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Degeneracy in T-cell antigen recognition – implications for the pathogenesis of autoimmune diseases
Journal of Neuroimmunology 107 (2000) 148–153 www.elsevier.com / locate / jneuroin
Degeneracy in T-cell antigen recognition – implications for the pathogenesis of autoimmune diseases Bernhard Hemmer*, Marc Jacobsen, Norbert Sommer Clinical Neuroimmunology Group, Department of Neurology, Philipps-University, Rudolf-Bultmann-Str. 8, 35033 Marburg, Germany
Abstract T-cells recognize by their T-cell receptor (TCR) short peptides presented by major histocompatibility complex (MHC) molecules. Based on functional and structural data, it has become widely accepted that this interaction is highly flexible thus allowing a specific TCR to interact with a broad range of different peptide ligands. Although cross-reactivity is essential for selection and maintenance of the T-cell repertoire, it also carries the danger of inducing autoreactivity following protective immune responses. This hypothesis has been supported by a large number of findings in vitro and in vivo experimental systems. Here we discuss recent findings on cross-recognition of T-cells and provide a new experimental approach to address specificity and cross-reactivity in autoimmune disorders. 2000 Elsevier Science B.V. All rights reserved. Keywords: T-cells; Combinatorial peptide libraries; T-cell receptor; Autoimmunity; Antigen recognition
1. The physiological need for flexibility in T-cell antigen recognition Most T-cell receptors (TCRs) are composed of a and b chains which are generated by a somatic rearrangement of germline variable, diversity, and junctional genes. This process leads to a highly diverse receptor repertoire with a broad range of specificites. The TCR interacts with a complex of a peptide and the major histocompatibility complex (MHC) molecule. The interaction is essential for maturation and selection of T-cells in the thymus, for maintenance of the T-cell repertoire, and for induction of protective immune responses. The wide range of functional activities that are induced after engagement of the TCR complex is puzzling. Functional, biochemical, and structural studies on the interaction of the TCR with its ligands have, however, contributed to the understanding of the paradigm. The first crystal structures of TCRs complexed with MHC–peptide molecules have shown that the MHC helices dominate the TCR MHC–peptide interface (reviewed in Garcia et al., 1999). This suggests that the TCR-binding energy must be primarily directed against the
*Corresponding author. Tel.: 149-6421-28-66419; fax: 149-6421-2866419. E-mail address: [email protected] (B. Hemmer).
most conserved features of the MHC molecule. In addition, the poor complementarity between peptide and TCR indicates the TCR’s ability to adapt to different bound ligands. Both observations suggest that the TCR is designed for recognizing many ligands. Few of them will allow an optimal fit of the TCR to its ligand but many will only contribute little to the preexisting binding energy between the MHC and the TCR (Garcia et al., 1999). Before the molecular structure of the trimolecular complex was resolved a variety of functional studies using in vitro assays with T-cell clones (TCC) or TCR–transgenic animal models had demonstrated flexibility in the interaction of the TCR with the MHC–peptide ligand. Experiments with peptide analogues proved that peptide antigens sharing only one amino acid or even no amino acid in corresponding positions were recognized by individual TCRs (Evavold et al., 1995; Hemmer et al., 1998a,b). Further studies employing a novel technique called soluble combinatorial peptide libraries in the positional scanning format (PS-SCL) showed an even higher degree of flexibility in TCR antigen recognition. Starting from peptide mixtures with only one defined amino acid, TCR motifs were determined and stimulatory peptide ligands identified. These studies revealed that a single TCR can recognize a broad spectrum of different ligands that includes peptides with quite distinct sequences (Hemmer et al., 1997). In contrast to the initial view of specific TCR recognition of single or few ligands, these experiments
0165-5728 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 00 )00226-5
B. Hemmer et al. / Journal of Neuroimmunology 107 (2000) 148 – 153
showed that some TCC respond to mixtures of 10 12 peptides containing each peptide at a concentration of 0.00258 fM. The response to those mixtures demonstrated that T-cells recognize thousands to millions of different peptides. Based on these and other experimental findings Mason (1998) proposed a mathematical model for degeneracy in T-cell antigen recognition. He calculated that each individual T-cell recognizes 10 6 –10 7 nonapeptides or 10 8 unadecapeptides. Although a high number of ligands are recognized by a single TCR they may differ over a wide range with respect to their stimulatory potency. Accordingly, only a few ligands will be able to activate a naive T-cell under physiological conditions, whereas many of those ligands will be potent enough to provide a signal of sufficient strength to support selection in the thymus and survival of T-cells in the periphery (Fig. 1). This view fits with the known properties of antigen presentation where most MHC molecules will be occupied by a repertoire of self peptides (Hunt et al., 1992). Those MHC–peptide complexes are probably responsible for positive selection in the thymus by providing weak TCR stimuli to immature T-cells (Robey and Fowlke, 1994). Similarly, the recognition of self MHC–peptide complexes mediates a survival signal to peripheral T-cells without reaching the threshold for induction of effector T-cell functions (Brocker, 1997; Kirberg et al., 1997; Tanchot et
