Myelin Basic Protein Epitope Research Articles

Role of myelin basic protein epitope MBP74-85 in experimental autoimmune encephalomyelitis: Elaboration of agonist and antagonist motifs

Conformational properties of the myelin basic protein epitope QKSQRSQDENPV (MBP 74- 85 ) which can initiate experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis, were investigated by semiempirical methods. Energy calculations were carried out on the full MBP 74-85 autoantigen and the antagonist analog [Ala 81 ]MBP 74-85 . These studies have revealed a low energy cyclic conformation for MBP 74-85 which is characterized by an agonist motif comprising an interaction of the sidechains of Arg 78 and Asp 81 of MBP 74-85 . Disruption of this agonist motif by removal of the residue 81 carboxylate, as in the antagonist [Ala 81 ]MBP 74-85 , invokes a compensatory rearrangement of the molecule resulting in interaction of the Arg 78 sidechain with the sidechain of Glu 82 together with Lys 75 . This antagonist motif, comprising guanidino, amino, and carboxylate groups, has been reproduced previously in semimimetic peptides having the general structure Ser-Arg-LINKER-Glu-NH 2 (where LINKER = one or more residues of aminocaproic acid or isonipecotic acid), which have preferred conformations characterized by interaction of the carboxylate with both the guanidino and amino groups in a similar manner to the antagonist motif in [Ala 81 ]MBP 74-85 . However, in EAE assays these small semimimetics turned out to be partial agonists, i.e., molecules with structures between agonists and antagonists. These findings provide insight into the design of small molecule (orally active) autoantigen antagonists for the treatment of autoimmune diseases such as MS.

Tolerance and autoimmunity in TCR transgenic mice specific for myelin basic protein.

Summary: T‐cell receptor (TCR) transgenic mice provide the ability to follow the maturation and fate of T cells specific for self‐antigens in vivo. This technology represents a major breakthrough in the study of autoimmune diseases in which specific antigens have been implicated. Proteins expressed within the central nervous system are believed to be important autoantigens in multiple sclerosis, TCR transgenic models specific for myelin basic protein (MBP) allowed us to assess the role of tolerance in providing protection from T cells with this specificity Our studies demonstrate that T cells specific for the immunodominant epitope of MBP do not undergo tolerance in vivo and that TCR transgenic mice are susceptible to spontaneous autoimmune disease. The susceptibility to spontaneous disease is dependent on exposure to microbial antigens, MBP TCR transgenic models expressing TCRs specific for the same epitope of MBP but utilizing different Vα genes exhibit differing susceptibilities to, spontaneous disease. These data support the idea that genetic and environmental differences play a role in susceptibility to autoimmunity MBP TCR transgenic models are playing an important role in defining mechanisms by which infectious agents trigger autoimmune disease as well as defining mechanisms by which tolerance is induced to distinct epitopes within self‐antigens.

Highly Cross-Reactive T Cell Responses to Myelin Basic Protein Epitopes Reveal a Nonpredictable Form of TCR Degeneracy

We identified two nonoverlapping epitopes in myelin basic protein presented by I-Au that are responsible for mediating tolerance induction to this self-Ag. A large number of T cells expressing diverse TCRs are strongly cross-reactive to both epitopes. Surprisingly, the TCR contact residues in each peptide are highly dissimilar. Furthermore, functional TCR contacts cannot be interchanged between the two epitopes, indicating that the TCR contacts in each peptide can only be recognized within the context of the other amino acids present in that peptide's sequence. This observation indicates that both buried and exposed residues of each peptide contribute to the sculpting of completely distinct antigenic surfaces. We propose that the cross-reactive TCRs adopt mutually exclusive conformations to recognize these dissimilar epitopes, adding a new dimension to TCR degeneracy. This unpredictable TCR plasticity indicates that using just the TCR contacts on a single epitope to define other cross-reactive peptides will identify only a subset of the complete repertoire of cross-reactive epitopes.

