-IE reactive T cells in these transgenic mice could be followed by anti-Vp6 monoclonal antibodies. According to this analysis, I E molecules had to be expressed on epithelial cells of the thymic cortex to assure effective positive selection (39). Further insight into the role which the different thymic cell types play in the selection processes may be obtained from class-I transgenic models. The H-2Kb gene was introduced in transgenic mice under the immuno globulin heavy-chain enhancer and promoter (40), under the CD2 pro-



303

moter (41), and under the keratin IV promoter (42). Expression was found in the first case in class-II positive stromal cells of the thymic medulla, in 25% of thymic lymphocytes, and in 5-10% of B220 negative cells in spleen and mesenteric lymph nodes (43). In the second case, the H-2Kb protein was expressed on all thymocytes and on almost all peripheral T and B lymphocytes (G. Schonrich, G. 1. Hiimmeriing, B. Arnold, unpublished). The keratin IV promoter, finally, directed expression of H-2Kb to a few epithelial cells in the medulla and to epithelial cells of hair follicles and of the tip of the tongue (B. Arnold, G. Schonrich, F. Momburg, G. 1. Hammeriing, unpublished). T cell-repertoire selection and immune reac tivity toward the H-2Kb antigen of these three types of transgenic mice are presently analyzed.

Expression of Soluble Class-I Proteins Does Not Induce Tolerance To the Membrane Form A prerequisite for tolerance induction by negative selection is the presence of the respective self-antigen in the thymus. How does the immune system establish and maintain tolerance to self-determinants which are only ex pressed outside the thymus? The definition of an extrathymic protein antigen is hampered by the uncertainty as to whether antigenic peptides of such proteins are present in the thymus (44). In fact, it has been suggested that all possible self-peptides could be present in the thymus (45). To address this issue of peripheral tolerance induction experimentally, several groups have established transgenic mice that express a "foreign" MHC allele under a tissue-specific regulatory element outside the thymus. The observed tolerance in these systems can, however, only be assigned to peripheral events as long as the possibility is excluded that such tissue specific MHC proteins could be shed and reach the thymus where these molecules could participate in the selection processes. The question was asked, whether soluble class-I protein can induce tolerance toward the membrane form of the class-I antigen. Transgenic (H-2b x H-2d)F 1 mice were produced which ex{}ressed a modified H-2Kk gene leading to secretion of soluble H-2Kk antigen (200 ng ml- 1 serum) (46). These mice could mount normal CTL and antibody responses against cell surface-bound H-2Kk antigen. Since the soluble H-2Kk protein was expressed in the thymus, this result suggested that the soluble form of a class-I antigen would not act as a tolerogen for CTL precursors directed to the membrane form, possibly due to the lack of cross-linking of T-cell receptors or/and interactions of other cell surface molecules. In contrast to normal litter mates, the transgenic mice failed to generate CTL recognizing soluble H2Kk presented by the H-2Db molecule (47). Thus, the transgenic mice were tolerant for an H-2Kk peptide presented by se\f-MHC protein.


304

ARNOLD

&

HAMMERLING

These data have recently been confirmed in studies of C57B1/6 mice transgenic for the soluble H-2Dd allele (48). Although expression of the transgene product was nearly lOOO -fold higher (0.1 mg ml-1 serum), the animals could still mount a CTL response to membrane H-2Dd and could still reject H-2Dd skin grafts (but with delayed kinetics compared to non transgenic littermates). On the other hand, these transgenic animals did not produce H-2Dd-specific antibodies, suggesting that the lack of B-cell tolerance in the soluble H-2Kk transgenic mice was due to the low expression of soluble H-2Kk. This interpretation is in agreement with data obtained with transgenic mice expressing the antibody 3-83 specific for H2K and D molecules of the k or b haplotypes (49). In the immunoglobulin transgenic mice expressing H-2 molecules of these haplotypes, self-reactive B cells were apparently deleted from peripheral lymphoid tissues (50). Mice expressing the soluble H-2Kk were mated with the 3-83 antibody transgenic animals to see if the soluble H-2Kk could have an effect similar to that of the membrane H-2Kk. However, no sign of B-cell tolerance could be seen in the double transgenic offsprings (D. Nemazee, personal communication). Taking into account the low expression of soluble H2Kk and the low binding constant of 3-83 Fab fragment to H-2Kk bearing cells (7.2 x 106 M-1), one could argue that the receptor occupancy might be too low to permit tolerance. This seems likely because in mice transgenic for an antilysozyme antibody with high affinity (2 x 1 09 M-1) and trans genic for hen egg lysozyme (20 ng ml-1 serum) B-cell tolerance could be observed (51). Antilysozyme B cells appeared to be rendered anergic but were not deleted from peripheral lymphoid tissue. Taken together, soluble class-I protein cannot act as a tolerogen for T cells specific for the intact membrane form. One could argue that soluble class-I protein is not a good correlate to vesicles containing class-I, which may be shed from cells and may fuse to thymic cells, constituting in this way sufficient but not measurable amounts of membrane-associated class I to influence the selection processes. Nevertheless, the results obtained with the previously mentioned IE class-II transgenic mice make such a mechanism unlikely. For instance, expression of I E protein in medullary cells only did not lead to positive selection, indicating that the I E molecules were apparently not exchanged among the different cell subsets in the thymus (39).