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al., 1997). The replacement of part of the self-peptide pool by non-self / foreign peptides will result in significant affinity changes for a few T-cells within the repertoire. Those T-cells will become activated by as few as 10–100 high affinity ligands per antigen presenting cell (Demotz et al., 1990; Sykulev et al., 1996). Following activation they will mediate effector functions and expand to large numbers. After removal of the invading non-self peptide from the MHC–peptide repertoire, those T-cells will return to the survival state and receive their low affinity TCR signal by the pool of self peptides. Due to quantitative and qualitative receptor changes those T-cells will have a much lower threshold for reactivation. As a consequence, the T-cells will respond vigorously upon re-exposure to their nominal ligand, i.e., a foreign antigen, but may also be prone to cross-reactivity with self antigens (Fig. 1). The two-step model for T-cell survival and T-cell activation is probably reflected by different signaling events. It is tempting to speculate that the incomplete TCR-associated signaling events (partial phosphorylation of TCR-z chain and no ZAP-70 phosphorylation) initially observed with partial agonists and antagonists (peptides that activate T-cell functions only partially or antagonize T-cell activation) are equivalent to a survival event, whereas the full signaling pattern (full phosphorylation of ZAP-70 and TCR-z) corresponds to physiological activation of the T-cell (Germain and Stefanova, 1999; Fig. 1).
Fig. 1. Outline of the hypothetical two-step model for T-cell survival, maturation, and activation. During thymic maturation positive selection is mediated by the mixture of self MHC–peptide complexes through weak stimulation of the T-cells. This process may involve a partial TCR signal. TCR with high affinity TCRs for single self MHC–peptide complexes within the mixture will become fully activated and undergo negative selection as a consequence of a full TCR signal. After maturation, T-cells will now receive a weak stimulus through the TCR by the self-MHC–peptide complexes. As a consequence of changes in the signaling machinery, full activation of the T-cell will now lead to expansion instead of deletion.
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B. Hemmer et al. / Journal of Neuroimmunology 107 (2000) 148 – 153
2. The pathological consequences of degenerate T-cell antigen recognition T-cell responses are crucial for the defense against microbial antigens as well as tumor cells. However, under certain conditions protective T-cell responses may result in autoimmune disease. Two not mutually exclusive models for the development of autoimmune responses exists that are based on either ‘molecular mimicry’ or ‘epitope spreading’. The latter model hypothesizes that the release of self-antigens after inflammatory organ diseases starts a self perpetuating cascade of autoimmune responses (Lehmann et al., 1992). The repeated priming of autoreactive T-cells eventually leads to a chronic organ specific autoimmune disease involving more and more self-epitopes. Some evidence from experimental autoimmune models supports this hypothesis (Lehmann et al., 1992), although the role of this mechanism in human autoimmune disorders remains to be established. In addition this model does not explain the ‘first hit’ that starts the cascade of events. The second concept for the development of autoimmune disorders that may explain the initial event is based on the mechanism of cross-reactivity and termed ‘molecular mimicry’. It has been shown that infectious agents can activate T- and B-cells that cross-react with self antigens and initiate autoimmune responses. The idea was established in animal models many years ago, when Fujinami and Oldstone (1985) demonstrated in rabbits the induction of autoimmunity after immunization with a hepatitis B virus (HBV) antigen. The HBV peptide used in those experiments had sequence identity in six positions with the myelin basic protein (MBP) peptide (66–75). Immunization with the peptide induced an antibody response to MBP and in some animals an inflammatory disease of the brain. For many years it was believed that sequence identity is a necessity for cross-recognition of T-cells. Accordingly, the search for cross-reactive ligands involved screening of protein databases for non-self peptides with sequence identity to self antigens. However, the emerging knowledge about the flexibility of the TCR and its degenerate recognition have changed this view (Kersh and Allen, 1996; Wucherpfennig and Strominger, 1995; Hemmer et al., 1998a,b). Studies by Allen and coworkers and other groups demonstrated how little sequence homology is necessary for cross-recognition of T-cells. Careful studies of the effect on single amino acid modifications led to the definition of MHC binding motifs and the identification of tolerated amino acids in TCR contact positions. As a result Allen and coworkers described for a hemoglobin-specific TCC a cross-reactive peptide that had only one amino acid in common with the original TCR ligand (Evavold et al., 1995). Wucherpfennig and Strominger (1995) used a similar approach in combination with a motif based database search and identified for two myelin basic protein
(MBP)-specific TCC stimulatory microbial peptide antigens with limited sequence homology. The peptides were derived from Epstein-Barr virus DNA polymerase and Herpes simplex virus terminase proteins. Both peptides were less potent than the MBP peptide. An additional finding for the understanding of antigen recognition of autoreactive T-cells was provided by extensive epitope mapping of TCC specific for MBP(83–99). In those studies it was demonstrated that autoreactive CD41 T-cell clones could recognize peptide analogues even better than the self-antigen used to establish the TCC. This indicated that the autoantigen was a suboptimal ligand for those T-cells. Although the peptide analogue approach was successful in identifying cross-reactive ligands it had some drawbacks. The approach is only applicable to TCC with known peptide specificity. In addition, for each epitope a large number of different peptide analogues is necessary to dissect recognition and determine TCR motifs. Both limitations established the need for more efficient methods to dissect T-cell recognition. Based