Autoaggressive T lymphocyte lines recognizing the encephalitogenic region of myelin basic protein: in vitro selection from unprimed rat T lymphocyte populations

Autoimmune T lymphocyte lines have been established from unprimed normal rat lymph node cell populations. In a first, negative-selection round, spontaneously proliferating (SMLR) T cells were eliminated by a pulse of BUdR followed by short wave light irradiation. In a second, positive-selection round, the SMLR-depleted populations were confronted with MBP presented by syngeneic spleen adherent cells. Reactive T cells were propagated until stable, permanent T lines were established. All lines were exclusively specific for the selecting antigen, MBP, and were restricted in recognition by determinants of the own MHC. All lines expressed the differentiation marker W3/25, but not OX8. Line vLe, which was derived from Lewis (LEW) rat lymphocytes, and which recognized the encephalitogenic sequence 48-88 of MBP, was extremely efficient in mediating EAE to normal untreated LEW rats. Doses of 1 X 10(6) and greater transferred lethal EAE, whereas transient although definite disease was caused by a minimum of 1 X 10(4) cells. Rats recovering from disease were resistant against subsequent active induction of EAE. In contrast, BN rat-derived line vBN was completely incapable of transferring EAE to syngeneic rats. This lack of encephalitogenicity was a property of the T line, because vLe cells transferred severe EAE to (LEW X BN)F1 hybrid rats, whereas none of hybrid rats injected with vBN cells showed any sign of disease. The data provide strong evidence in favor of the presence of potentially autoaggressive T clones in the normal immune system, and they might suggest that the actual proportion of these clones within the natural T cell repertoire is genetically determined.     

Specificity of zinc binding to myelin basic protein

Z²⁺ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of⁶⁵Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.     

Phosphorylation of myelin basic protein and peptides by ganglioside-stimulated protein kinase

Rabbit myelin basic protein (MBP) was phosphorylated by a ganglioside-stimulated protein kinase to a stoichiometry of 1.4 and 2.1 mol phosphate/mol MBP in the presence and absence of GTlb, respectively. Two-dimensional peptide mapping analyses revealed that two of the sites of phosphorylation were distinct from those catalyzed by cAMP-dependent protein kinase or protein kinase C. Phosphorylation of one of these sites by ganglioside-stimulated protein kinase was inhibited by GTlb, suggesting that the inhibitory effect of gangliosides on MBP phosphorylation may be substrate-directed. Although ganglioside-stimulated protein kinase did not phosphorylate MBP at a domain containing residues 82-117, a synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys corresponding to residues 111-120 was phosphorylated by the kinase in a ganglioside-stimulated manner. These findings suggest that the conformation of MBP may be important in determining its phosphorylatability.     

A common epitope on human myelin basic protein and the human T lymphocyte CD3 molecule

In this report, we describe for the first time an epitope common to human myelin basic protein (H.MBP), a structural component of central nervous system myelin, and T lymphocyte CD3, an activation molecule important in signal transduction. This cross-reactive determinant was recognized by a murine mAb WW.B1, which was raised against H.MBP. WW.B1 recognized PBMC and the Jurkat T leukemic cell line, immunoprecipitated both H.MBP and a complex indistinguishable from CD3, and possessed the same biologic properties--induction of T lymphocyte proliferation and inhibition of CTL function--as commercially available anti-CD3 antibodies. It is likely, however, that the epitope recognized by WW.B1 is distinct from those recognized by the anti-CD3 mAb OKT3 and anti-Leu-4. Although the biologic importance of this common determinant awaits further clarification, it is conceivable that autoimmunization to MBP could induce similar immunoregulatory antibody specificities.     

