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The multiple sclerosis (MS)-associated HLA major histocompatibility complex (MHC) class II alleles DRB1*1501, DRB5*0101 and DQB1*0602 are in strong linkage disequilibrium, making it difficult to determine which is the principal MS risk gene. Here we show that together the DRB1 and DRB5 loci may influence susceptibility to MS. We demonstrate that a T cell receptor (TCR) from an MS patient recognized both a DRB1*1501-restricted myelin basic protein (MBP) and DRB5*0101-restricted Epstein-Barr virus (EBV) peptide. Crystal structure determination of the DRB5*0101-EBV peptide complex revealed a marked degree of structural equivalence to the DRB1*1501-MBP peptide complex at the surface presented for TCR recognition. This provides structural evidence for molecular mimicry involving HLA molecules. The structural details suggest an explanation for the preponderance of MHC class II associations in HLA-associated diseases.

Susceptibility to multiple sclerosis (MS) is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 haplotype, suggesting that major histocompatibility complex class II-restricted presentation of central nervous system-derived antigens is important in the disease process. Antibodies specific for defined HLA-DR2-peptide complexes may therefore be valuable tools for studying antigen presentation in MS. We have used phage display technology to select HLA-DR2-peptide-specific antibodies from HLA-DR2-transgenic mice immunized with HLA-DR2 molecules complexed with an immunodominant myelin basic protein (MBP) peptide (residues 85-99). Detailed characterization of one clone (MK16) demonstrated that both DR2 and the MBP peptide were required for recognition. Furthermore, MK16 labeled intra- and extracellular HLA-DR2-MBP peptide complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBP-specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85-99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2-MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.

Monovalent major histocompatibility complex-peptide complexes dissociate within seconds from the T-cell receptor (TCR), indicating that dimerization/multimerization may be important during early stages of T-cell activation. Soluble bivalent HLA-DR2.myelin basic protein (MBP) peptide complexes were expressed by replacing the F(ab) arms of an IgG2a antibody with HLA-DR2.MBP peptide complexes. The binding of bivalent HLA-DR2.peptide complexes to recombinant TCR was examined by surface plasmon resonance. The bivalent nature greatly enhanced TCR binding and slowed dissociation from the TCR, with a t((1)/(2)) of 2.1 to 4.6 min. Soluble bivalent HLA-DR2.MBP peptide complexes activated antigen-specific T-cells in the absence of antigen presenting cells. In contrast, soluble antibodies to the TCR.CD3 complex were ineffective, indicating that they failed to induce an active TCR dimer. TCR/CD3 antibodies induced T-cell proliferation when bound by antigen presenting cells that expressed Fc receptors. In the presence of dendritic cells, bivalent HLA-DR2. MBP peptide complexes induced T-cell activation at >100-fold lower concentrations than TCR/CD3 antibodies and were also superior to peptide or antigen. These results demonstrate that bivalent HLA-DR. peptide complexes represent effective ligands for activation of the TCR. The data support a role for TCR dimerization in early TCR signaling and kinetic proofreading.

Susceptibility to multiple sclerosis is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 (DRB1*1501) haplotype. The structure of HLA-DR2 was determined with a bound peptide from human myelin basic protein (MBP) that is immunodominant for human MBP-specific T cells. Residues of MBP peptide that are important for T cell receptor recognition are prominent, solvent exposed residues in the crystal structure. A distinguishing feature of the HLA-DR2 peptide binding site is a large, primarily hydrophobic P4 pocket that accommodates a phenylalanine of the MBP peptide. The necessary space for this aromatic side chain is created by an alanine at the polymorphic DRbeta 71 position. These features make the P4 pocket of HLA-DR2 distinct from DR molecules associated with other autoimmune diseases.

HLA-DR2 is associated with susceptibility to multiple sclerosis (MS). A peptide from human myelin basic protein (MBP, residues 85-99) was previously found to bind to purified HLA-DR2 (DRA, DRB1*1501) and to be recognized by human MBP-specific T cell clones. Soluble HLA-DR2 was expressed in the baculovirus system by replacing the hydrophobic transmembrane regions and cytoplasmic segments of DRalpha and DRbeta with leucine zipper dimerization domains from the transcription factors Fos and Jun. In the expression construct, the MBP(85-99) sequence was covalently linked to the N terminus of the mature DRbeta chain. The recombinant protein was secreted by Sf9 cells infected with the recombinant baculovirus and purified by affinity chromatography. The leucine zipper dimerization domains were then cleaved from the assembled HLA-DR2/MBP peptide complex with V8 protease, and the protein was further purified by anion-exchange HPLC. Analysis by HPLC gel filtration indicated that the HLA-DR2/MBP peptide complex did not have a tendency to aggregate. The purified HLA-DR2/MBP peptide complex readily crystallized by the hanging drop method in 15-18% polyethylene glycol 6000/100 mM glycine, pH 3.5. At a synchrotron radiation source, a crystal with a tetragonal space group diffracted to a resolution of 2.6 A. The expression of such homogenous HLA-DR/peptide complexes may facilitate cocrystallization with T cell receptors as well as other molecules involved in T cell receptor recognition and signaling.