on the observation that each single amino acid within the peptide sequence contributes largely independently to the interaction of the TCR with its MHC–peptide ligand (Hemmer et al., 1998a,b), soluble combinatorial peptide libraries were introduced to determine antigen recognition of T-cells. The PS-SCL approach to examine TCR recognition offered significant advantages over previously used procedures. The method was introduced to determine antigen recognition of antigen-specific and alloreactive CD8- and CD4- positive T-cell clones. (Udaka et al., 1995; Gundlach et al., 1996; Hemmer et al., 1997; Hiemstra et al., 1998). Cross-reactive ligands for autoreactive T-cells were easily determined by using this approach. For several human MBP-specific TCC cross-reactive antigens from a variety of microbial and self-peptides were identified. Those involved peptides derived from the human Cytomegalovirus and Salmonella typhimurium proteins (Hemmer et al., 1997). In addition, these studies determined another feature of autoreactive T-cells that may change our view of crossrecognition and molecular mimicry. In contrast to TCC with non-self specificities (R. Kubota, B. Hemmer, R. Martin, S. Jacobson, unpublished) all autoreactive TCC examined so far recognized the autoantigen rather inefficiently (Fig. 2). The PS-SCL and the peptide analogue approach identified for those TCC peptide ligands that elicited much stronger responses than the autoantigen that was used to establish the TCC (Hemmer et al., 1997; Vergelli et al., 1997; Hemmer et al., unpublished). Some of those peptides that were termed superagonist ligands were deduced from microbial antigens. These results suggest that autoreactive cells do not recognize the autoantigen with optimal fit. It is tempting to speculate that autoreactive T-cells, although present in the peripheral blood of patients with autoimmune diseases, were initially activated
B. Hemmer et al. / Journal of Neuroimmunology 107 (2000) 148 – 153
Fig. 2. Differences in antigen recognition between T-cells that recognize self-peptides versus those that recognize non-self peptides. TCC that were in vivo selected by immunodominant antigens showed a much better fit with the PS-SCL TCR motif than TCCs generated with MBP.
and expanded in vivo by cross-reactive ligands. Candidate antigens are peptides from the pool of microbial proteins.
3. The use of combinatorial peptide libraries to identify relevant antigen epitopes in chronic Lyme disease Although T-cell recognition is highly degenerate, it became clear from those studies that PS-SCL are promising tools to identify optimal ligands for a given TCR. This notion was supported by preliminary results with virusspecific TCCs. In contrast to autoreactive T-cells, TCCs generated with immunodominant peptides from patients after influenza virus or HTLV-I infection (Y. Kubota, B. Hemmer, R. Martin, S. Jacobsen, personal communication) seem to recognize the antigen with a better fit. The TCR motifs established by the PS-SCL approach contained the entire peptide sequence. A database search with the motif would have disclosed the viral antigens as likely candidate antigens. These results indicate that the PS-SCL approach can identify for a given T-cell peptide antigens that are likely to have expanded the cells in vivo. To support this hypothesis of T-cell responses in chronic Lyme diesease, an inflammatory organ disorder following infection with the spirochete Borrelia burgdorferi (B.b.) were investigated. B.b. may spread to different organism including skin, joints, or nervous system in the early stages of the bacteremia. Sometimes the inflammation persists, although the bacterium may no longer be present in the organ compartment. This suggests that an immune response initiated by the bacterium can persist in the absence of the original antigen. One likely mechanism is crossrecognition of B.b.-specific T-cells with self antigens in the
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organ compartment. To follow this hypothesis T-cells were isolated from the cerebrospinal fluid (CSF) of a patient who developed chronic encephalomyelitis after B.b. infection (Hemmer et al., 1999). CSF cells were analyzed by flow cytometry and RT-PCR for the presence of expanded T-cell populations by their TCR-Vb chain expression in comparison to peripheral blood T-cells. In addition, single stranded conformational polymorphism analysis of TCRVb chains was performed to identify T-cell clonotypes. Using both methods, expansion of several TCR-Vb chains was identified. In parallel, TCC were established with whole Borrelia lysate from CSF T-cells. The TCC were then analyzed for their function and TCR-Vb expression. One in vivo expanded T-cell clonotype expressed the TCR of a cultured TCC. Further studies focused on this TCC. The clone was tested for its response to a decapeptide PS-SCL. A highly discriminative response to the peptide library was observed that allowed a detailed analysis for peptide specificity of the TCC. Using the TCR motif of the TCC protein, databases were searched to identify potential peptide ligands for the TCC. The peptides with the highest potency were derived from B.b. itself, whereas a large number of self-peptides or peptides derived from other microbes with lower potency were identified (Hemmer et al., 1999). Interestingly one peptide was derived from a myelin protein. These results demonstrate for the first time that peptide libraries can identify high and low affinity ligands for T-cells with unknown peptide specificity (Fig. 3). It can be hypothesized that the in vivo expanded TCC was activated in the first instance by the identified Borrelia antigens in the lymphatic system or even the brain. After clearing the CNS from the microbe, other antigens might have continued to stimulate the TCC. Likely candidates are some of the self-peptides that were discovered by the PS-SCL approach. This finding, although only established with one TCC supports the concept of molecular mimicry as a potential pathogenic mechanism in the development of autoimmune responses after infectious diseases.
