A polyalanine peptide with only five native myelin basic protein residues induces autoimmune encephalomyelitis.

The minimum structural requirements for peptide interactions with major histocompatibility complex (MHC) class II molecules and with T cell receptors (TCRs) were examined. In this report we show that substituting alanines at all but five amino acids in the myelin basic protein (MBP) peptide Ac1-11 does not alter its ability to bind A alpha uA beta u (MHC class II molecules), to stimulate specific T cells and, surprisingly, to induce experimental autoimmune encephalomyelitis (EAE) in (PL/J x SJL/J)F1 mice. Most other amino acid side chains in the Ac1-11 peptide are essentially irrelevant for T cell stimulation and for disease induction. Further analysis revealed that binding to A alpha uA beta u occurred with a peptide that consists mainly of alanines and only three of the original residues of Ac1-11. Moreover, when used as a coimmunogen with MBP Ac1-11, this peptide inhibited EAE. The finding that a specific in vivo response can be generated by a peptide containing only five native residues provides evidence that disease-inducing TCRs recognize only a very short sequence of the MHC-bound peptide.

Lewis rat lymphoid dendritic cells can efficiently present homologous myelin basic protein to encephalitogenic lymphocytes.

Lymphocytes isolated from Lewis rats immunised with protein antigen in adjuvant were stimulated to proliferate in vitro by splenic dendritic cells (DC) which had been pulsed with purified homologous myelin basic protein (MBP). By contrast, in parallel experiments, lymphocytes did not respond to ovalbumin unless the protein was first processed by macrophages by a chloroquine-sensitive mechanism. DC, pulsed with rat MBP at concentrations as low as 6 micrograms/ml, activated lymphocytes for transfer of severe experimental autoimmune encephalomyelitis (EAE). MBP dissociating from myelin membranes in physiological medium, as well as MBP purified from highly acidic extracts of myelin, was effective for pulsing DC; preincubating myelin with macrophages led to a reduction rather than an enhancement in the severity of the EAE transferred. It is concluded that macrophage-mediated antigen processing is not required for immunogenic presentation of the determinants of MBP which cause EAE in Lewis rats. Furthermore, MBP-pulsed DC may prove useful in experiments requiring activation of encephalitogenic T cells.

Competitive dissociation of encephalitogenic complexes between antigen presenting cells and myelin basic protein.

Splenic T cells from myelin basic protein (MBP)-immunised Lewis rats were activated to transfer experimental autoimmune encephalomyelitis (EAE) by co-culture with MBP-pulsed lymphoid dendritic cells (DC). MBP-pulsed DC could be kept for at least 24 h at 37 degrees C in antigen-free medium without affecting their ability subsequently to activate encephalitogenic T cells. However, MBP-pulsed DC were rendered much less stimulatory after a 6 h, but not 2 h, secondary incubation with ovalbumin. Thus, although encephalitogenic complexes between MBP and DC appear very stable in the absence of competing antigens, in their presence, antigen exchange can take place over a period of a few hours; this has positive implications for therapy of EAE by antigen competition.

Autoantibodies to glycoprotein antigens mediate subacute demyelinating encephalomyelitis in the Lewis rat.

Serum autoantibodies were induced in Lewis rats by immunization with a mixture of lentil lectin-binding glycoproteins isolated from bovine brain myelin. Intraperitoneal administration of 2-10 million syngeneic myelin basic protein-activated spleen cells to these rats led within 4-5 days to paralysis which, in most cases, persisted for several weeks. The major neuropathological features of the disease were numerous macrophages in both brain and spinal cord and large areas of demyelination, generally with axon preservation, particularly adjacent to the pial surfaces of the cord. This model is easily induced and will be useful for studies of demyelination and remyelination.

EAE: a model for immune intervention with synthetic peptides.