A myelin basic protein-specific T lymphocyte line that mediates experimental autoimmune encephalomyelitis

A T lymphocyte line, BP-1, expressing the T helper phenotype was selected from Lewis rats immunized with guinea pig myelin basic protein (GP-BP) in complete Freund's adjuvant (CFA). The BP-1 line responded specifically to GP-BP but not to PPD after the first round of selection, and responded to rat but not human or bovine BP. When injected i.p. into histocompatible Lewis or F1 (Lewis X P2) recipients, the BP-1 line induced both clinical signs of experimental autoimmune encephalomyelitis (EAE) and delayed type hypersensitivity (DTH) reactions in ears challenged intradermally with GP-BP but not PPD. The severity of clinical signs and the degree of ear swelling were dependent on the dose of BP-1 cells injected. Both activities were detectable with as few as 0.1 X 10(6) BP-1 line cells and required prior activation of the line cells with GP-BP presented by accessory cells. Lewis rats that had recovered from EAE induced by injection of GP-BP in CFA were more susceptible than naive rats to BP-1 line-mediated disease, requiring as few as 0.03 X 10(6) line cells. Clinical EAE and DTH could be serially transferred into F1 (Lewis X P2) recipients with BP-1 cells and back to nonirradiated Lewis parents with activated splenocytes, suggesting that BP-1 cells persist in recipient rats. These results demonstrate the potent biologic activities of an autoreactive BP-specific T lymphocyte line. This line possesses properties similar to BP lines described previously as well as to culture-conditioned splenic T effector cells; thus, the data presented here bridge the gap between these two approaches for studying T effector lymphocyte functions.     

Cell-mediated immunoreactivity of tumor-bearing mice with myelin basic protein and related peptides

Spleen cells (SC) from tumor-bearing mice and mice immunized with porcine myelin basic protein (MBP) reacted in vitro in E-rosette augmentation assays with MBP and certain of its constituent peptides. Peptides 1-115, 43-169, 64-83, 113-121, and 153-161 reacted significantly with both types of SC, while peptide 1-19 reacted only with SC from MBP-immunized mice. The phenomenon of specific inhibition of peptide reactivity by a moderate excess of a related protein was used to identify peptides as accessible epitopes of that protein. Peptide 113-121 was specifically inhibited by excess MBP when reacted with both types of SC, whereas peptide 64-83 was inhibited by excess MBP only when reacted with SC from MBP-immunized mice. These reactions suggest that the immunizing antigen in tumor-bearing mice is related to MBP but differs in epitopes associated with peptides 1-19 and 64-83.     

Enzymic O-glycosylation of synthetic peptides from sequences in basic myelin protein.

Nine synthetic peptides containing sequences in the region of a threonine residue at position 98 of bovine basic myelin protein were prepared by the Merrifield solid-phase method and tested for their ability to be glycosylated with [14C]uridinediphospho-N-acetylgalactosamine and a crude detergent-solubilized preparation of uridinediphospho-N-acetylgalactosamine:mucin polypeptide N-acetylgalactosaminyltransferase obtained from porcine submaxillary glands. The tetrapeptide Thr-Pro-Pro-Pro and all larger peptides containing this sequence were glycosylated. The glycosylation was greater for peptides containing residues N-terminal to the Thr-Pro-Pro-Pro. Under the conditions used, the peptide Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro was glycoslyated twice as much as bovine basic myelin protein. Thr-Pro and Thr-Pro-Pro, as well as 10 other synthetic peptides which did not contain the Thr-Pro-Pro-Pro sequence, were not glycosylated. Treatment of the glycopeptide of Phe-Lys-Asn-Leu-Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro-Ser with an alpha-N-acetylgalactosaminidase released N-acetylgalactosamine from the peptide, indicating that the hexosamine was covalently bonded to the peptide in an alpha linkage.     

Autoimmune encephalomyelitis (EAE) mediated or prevented by T lymphocyte lines directed against diverse antigenic determinants of myelin basic protein. Vaccination is determinant specific

Lines of T lymphocytes reactive against the basic protein of myelin (BP) were found in previous studies to mediate experimental autoimmune encephalomyelitis (EAE) in rats. Moreover, inoculation of rats with attenuated anti-BP line cells vaccinated them against subsequent attempts to induce active EAE by injection of BP in adjuvant. In the present study, we investigated the effects of T lymphocyte lines reactive to different antigenic determinants on the BP molecule, they are the major encephalitogenic peptide (EP) determinant present on guinea pig BP (G-BP), and minor, non-EP determinants present on bovine BP (B-BP). We found that both lines of T lymphocytes could mediate EAE. Resistance to active EAE acquired by spontaneous recovery from line mediated EAE or by vaccination with attenuated cells, however, was found to be specific for the particular BP determinant. Thus, EAE may be mediated by lines of T lymphocytes reactive to different determinants on the BP molecule, but the resistance to EAE acquired by exposure to line cells is determinant specific. This suggests that acquired resistance to EAE is directed by the receptor specificity of the autoimmune anti-BP T cells.     