4. Perspectives for the study of T-cell responses in human diseases These results demonstrate that T-cell responses in a patient with chronic Neuroborreliosis allow disease related antigens to be discovered by the specificity of the organ infiltrating lymphocytes. Since T-cells play a significant role in most inflammatory and neoplastic diseases, we believe that such an approach could help to identify relevant antigens in those disorders by determining antigen specificity of expanded T-cell populations. The technique may replace large scale peptide synthesis or protein expression experiments that have been performed to discover candidate antigens in those disorders. After identification of dominant T-cell responses the approach
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the clinical relevance of this mechanism in the pathogenesis of autoimmune diseases such as multiple sclerosis.
Acknowledgements The work was supported by the Deutsche Forschungsgemeinschaft (He 2368 / 2-1). Bernhard Hemmer was in ¨ part sponsored by the Gemeinnutzige Hertie-Stiftung.
References
Fig. 3. Schematic protocol for defining potential ligands for a T-cell clone with unkown specificity.
may also help to design highly specific immunotherapies based on those antigenic determinants.
5. Conclusions Recent studies on T-cell antigen recognition have shown that a single TCR can recognize many thousands of different peptides, demonstrating the flexibility of the cellular immune system in recognizing a broad and variable spectrum of antigens. While this flexibility is fundamental for efficient host defense, however, it imposes constant danger to the host by generating potentially autoaggressive immune reactivity. Degeneracy in T-cell recognition is therefore a prerequisite for the initiation of autoimmunity, supporting and extending the concept of ‘molecular mimicry’. Further studies will have to clarify
Brocker, T., 1997. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells. J. Exp. Med. 18, 1223–1232. Demotz, S., Grey, H.M.S., Sette, A., 1990. The minimal number of class II MHC–antigen complexes needed for T-cell activation. Science 249, 1028–1030. Evavold, B.D., Sloan-Lancaster, J., Wilson, K.J., Rothbard, J.B., Allen, P.M., 1995. Specific T-cell recognition of minimally homologous peptides: evidence for endogenous ligands. Immunity 2, 655–663. Fujinami, R.S., Oldstone, M.B.A., 1985. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science 230, 1043–1045. Garcia, K.C., Teyton, L., Wilson, I.A., 1999. Structural basis of T-cell recognition. Annu. Rev. Immunol. 17, 369–397. Germain, R.N., Stefanova, I., 1999. The dynamics of T-cell receptor signaling: complex orchestration and the key roles of tempo and cooperation. Annu. Rev. Immunol. 17, 467–522. ¨ Gundlach, B.R., Wiesmuller, K.-H., Junt, T., Kienle, S., Jung, G., Walden, P., 1996. Specificity and degeneracy of minor histocompatibility antigen-specific MHC-restricted CTL. J. Immunol. 156, 3645–3651. Hemmer, B., Fleckenstein, B.T., Vergelli, M., Jung, G., McFarland, H., Martin, R., Wiesmuller, K.H., 1997. Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T-cell clone. J. Exp. Med. 185, 1651–1659. Hemmer, B., Vergelli, M., Gran, B., Ling, N., Conlon, P., Pinilla, C., Houghten, R., McFarland, H.F., Martin, R., 1998a. Cutting edge: predictable TCR antigen recognition based on peptide scans leads to the identification of agonist ligands with no sequence homology. J. Immunol. 160, 3631–3636. Hemmer, B., Vergelli, M., Pinilla, C., Houghten, R., Martin, R., 1998b. Probing degeneracy in T-cell recognition using peptide combinatorial libraries. Immunol. Today 19, 163–168. Hemmer, B., Gran, B., Zhao, Y., Marques, A., Pinilla, C., Pascal, J., Tzou, A., Kondo, T., Cortese, I., Bielekova, B., Straus, S., McFarland, H.F., Houghten, R., Simon, R., Martin, R., 1999. Identification of candidate T-Cell epitopes and molecular mimics in chronic Lyme disease. Nature Med. 5, 1375–1382. Hiemstra, H.S., van Veelen, P.A., Schloot, N.C., Geluk, A., van Meijgaarden, K.E., Willemen, S.J., Leunissen, J.A., Benckhuijsen, W.E., Amons, R., de Vries, R.R., Roep, B.O., Ottenhoff, T.H., Drijfhout, J.W., 1998. Definition of natural T-cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries. J. Immunol. 161, 4078–4082. Hunt, D.F., Henderson, R.A., Sharanowitz, J., Sakaguchi, K., Michel, H., Sevilir, N., Cox, A.L., Appella, E., Engelhard, V.H., 1992. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255, 1261–1263. Kersh, G.J., Allen, P.M., 1996. Essential flexibility in the T-cell recognition of antigen. Nature 380, 495–498. Kirberg, J., Berns, A., von Boehmer, H., 1997. Peripheral T-cell survival
