Understanding the roles of activation threshold and infections in the dynamics of autoimmune disease.

Onset and development of autoimmunity have been attributed to a number of factors, including genetic predisposition, age and different environmental factors. In this paper we discuss mathematical models of autoimmunity with an emphasis on two particular aspects of immune dynamics: breakdown of immune tolerance in response to an infection with a pathogen, and interactions between T cells with different activation thresholds. We illustrate how the explicit account of T cells with different activation thresholds provides a viable model of immune dynamics able to reproduce several types of immune behaviour, including normal clearance of infection, emergence of a chronic state, and development of a recurrent infection with autoimmunity. We discuss a number of open research problems that can be addressed within the same modelling framework.

Tumour necrosis factor-mediated macrophage activation in the target organ is critical for clinical manifestation of uveitis.

Clinically available anti-tumour necrosis factor (TNF) biologics, which inhibit both soluble (sTNF) and transmembrane forms (tmTNF) of TNF, eliminating all TNF signalling, have successfully treated autoimmune diseases including uveitis. These have potentially serious side effects such as reactivation of latent Mycobacterium tuberculosis and, therefore, more specific inhibition of TNF signalling pathways may maintain clinical efficacy while reducing adverse effects. To determine the effects of specific pharmacological inhibition of sTNF on macrophage activation and migration, we used a mouse model of uveitis (experimental autoimmune uveoretinitis; EAU). We show that selective inhibition of sTNF is sufficient to suppress EAU by limiting inflammatory CD11b(+) macrophages and CD4(+) T cell migration into the eye. However, inhibition of both sTNF and tmTNF is required to inhibit interferon-γ-induced chemokine receptor 2, CD40, major histocompatibility complex class II and nitric oxide (NO) up-regulation, and signalling via tmTNF is sufficient to mediate tissue damage. In confirmation, intravitreal inhibition of sTNF alone did not suppress disease, and inflammatory cells that migrated into the eye were activated, generating NO, thus causing structural damage to the retina. In contrast, intravitreal inhibition of both sTNF and tmTNF suppressed macrophage activation and therefore disease. We conclude that sTNF is required for inflammatory cell infiltration into target tissue, but at the tissue site inhibition of both sTNF and tmTNF is required to inhibit macrophage activation and to protect from tissue damage.

The role of tunable activation thresholds in the dynamics of autoimmunity.

It has been known for some time that human autoimmune diseases can be triggered by viral infections. Several possible mechanisms of interactions between a virus and immune system have been analysed, with a prevailing opinion being that the onset of autoimmunity can in many cases be attributed to "molecular mimicry", where linear peptide epitopes, processed from viral proteins, mimic normal host self-proteins, thus leading to a cross-reaction of immune response against virus with host cells. In this paper we present a mathematical model for the dynamics of an immune response to a viral infection and autoimmunity, which takes into account T cells with different activation thresholds. We show how the infection can be cleared by the immune system, as well as how it can lead to a chronic infection or recurrent infection with relapses and remissions. Numerical simulations of the model are performed to illustrate various dynamical regimes, as well as to analyse the potential impact of treatment of autoimmune disease in the chronic and recurrent states. The results provide good qualitative agreement with available data on immune responses to viral infections and progression of autoimmune diseases.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein.

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti- B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire.

The autoreactive T cells that escape central tolerance and form the peripheral self-reactive repertoire determine both susceptibility to autoimmune disease and the epitope dominance of a specific autoantigen. SJL (H-2(s)) mice are highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) with myelin proteolipid protein (PLP). The two major encephalitogenic epitopes of PLP (PLP 139-151 and PLP 178-191) bind to IA(s) with similar affinity; however, the immune response to the PLP 139-151 epitope is always dominant. The immunodominance of the PLP 139-151 epitope in SJL mice appears to be due to the presence of expanded numbers of T cells (frequency of 1/20,000 CD4(+) cells) reactive to PLP 139-151 in the peripheral repertoire of naive mice. Neither the PLP autoantigen nor infectious environmental agents appear to be responsible for this expanded repertoire, as endogenous PLP 139-151 reactivity is found in both PLP-deficient and germ-free mice. The high frequency of PLP 139-151-reactive T cells in SJL mice is partly due to lack of thymic deletion to PLP 139-151, as the DM20 isoform of PLP (which lacks residues 116-150) is more abundantly expressed in the thymus than full-length PLP. Reexpression of PLP 139-151 in the embryonic thymus results in a significant reduction of PLP 139-151-reactive precursors in naive mice. Thus, escape from central tolerance, combined with peripheral expansion by cross-reactive antigen(s), appears to be responsible for the high frequency of PLP 139-151-reactive T cells.