Peptide-based tolerance induction may be useful for antigen-specific immunotherapy of human autoimmune diseases. Induction of tolerance to myelin basic protein (MBP) was examined in a Phase I clinical trial in multiple sclerosis (MS) patients with chronic progressive disease using a peptide that is immunodominant for MBP specific T cells and B cells. Tolerance induction was monitored by quantification of MBP specific autoantibodies in cerebrospinal fluid (CSF). The route of peptide administration was important since only intravenous but not intrathecal or subcutaneous injection induced tolerance to MBP. Following a single intravenous injection of a peptide containing epitope P85VVHFFKNIVTP96, MBP autoantibodies were undetectable for three to four months. Tolerance was more prolonged following a second injection since autoantibodies were low or undetectable after one year in the majority of patients. Duration of tolerance to MBP depended on MHC class II haplotypes of patients; tolerance was long-lived in all patients with disease associated HLA-DR2. No neurological or systemic side effects were observed, regardless of the route of peptide administration. These data demonstrate that intravenous administration of a soluble peptide can result in long-lasting tolerance to an autoantigen in humans.

Myelin basic protein (MBP) may be an important autoantigen in multiple sclerosis (MS), with the MBP(82-100) region being immunodominant for T cells and autoantibodies. The structural requirements for autoantibody recognition were compared to those previously defined for MBP-specific T cell clones. MBP autoantibodies were affinity-purified from central nervous system lesions of 11/12 postmortem cases studied. The MBP(83-97) peptide was immunodominant in all 11 cases since it inhibited autoantibody binding to MBP > 95%. Residues contributing to autoantibody binding were located in a 10-amino acid segment (V86-T95) that also contained the MHC/T cell receptor contact residues of the T cell epitope. In the epitope center, the same residues were important for antibody binding and T cell recognition. Based on the antibody-binding motif, microbial peptides were identified that were bound by purified autoantibodies. Autoantibody binding of microbial peptides required sequence identity at four or five contiguous residues in the epitope center. Microbial peptides previously found to activate T cell clones did not have such obvious homology to MBP since sequence identity was not required at MHC contacts. The similar fine specificity of B cells and T cells may be useful for tolerance induction to MBP in MS.

T-cell receptors (TCRs) recognize peptide bound within the relatively conserved structural framework of major histocompatibility complex (MHC) class I or class II molecules but can discriminate between closely related MHC molecules. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes. Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction--i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but a similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP-(85-99)-specific TCRs therefore appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.

Understanding the immune response to myelin antigens in regard to the peptide/MHC/TCR complex is important in defining pathogenesis of demyelinating autoimmune diseases and in developing antigen-specific therapies. We previously reported that individual multiple sclerosis patients may use certain dominant TCR V beta chains to recognize immunodominant MBP peptides. In examining the TCR beta chain usage, we observed repeated TCR VDJ sequences among different T-cell lines isolated from the same patient. This suggested that a few expanded T-cell clones may dominate the immune response to immunodominant MBP peptides. Here, we report experiments where TCR rearrangements were used as a probe for the clonal origin of MBP specific T-cells cultured from blood lymphocytes of MS patients and normal subjects. In two patients with the DR2 haplotype that were analyzed in detail, the T-cell response to MBP was focused on the MBP (84-102) peptide and in vivo expanded population(s) dominated the response to the MBP (84-102) peptide. Two MBP (84-102) specific T-cell clones from a normal subject with the DR2 haplotype were also found to have identical TCR sequences. Clonality was proven by demonstrating that independent clones had identical TCR alpha and beta chain sequences as well as identical sequences of a TCR gamma chain or of a second TCR alpha chain rearrangement. These data suggest that the response to human MBP is dominated in at least some subjects by expanded clones that may persist in vivo for relatively long periods of time.