Extrathymic Mechanisms Contribute to Self Tolerance: f3 Islet Cell Specific Expression The rat insulin promoter was shown to direct expression of genes exclus ively to the pancreatic-islet f3 cells (except a weak expression in kidneys) (52). This promoter was, therefore, used by several groups to express class-



305

I and class-II alloantigens in transgenic mice, expecting an easy readout system for possible peripheral tolerance mechanisms.In the case of absence of peripheral tolerance, destruction of f3 cells by autoreactive T cells should occur, leading to insulin-dependent diabetes (JDDM). If, however, toler ance could be induced outside the thymus, the transgenic animals should stay healthy. Surprisingly, expression of H-2Kb (53), lAd (54), or I E/, lEad (55) under the rat insulin promoter led in syngeneic transgenic mice to destruction of the f3 cells and IDDM at a very early age. No signs of an autoimmune reaction such as infiltration of mononuclear cells could be seen. Diabetes even developed in transgenic mice depleted of T lympho cytes by neonatal thymectomy, and in transgenic nude mice (53, 55). In only one case (56), in which an IAk cDNA was used, was diabetes not observed. This could be due to the lower level of MHC antigen expression in comparison to the other three systems. Despite the unexpected f3 islet destruction, these three transgenic systems have been very valuable in showing that peripheral tolerance mechanisms exist. Expression of both c1ass-I and class-II al\oantigens on the f3 cells led to nonresponsiveness in vivo. Tolerance could not be overcome by immunization of the transgenic mice with the relevant alloantigen. On the other hand, expression of the class-lor class-II transgene products was high enough to be detected by T cells, because injection of alloantigen specific T cells resulted in a strong T-cell infiltration of the pancreas. In case of the H-2Kb transgenic mice it could be shown that tolerance occurred only as long as the transgene product was present in the mice. Spleen cells of 17 week old transgenic mice could respond normally to H-2Kb. At this age most f3 cells were depleted (57). To understand by which mechanism tolerance is established in these transgenic models, in vitro assays were employed. Stimulation of spleen cells from H-2Kb transgenic, prediabetic mice with irradiated H-2Kb, Dd spleen cells resulted in CTL capable of killing H-2Dd target cells, but nonresponsive to H-2Kb targets. This anti-H-2Kb nonresponsiveness

could, however, be overcome by the addition of recombinant IL-2 (57). This result points to a tolerance induction other than clonal elimination of the specific CTL. Instead, it has been suggested (58) that "helper" cells are eliminated which deliver IL-2 to the CTL, like the class II-restricted, CD4 + helper cells, which in other systems provide help for class I-specific CTL (59). On the other hand, helper tolerance for a class-I antigen can be reversed by helper activity specific for another c1ass-I antigen, if both c1ass

I antigens are present on the same stimulator cell (60). According to these results sufficient help should be present for the anti-H-2Kb CTL precursor by anti-H-2Dd helper cells, because BIO.A(5R) (Kb, Dd) stimulator cells were used. If this interpretation is correct, the observed tolerance may not


