Defective T helper cell epitope responsible for the failure of region 69-84 of the human myelin basic protein to induce experimental allergic encephalomyelitis in the Lewis rat.

Studies from our laboratory have shown that region 69-84 (synthetic peptide S49S) of myelin basic protein (MBP) defines an encephalitogenic sequence for experimental allergic encephalomyelitis (EAE) in Lewis rats. The most potent EAE inducers are the guinea pig MBP (Gp-MBP) and region 69-84, known as synthetic peptide Gp-S49S: (See text: formula). Human (H-MBP) was considerably less potent than Gp-MBP, and region 69-84 (H-S49S) of H-MBP did not induce hind leg paralysis or any histological signs of EAE. Since the development of EAE requires the expression of specific T and B cell epitopes, sequence analysis of H-S49S and Gp-S49S revealed phylogenetic variations in the H-S49S sequence, characterized by positions 77 and 78, and substitution of Ser with Thr at position 80: (See text: formula). Like Gp-S49S, peptide H-S49S induced the formation of antibodies with specificities directed against the C-terminal of the H-S49S, Gp-S49S, and homologous sequences. In contrast to Gp-S49S, neither II-S49S nor shorter peptides induced clonal T cell expansion when either of the peptides was added to encephalitogenic T cell clone D in culture. Clone D, which expresses T helper phenotype, was selected from encephalitogenic peptide-primed Lewis rats. The results of the study show that the failure of H-S49S to induce EAE is related to sequence alterations in the T helper cell epitope but not in the B cell epitope located in the N- and C-terminal portions of the S49S sequence, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of antibodies in T cell-mediated experimental allergic encephalomyelitis.

The role of the humoral phase of the immune response in development of T cell-mediated experimental allergic encephalomyelitis (EAE) had not been clearly defined previously even though studies of the myelin basic protein (MBP) molecule had demonstrated the presence not only of T cell but also B cell epitopes capable of inducing cell-mediated immunity and antibody formation. Particularly relevant to this report are the immunological expressions of the region which induces EAE in the Lewis rat. The development of primary demyelination in Lewis rats is preceded by a cell-mediated immune response as well as antibody formation, both of which are highly specific to the encephalitogenic 14-residue peptide that defines the 69-84 region of the parent MBP. Our results are consistent with the dogma that EAE is a T cell-mediated disease, but they also clearly demonstrate an important role for specific antibodies in the development of these T cells responsible for demyelination. The antibody response, which may be heteroclitic, is necessary for T cells to develop into an effector T cell subset. Without this B cell response the subsequent T cell response does not lead to demyelination. In this report we shall discuss these findings and further show that the T cell and B cell epitopes, which are located within the 14-residue sequence, are physically separated and dependent upon the form of synthetic peptides known to induce T cell-mediated and/or humoral immunity.(ABSTRACT TRUNCATED AT 250 WORDS)

A major epitope of synthetic rabbit encephalitogen S24 disclosed through the use of a tritiated peptide probe.

The antibody recognition of synthetic rabbit encephalitogen S24 (TTHYGSLPQKG), which had been inactivated by extensive iodination, was fully restored by tritium-iodine exchange during boron hydride reduction. A highly active form of 3H-S24 was obtained by reverse-phase high-performance liquid chromatography (HPLC) purification of the crude product and could be further separated into three continuous subfractions, the latter two of which were fully active from an immunochemical point of view. A major epitope was disclosed through use of the 3H-S24 probe, which required the intact S24 peptide for its expression. Confirmation of two linear epitopes previously encountered, GSLPQK and THYGSL, was also obtained, the latter through the use of the third HPLC subfraction of 3H-S24.

Synthetic peptide analogs to probe the immunological expression of the rat encephalitogenic neuropeptide.

The rationale for designing and synthesizing 24 different peptide analogs and subpeptides of residues 69-84 of guinea pig myelin basic protein is presented. The encephalitogenic potential of 19 of these peptides in Lewis rats is also given.

The polyclonal antibody responses of Lewis rats to the synthetic encephalitogenic neuropeptide S55S (residues 72-84 of guinea pig myelin basic protein) and its analogs.