Preparation and properties of monoclonal antibodies to myelin basic protein and its peptides

Techniques are described which have enabled the production and characterisation of monoclonal antibodies to myelin basic protein. These are shown by enzyme immunoassay to react with six different epitopes. Two of these are to peptide 82–91, a region claimed to be present in the spinal fluid of patients with demyelinating disease. One of these, an IgG_2a, is shown to react only with peptides in which the 91–92 phe-phe bond has broken. The other, an IgM, also reacts with whole myelin basic protein. The IgG_2a antibody is shown to have an affinity suitable for use in immunoassay of peptide 82–91. The enzyme immunoassay procedures described help to minimise the work load involved in the preparation and characterisation of monoclonal antibodies to this protein.     

On the spontaneous adherence of myelin basic protein to T lymphocytes.

We have applied a double tagging system in order to study whether purified myelin basic protein is able to adhere to normal human peripheral T lymphocytes without the need to purify cells. Evaluation of myelin basic protein adherence to peripheral blood mononuclear cells was determined with biotinylated myelin basic protein and fluoresceinated avidin, and lymphocyte population was identified by the corresponding phycoerythrinated monoclonal antibody. The observed adherence of myelin basic protein to T lymphocytes was found to depend on protein conformation.     

An inhibition enzyme immunoassay for myelin basic protein

An improved Enzyme Immunoassay for Myelin Basic Protein is described. Myelin Basic Protein covalently attached to glass balls, and Myelin Basic Protein in samples compete with each other for binding of a peroxidase conjugated anti Myelin Basic Protein antibody. The peroxidase activity on the balls is then inversely proportional to the amount of Myelin Basic Protein in the sample. A detection limit of 0.6 ng/ml is demonstrated for diluent or spinal fluid. For plasma a dilution step increases this to 1.8 ng/ml. Both the coated balls and the peroxidase conjugate are stable for long periods. The assay requires no expensive equipment. Although the assay appears to be valid for subcellular fractions spinal fluid and plasma, successful detection of Myelin Basic Protection peptides in clinical samples may require careful selection of suitable antisera. The assay would be very suitable for eventual use with an appropriate monoclonal antibody.     

D-aspartic acid in purified myelin and myelin basic protein

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.     

Recognition and degradation of myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis

The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant "epitope library" covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48-70 and 85-170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43-68 and 146-170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81-103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.     

Experimental autoimmune encephalomyelitis mediated by T lymphocyte lines: genotype of antigen-presenting cells influences immunodominant epitope of basic protein

Lewis rats are susceptible to experimental autoimmune encephalomyelitis (EAE), and their T lymphocytes recognize epitopes in the 68-88 sequence of guinea pig myelin basic protein (BP). BN rats are resistant to EAE, and their T lymphocytes recognize epitopes outside of the 68-88 sequence, probably in the 43-67 portion of BP. To investigate the influence of the genome of antigen-presenting cells (APC) on the dominance of BP epitopes for T lymphocyte lines, we selected anti-BP lines from (Lewis X BN)F1 rats by using the APC of Lewis, BN, or F1 origin. We now report that the F1/Lewis and F1/F1 lines recognized the 68-88 epitopes and were highly encephalitogenic in F1 rats, whereas the F1/BN line recognized the 43-67 epitopes and was only weakly encephalitogenic. Thus, the genotype of the APC can influence the immunologic dominance for T lymphocytes of BP epitopes, and this dominance in turn can influence the expression of disease.     

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used conventional strategies in our search for a suitable composition or a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization-homogeneity of tertiary structure-is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.