B. Hemmer et al. / Journal of Neuroimmunology 107 (2000) 148 – 153 requires continual ligation of the T-cell receptor to major histocompatibility complex-encoded molecules. J. Exp. Med. 186, 1269–1275. Lehmann, P.V., Forsthuber, T., Miller, A., Sercarz, E.E., 1992. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 358, 155–157. Mason, D., 1998. A very high level of cross-reactivity is an essential feature of the T-cell receptor. Immunol. Today 19, 395–404. Robey, E., Fowlke, B.J., 1994. Selective events in T-cell development. Annu. Rev. Immunol. 12, 675–705. Sykulev, Y., Joo, M., Vturina, I., Tsomides, T.J., Eisen, H.N., 1996. Evidence that a single peptide–MHC complex on a target cell can elicit a cytolytic T-cell response. Immunity 4, 565–571. Tanchot, C., Lemonnier, F.A., Perarnau, B., Freitas, A.A., Rocha, B., 1997. Differential requirements for survival and proliferation of CD8 naive or memory T-cells. Science 276, 2057–2062.
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Degeneracy in T-cell antigen recognition – implications for the pathogenesis of autoimmune diseases Bernhard Hemmer*, Marc Jacobsen, Norbert Sommer Clinical Neuroimmunology Group, Department of Neurology, Philipps-University, Rudolf-Bultmann-Str. 8, 35033 Marburg, Germany
Abstract T-cells recognize by their T-cell receptor (TCR) short peptides presented by major histocompatibility complex (MHC) molecules. Based on functional and structural data, it has become widely accepted that this interaction is highly flexible thus allowing a specific TCR to interact with a broad range of different peptide ligands. Although cross-reactivity is essential for selection and maintenance of the T-cell repertoire, it also carries the danger of inducing autoreactivity following protective immune responses. This hypothesis has been supported by a large number of findings in vitro and in vivo experimental systems. Here we discuss recent findings on cross-recognition of T-cells and provide a new experimental approach to address specificity and cross-reactivity in autoimmune disorders. 2000 Elsevier Science B.V. All rights reserved. Keywords: T-cells; Combinatorial peptide libraries; T-cell receptor; Autoimmunity; Antigen recognition
1. The physiological need for flexibility in T-cell antigen recognition Most T-cell receptors (TCRs) are composed of a and b chains which are generated by a somatic rearrangement of germline variable, diversity, and junctional genes. This process leads to a highly diverse receptor repertoire with a broad range of specificites. The TCR interacts with a complex of a peptide and the major histocompatibility complex (MHC) molecule. The interaction is essential for maturation and selection of T-cells in the thymus, for maintenance of the T-cell repertoire, and for induction of protective immune responses. The wide range of functional activities that are induced after engagement of the TCR complex is puzzling. Functional, biochemical, and structural studies on the interaction of the TCR with its ligands have, however, contributed to the understanding of the paradigm. The first crystal structures of TCRs complexed with MHC–peptide molecules have shown that the MHC helices dominate the TCR MHC–peptide interface (reviewed in Garcia et al., 1999). This suggests that the TCR-binding energy must be primarily directed against the
*Corresponding author. Tel.: 149-6421-28-66419; fax: 149-6421-2866419. E-mail address: [email protected] (B. Hemmer).