Immunochemical analysis of polyclonal antisera from Lewis rats immunized with peptide analogs and subpeptides of residues 72-84 of guinea pig myelin basic protein (MBP) indicated that there were four main epitopic specificities involved: one represented by residues 76-80, one by residues 72-74, one by 81-84-Gly, and one involving 74-76 in a heteroclitic way even when the immunogen contained a tetraleucine spacer between residues 74 and 75. The affinities were all relatively low, ranging from 3 x 10(4) M-1 to 7.2 x 10(6) M-1, and each affinity population was very restricted with respect to heterogeneity. The results were commensurate with our hypothesis that autoimmune responses to homologous encephalitogenic neuropeptide determinants reflect an evolutionarily imposed degeneration of the overall response, and were also in agreement with our hypothesis that the affinities of autoantibodies against epitopes in the molecular neighborhood of an encephalitogen would be necessarily low. It was also found that peptide 69-81-Gly (S67) in solution would not interact in liquid phase radioimmunoassays with any of the above antibody populations, thus indicating that its preferred conformation did not allow the expression of autoreactive epitopes. There was no evidence that S67 in solution. even in the presence of S53, would express any epitopes reactive with autoantibodies. How this finding for a B cell product correlates with T cell-mediated immunologic responses is not totally understood, but it will be recalled that S67 by itself does not induce T cell-mediated experimental allergic encephalomyelitis (EAE), that S53 (residues 75-84-Gly) induces only clinical symptoms of EAE without attendant pathological disease, but that the two together induce classical EAE.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental allergic encephalomyelitis (EAE): role of B cell and T cell epitopes in the development of EAE in Lewis rats.

Studies from our laboratory have shown that classical clinical and histological signs of experimental allergic encephalomyelitis (EAE) may be induced in Lewis rats by synthetic peptides S49 or S55. Peptides S49S and S55S are defined by residues 69-84 and 72-84 of the guinea pig myelin basic protein (MBP), respectively. Peptide S53 (residues 75-84 of the guinea pig MBP), six residues shorter than S49S at the N-terminal end, induced mild clinical signs of disease unaccompanied by hind leg paralysis, incontinence, or central nervous system pathology. In contrast, peptide S67 (residues 69-81 of the guinea pig MBP), three residues shorter than S49S at the C-terminal end, did not induce either clinical or histological signs of EAE despite the fact that the S67-sequence houses an epitope known to induce cell-mediated immunity. Peptides S49S, S55S, and S53 are antigenic and gave rise to antibodies that recognized either of the three peptide sequences. In this report we explore the interrelationship between cellular immunity induced by the S67 sequence and humoral immunity, induced by the S53 sequence and the development of classical clinical and histological signs of EAE. The results show that the nonencephalitogenic sequence of S67 may be rendered encephalitogenic in the presence of antibody directed against the S53 sequence. Lewis rats immunized with S53 developed pathological signs of EAE only after they were challenged with S67. The fact that a simultaneous challenge with S67 and S53 was as effective in inducing EAE pathology as a delayed one (up to 40 days) suggests that the cellular response to S67 is dependent upon the humoral response to S53.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical specificity of antisera raised against the synthetic encephalitogenic peptide SH624, residues 59-74 of the myelin basic protein.

Synthetic peptide SH624 (SHHPARTAHYGSLPQK), residues 59-74 of human myelin basic protein (MBP) was found to be encephalitogenic in the rabbit. Four antisera raised, against the peptide were employed in a liquid-phase equilibrium competitive radioimmunoassay with a series of synthetic peptide analogs of the region to probe the structural requirements of the B-cell determinant subsumed within SH624. The cross-reactivities of the four antisera with intact MBP were also examined. Immunochemical analyses of the four antisera suggested specificities directed against a conformational determinant dependent upon residues from the more phylogenetically conserved carboxyl C-terminal region, residues 65-74 (TAHYGSLPQK) of the synthetic immunogen. Peptide analogs shorter than SH624 from the C-terminal end showed no cross-reactivity with any of the reagent antisera while analogs shorter from the N-terminal end and including the encephalitogenic sequence TTHYGSLPQK, as well as, HYGSLPQK were reactive under equilibrium competitive conditions. SH624-reactive antibodies, cross-reactive with purified heterologous MBPs from 10 different species were also identified in all four reagent antisera. The results of these experiments support previous investigations demonstrating the accessibility of the encephalitogenic 65-74 region in intact MBP. They also underscore the importance of B-cell recognition of organ specific antigenic determinants with respect to MBP immunology and, in particular, the recognition of autoreactive determinants in the neighborhood of encephalitogenic centers.