A major influence of the T cell receptor repertoire as compared to antigen processing–presentation in the selection of myelin basic protein epitopes in multiple sclerosis

We selected two multiple sclerosis (MS) patients, compatible for HLA-DR2 subtype, and differing for HLA-DM haplotype as well as for the myelin basic protein (MBP) epitope recognized by the vast majority of their T cell lines (TCL) (residues 16–38 and 86–99, respectively). TCL sharing the same restriction element were re-assayed in the presence of reciprocally mismatched antigen-presenting cells (APC). The TCL recognized both the whole MBP and the relevant peptide also in the presence of non-autologous APC, (compatibility for processing, despite a difference in the DM haplotype). The same protocol, performed in serum-free pulsing experiments or in the presence of `fixed' APC, excluded extracellular processing or mutual T cell presentation, and confirmed the need for MBP processing in our system. The finding, that only TCL recognizing MBP peptide 16–38 (a region not previously related to the DR2 haplotype) used a novel Vβ, supports the importance of the TCR repertoire over the processing–presentation machinery in the selection of MBP epitopes in MS.

Microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis.

Molecular mimicry refers to structural homologies between a self-protein and a microbial protein. A major epitope of myelin basic protein (MBP), p87-99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE). VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex. Peptides from papilloma virus strains containing the motif VHFFK induce EAE. A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE. A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE. T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE. Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87-99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease.

Design and synthesis of a potent cyclic analogue of the myelin basic protein epitope MBP72-85: importance of the Ala81 carboxyl group and of a cyclic conformation for induction of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP), such as guinea pig sequence MBP72-85. Two linear and one cyclic analogues based on MBP72-85 have been synthesized and evaluated for EAE induction in Lewis rats. The linear peptide Gln1-Lys2-Ser3-Gln4-Arg5-Ser6-Gln7-+ ++Asp8-Glu9-Asn10-Pro11-Val12 (1) was found to induce EAE, while substitution of the Asp residue at position 8 with Ala resulted in an analogue (2) which suppressed the induction of EAE by its parent peptide. Nuclear magnetic resonance studies of analogue 1 in dimethyl sulfoxide (DMSO) using TOCSY/ROESY techniques revealed a head-to-tail intramolecular interaction (ROE connectivity between betaVal12-gammaGln1), indicating a pseudocyclic conformation for the immunogenic peptide 1. A conformational model was developed using NMR constraints and molecular dynamics. Based on this model, a novel amide-linked cyclic analogue has been designed and synthesized by connecting the side-chain amino and carboxyl groups of Lys and Glu at positions 2 and 9, respectively, of linear analogue 1. The cyclic analogue (3) had similar activity to the linear peptide 1, and the EAE effects induced by cyclic analogue 3 were completely suppressed by co-injection with the Ala81-substituted analogue 2 in Lewis rats. The similar potencies of analogues 1 and 3 support the proposed cyclic comformation suggested for analogue 1 from NMR studies and computer modeling and provides the basis for designing more potent molecules with improved properties such as increased resistance to degradation.15 The present findings suggest that a cyclic conformation for the MBP72-85 epitope positions the carboxyl group of Asp81 correctly and presumably other side groups of the peptide such as Arg78 in a manner which enables functional binding of the trimolecular complex MHC-peptide-T cell receptor resulting in EAE.

Diversity of the B Cell Repertoire to Myelin Basic Protein in Rat Strains Susceptible and Resistant to EAE

Myelin basic protein (MBP) is a major protein of central nervous system myelin which can induce experimental autoimmune encephalomyelitis (EAE) in susceptible laboratory animals. The role of T cells in the induction of EAE has been extensively studied, but the antibody response to MBP has not been well characterized. In the present work, we immunized rats with encephalitogenic guinea-pig MBP and mapped autoreactive antibodies binding to peptides in the rat MBP sequence. We studied the responses of the Lewis rat strain, susceptible to EAE, and the responses of the Fischer and Brown–Norway (BN) rats, resistant to EAE. We found that Lewis rats immunized to guinea-pig MBP develop antibodies to a diversity of MBP epitopes with a dominance of MBP peptide p11–30 and peptides in the 71–140 region. Fischer rats showed a similar pattern of antibody specificities, but with higher titers than the Lewis rats. BN rats, in contrast, developed a very low titer of antibodies and lacked a response to p11–30. Thus, there is no clear correlation between the nature of the anti-MBP antibody response and the state of susceptibility or resistance to EAE induction in the different rat strains.