most conserved features of the MHC molecule. In addition, the poor complementarity between peptide and TCR indicates the TCR’s ability to adapt to different bound ligands. Both observations suggest that the TCR is designed for recognizing many ligands. Few of them will allow an optimal fit of the TCR to its ligand but many will only contribute little to the preexisting binding energy between the MHC and the TCR (Garcia et al., 1999). Before the molecular structure of the trimolecular complex was resolved a variety of functional studies using in vitro assays with T-cell clones (TCC) or TCR–transgenic animal models had demonstrated flexibility in the interaction of the TCR with the MHC–peptide ligand. Experiments with peptide analogues proved that peptide antigens sharing only one amino acid or even no amino acid in corresponding positions were recognized by individual TCRs (Evavold et al., 1995; Hemmer et al., 1998a,b). Further studies employing a novel technique called soluble combinatorial peptide libraries in the positional scanning format (PS-SCL) showed an even higher degree of flexibility in TCR antigen recognition. Starting from peptide mixtures with only one defined amino acid, TCR motifs were determined and stimulatory peptide ligands identified. These studies revealed that a single TCR can recognize a broad spectrum of different ligands that includes peptides with quite distinct sequences (Hemmer et al., 1997). In contrast to the initial view of specific TCR recognition of single or few ligands, these experiments
0165-5728 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 00 )00226-5
B. Hemmer et al. / Journal of Neuroimmunology 107 (2000) 148 – 153
showed that some TCC respond to mixtures of 10 12 peptides containing each peptide at a concentration of 0.00258 fM. The response to those mixtures demonstrated that T-cells recognize thousands to millions of different peptides. Based on these and other experimental findings Mason (1998) proposed a mathematical model for degeneracy in T-cell antigen recognition. He calculated that each individual T-cell recognizes 10 6 –10 7 nonapeptides or 10 8 unadecapeptides. Although a high number of ligands are recognized by a single TCR they may differ over a wide range with respect to their stimulatory potency. Accordingly, only a few ligands will be able to activate a naive T-cell under physiological conditions, whereas many of those ligands will be potent enough to provide a signal of sufficient strength to support selection in the thymus and survival of T-cells in the periphery (Fig. 1). This view fits with the known properties of antigen presentation where most MHC molecules will be occupied by a repertoire of self peptides (Hunt et al., 1992). Those MHC–peptide complexes are probably responsible for positive selection in the thymus by providing weak TCR stimuli to immature T-cells (Robey and Fowlke, 1994). Similarly, the recognition of self MHC–peptide complexes mediates a survival signal to peripheral T-cells without reaching the threshold for induction of effector T-cell functions (Brocker, 1997; Kirberg et al., 1997; Tanchot et
149
al., 1997). The replacement of part of the self-peptide pool by non-self / foreign peptides will result in significant affinity changes for a few T-cells within the repertoire. Those T-cells will become activated by as few as 10–100 high affinity ligands per antigen presenting cell (Demotz et al., 1990; Sykulev et al., 1996). Following activation they will mediate effector functions and expand to large numbers. After removal of the invading non-self peptide from the MHC–peptide repertoire, those T-cells will return to the survival state and receive their low affinity TCR signal by the pool of self peptides. Due to quantitative and qualitative receptor changes those T-cells will have a much lower threshold for reactivation. As a consequence, the T-cells will respond vigorously upon re-exposure to their nominal ligand, i.e., a foreign antigen, but may also be prone to cross-reactivity with self antigens (Fig. 1). The two-step model for T-cell survival and T-cell activation is probably reflected by different signaling events. It is tempting to speculate that the incomplete TCR-associated signaling events (partial phosphorylation of TCR-z chain and no ZAP-70 phosphorylation) initially observed with partial agonists and antagonists (peptides that activate T-cell functions only partially or antagonize T-cell activation) are equivalent to a survival event, whereas the full signaling pattern (full phosphorylation of ZAP-70 and TCR-z) corresponds to physiological activation of the T-cell (Germain and Stefanova, 1999; Fig. 1).
Fig. 1. Outline of the hypothetical two-step model for T-cell survival, maturation, and activation. During thymic maturation positive selection is mediated by the mixture of self MHC–peptide complexes through weak stimulation of the T-cells. This process may involve a partial TCR signal. TCR with high affinity TCRs for single self MHC–peptide complexes within the mixture will become fully activated and undergo negative selection as a consequence of a full TCR signal. After maturation, T-cells will now receive a weak stimulus through the TCR by the self-MHC–peptide complexes. As a consequence of changes in the signaling machinery, full activation of the T-cell will now lead to expansion instead of deletion.