306

ARNOLD

&

HAMMERLING

necessarily be due to a defect in the T helper cell compartment; the tol erance could be based on the inability of the CTL to be activated by normal amounts of IL-2. These considerations concern only the previously described in vitro mixed lymphocyte reaction and may not reflect the mechanisms that main tain tolerance in vivo. It has been suggested for a long time that a second signal in addition to antigen may be required for T cell activation, although this signal has not been identified (61). In contrast to macrophages and dendritic cells, fJ cells may not be able to transmit this second signal and may, therefore, induce tolerance (62). This hypothesis is based on experiments with the IE class II transgenic mice (62). In vivo, grafts of IE positive transgenic islets into IE negative naive hosts were not rejected unless the host was primed by an injection of IE positive spleen cells. In vitro, the IE-expressing fJ cells were unable to stimulate T cells reactive to IE plus antigen peptide; they rather induced antigen-specific unresponsiveness. The given interpretation of these results may have to be reconsidered on the basis of very recently published experiments. The rat insulin promoter was also used to express the influenza virus hemagglutinin in fJ islet cells (63). These mice developed IDDM caused by an autoimmune response. Lymphocyte infiltration of the islets containing 70% T cells and a humoral response against fJ-ceIl antigens including hemagglutinin were found. Further analyses of these transgenic systems should clarify why expression of MHC antigens results in tolerance of the specific T cells whereas expression of influenza virus hemagglutinin causes autoimmunity. In summary, expression of MHC proteins on {J islet cells resulted in tolerance to the respective MHC antigens. The specific T cells are still present in the peripheral lymphoid tissues, but they are nonreactive. The mechanisms, however, that lead to this so-called anergy (64) have still to be elucidated.

Liver Cell-Specific Expression In a related set of experiments, transgenic mice were analyzed that ex pressed the class-I antigen only (65) or predominantly (66) in the liver. The QlO class-I protein is expressed only in the liver as a secreted protein. Secretion is a result of a mutation in the exon coding for the trans membrane region, leading to several polar and charged amino acids. Replacement of the exons coding for the transmembrane and cytoplasmic portion by the equivalent coding region of the H-2Ld gene gave rise to a membrane-associated QI 0 protein (67). Introduction of this gene construct into mice led to liver-specific expression of the hybrid antigen (65). In the other transgenic system the sheep metallothionein promoter was used to



307

direct expression of the H-2Kb gene to the liver (66). Administration of zinc enhanced H-2Kb expression in the liver and also in kidney and exocrine pancreas. In both types of transgenic mice, tolerance was observed in vivo, but CTL specific to the relevant antigen could be obtained in vitro. This again points to a tolerance mechanism other than clonal deletion. Recon stitution of sublethally irradiated metallothionein/H-2Kb transgenic mice with T cell-depleted, syngeneic bone marrow cells resulted in tolerance to H-2Kb. In contrast, reconstitution with spleen cells led to a graft-versus host disease (GVHD) in the animals. GVHD lesions could only be detected in organs expressing the transgene product (liver, kidney, pancreas), while other organs like thymus and spleen were histologically normal (66). Thus, in contrast to the previously discussed {3 islet cells expressing IE, liver cells expressing H-2Kb can stimulate mature T cells, suggesting a different mechanism for the observed tolerance. Another interesting aspect of this system is the finding that the signs of GVHD progressively diminished with increasing time after reconstitution. This suggests that if a graft can survive the initial onslaught of an immune response, tolerance mechanisms may ensure its survival (66).

Downregulation of T Cell Receptor and CD8 on Self-reactive T Cells Contributes to Peripheral Tolerance The glial fibrillary acidic protein (GFAP) promoter (generously provided by N. Cowan, New York) was taken to restrict expression of the H-2Kb molecule to astrocytes in (C57Bl/6 x DBAj2)F I mice (unpublished data of the author's laboratory). Expression of the transgene was verified on the mRNA level by cDNA synthesis and PCR analysis using allele-specific oligonucleotides and on the protein level by immunohistology. H-2Kb expression was also found on Schwann cells in the intestine as well as on brain cells like astrocytes, ependymal cells, and some epithelial cells of choroid plexus. The transgenic mice were tolerant in vivo to the transgene product, as judged by H-2Kb skin graft and tumor cell acceptance. Thus, this is another example that expression of an alloantigen on very few cells in a certain tissue causes nonresponsiveness to this antigen. To determine the basis of the observed tolerance, T-cell receptor (TCR) transgenic mice were developed with anti-H-2Kb specificity. The a, {3, TCR genes had been isolated from the CTL clone KB5.C20 of B20.BR (H-2k) origin (68), specific for the H-2Kb alIaantigen (10). The KB5.C20 TCR can be identified by the monoclonal, anticlonotypic antibody Desire-l (69). With this antibody the fate of a monoclonal H-2Kb-specific T-cell population could be followed in mice double transgenic for GFAP-H2Kb and KB5.C20 TCR. In some of the systems for peripheral tolerance discussed so far (53, 65, 66), T cells with the respective specificity were still