Systemic and local anti-C5 therapy reduces the disease severity in experimental autoimmune uveoretinitis.

Activation of complement occurs during autoimmune retinal and intraocular inflammatory disease as well as neuroretinal degenerative disorders. The cleavage of C5 into fragments C5a and C5b is a critical event during the complement cascade. C5a is a potent proinflammatory anaphylatoxin capable of inducing cell migration, adhesion and cytokine release, while membrane attack complex C5b-9 causes cell lysis. Therapeutic approaches to prevent complement-induced inflammation include the use of blocking monoclonal antibodies (mAb) to prevent C5 cleavage. In these current experiments, the rat anti-mouse C5 mAb (BB5.1) was utilized to investigate the effects of inhibition of C5 cleavage on disease progression and severity in experimental autoimmune uveoretinitis (EAU), a model of organ-specific autoimmunity in the eye characterized by structural retinal damage mediated by infiltrating macrophages. Systemic treatment with BB5.1 results in significantly reduced disease scores compared with control groups, while local administration results in an earlier resolution of disease. In vitro, contemporaneous C5a and interferon-gamma signalling enhanced nitric oxide production, accompanied by down-regulation of the inhibitory myeloid CD200 receptor, contributing to cell activation. These experiments demonstrate that C5 cleavage contributes to the full expression of EAU, and that selective C5 blockade via systemic and local routes of administration can suppress disease. This presents great therapeutic potential to protect against tissue damage during autoimmune responses in the retina or inflammation-induced degenerative disease.

Manipulation of the Th1/Th2 balance in autoimmune disease.

Many autoimmune diseases are caused by autopathogenic Th1 cells. Because in vitro Th1 and Th2 cells cross-regulate each other, it is likely that the induction of self-antigen-specific Th2 cells can prevent autoimmune disease. In the past year, investigators have further defined the role of Th1 and Th2 cytokines in the induction and regulation of autoimmunity. Furthermore, the role of MHC-antigen-T-cell avidity (strength of signal) in inducing such protective immune responses has been elucidated.

T cell recognition of self and altered self antigens.

T lymphocytes bearing alpha/beta TCR recognize antigens in the context of self MHC molecules, and this recognition leads to growth, differentiation, and effector functions. Recently, it has become clear that altered peptides generated by single amino acid substitution of the antigenic peptide can alter the patterns of differentiation and effector functions of the responding T lymphocytes. By defining the pattern of recognition and residues of the cognate ligand that bind to the TCR, altered peptide ligands (APLs) have been generated by selectively substituting the TCR contact residues in the antigenic peptide. These APLs have been utilized in vitro to study the biology of T cell function and alterations in the T cell signaling pathway. In vivo APLs have been utilized to study the mechanism of positive selection in the thymus and in regulation of autoimmune diseases. With this basic knowledge, APLs that can either hypo- or hyper-stimulate T cell function can be generated that can specifically alter (inhibit or enhance) immune responses in vivo in autoimmune diseases and cancers.

The role of lipoprotein-associated phospholipase A2 in a murine model of experimental autoimmune uveoretinitis.