Polyclonal antibodies to the encephalitogenic neighborhoods of myelin basic protein: singular affinity populations neutralized by specific synthetic peptide probes.

Specific ligand neutralization was used to probe the extent to which singular antibody affinity populations signified specific determinants in the neighborhood myelin basic protein (MBP) encephalitogens. The probes were individual members of a panel of synthetic peptide analogs subsuming encephalitogenic regions. Comparative Scatchard analyses of neutralized and unneutralized antisera helped to identify the particular peptide determinants involved in the original polyclonal antibody responses to the multiple antigenic determinants of encephalitogenic peptides. The range of affinities for an antibody population against a singular MBP peptide determinant was found to be relatively restricted while the range of affinities overall for all populations within a given antipeptide antiserum was found to be relatively wide and invariably discontinuous. Consequently, the individual discontinuous affinity populations could readily be dissected by application of the Rosenthal method of Scatchard curve analysis. It was found that the singular high affinity antibody population (5.6 x 10(7) M-1) of a Lewis rat antiserum to rat encephalitogenic GSLPQKAQRPQDENG (S49) was against a determinant near the N-terminal non-encephalitogenic end of the peptide. Only the low affinity antibody populations were found that had reactivity for determinants within the encephalitogenic region itself. The singular high affinity antibody population (5.97 x 10(7) M-1) of a rabbit antiserum to rabbit encephalitogenic TTHYGSLPQKAQGHRPQDEG (S82) was against a determinant centered about the tyrosyl residue, within the encephalitogenic region for the rabbit, but was completely cross-reactive with a specific circulating endogenous inhibitor. The results obtained with the rat and rabbit EAE sera were consistent with a previously advanced hypothesis that antibodies to determinants within encephalitogenic neighborhoods would effectively block the onset of EAE if high enough in affinity and not neutralized by an endogenous inhibitor.

Heteroclitic antibodies in Fischer 344 rats to a synthetic encephalitogenic myelin basic protein peptide.

Fischer 344 rats, immunized with the synthetic encephalitogenic myelin basic protein peptide YS49 (YGSLPQKAQRPQDENG), produced heteroclitic antibodies that reacted much more extensively and with a much higher affinity with the cross-reacting encephalitogenic guinea pig sequence S49S (GSLPQKSQRSQDENG) than they did with the immunogenic YS49. On the other hand, antisera against S49S reacted in a normal manner with homologous S49S and cross-reacted only poorly with YS49. The phenomenon of heteroclisis in Fischer 344 rats correlated with the greater encephalitogenic potency of the cross-reacting entity. Kibler et al. (J. Exp. Med., 146 (1977) 1323-1331), by comparing the encephalitogenic guinea pig sequence to a less potent analog, had also previously observed what now would be termed a heteroclitic phenomenon at the T cell level in Lewis rats. In their hands, however, as well as in ours Lewis rat antisera against the encephalitogenic peptide region were much too complex to be analyzed with respect to heteroclisis. It was shown in the present experiments that by utilizing the Fischer 344 system one may also readily obtain heteroclisis at the B cell level against encephalitogenic peptides. Neither YS49 nor S49S as immunogen produced detectable antibody in Brown Norway (BN) rats with exception of two immunized with YS49. In those two cases heteroclitic antibodies were obtained that had a very low significant (greater than 3 SD above baseline) antigen binding capacity for S49S and no detectable reactivity for the homologous YS49 ligand.

Monoclonal and polyclonal antibodies to myelin basic protein determinants.