The cellular and molecular requirements for the autoimmune disease EAE are being defined in increasing detail through intense scrutiny of critical autoantigenic peptides, class II MHC molecules, and alpha beta TCRs involved in the disease process. This study has led to novel immunotherapeutic approaches, many of which are based on the administration of synthetic peptides. Since short peptides are understood to be the minimal antigenic units bound by MHC molecules for recognition by T cells, they are attractive experimental tools for finely modulating specific immune responses. It is clear that a large number of defined peptides can dramatically influence the course of EAE. Table IV lists a number of potential mechanisms which may mediate disease prevention. Increasing evidence supports the idea that prevention of autoimmune disease can result from MHC-blockade by peptides which competitively bind to class II molecules. However, for some peptides such as the perplexing partial agonist Ac1-11[4A], the mechanism by which these precisely defined units act is not yet fully understood. Numerous hurdles hinder immediate clinical application of peptide-based immunotherapy. Nevertheless, the knowledge gained by probing experimental autoimmunity with defined peptides promises to inspire original and practical approaches to treating human autoimmune disease.

Self and non-self peptides treat autoimmune encephalomyelitis: T cell anergy or competition for major histocompatibility complex class II binding?

In susceptible strains of mice, myelin basic protein (MBP) peptide Acl-ll induces experimental autoimmune encephalomyelitis (EAE) providing a useful model for human multiple sclerosis. Acl-11 binds major histocompatibility complex (MHC) class II molecules A alpha upsilon A beta upsilon. Here, we show that the Acl-11 peptide, when administered intraperitoneally in incomplete Freund's adjuvant (IFA) emulsion, can effectively treat Acl-11-induced EAE in mice. Treatment with Acl-11/IFA 9 days after initial immunization with Acl-11 in complete Freund's adjuvant (CFA) results in a loss of T cell proliferation to MBP Acl-11. This lack of T cell proliferation is not due to T cell anergy and is not specific. A similar lack of T cell proliferation and inhibition of EAE is observed when an ovalbumin peptide OVA323-339 or a sperm whale myoglobin peptide SWM110-121 are used to treat mice immunized with Acl-11. Interestingly, we show that previously unresponsive lymph node cells from treated mice respond normally if Acl-11 is presented by fresh antigen-presenting cells taken from normal mice. These results argue that the lack of T cell proliferation and inhibition of EAE is not due to specific T cell anergy as suggested by others. Instead this appears to be due to blocking of MHC class II molecules A alpha upsilon A beta upsilon by the treating peptides.

Competition between foreign and self proteins in antigen presentation. Ovalbumin can inhibit activation of myelin basic protein-specific T cells.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory neurological disease initiated by activated T cells specific for the autoantigen, myelin basic protein (MBP). The ability of Lewis rat splenic T cells to transfer EAE after in vitro incubation with MBP-pulsed dendritic cells (DC) was used as an index of MBP-specific T cell activation. OVA, previously processed by macrophages, was incubated with MBP and DC at the pulsing stage to determine whether it could inhibit presentation of the autoantigen. At molar equivalents of 2.5:1 and 20:1 relative to MBP, processed OVA increasingly inhibited the ability of DC to activate MBP-specific T cells for EAE transfer. Unprocessed OVA, which cannot be presented immunogenically by Lewis rat DC, was much less effective. However, processed OVA added to DC after they had been pulsed with MBP could not compete. OVA also blocked appearance of EAE when mixed with MBP/CFA in the inoculum used for active induction of the disease. Splenic T cells from MBP + OVA/CFA-immunized rats transferred EAE with a substantially delayed onset, suggesting that a reduced number of MBP-specific T cells was generated by immunizing with the OVA + MBP mixture compared with MBP alone. Overall, the data indicate that fragments of a foreign protein, OVA, which can be bound by APC, can also inhibit presentation of encephalitogenic determinants of MBP to T cells.