Copolymer 1 acts against the immunodominant epitope 82-100 of myelin basic protein by T cell receptor antagonism in addition to major histocompatibility complex blocking.

The synthetic random amino acid copolymer Copolymer 1 (Cop 1, Copaxone, glatiramer acetate) suppresses experimental autoimmune encephalomyelitis, slows the progression of disability, and reduces relapse rate in multiple sclerosis (MS). Cop 1 binds to various class II major histocompatibility complex (MHC) molecules and inhibits the T cell responses to several myelin antigens. In this study we attempted to find out whether, in addition to MHC blocking, Cop 1, which is immunologically cross-reactive with myelin basic protein (MBP), inhibits the response to this autoantigen by T cell receptor (TCR) antagonism. Two experimental systems, "prepulse assay" and "split APC assay," were used to discriminate between competition for MHC molecules and TCR antagonism. The results in both systems using T cell lines/clones from mouse and human origin indicated that Cop 1 is a TCR antagonist of the 82-100 epitope of MBP. In contrast to the broad specificity of the MHC blocking induced by Cop 1, its TCR antagonistic activity was restricted to the 82-100 determinant of MBP and could not be demonstrated for proteolipid protein peptide or even for other MBP epitopes. Yet, it was shown for all the MBP 82-100-specific T cell lines/clones tested that were derived from mice as well as from an MS patient. The ability of Cop 1 to act as altered peptide and induce TCR antagonistic effect on the MBP p82-100 immunodominant determinant response elucidates further the mechanism of Cop 1 therapeutic activity in experimental autoimmune encephalomyelitis and MS.

Design and synthesis of small semi-mimetic peptides with immunomodulatory activity based on myelin basic protein (MBP).

Experimental allergic encephalomyelitis (EAE) is induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP). Analogs of these disease-associated peptides have been identified with disease progression upon coimmunization. Usage of peptides, with disease-specific immunomodulatory capacity in vivo is limited, however, due to their sensitivity to proteolytic enzymes. Alternative approaches include the development of mimetic molecules which maintain the biological function of an original peptide, yet are stable and able to elicit their response in pharmacological quantities. A novel technique was employed to design a series of semi-mimetic peptides, based on the guinea pig MBP72-85 peptide used to induce EAE in Lewis rats. We used isonipecotic (iNip) and aminocaproic (Acp) acids as templates. Acp-MBP72-85 peptide derived analogues were effective in inducing EAE compared to iNip-peptide analogues which were ineffective at 350 micrograms. These findings suggest that the design and synthesis of semi-mimetic peptide molecules with immunomodulatory potential is possible and that eventually these molecules may form the basis for the development of novel and more effective disease-specific therapeutic agents.

Neutralizing Antibodies to IFN-γ-Inducing Factor Prevent Experimental Autoimmune Encephalomyelitis

AbstractSpecific oligonucleotide primers were used to identify and isolate IFN-γ-inducing factor (IGIF) from the brain of rats with developing experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as a model for multiple sclerosis. IGIF was highly transcribed in the brain at the onset and during the course of active EAE. PCR products encoding rat IGIF were used to generate the recombinant protein that was used to induce anti-IGIF neutralizing Abs. These Abs significantly reduced the production of IFN-γ by primed T cells proliferating in response to their target myelin basic protein epitope and by Con A-activated T cells from naive donors. When administered to rats during the development of either active or transferred EAE, these Abs significantly blocked the development of disease. Splenic T cells from protected rats were cultured with the encephalitogenic myelin basic protein epitope and evaluated for production of IL-4 and IFN-γ. These cells, which proliferated, exhibited a profound increase in IL-4 production that was accompanied by a significant decrease in IFN-γ and TNF-α production. Thus, we suggest that perturbation of the Th1/Th2 balance toward Th2 cells is the mechanism underlying EAE blockade by anti-IGIF immunotherapy.