Macrophage activation is, in part, regulated via hydrolysis of oxidised low density lipoproteins by Lipoprotein-Associated phospholipase A2 (Lp-PLA2), resulting in increased macrophage migration, pro-inflammatory cytokine release and chemokine expression. In uveitis, tissue damage is mediated as a result of macrophage activation; hence inhibition of Lp-PLA2 may limit macrophage activation and protect the tissue. Utilising Lp-PLA2 gene-deficient (KO) mice and a pharmacological inhibitor of Lp-PLA2 (SB-435495) we aimed to determine the effect of Lp-PLA2 suppression in mediating retinal protection in a model of autoimmune retinal inflammation, experimental autoimmune uveoretinitis (EAU). Following immunisation with RBP-3 (IRBP) 1-20 or 161-180 peptides, clinical disease was monitored and severity assessed, infiltrating leukocytes were enumerated by flow cytometry and tissue destruction quantified by histology. Despite ablation of Lp-PLA2 enzyme activity in Lp-PLA2 KO mice or wild-type mice treated with SB-435495, the number of infiltrating CD45+ cells in the retina was equivalent to control EAU animals, and there was no reduction in disease severity. Thus, despite the reported beneficial effects of therapeutic Lp-PLA2 depletion in a variety of vascular inflammatory conditions, we were unable to attenuate disease, show delayed disease onset or prevent progression of EAU in Lp-PLA2 KO mice. Although EAU exhibits inflammatory vasculopathy there is no overt defect in lipid metabolism and given the lack of effect following Lp-PLA2 suppression, these data support the hypothesis that sub-acute autoimmune inflammatory disease progresses independently of Lp-PLA2 activity.

The thyrotrophin hormone receptor of Graves' disease: overexpression of the extracellular domain in insect cells using recombinant baculovirus, immunoaffinity purification and analysis of autoantibody binding.

Since the cloning of the TSH receptor (TSH-R), the target autoantigen of Graves' disease, the receptor has been expressed in a variety of eukaryotic cells to obtain a functional molecule. Despite this success, the levels of receptor expression have been marginally higher than the extremely low levels found in thyroid cells, preventing any progress on the purification of the molecule. In this study, the large extracellular region of the TSH-R, without the membrane spanning segments, has been expressed in insect cells using recombinant baculovirus to generate substantial quantities of the receptor protein. A monoclonal antibody previously generated to a bacterial TSH-R fusion protein was used to characterize and monitor the expression of the truncated receptor in insect cells. Two polypeptides of 63 and 49 kDa were recognized as the components of the truncated recombinant receptor. The 63 kDa protein was shown to be the glycosylated form of the smaller, 49 kDa, component. Expression in different insect cell lines showed that an increase in expression of approximately tenfold was apparent in High Five cells when compared with Sf21 cells. Very small quantities of the truncated receptor were secreted by the three insect cell lines examined, with the majority of the molecule being retained within the cells. Immunoaffinity purification of milligram quantities of the truncated receptor was achieved using the monoclonal antibody. The availability of the purified TSH-R has allowed the establishment of an enzyme-linked immunosorbent assay to measure autoantibodies in the sera of patients with Graves' disease. Although the truncated receptor interacts with autoantibodies, our results show that it does not bind TSH and differs in this respect from other glycoprotein hormone receptors.

Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells.

We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.

The role of cytokines in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of the demyelinating disease multiple sclerosis. In EAE cytokines play a critical role in defining the Th1 or Th2 nature of the autoantigen directed immune response, and in propagating and regulating inflammation within the central nervous system. In this review we summarize some of the recent developments in the field of cytokine research that relate to this model of human disease, focusing principally on disease induced with the autoantigens myelin proteolipid protein and myelin oligodendrocyte protein.

Tuning T cell activation threshold and effector function with cross-reactive peptide ligands.