HLA, molecular mimicry and multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system in which an autoimmune response most probably contributes to pathogenesis. To date, the best characterized susceptibility-associated gene has been mapped to the HLA complex. The HLA-DRB1*1501 - DRB5*0101 - DQA1*0102 - DQB1*0602 haplotype is both associated and linked to MS in different ethnic groups. The locus within the HLA class II region encoding the MS-susceptibility gene is under intensive investigation. Epidemiological studies, however, have suggested that environmental antigens also play a critical role in MS pathogenesis. One of the ways a pathogen could trigger autoimmune disease is via immunological cross-reactivity or molecular mimicry. This concept argues that a microbial peptide with certain degree of homology to a self peptide can stimulate pathogenic self-reactive specific T cells to cause an autoimmune disease. Many microbial agents have regions of sequences that may serve as binding motifs for HLA-DR2. HLA genetics and molecular mimicry may therefore be intimately interlinked in the disease process. In the present review, we focus on the HLA association with MS and the role of microbial antigens in MS, with special reference to the molecular mimicry hypothesis.

Antigen presentation by MHC class II molecules.

The treamendous explosion in the field of MHC research in the last 5 years has significantly advanced our understanding of antigen processing pathways, particularly with regard to details of MHC class II-mediated antigen presentation. MHC class II molecules at the surface of antigen presenting cells present antigenic peptides to CD4+ T helper cells. However for effective cell surface antigen presentation, a number of highly synchronized events must first take place intracellulary. The monomorphic protein, invariant chain (Ii), is a crucial participant in MHC class II antigen presentation. Acting as a molecular chaperone, this molecule escorts the newly synthesized class II heterodimers from the endoplasmic reticulum into the endosomal system. During this manoeuvre, the interaction of li with class II serves to prevent premature association of antigenic peptide. Once the complex reaches the acidic environment of the endosomes, li is proteolytically degraded and dissociates, leaving the class II binding site available for binding antigenic peptide derived from exogenous proteins. The final Ii fragment to be displaced. CLIP (class II-associated invariant chain peptides), must be physically removed from the class II binding groove with assistance from another MHC-encoded molecule, DM. The interaction of DM with class II also aids in the subsequent rapid loading of high-affinity antigen-derived peptides into the MHC class II groove. The stable peptide-loaded complexes are now ready to exit the endocytic compartments to present their peptide antigen to specific T helper cells at the cell surface.

A continuous central motif of invariant chain peptides, CLIP, is essential for binding to various I-A MHC class II molecules.

Invariant chain (li) associates with MHC class II molecules and performs a number of crucial functions in antigen presentation. A nested set of class II-associated li peptides (CLIP) has been isolated, comprising the li sequence between residues 82 and 107. Recently, X-ray crystallographic analysis has revealed that residues 87-101 occupy the HLA-DR3 peptide-binding groove. Based on our previous results, Lee and McConnell have also proposed a model for the binding of CLIP to various mouse I-A molecules in the binding groove. CLIP sequences are able to bind many MHC class II molecules but the molecular basis of this promiscuity has not yet been resolved. We have shown recently that CLIP binding to I-A class II molecules is generally tolerant to side chain substitutions, suggesting that the backbone structure of CLIP may provide the features critical for its interaction with class II. In pursuit of this, backbone stereochemical disruptions by serial D-alanine substitutions in CLIP86-104 have been used in competitive binding assays to I-A class II molecules. These studies have revealed that the phylogenetically conserved central continuous region, CLIP91-99, is intolerant to such configurational substitutions. Experiments with truncated and frame-shift analogues of CLIP showed that for effective binding to class II, the sequence element CLIP90-100 must be incorporated into a peptide of 13 or more residues including at least three residues N-terminal to this motif. Additionally, it appears that different I-A molecules accommodate CLIP in different binding frames. These investigations of the relationship between the structure and binding of CLIP analogues lead us to propose that there is a general backbone motif of a periodic nature within the CLIP sequence that minimizes deleterious contacts and allows promiscuous binding to class II molecules.

Development of insulitis and diabetes in B cell-deficient NOD mice.

Insulin-dependent diabetes mellitus (IDDM) is believed to be an autoimmune disease that results from autoimmune destruction of the insulin-secreting beta-cells of the pancreas. In addition to a lymphocytic infiltration (insulitis) of the islets, patients with IDDM have autoantibodies directed against the components of the islet cells. Several beta-cell proteins have been identified as candidate autoantigens. The non-obese diabetic (NOD) mouse is a murine model for spontaneous IDDM. It is generally accepted that IDDM in patients and NOD mice results from the T lymphocyte-mediated destruction of beta-cells. However, the direct role of B lymphocytes in the disease process has not yet been clarified. To test directly the role of B cells in IDDM, we have generated B cell-deficient NOD mice by backcrossing the microMT-/- B cell 'knockout mice' onto the NOD background. The mice had no evidence of functional B cells as determined by flow cytometry and antibody production. We show that two out of seven of these mice developed insulitis and diabetes. These results suggest that despite an absence of B cells some NOD mice can still develop insulitis and diabetes.