Astrocytes Express Elements of the Class II Endocytic Pathway and Process Central Nervous System Autoantigen for Presentation to Encephalitogenic T Cells

Astrocytes are nonprofessional APCs that may participate in Ag presentation and activation of pathogenic CD4+ T cells involved in central nervous system (CNS) inflammatory diseases. Using immortalized pure astrocytes as a complement to the study of primary astrocytes, we investigated whether these astrocytes express elements involved in the class II endocytic pathway and if they are capable of processing native myelin basic protein (MBP), a step that could be necessary for initiating or perpetuating T cell recognition of this self-Ag in vivo. Upon IFN-gamma-stimulation, primary and immortalized astrocytes up-regulate class II transactivator (CIITA), invariant chain (Ii) (p31 and p41), H-2Ma, and H-2Mb. Analysis of CIITA cDNA sequences demonstrated that CIITA transcription in astrocytes is directed by a promoter (type IV) that mediates IFN-gamma-inducible CIITA expression and encodes a CIITA protein that differs in its N-terminal sequence from CIITA reported in professional APC. Comparing live and fixed APC for Ag presentation, we show that Ag processing by APC is required for presentation of native MBP to autopathogenic T cells specific for the major MBP epitope, Acl-11. We have observed that primary astrocytes and some, but not all, astrocyte lines in the absence of contaminating microglia are capable of processing and presenting native MBP, suggesting that there may be heterogeneity. Our study provides definitive evidence that astrocytes are capable of processing CNS autoantigen, indicating that astrocytes have potential for processing and presentation of CNS autoantigen to proinflammatory T cells in CNS autoimmune disease.

Treatment of murine experimental autoimmune encephalomyelitis with a myelin basic protein peptide analog alters the cellular composition of leukocytes infiltrating the cerebrospinal fluid

Experimental autoimmune encephalomyelitis (EAE) can be effectively treated during disease exacerbation by administration of a peptide corresponding to the major T cell epitope of myelin basic protein (MBP), but the mechanism by which T cell tolerance leads to clinical improvement is not well-defined. Acute exacerbations of EAE are accompanied by an infiltration of blood-borne leukocytes into the brain and spinal cord, where they mediate inflammation and demyelination. To investigate peptide effects on infiltrating cells, we collected cerebrospinal fluid (CSF) from (PL/JxSJL)F 1 mice with MBP-induced EAE. Pleiocytosis by lymphocytes, neutrophils, and macrophages was seen throughout the course of relapsing–remitting disease. A single administration of the MBP peptide analog, Ac1-11[4Y], reduced disease severity, accompanied by a dramatic and selective loss of neutrophil pleiocytosis. A longer course of peptide therapy resulted in complete recovery from clinical signs of disease, and decreased pleiocytosis by all cell types. Clinical severity throughout the course of disease and therapy was directly related to the degree of infiltration by neutrophils and macrophages, and the clinical improvement following peptide therapy was accompanied by decreased central nervous system (CNS) expression of chemoattractants for these cell types. These observations support a model of disease exacerbation mediated by phagocytic cellular infiltration under the ultimate control of T cell-derived factors, amenable to treatment by down-regulation of the T cell activation state.

Fine Specificity of the Myelin-Reactive T Cell Repertoire: Implications for TCR Antagonism in Autoimmunity

The use of altered peptide ligands (APL) to modulate T cell responses has been suggested as a means of treating T cell-mediated autoimmune disorders. We have assessed the therapeutic potential of TCR antagonist peptides in autoimmunity using murine experimental autoimmune encephalomyelitis (EAE) as a model. The Tg4 transgenic mouse expresses an MHC class II-restricted TCR specific for the immunodominant encephalitogenic epitope of myelin basic protein, Ac1-9 (acetylated N-terminal nonamer). We have used T cell lines derived from Tg4 mice to define the TCR contact residues within Ac1-9. APL with appropriate substitutions at the primary TCR contact residue were effective antagonists of Tg4 T cells. These antagonist APL, however, were found to induce EAE in susceptible, nontransgenic strains of mice. Underlying this, the Ac1-9-specific T cell repertoire of normal mice, rather than reflecting the Tg4 phenotype, showed considerable diversity in fine specificity and was able to respond to the Tg4 antagonist APL. Defining antagonist APL in vitro using T cell clones, therefore, was not a reliable approach for the identification of APL with EAE-suppressing potential in vivo. Our findings highlight the complexities of the autoreactive T cell repertoire and have major implications for the use of APL in autoimmune diseases.