A detailed immunochemical examination of monoclonal and polyclonal antibody responses to myelin basic protein (MBP) and its peptides has revealed the existence of as many as 27 antigenic determinants, many of them conformational. Topological mapping of the potential antigenic determinants onto a model of MBP secondary structure places these determinants within 11 separate regions of the molecule, including those portions that have been found to be encephalitogenic. MBP and its peptides, therefore, fall under the umbrella of the Multideterminant-Regulatory Model of Benjamin et al. (1984). However, in the case of MBP, multideterminant immunogenicity appears to represent mainly an escape from tight regulation through the avenue of conformational change.

An immunochemical analysis of a myelin basic protein serum factor: cross reactivity with residues 69-71 of the rabbit encephalitogenic sequence 65-74 of myelin basic protein.

A partially purified myelin basic protein serum factor (MBP-SF), cross-reactive with residues 65-74 (TTHYGSLPQK) of myelin basic protein, has been employed in an immunochemical study to identify the nature of the cross-reacting determinant more precisely. To probe the structural requirements of this determinant, Scatchard inhibition analyses and competitive peptide inhibition radioimmunoassays were employed with a series of peptide analogs of the 65-74 region and with three different reagent antisera: a rabbit anti-rat myelin antiserum (#My05) and two antisera, one rabbit (#162) and one chicken (#66), raised against synthetic peptide S24 (TTHYGSLPQKG). Scatchard inhibition analyses with MBP-SF revealed specific inhibition of binding of 125I-S24 to #162 and #My05, but not to #66. Further delineation of the structural requirements of the cross-reactive determinant, employing a liquid-phase radioimmunoassay, revealed a unique reactivity pattern for the chicken anti-S24 antiserum which, unlike #162 and #My05, did not cross-react under high-affinity conditions with synthetic peptide S20 (GSLPQK, representing the C-terminal half of S24). This, in concert with the Scatchard data, is suggestive of the presence of a cross-reactive determinant centered around residues 69-71 of MBP.

Identification of an antigenic determinant within the phylogenetically conserved triprolyl region of myelin basic protein.

Synthetic peptide SP6 (RTPPPSG), comprising amino acid residues 98-103-Gly of myelin basic protein (MBP), and a series of peptide analogs were used to probe the structural requirements for antigenicity of a highly conserved region of a self protein. By means of a liquid-phase radioimmunoassay, antibody responses directed toward this determinant in both multi-specific anti-MBP and monospecific anti-peptide antisera were measured. The specificities of the antibodies present in the anti-MBP and anti-peptide antisera were examined by an equilibrium competitive inhibition radioimmunoassay by using the set of related peptides, as well as intact MBP from different species. Although the fine specificities of the reagent antisera differed, competitive inhibition analyses with intact MBP revealed a cross-reactive determinant involving residues 99-100 (Thr-Pro). This suggests that the neighborhood of the triprolyl region of MBP, despite its strong phylogenetic conservation, serves as an immunogen for humoral responses whether presented as a hapten-carrier conjugate or in the context of intact MBP. The latter supports the contention that the general antigenicity of a protein need not require sequence differences between the immunizing protein and its counterpart in the host.

Biological activity of region 65-102 of the myelin basic protein.