Binding of an invariant-chain peptide, CLIP, to I-A major histocompatibility complex class II molecules.

Invariant chain (Ii) associates with major histocompatibility complex (MHC) class II molecules and is crucial for antigen presentation by class II molecules. The exact nature of Ii interaction with MHC class II molecules remains undefined. A nested set of Ii peptides, CLIPs (class II-associated Ii peptides), have been eluted from various MHC class II molecules, suggesting that CLIPs correspond, at least in part, to the Ii motif which blocks the conventional peptide binding site in MHC class II molecules. Here we report how CLIPs interact with class II MHC molecules, I-A. We have identified regions critical for binding of CLIPs and I-A class II molecules. In most cases, the binding of CLIPs to a number of I-A molecules is modulated by the steric bulk of methionine residues at positions 93 and 99. In addition, the binding of CLIPs to an I-A molecule, I-Au, is sensitive to substitutions at aspartic acid-59 in the alpha chain and threonine-86 in the beta chain, whereas the binding of an antigen-derived peptide is not. Taken together, these results provide an insight as to how CLIPs bind to MHC class II heterodimers.

Nonobese diabetic mice display elevated levels of class II-associated invariant chain peptide associated with I-Ag7 on the cell surface.

Peptide presentation by MHC class II molecules plays a pivotal role in determining the peripheral T cell repertoire as a result of both positive and negative selection in the thymus. Homozygous I-A(g7) expression imparts susceptibility to autoimmune diabetes in the nonobese diabetic mouse, and recently, it has been proposed that this arises from ineffectual peptide binding. Following biosynthesis, class II molecules are complexed with class II-associated invariant chain peptides (CLIP), which remain associated until displaced by Ag-derived peptides. If I-A(g7) is a poor peptide binder, then this may result in continued occupation by CLIP to the point of translocation to the cell surface. To test this hypothesis we generated affinity-purified polyclonal antisera that recognized murine CLIP bound to class II molecules in an allele-independent fashion. We have found abnormally high natural levels of cell surface class II occupancy by CLIP on nonobese diabetic splenic B cells. Experiments using I-A-transfected M12.C3 cells showed that I-A(g7) alone was associated with elevated levels of CLIP, suggesting that this was determined solely by the amino acid sequence of the class II molecule. These results indicated that an intrinsic property of I-A(g7) would affect both the quantity and the repertoire of self-peptides presented during thymic selection.

Antigen presentation and assembly by mouse I-Ak class II molecules in human APC containing deleted or mutated HLA DM genes.

The behavior of mouse I-Ak molecules was studied in the human Ag presentation mutants T2 and 9.5.3, which contain deleted or mutated HLA DM genes. HLA class II molecules expressed by these APC are defective in presentation of native Ag and are mostly complexed with class II-associated invariant chain peptides (CLIP). In contrast to human class II molecules, a significant proportion of mouse I-Ak molecules expressed in T2 and 9.5.3 were associated with antigenic peptides, indicating that I-Ak/peptide assembly is possible in the absence of the Dm proteins. Thus, the presentation of determinants derived from hen egg lysozyme (HEL), keyhole limpet hemocyanin, and conalbumin was normal in 9.5.3Ak and a conalbumin determinant was presented normally by T2.Ak. However, the keyhole limpet hemocyanin determinant was not presented by T2.Ak, and HEL46-61 was only presented at a low level by these APC. SDS-stable, dimeric I-Ak molecules were expressed by both T2.Ak and 9.5.3Ak and formed late in their intracellular transport. Presentation of HEL46-61 was partially inhibited by disrupting vacuolar acidification in 9.5.3Ak, consistent with I-Ak/peptide assembly in a post-Golgi endosomal compartment. Accordingly, Dm is not an obligatory requirement for MHC class II/peptide assembly. We propose that Dm influences the displacement of CLIP from recently synthesized class II molecules, a process that is likely to be less critical for I-Ak because of its low affinity for CLIP.