Structural similarity between viral T cell epitopes and self-peptides could lead to the induction of an autoaggressive T cell response. Based on the structural requirements for both MHC class II binding and TCR recognition of an immunodominant myelin basic protein (MBP) peptide, criteria for a data base search were developed in which the degeneracy of amino acid side chains required for MHC class II binding and the conservation of those required for T cell activation were considered. A panel of 129 peptides that matched the molecular mimicry motif was tested on seven MBP-specific T cell clones from multiple sclerosis patients. Seven viral and one bacterial peptide efficiently activated three of these clones. Only one peptide could have been identified as a molecular mimic by sequence alignment. The observation that a single T cell receptor can recognize quite distinct but structurally related peptides from multiple pathogens has important implications for understanding the pathogenesis of autoimmunity.

TCR rearrangements were used to probe the clonal origin of myelin basic protein (MBP)-reactive T cells from patients with multiple sclerosis (n = 7) and normal subjects (n = 3). The majority of MBP-specific T cell lines were specific for the immunodominant MBP(84-102) and MBP(143-168) peptides and were restricted by HLA-DR molecules. In two patients with the DR2 haplotype, the T cell response to MBP was focused on the MBP(84-102) peptide. In both patients, in vivo expanded population(s) (three expanded populations in the first patient, one expanded population in the second patient) dominated the response to the MBP(84-102) peptide. Two MBP(84-102)-specific T cell clones from a normal subject with the DR2 haplotype were also found to have identical TCR sequences. Clonality was proven by demonstrating that independent clones had identical TCR alpha- and TCR beta-chain sequences as well as identical sequences of a TCR gamma-chain or of a second TCR alpha-chain rearrangement. Repeated analysis of one patient after 13 mo demonstrated that the three expanded clones had persisted in vivo. A representative of one of the expanded clones was again obtained after 31 mo by IL-2 stimulation suggesting that this clone was activated in vivo. These data suggest that the response to human MBP is dominated in at least some subjects by expanded clones that may persist in vivo for relatively long periods of time.

Immunodominant T cell epitopes of myelin basic protein (MBP) may be target antigens for major histocompatibility complex class II-restricted, autoreactive T cells in multiple sclerosis (MS). Since susceptibility to MS is associated with the DR2 haplotype, the binding and presentation of the immunodominant MBP(84-102) peptide by DR2 antigens were examined. The immunodominant MBP(84-102) peptide was found to bind with high affinity to DRB1*1501 and DRB5*0101 molecules of the disease-associated DR2 haplotype. Overlapping but distinct peptide segments were critical for binding to these molecules; hydrophobic residues (Val189 and Phe92) in the MBP(88-95) segment were critical for peptide binding to DRB1*1501 molecules, whereas hydrophobic and charged residues (Phe92, Lys93) in the MBP(89-101/102) sequence contributed to DRB5*0101 binding. The different registers for peptide binding made different peptide side chains available for interaction with the T cell receptor. Although the peptide was bound with high affinity by both DRB1 and DRB5 molecules, only DRB1 (DRB1*1501 and 1602) but not DRB5 molecules served as restriction elements for a panel of T cell clones generated from two MS patients suggesting that the complex of MBP(84-102) and DRB1 molecules is more immunogenic for MBP reactive T cells. The minimal MBP peptide epitope for several T cell clones and the residues important for binding to DRB1*1501 molecules and for T cell stimulation have been defined.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system which has been hypothesized to be autoimmune in nature. To test whether this is the case, Kai Wucherpfennig and colleagues have developed a set of criteria that must be met to satisfy the hypothesis. Here, they present these criteria and assess the extent to which studies to date satisfy them.

Multiple sclerosis (MS) may be an autoimmune disease mediated by T cells specific for a myelin protein. Investigations have demonstrated myelin basic protein (MBP)-reactive T cells that were activated in vivo in MS patients, suggesting that MBP may be a target antigen in MS. The variable (V) region of the T cell receptor (TCR) beta chain was examined among 83 T cell lines from both MS patients and healthy subjects that were reactive with the immunodominant region of human MBP (residues 84 to 102) or with a second immunodominant region of MBP (143 to 168). V beta 17 and to a lesser extent V beta 12 were frequently used in recognition of MBP(84-102) among different individuals. In contrast, V beta 17 was very infrequent among lines reactive with MBP (143-168). These data demonstrate shared TCR V beta gene usage for the recognition of immunodominant regions of the human autoantigen MBP. Such TCR structures may be used as targets for specific immunotherapy in MS.