Region 65-102 of the myelin basic protein (MBP) houses a number of antigenic determinants known to induce delayed-type hypersensitivity, experimental allergic encephalomyelitis (EAE), suppressor cell function, and antibodies. In this report we describe the biological activity of synthetic peptides S53, S55, and S49 with sequence homology to region 69-84 of the rat, guinea pig, and bovine MBP. Peptide S53-A, defined by residues 75-84 of the guinea pig (SQRSQDEN) and of the rat (SQRTQDEN) MBP induced clinical signs of disease in Lewis rats. These included weight loss, flaccid tail, "muscle wasting," and hind-leg weakness. Histological examination of brain, spinal cord, and sciatic nerve sections of diseased rats revealed the complete absence of focal and perivascular lymphocytic infiltrates characteristics of demyelinating EAE lesions. Elongation of peptide S53 by three or six residues to residue sequences naturally found at its N-terminal end gave rise to peptides S55S (PQKSQRSQDEN) and S49S (GSLPQKSQRSDQDEN), respectively. Lewis rats challenged with either S55S or S49S developed classical clinical and histological signs of EAE. Severe hind-leg paralysis was accompanied by incontinence and sometimes death. Injected in the form of carrier-free peptide, S53 was a meager B cell immunogen. S53 conjugated with methylated-bovine serum albumin was also a potent immunogen and produced clinical signs of disease without CNS pathology. By comparison, carrier-free S55S and S49S were potent immunogens giving rise to antibodies that cross reacted completely and competitively with S55S but considerably less so with S53. The results show that the sequence of S53 defines an epitope responsible for the formation of anti-S53 antibodies. Elongation of the S53 sequence at its N-terminal end generated an additional epitope which induced cell-mediated immunity responsible for the concomitant development of pathological signs of EAE. It may be concluded that the induction of classical signs of EAE requires specific and defined sequences capable of expressing both B cell and T cell functions.

Immunochemical analysis of Lewis rat antisera to the synthetic encephalitogenic peptide S49.

Discrete populations of anti-S49 antibodies were found in the antisera of Lewis rats recovered from S49-induced experimental allergic encephalomyelitis (EAE). A potent inducer of EAE in Lewis rats, S49 is a synthetic peptide representing residues 69-84 of bovine myelin basic protein but with deletions at Gly-77 and His-78 to form an analogue of guinea pig or rat 69-84, GSLPQKAQRPQDENG. Each population within a given antiserum, as identified by Scatchard and Sipsian window analysis, was found to exhibit reactivity for a different S49 determinant, and the affinities of each population were relatively restricted and discontinuous. The high affinity populations (10(7)-10(8) M-1) were cross-reactive with YS8 (YGSLPQKAQGHRPQDENG) in equilibrium competitive inhibition reactions whereas the low affinity populations (10(5)-10(6) M-1) were reactive only with S49 and YS49 among a panel of peptide analogues. Of the YS8 cross-reactive antibodies the highest affinity (10(8) M-1) were also cross reactive with S81 (YGSLPQKAQGHRPQDEG) but not S49 (69-84-Gly), thus emphasizing the need for Tyr-68 for format stability of the determinant involved. The other YS8 cross-reactive population (10(7) M-1) was completely reactive with S49 but totally unreactive with S81 in equilibrium reactions, thus emphasizing the requirement for Asn-84 but not Tyr-68 for the determinant's topographic stability. Peptides shorter than S49 from the N-terminal end, but retaining the sequences AQRPQDEN or SQRSQDEN (suspected residence of minimal encephalitogenic determinants), reacted only under conditions of two-step non-equilibrium competitive inhibition assays. Such reactions would occur only at very low affinity (less than 10(5) M-1) with the anti-S49 antibodies. It was hypothesized that the encephalitogenic T-cell determinant for Lewis rats, although permitting B-cell responses at very low affinity, may exclude high affinity responses in susceptible animals.

Lipid-induced recognition of a conformational determinant (residues 65 to 83) in myelin basic protein.

The precipitation by antibodies to intact myelin basic protein (BP) and to synthetic peptides containing a sequence based on the region 65 to 83 of bovine BP, S82, S81, S79, and S24, of intact BP in solution or bound to lipid vesicles was compared, using 125I-BP or 14C-DPPC-labeled lipid-BP vesicles. The antipeptide antibodies were shown earlier to recognize conformational determinants which are not expressed in the intact protein in solution. Several anti-BP antibodies precipitated more of the BP free in solution than when bound to lipid vesicles, suggesting that some of the determinants recognized by these antibodies were either sequestered in the bilayer or were altered in conformation. In contrast, one anti-peptide antisera, which had a high titer for the conformational determinant in two of these peptides, S82 and S81, precipitated the protein to a significant degree when it was bound to PG vesicles, even though it did not react with the intact protein in solution. These results indicated that PG was able to confer on the protein the unique peptide conformation recognized by this antibody. PS was less effective, and other lipids were ineffective at conferring this conformation on the protein, supporting earlier results which showed that the conformation of the protein is influenced by the lipid composition of its environment. None of the other anti-peptide antibodies studied bound to the protein either in solution or in lipid vesicles. These results indicate that the lipid environment can sequester or alter the conformation of some antigenic determinants, preventing recognition by some anti-BP antibodies, and can expose or generate other conformational determinants, allowing recognition by an anti-peptide antiserum.