150
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2. The pathological consequences of degenerate T-cell antigen recognition T-cell responses are crucial for the defense against microbial antigens as well as tumor cells. However, under certain conditions protective T-cell responses may result in autoimmune disease. Two not mutually exclusive models for the development of autoimmune responses exists that are based on either ‘molecular mimicry’ or ‘epitope spreading’. The latter model hypothesizes that the release of self-antigens after inflammatory organ diseases starts a self perpetuating cascade of autoimmune responses (Lehmann et al., 1992). The repeated priming of autoreactive T-cells eventually leads to a chronic organ specific autoimmune disease involving more and more self-epitopes. Some evidence from experimental autoimmune models supports this hypothesis (Lehmann et al., 1992), although the role of this mechanism in human autoimmune disorders remains to be established. In addition this model does not explain the ‘first hit’ that starts the cascade of events. The second concept for the development of autoimmune disorders that may explain the initial event is based on the mechanism of cross-reactivity and termed ‘molecular mimicry’. It has been shown that infectious agents can activate T- and B-cells that cross-react with self antigens and initiate autoimmune responses. The idea was established in animal models many years ago, when Fujinami and Oldstone (1985) demonstrated in rabbits the induction of autoimmunity after immunization with a hepatitis B virus (HBV) antigen. The HBV peptide used in those experiments had sequence identity in six positions with the myelin basic protein (MBP) peptide (66–75). Immunization with the peptide induced an antibody response to MBP and in some animals an inflammatory disease of the brain. For many years it was believed that sequence identity is a necessity for cross-recognition of T-cells. Accordingly, the search for cross-reactive ligands involved screening of protein databases for non-self peptides with sequence identity to self antigens. However, the emerging knowledge about the flexibility of the TCR and its degenerate recognition have changed this view (Kersh and Allen, 1996; Wucherpfennig and Strominger, 1995; Hemmer et al., 1998a,b). Studies by Allen and coworkers and other groups demonstrated how little sequence homology is necessary for cross-recognition of T-cells. Careful studies of the effect on single amino acid modifications led to the definition of MHC binding motifs and the identification of tolerated amino acids in TCR contact positions. As a result Allen and coworkers described for a hemoglobin-specific TCC a cross-reactive peptide that had only one amino acid in common with the original TCR ligand (Evavold et al., 1995). Wucherpfennig and Strominger (1995) used a similar approach in combination with a motif based database search and identified for two myelin basic protein
(MBP)-specific TCC stimulatory microbial peptide antigens with limited sequence homology. The peptides were derived from Epstein-Barr virus DNA polymerase and Herpes simplex virus terminase proteins. Both peptides were less potent than the MBP peptide. An additional finding for the understanding of antigen recognition of autoreactive T-cells was provided by extensive epitope mapping of TCC specific for MBP(83–99). In those studies it was demonstrated that autoreactive CD41 T-cell clones could recognize peptide analogues even better than the self-antigen used to establish the TCC. This indicated that the autoantigen was a suboptimal ligand for those T-cells. Although the peptide analogue approach was successful in identifying cross-reactive ligands it had some drawbacks. The approach is only applicable to TCC with known peptide specificity. In addition, for each epitope a large number of different peptide analogues is necessary to dissect recognition and determine TCR motifs. Both limitations established the need for more efficient methods to dissect T-cell recognition. Based on the observation that each single amino acid within the peptide sequence contributes largely independently to the interaction of the TCR with its MHC–peptide ligand (Hemmer et al., 1998a,b), soluble combinatorial peptide libraries were introduced to determine antigen recognition of T-cells. The PS-SCL approach to examine TCR recognition offered significant advantages over previously used procedures. The method was introduced to determine antigen recognition of antigen-specific and alloreactive CD8- and CD4- positive T-cell clones. (Udaka et al., 1995; Gundlach et al., 1996; Hemmer et al., 1997; Hiemstra et al., 1998). Cross-reactive ligands for autoreactive T-cells were easily determined by using this approach. For several human MBP-specific TCC cross-reactive antigens from a variety of microbial and self-peptides were identified. Those involved peptides derived from the human Cytomegalovirus and Salmonella typhimurium proteins (Hemmer et al., 1997). In addition, these studies determined another feature of autoreactive T-cells that may change our view of crossrecognition and molecular mimicry. In contrast to TCC with non-self specificities (R. Kubota, B. Hemmer, R. Martin, S. Jacobson, unpublished) all autoreactive TCC examined so far recognized the autoantigen rather inefficiently (Fig. 2). The PS-SCL and the peptide analogue approach identified for those TCC peptide ligands that elicited much stronger responses than the autoantigen that was used to establish the TCC (Hemmer et al., 1997; Vergelli et al., 1997; Hemmer et al., unpublished). Some of those peptides that were termed superagonist ligands were deduced from microbial antigens. These results suggest that autoreactive cells do not recognize the autoantigen with optimal fit. It is tempting to speculate that autoreactive T-cells, although present in the peripheral blood of patients with autoimmune diseases, were initially activated
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Fig. 2. Differences in antigen recognition between T-cells that recognize self-peptides versus those that recognize non-self peptides. TCC that were in vivo selected by immunodominant antigens showed a much better fit with the PS-SCL TCR motif than TCCs generated with MBP.
and expanded in vivo by cross-reactive ligands. Candidate antigens are peptides from the pool of microbial proteins.