30S

ARNOLD & HAMMERLING

present due to their in vitro responsiveness. The processes occurring in the animals, however, could not be studied. In the IE class-II transgenic mice, I E reactive T cells could be directly demonstrated by anti-Vp17a antibodies (64). In all cases, however, a contribution ofT-cell deletion to the observed tolerance could not be excluded, because the studied T-cell responses were polyclonal. T cells with high affinity to liver or pancreatic peptides in the context of the chosen MHC molecules could have been deleted, whereas the responses obtained were based on T cells reactive to spleen peptides in context of the MHC proteins. T-cell reactivity to tissue-specific peptides presented by MHC antigens has been reported (70). The fact that the (GFA P-H-2Kb x KB5.C20 TCR) F 1 mice were tolerant to H-2Kb indicates that the KB5.C20 TCR does not recognize a tissue-specific peptide in context with H-2Kb. Three-color fiow-cytometric analysis of thymocytes allowed the identi fication of clonotype+, CDS+, CD4 - T cells. This cell population was deleted in H_2kxb TCR transgenic mice. No alteration was found, however, in double transgenic H_2kxd in comparison to single TCR transgenic H2kxd mice (Figure l a). It can, therefore, be concluded, that no negative selection occurred in the thymus of (GFAP-H-2Kb x TCR) mice. In spleens and lymph nodes of double transgenic animals, however, the number of clonotype+, CDS+ T cells was strongly reduced in comparison to the numbers found in TCR mice (Figure Ib). On the other hand the number of clonotype+, CD4+ T cells was only slightly diminished. Stimulation of spleen cells of double transgenic mice with irradiated C57Bl/6 spleen cells in vitro led to a H-2Kb-specific CTL response, which was inhibitable by anticlonotypic antibodies. Two interpretations of these results are appar ent. Either the majority of clonotype+, CDS+ T cells were deleted, and the remaining cells could be activated in vitro, or CDS and TCR molecules on the T cells were downregulated in the animal and upregulated in vitro, leading to reactive CTL. To distinguish these two possibilities, splenic T cells of double transgenic animals were stained with anticlonotypic antibodies and separated in clonotype+ and clonotype- T cells, by means of a fluorescence activated cell sorter. Both populations were then stimu lated in vitro with C57Bl/6 spleen cells and tested 6 days later for clonotype expression and anti-H-2Kb CTL activity. In both cases clonotype+ cells were obtained, which could kill H-2Kb target cells in a clonotype-depen dent way. These results indicate that downregulation of CDS and TCR molecules on the antigen-specific T cells is one mechanism by which per ipheral tolerance to this antigen can be established. Taken together, these transgenic mice systems have proven to be valu able models to approach the various mechanisms leading to self-tolerance. One could expect that new transgenic models with other promoter clements


CONTROL A,

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_....:..:.. H --= _ 2=.-

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d ---.:. H_ ..: _ ..::;2�

309

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Tolerance and MHC restriction in transgenic mice expressing a MHC class I gene in erythroid cells.

DR1) transgenic mice for vaccine evaluation.

Inhibition of class II MHC gene expression by anti-sense RNA in transgenic mice.

Of mice and the MHC.

Cryopreservation of transgenic mice.

Retinoblastoma in transgenic mice.

Ocular inflammation in HLA-B27 transgenic mice reveals a potential role for MHC class I in corneal immune privilege.

Mice lacking MHC class II molecules.

Transgenic mice: 'new wave' immunogenetics.

Malignant melanoma in transgenic mice.

Reverse genetics using transgenic mice.

Transgenic mice susceptible to poliovirus.

Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice.

Skeletal defects in Osterix-Cre transgenic mice.

Transgenic oncogene mice. Tumor phenotype predicts genotype.

Human sickle hemoglobin in transgenic mice.

Melanosis and associated tumors in transgenic mice.

Eosinophilia in transgenic mice expressing interleukin 5.

abl transgenic mice.

Partial tolerance in beta-galactosidase-transgenic mice.

The Hox gene family in transgenic mice.

Transgenic mice and age-related mutations.

Transgenic mice. Gene expression and hair-loss.

Transgenic mice as immune system models.