We have generated a panel of cross-reactive T cells by immunizing SJL mice (I-A(s)) with Q144 peptide, an analog of an autoantigenic peptide (W144) of myelin proteolipid protein (PLP) 139-151 (HSLGKWLGHPDKF) in which W was replaced by Q at position 144. Following immunization with Q144, T cells were expanded in vitro with W144, which is a cross-reactive, suboptimal ligand, for Q144-specific T cells. The T cell clones responded to both ligands and grew normally on the peptide W144, but were hyperstimulated when activated by Q144 in vitro. This hyperstimulation results in a heteroclitic proliferative response with secretion of additional cytokines not induced by W144. Thus expansion of T cells by a suboptimal cross-reactive ligand effectively lowers the activation threshold so that the immunizing antigen becomes a hyperstimulating ligand for the clones. Surprisingly, when the T cell clones are grown on the hyperstimulating ligand Q144, some adapt by increasing their activation threshold. This desensitization results in a loss of response to a number of cross-reactive ligands and the appearance of a more specific T cell response. Long-term culture with the hyperstimulating ligand is sometimes associated with down-regulation of CD4 expression. These results provide an explanation for the common finding of T cell heteroclicity, and suggest that although the specificity and hierarchy of the response of T cells to peptides is determined by the TCR, activation threshold and effector functions are modified by exposure to cross-reactive ligands. This observation has implications for the development and regulation of autoimmune disease.

The origin and regulation of autopathogenic T cells.

A clear understanding of the events surrounding the selection of autoreactive T cells in the thymus and their regulation in the periphery has eluded immunologists for years. However, recent work examining the expression of tissue-specific antigens in the thymus and the biochemistry of disease associated MHC alleles has provided important clues into the generation of the autoreactive T cell repertoire in the thymus. In addition, recent studies focusing on the role of immunoregulatory cytokines and cross-reactive peptide ligands has provided information regarding both the regulation and activation of autoreactive cells in the periphery. An improved understanding of the selection and regulation of autoreactive T cells will undoubtedly aid in the development of strategies for treating autoimmune disease.

Analysis of human T-cell receptor V beta gene usage following immunization to tetanus toxoid in vivo.

The human T-cell antigen receptor (TcR) V beta repertoire was investigated following in vivo reimmunization with tetanus toxoid (TT). Four healthy subjects were immunized subcutaneously with TT, and 24 samples of peripheral blood T cells were taken at intervals over several weeks and used to generate TcR-C beta chain-specific first-strand cDNA. A semi-quantitative assay utilizing the polymerase chain reaction (PCR) was used to measure the amount of 22 different TcR-V beta gene transcripts in the cDNA. A peak increase in the amount of V beta 2, 4, 6, 13.1 and 14 occurred 14 days post-immunization, with each V beta increased in at least two of the four subjects. No obvious changes in the other 17 V beta genes were found. A secondary antibody response to TT occurred in all subjects by day 14. These results show that it is now possible to characterize the in vivo kinetics of the human TcR repertoire following stimulation with a conventional antigen.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens.

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139-151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139-151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178-191) and myelin oligodendrocyte protein (MOG) residues 92-106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139-151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139-151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Autoantigen-responsive T cell clones demonstrate unfocused TCR cross-reactivity toward multiple related ligands: implications for autoimmunity.

It has been suggested that the cross-reaction of a single T cell receptor with multiple different peptide ligands is a mechanism for maintaining a diverse yet compact immune repertoire. In the context of autoimmune disease it is important to understand how this property is balanced against the maintenance of self-tolerance. Specifically, whether the cross-reactivity inherent in the immune system is focused or unfocused will have important consequences for the development of autoimmune disease. If cross-reactivity is "focused," then in an immune response to a foreign antigen all T cell receptors that recognize the foreign antigen will cross-react with a specific autoantigenic peptide. However, if cross-reactivity is "unfocused," an immune response to a foreign antigen will result in the activation of a small number of self-reactive cells within a larger pool of cells specific for the foreign antigen. We have tested whether cross-reactivity is focused or unfocused by generating a panel of T cell clones that respond to two closely related ligands. W144 is an autoantigenic peptide of myelin proteolipid protein, PLP 139-151 (HSLGKWLGHPDKF), and Q144 is an altered peptide of PLP 139-151 bearing a glutamine for tryptophan substitution at position 144. The Q144-responsive clones have a broad degree of cross-reactivity with other position 144 substituted peptides. We find that despite their characteristic responses to Q144 and W144, the patterns of responses of these clones to other structurally related ligands are random, demonstrating that cross-reactivity is unfocused in the absence of selection. Maintaining a diverse range of cross-reactive interactions may limit nonspecific responses to autoantigens.