3. The use of combinatorial peptide libraries to identify relevant antigen epitopes in chronic Lyme disease Although T-cell recognition is highly degenerate, it became clear from those studies that PS-SCL are promising tools to identify optimal ligands for a given TCR. This notion was supported by preliminary results with virusspecific TCCs. In contrast to autoreactive T-cells, TCCs generated with immunodominant peptides from patients after influenza virus or HTLV-I infection (Y. Kubota, B. Hemmer, R. Martin, S. Jacobsen, personal communication) seem to recognize the antigen with a better fit. The TCR motifs established by the PS-SCL approach contained the entire peptide sequence. A database search with the motif would have disclosed the viral antigens as likely candidate antigens. These results indicate that the PS-SCL approach can identify for a given T-cell peptide antigens that are likely to have expanded the cells in vivo. To support this hypothesis of T-cell responses in chronic Lyme diesease, an inflammatory organ disorder following infection with the spirochete Borrelia burgdorferi (B.b.) were investigated. B.b. may spread to different organism including skin, joints, or nervous system in the early stages of the bacteremia. Sometimes the inflammation persists, although the bacterium may no longer be present in the organ compartment. This suggests that an immune response initiated by the bacterium can persist in the absence of the original antigen. One likely mechanism is crossrecognition of B.b.-specific T-cells with self antigens in the
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organ compartment. To follow this hypothesis T-cells were isolated from the cerebrospinal fluid (CSF) of a patient who developed chronic encephalomyelitis after B.b. infection (Hemmer et al., 1999). CSF cells were analyzed by flow cytometry and RT-PCR for the presence of expanded T-cell populations by their TCR-Vb chain expression in comparison to peripheral blood T-cells. In addition, single stranded conformational polymorphism analysis of TCRVb chains was performed to identify T-cell clonotypes. Using both methods, expansion of several TCR-Vb chains was identified. In parallel, TCC were established with whole Borrelia lysate from CSF T-cells. The TCC were then analyzed for their function and TCR-Vb expression. One in vivo expanded T-cell clonotype expressed the TCR of a cultured TCC. Further studies focused on this TCC. The clone was tested for its response to a decapeptide PS-SCL. A highly discriminative response to the peptide library was observed that allowed a detailed analysis for peptide specificity of the TCC. Using the TCR motif of the TCC protein, databases were searched to identify potential peptide ligands for the TCC. The peptides with the highest potency were derived from B.b. itself, whereas a large number of self-peptides or peptides derived from other microbes with lower potency were identified (Hemmer et al., 1999). Interestingly one peptide was derived from a myelin protein. These results demonstrate for the first time that peptide libraries can identify high and low affinity ligands for T-cells with unknown peptide specificity (Fig. 3). It can be hypothesized that the in vivo expanded TCC was activated in the first instance by the identified Borrelia antigens in the lymphatic system or even the brain. After clearing the CNS from the microbe, other antigens might have continued to stimulate the TCC. Likely candidates are some of the self-peptides that were discovered by the PS-SCL approach. This finding, although only established with one TCC supports the concept of molecular mimicry as a potential pathogenic mechanism in the development of autoimmune responses after infectious diseases.
4. Perspectives for the study of T-cell responses in human diseases These results demonstrate that T-cell responses in a patient with chronic Neuroborreliosis allow disease related antigens to be discovered by the specificity of the organ infiltrating lymphocytes. Since T-cells play a significant role in most inflammatory and neoplastic diseases, we believe that such an approach could help to identify relevant antigens in those disorders by determining antigen specificity of expanded T-cell populations. The technique may replace large scale peptide synthesis or protein expression experiments that have been performed to discover candidate antigens in those disorders. After identification of dominant T-cell responses the approach
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the clinical relevance of this mechanism in the pathogenesis of autoimmune diseases such as multiple sclerosis.
Acknowledgements The work was supported by the Deutsche Forschungsgemeinschaft (He 2368 / 2-1). Bernhard Hemmer was in ¨ part sponsored by the Gemeinnutzige Hertie-Stiftung.
References
Fig. 3. Schematic protocol for defining potential ligands for a T-cell clone with unkown specificity.
may also help to design highly specific immunotherapies based on those antigenic determinants.
5. Conclusions Recent studies on T-cell antigen recognition have shown that a single TCR can recognize many thousands of different peptides, demonstrating the flexibility of the cellular immune system in recognizing a broad and variable spectrum of antigens. While this flexibility is fundamental for efficient host defense, however, it imposes constant danger to the host by generating potentially autoaggressive immune reactivity. Degeneracy in T-cell recognition is therefore a prerequisite for the initiation of autoimmunity, supporting and extending the concept of ‘molecular mimicry’. Further studies will have to clarify
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