Astrocytes express elements of the class II endocytic pathway and process central nervous system autoantigen for presentation to encephalitogenic T cells

Astrocytes are nonprofessional APCs that may participate in Ag presentation and activation of pathogenic CD4 1 T cells involved in central nervous system (CNS) inflammatory diseases. Using immortalized pure astrocytes as a complement to the study of primary astrocytes, we investigated whether these astrocytes express elements involved in the class II endocytic pathway and if they are capable of processing native myelin basic protein (MBP), a step that could be necessary for initiating or perpetuating T cell recognition of this self-Ag in vivo. Upon IFN-g-stimulation, primary and immortalized astrocytes up-regulate class II transactivator (CIITA), invariant chain (Ii) (p31 and p41), H-2Ma, and H-2Mb. Analysis of CIITA cDNA sequences demonstrated that CIITA transcription in astrocytes is directed by a promoter (type IV) that mediates IFN-g-inducible CIITA expression and encodes a CIITA protein that differs in its N-terminal sequence from CIITA reported in professional APC. Comparing live and fixed APC for Ag presentation, we show that Ag processing by APC is required for presentation of native MBP to autopathogenic T cells specific for the major MBP epitope, Ac1-11. We have observed that primary astrocytes and some, but not all, astrocyte lines in the absence of contaminating microglia are capable of processing and presenting native MBP, suggesting that there may be heterogeneity. Our study provides definitive evidence that astrocytes are capable of processing CNS autoantigen, indicating that astrocytes have potential for processing and presentation of CNS autoantigen to proinflammatory T cells in CNS autoimmune disease. The Journal of Immunology, 1998, 161: 5959 ‐5966.

Myelin protein expression in lymphoid tissues: implications for peripheral tolerance.

During chronic relapsing experimental autoimmune encephalomyelitis (EAE), T lymphocytes specific for myelin protein epitopes are stimulated in vivo. When epitopes are unique from the disease-initiating myelin protein epitope, this phenomenon has been termed "epitope spreading". These T-lymphocyte responses have been detected primarily in lymph node and spleen during the relapsing phase of disease. If myelin proteins are sequestered behind the blood brain barrier, a fundamental question arises: where does the in vivo stimulation of T lymphocytes occur during relapsing EAE? While it has been thought that epitope spreading may occur within the central nervous system (CNS), here we present data supporting a novel hypothesis. Epitope spreading during EAE may not occur within the CNS, but rather within lymphoid tissues. Both myelin basic protein (MBP) and proteolipid protein (PLP) are expressed at the RNA and protein level in lymph node, thymus and spleen of SJL mice with relapsing EAE. This myelin protein expression occurs within T lymphocytes, B lymphocytes and macrophages. Further, T-lymphocyte lines from SJL mice specific for the immunodominant and subdominant epitopes of MBP and PLP can recognize endogenous protein within cells derived from lymphoid tissues. Thus, immunologically relevant myelin proteins are endogenously produced and presented within lymphoid tissues. The hypothesis that epitope spreading occurs within lymphoid tissues would explain how myelin protein-specific T lymphocytes become activated outside the CNS to allow their passage through the blood brain barrier to form new CNS lesions during relapses.