Unaltered thyroid function in mice responding to a highly immunogenic thyrotropin receptor: implications for the establishment of a mouse model for Graves' disease.

Grave's disease (GD) is a common disorder characterized by the presence of autoantibodies to the thyrotropin receptor. In the past, the exceedingly low expression of the thyrotropin receptor on thyrocytes has not allowed its purification in quantities sufficient to investigate the establishment of an animal model for this disease. In this study, we have purified the 398-amino acid, extracellular region of the human thyrotropin receptor (TSH-R.E) from insect cells using recombinant baculovirus, and explored its immunopathogenic properties in H-2b,d,q,k,s strains of mice. The receptor preparation was highly immunogenic since it elicited strong specific proliferative T cell responses as well as IgG responses in all strains tested. In addition, hyperimmunization with TSH-R.E induced (i) serum antibodies that blocked the binding of 125I-TSH to its receptor, a common feature of GD autoantibodies; and (ii) IgG that reacted with a synthetic peptide (residues 32-54) from the N-terminus of the receptor, a region implicated in the binding of thyroid stimulating antibodies. In SJL animals only, a weak antibody response to two other thyroid antigens, thyroglobulin and thyroid peroxidase, was also observed. The presence of these antibodies, however, was not accompanied by a detectable alteration in thyroid function as assessed by the measurement of serum TSH, T4 and iodine levels. Also mononuclear infiltration of the thyroid gland or morphological changes compatible with an activation state of thyrocytes were not apparent in TSH-R-challenged mice. In contrast, mice treated with the anti-oxidant aminotriazole showed a dramatic increase in serum TSH levels and an activated follicular epithelium. These data demonstrate that a highly immunogenic TSH-R.E in mice does not necessarily provide a proper stimulus for the induction of a hyper- or hypothyroid status as defined by hormonal or histological criteria. Main reasons for the inability to induce receptor-specific antibodies that affect thyroid function such as those generated in GD are likely to be the inappropriate folding of the recombinant extracellular domain of the receptor, or the xenogeneic nature of the autoantigen.

An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) peptide 139-151, we have previously shown that the disease is mediated by Th1 cells, which recognize tryptophan 144 as the primary TCR contact point. Here we describe an altered peptide ligand (APL), generated by a single amino acid substitution (tryptophan to glutamine) at position 144 (Q144), which inhibits the development of EAE induced with the native PLP 139-151 peptide (W144). We show that the APL induces T cells that are cross-reactive with the native peptide and that these cells produce Th2 (IL-4 and IL-10) and Th0 (IFN gamma and IL-10) cytokines. Adoptive transfer of T cell lines generated with the APL confer protection from EAE. These data show that changing a single amino acid in an antigenic peptide can significantly influence T cell differentiation and suggest that immune deviation may be one of the mechanisms by which APLs can inhibit an autoimmune disease.

Expansion by self antigen is necessary for the induction of experimental autoimmune encephalomyelitis by T cells primed with a cross-reactive environmental antigen.

Cross-reactivity with environmental antigens has been postulated as a mechanism responsible for the induction of autoimmune disease. Experimental autoimmune encephalomyelitis is a T cell-mediated autoimmune disease model inducible in susceptible strains of laboratory animals by immunization with protein constituents of myelin. We used myelin proteolipid protein (PLP) peptide 139-151 and its analogues to define motifs to search a protein database for structural homologues of PLP139-151 and identified five peptides derived from microbial Ags that elicit immune responses that cross-react with this self peptide. Exposure of naive SJL mice to the cross-reactive environmental peptides alone was insufficient to induce autoimmune disease even when animals were treated with Ag-nonspecific stimuli (superantigen or LPS). However, immunization of SJL mice with suboptimal doses of PLP139-151 after priming with cross-reactive environmental peptides consistently induced experimental autoimmune encephalomyelitis. Furthermore, T cell lines from mice immunized with cross-reactive environmental peptides and restimulated in vitro with PLP139-151 could induce disease upon transfer into naive recipients. These data suggest that expansion by self Ag is required to break the threshold to autoimmune disease in animals primed with cross-reactive peptides.