A viral peptide with limited homology to a self peptide can induce clinical signs of experimental autoimmune encephalomyelitis.

Molecular mimicry has been suggested as a mode of autoreactive T cell stimulation in autoimmune diseases. Myelin basic protein (MBP) peptide 1-11 induces experimental autoimmune encephalomyelitis (EAE) in susceptible strains of mice. Here we show that a herpesvirus Saimiri (HVS) peptide, AAQRRPSRPFA, with a limited homology to MBP1-11 peptide, ASQKRPSQRHG (underlined letters showing homology), can stimulate a panel of MBP-11-specific T cell hybridomas and more importantly cause EAE in mice. We demonstrate that this is due to cross-recognition of these two peptides by TCRs. Results presented in this communication are the first demonstration that a viral peptide with homology at just 5 amino acids with a self peptide can induce clinical signs of EAE in mice. These findings have important implications in understanding the breakdown of T cell tolerance to self Ags in autoimmune diseases by means of cross-reactivity with unrelated peptides.

Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner.

Invariant chain (Ii) associates with class II MHC molecules and is crucial for Ag presentation by class II molecules. A general explanation for how invariant chain (Ii) associates with polymorphic MHC class II molecules has been suggested by the crystallographic structure of CLIP (class II-associated Ii peptide) complexed with an HLA class II molecule, HLA-DR3. We show here that methionine residues at positions 93 and 99 in Ii are important in MHC class II-mediated Ag presentation, but function in an allele-dependent manner. Introduction of a Met-->Ala mutation at position 99 in Ii (M99AIi) impaired presentation of peptides derived from exogenous proteins by I-Ad and I-Au class II molecules. Mutating Met-->Ala in Ii at position 93 (M93AIi) abrogated presentation by I-Au molecules, but not by I-Ad. Impaired Ag presentation was associated with conformationally altered expression of I-A molecules on the surface of cells expressing mutated Ii. Cell surface CLIP staining and immunoprecipitation studies showed that both I-Ad and I-Au molecules were associated with an increased abundance of Ii peptides, CLIP, in cells expressing mutated Ii. These results show that methionine 93 and methionine 99 play an important physiologic role in Ii association with class II molecules by regulating release of CLIP from class II in the endocytic compartments to allow binding of cognate peptides.

Minimum structural requirements for peptide presentation by major histocompatibility complex class II molecules: implications in induction of autoimmunity.

The precise mechanisms of failure of immunological tolerance to self proteins are not known. Major histocompatibility complex (MHC) susceptibility alleles, the target peptides, and T cells with anti-self reactivity must be present to cause autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a murine model of a human autoimmune disease, multiple sclerosis. In EAE, residues 1-11 of myelin basic protein (MBP) are the dominant disease-inducing determinants in PL/J and (PL/J x SJL/J)F1 mice. Here we report that a six-residue peptide (five of them native) of MBP can induce EAE. Using peptide analogues of the MBP-(1-11) peptide, we demonstrate that only four native MBP residues are required to stimulate MBP-specific T cells. Therefore, this study demonstrates lower minimum structural requirements for effective antigen presentation by MHC class II molecules. Many viral and bacterial proteins share short runs of amino acid similarity with host self proteins, a phenomenon known as molecular mimicry. Since a six-residue peptide can sensitize MBP-specific T cells to cause EAE, these results define a minimum sequence identity for molecular mimicry in autoimmunity.