A serum factor cross-reactive with antibodies to a determinant of rabbit encephalitogenic sequence 65-74 of myelin basic protein.

A serum factor, cross-reactive with antibodies to a defined determinant of myelin basic protein (residues 66-71), has been found in the sera of nine mammalian species where it may function as a specific neuroautotolerogen. In equilibrium competitive inhibition radioimmunoassays the factor appears to be completely competitive with synthetic peptide S24 (TTHYGSLPQKG) at high affinity and is therefore termed MBP-SF-24 (myelin basic protein serum factor of the S24 type). The bulk of the activity can be recovered by ammonium sulfate fractionation at 61.1% saturated ammonium sulfate (SAS), pH 7, (fraction E) after removal by precipitation at pH 7 of the 37.5, 42.6, 47.5, and 51.4% SAS fractions (fractions A-D), including the immunoglobulins, and before removal by precipitation at pH 5 of the albumin fraction (fraction F). The factor, by its retention on XM300 during ultrafiltration of fraction E, can be purified 20-fold from serum proteins without much loss through a combination of SAS fractionation and ultrafiltration. The yield of MBP-SF-S24 in fraction E may range from a low 26 pmol S24 equivalents from 10 ml in sheep serum to a high 1.7 nmoles from 10 ml rat serum. The serum factor is reactive at high affinity with each of two populations of S24-reactive antibodies in one rabbit reagent antiserum and with one of two populations of S24-reactive antibodies in another. It appears to express a determinant involving residues THYGSL (66-71) of myelin basic protein with the same conformation as found in intact S24.

Format determinants of synthetic myelin basic protein peptide S82 mimicked by a mixture of synthetic peptides S8 and S79.

Antibodies to synthetic myelin basic protein peptide S82 (TTHYG-SLPQKAQGHRPQDEG) did not react with synthetic peptide S8 (GSLPQKAQGHRPQDENG) and only partially so with synthetic peptide S79 (AQGHRPQDEG); however, the antibodies did react to a considerable extent with an equimolar mixture of S8 and S79. Since the anti-S82 antibodies had previously been shown to be directed to a non-sequential format determinant dependent on the conformation of secondary structure, it seems probable that the mixture of S8 and S79 assumed a format that neither one individually possessed to any great degree.

Affinity purification of two populations of antibodies against format determinants of synthetic myelin basic protein peptide S82 from S82-AH- and S82-CH-Sepharose 4B columns.

Two different kinds of immunosorbents were prepared that contained the synthetic myelin basic protein didecapeptide S82 (TTHYGSLPQKAQGHRDQDEG)--one coupled with AH-Sepharose 4B through hexanoate spacers to the C-terminal glycyl residue; the other, with CH-Sepharose 4B through hexanoate spacers to the N-terminal threonine residue. An antiserum rich in antibodies to a format determinant of S82 was passed through each column, and, by means of affinity purification, two homogeneous populations of anti-format antibodies were obtained, each with a binding affinity of 1 X 10(8)M-1 for S82. The population recovered from S82-AH-Sepharose 4B cross-reacted to a considerable extent with synthetic peptide S8 (GSLPQKAQGHRPQDENG) but only to a limited extent with S79 (AQGHRPQDEG). The population recovered from S82-CH-Sepharose 4B cross-reacted poorly, if at all, with S8. An equimoler mixture of S8 + S79, however, reacted well with either population of anti-format antibodies, thus showing that the mixture could mimic the format of S82. It was concluded that secondary structural conformation of S82 could be preserved during the coupling procedure and that the resulting immunosorbents could be used for the affinity purification of anti-S82 antibodies to the format determinants.