Differential recognition of MBP epitopes in BALB/c mice determines the site of inflammatory disease induction

Although myelin basic protein (MBP)-recognizing T cells are not readily obtained after immunization of BALB/c mice with MBP (reflecting the BALB/c resistance to actively induced experimental autoimmune encephalomyelitis (EAE)), they can be expanded and cloned after several rounds of in vitro culture. The majority of BALB/c-derived clones recognize an epitope defined by MBP peptide 59–76. When transferred to naive BALB/c recipients, these clones cause classical EAE, with characteristic inflammation and demyelination of the central nervous system (CNS). We previously showed that two related clones recognizing a minor epitope, defined by MBP peptide 151–168, cause inflammation and demyelination preferentially of the peripheral nervous system (PNS). Because MBP has alternatively spliced isoforms, residues 151–168 are not present contiguously in all MBP isoforms. In order to determine whether induction of PNS disease is idiosyncratic to these sister clones, or related to their properties of epitope recognition, an independent T-cell line with similar recognition properties was studied. Clone 116F, derived from a BALB/c shiverer mouse, expresses a different T-cell receptor (TCR), with distinct TCR contact residues, but like the previously described T cells, this clone requires residues from both exons 6 and 7 for optimal stimulation. When adoptively transferred to BALB/c recipients, this clone preferentially induces disease of the PNS. A control BALB/c shiverer-derived MBP 59–76-recognizing clone, in contrast, induces CNS disease. These data strongly suggest that the site of disease initiation may correlate with epitope recognition, particularly when alternative isoforms are involved.

Delineation of two encephalitogenic myelin basic protein epitopes for DA rats

We studied synthetic peptides that correspond to two regions of the guinea pig myelin basic protein (MBP) molecule which elicit experimental autoimmune encephalomyelitis (EAE) in DA rats. Using truncated peptides, we determined that two encephalitogenic epitopes reside within MBP63–81, a major determinant defined by MBP residues, 63–76, and a minor encephalitogenic epitope defined by residues, 66–81. Experiments with alanine-substituted analogs of MBP63–76 revealed that the HYGSLP sequence is critical for encephalitogenicity. The core epitope within a second encephalitogenic region, MBP101–120, was defined by residues, 106–119. Studies with analogs of this sequence indicated that residues, Leu 111, Phe 114 and Trp 116 are important for T-cell responsiveness.

Design and synthesis of potent cyclic analogues and mimetics of myelin basic protein epitope MBP87-99 for suppression of experimental allergic encephalomyelitis (EAE)

Design and synthesis of potent cyclic analogues and mimetics of myelin basic protein epitope MBP87-99 for suppression of experimental allergic encephalomyelitis (EAE)

Short Peptide-Based Tolerogens Without Self-Antigenic or Pathogenic Activity Reverse Autoimmune Disease

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in brain lesions of multiple sclerosis (MS), and this peptide can trigger experimental autoimmune encephalomyelitis (EAE). We designed truncated peptides based on this pathogenic 13-mer that are not antigenic. These short peptides reduced production of IFN-gamma and TNF-alpha in vivo. Moreover, paraplegic rats given the 7-mer FKNIVTP in soluble form showed total reversal of paralysis in 24 h. Truncated peptides that are too small to stimulate antigenic responses to pathogenic regions of myelin basic protein are nevertheless effective tolerogens and are able to anergize autoreactive T cells. Short peptide-based tolerogens, devoid of immunogenic and pathogenic potential, may be attractive for therapy of autoimmune diseases.

Differential Tolerance Is Induced in T Cells Recognizing Distinct Epitopes of Myelin Basic Protein

Experimental allergic encephalomyelitis (EAE) is induced by T cell–mediated immunity to central nervous system antigens. In H-2 u mice, EAE is mediated primarily by T cells specific for residues 1–11 of myelin basic protein (MBP). We demonstrate that differential tolerance to MBP1-11 versus epitopes in MBP121-150 is induced by expression of endogenous MBP, reflecting extreme differences in stability of peptide/MHC complexes. The diverse MBP121-150–specific TCR repertoire can be divided into three fine specificity groups. Two groups were identified in wild-type mice despite extensive tolerance, but the third group was not detected. Activated MBP121-150–specific T cells induce EAE in wild-type mice. Thus, encephalitogenic T cells that escape tolerance either recognize short-lived peptide/MHC complexes or express TCRs with unique specificities for stable complexes.