Inhibition of experimental autoimmune encephalomyelitis by a nonimmunogenic non-self peptide that binds to I-Au.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory neurologic disease initiated by myelin basic protein-reactive CD4+ T cells, which are restricted by a particular MHC class II molecule. Recent studies have utilized inhibitor peptides that bind to restricting MHC class II molecules in order to inhibit EAE, presumably by means of competing with encephalitogenic epitopes. However, these studies leave open the possibility of alternative explanations, such as Ag-specific nonresponsiveness and immunodominance. In order to demonstrate that competition for MHC binding alone can inhibit EAE, the inhibitor peptide should ideally be structurally unrelated and nonimmunogenic yet physically associate with the MHC class II molecule. In this study, we show that the OVA-323-339 peptide, which is unrelated to the disease-inducing peptide, binds to A alpha uA beta u. However, although OVA-323-339 is extremely immunogenic in A alpha dA beta d-expressing BALB/c mice, it is nonimmunogenic in (PL/J x SJL)F1 and PL/J mice expressing A alpha uA beta u. When administered as a coimmunogen with Ac1-11, OVA-323-339 inhibited induction of EAE in (PL/J x SJL)F1 mice. Myelin basic protein-89-101, which does not bind A alpha uA beta u, had no effect on the disease process. This study provides evidence that MHC class II binding alone can modulate the induction of EAE. The use of a nonimmunogenic non-self peptide to modulate an autoimmune disease minimizes the potential complications of immunodominance or alternative regulatory mechanisms associated with immunogenic peptide therapies and further confirms the MHC-blocking model of immunosuppression.

A small number of residues in the class II molecule I-Au confer the ability to bind the myelin basic protein peptide Ac1-11.

The N-terminal peptide Ac1-11 of myelin basic protein induces experimental autoimmune encephalomyelitis in H-2(u) and (H-2(u) x H-2(s)) mice but does not in H-2(s) mice. Ac1-11 binds weakly to the class II major histocompatibility complex (MHC) molecule I-Au but not at all to I-As. We have studied the interaction of Ac1-11 and I-Au as a model system for therapeutic intervention in the autoimmune response seen in experimental autoimmune encephalomyelitis. Two polymorphic residues that differ between I-Au and I-As, Y26beta and T28beta, and one conserved residue, E74beta, confer specific binding of Ac1-11 to I-Au. A fourth residue, R70beta in I-Au, affects both peptide binding and T cell recognition. These results are consistent with a model that places arginine at position five of Ac1-11 in pockets 4 and 7 of the MHC groove, which is formed in part by residues 26, 28, 70, and 74 of Abetau and places lysine at position four of Ac1-11, previously shown to be a major MHC contact, in hydrophobic pocket 6. The data indicate that the primary region of I-Au that confers specific binding of Ac1-11 lies in the center of the peptide binding groove rather than in the region that contacts the N terminus of the peptide, as has been shown for HLA DR and the homologous I-E molecules.

Impaired antigen presentation by murine I-Ad class II MHC molecules expressed in normal and HLA-DM-defective human B cell lines.

The inability of certain antigen processing mutant cell lines to present intact proteins to T cells and to form SDS-stable MHC class II dimers has been shown to result from defective expression of HLA-encoded DMA and DMB genes. We have utilized some of these mutants to determine species compatibility of antigen presentation components. Mouse MHC class II I-Ad cDNA was transfected into the human B cell lymphoblastoid cell lines 8.1.6, 7.9.6 (a mutant cell line derived from 8.1.6) and an independent deletion mutant T2 (called 8.1.6d, 7.9.6d and T2.d respectively). These cells were than examined for various functions in antigen presentation. Interestingly, none of the cells transfected with I-Ad presented peptides derived from intact proteins to specific T cell hybridomas. However, presentation of synthetic peptides by these cells was normal. The ability to form SDS-stable dimers was dramatically reduced in the transfectants. In addition, I-Ad molecules at the cell surface appeared loaded predominantly with the invariant chain peptides, CLIP. These properties of the I-Ad transfectants are identical to those described for HLA class II molecules expressed in HLA-DM mutants. Perhaps the most interesting finding was the inability of I-Ad in 8.1.6 to present protein antigens. Since 8.1.6 cells present antigens to HLA-DR, DP, DQ-restricted T cells and also have intact HLA-DM and invariant chain (II) functions, these results argue that some component of human antigen processing machinery is incompatible with I-Ad molecules.